WO1999019347A1 - Synthetic genes with immunomodulatory effects - Google Patents

Synthetic genes with immunomodulatory effects Download PDF

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Publication number
WO1999019347A1
WO1999019347A1 PCT/SE1998/001801 SE9801801W WO9919347A1 WO 1999019347 A1 WO1999019347 A1 WO 1999019347A1 SE 9801801 W SE9801801 W SE 9801801W WO 9919347 A1 WO9919347 A1 WO 9919347A1
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Prior art keywords
pro
cys
leu
gly
seq
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PCT/SE1998/001801
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French (fr)
Inventor
Håkan BERGSTRAND
Tomas Eriksson
Magnus Lindvall
Bengt Särnstrand
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Astrazeneca Ab
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Priority to AU96556/98A priority Critical patent/AU9655698A/en
Publication of WO1999019347A1 publication Critical patent/WO1999019347A1/en

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    • 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
    • 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.
  • 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. Where global stimulation of the immune system is desired, this can sometimes be achieved with nonspecific agents such as adjuvants, interleukins, interferons, and colony-stimulating factors.
  • the immune system loses its ability to distinguish self from non-self.
  • the individual may develop an autoimmune disease such as systemic lupus erythrematosis, Type I diabetes, or rheumatoid arthritis.
  • autoimmune disease such as systemic lupus erythrematosis, Type I diabetes, or rheumatoid arthritis.
  • the immune 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.
  • 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 protein targeting sequence.
  • the DNA molecule of the invention encodes an immunoactive 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, He, Gly, Asp, Glu, Asn, Gin,
  • Y is selected from the group consisting Ala, Val, Leu, He, Gly, Ser, Thr, Asp, Glu,
  • n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-six, inclusive; where the formula I immunoactive peptide is linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide.
  • a protein targeting sequence e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence
  • a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
  • protein targeting sequence is meant any amino acid sequence which directs a polypeptide to which it is linked to a specific cellular compartment. Such compartments include, but are not limited to, the extracellular space, endoplasmic reticulum, mitochondria, nucleus, and plasma membrane (e.g., via a transmembrane sequence or a GPI linkage).
  • GPI attachment sequences are described in Crise et al., J Virol 63:5328- 5333 (1989) and Salzwedel et al., J Virol 67:5279-5288 (1993).
  • a "glycosyl- phosphatidylinositol attachment signal” is meant an amino acid sequence within a polypeptide which leads to a covalent bond between the polypeptide and aglycolipid on the surface of a lipid bilayer: e.g., a cell membrane.
  • the nucleic acid can further encode a cytokine such as an interferon or interleukin; e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • a cytokine such as an interferon or interleukin; e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • X is Gly, Pro, He, Val, Asp, Leu, Glu, Gin, or Ala
  • Y is Gly, Pro, He, Val,
  • n and m are zero to eleven, inclusive.
  • X is Gly and Y is Gly
  • X is Pro and Y is Pro, X is Pro and Y is Val,
  • X is Pro and Y is Glu
  • X is Glu and Y is Tyr
  • X is Pro and Y is Phe, X is Glu and Y is Phe,
  • X is Ala and Y is Val
  • X is Gin and Y is Ser
  • X is He and Y is Thr, X is Leu and Y is Asp, or X is Asp and Y is He; 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 immunoactive peptide, and a protein targeting sequence.
  • immunoactive peptides of Formula I include the following:
  • A is preferably Gly, Lys, Arg, Cys, Ser, Val, Ala, Thr, Glu, Pro, Trp, Leu, Asp, Phe, or He; B is Leu, Arg, He, 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.
  • A can be Pro, Gly, Glu, Ala, Val, Lys, Thr, Leu, or Ser; B can be Tyr, Pro, Gly, Thr, Arg, Val, Ala, Leu, Lys, or He; n is one; and m is one.
  • the DNA molecule of the invention may encode an immunoactive 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: A n -X-Cys-Z-Cys-Y-B m (Formula II) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids; X is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Asn, Gin,
  • Z is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Lys, His. Phe, Tyr, Arg, and Pro;
  • Y is selected from the group consisting of Ala, Val, Leu, He, 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; the formula II immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide.
  • a protein targeting sequence e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence
  • a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
  • the nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • X is Gly, Pro, He, Val, Asp, Leu, Glu, Gin, or Ala
  • Y is selected from the group consisting of Gly, Glu, Val, Gin, Arg, Leu, Tyr, Phe, He, Ser, Thr, Asp, and Pro
  • Z is selected from the group consisting of He, Gly, Thr, Ala, Arg, and Lys
  • the sum of n and m is zero to ten, inclusive. More preferably,
  • X is Gly and Y is Gly
  • X is Pro and Y is Pro
  • X is He and Y is Leu, X is Pro and Y is Glu,
  • X is Glu and Y is Tyr, X is Pro and Y is Phe,
  • X is Glu and Y is Phe
  • X is Ala and Y is Val
  • X is Val and Y is He, X is Gin and Y is Ser,
  • X is Leu and Y is Asp, or
  • X is Asp and Y is He; Z is He, Gly, Thr, Ala, or Lys; A and B vary according to the parameters above; and the sum of n and m is zero to ten, inclusive.
  • Examples of such peptides of Formula II include the following:
  • Ala-Cys-Lys-Cys-Gln (SEQ ID NO.:26), and Gly-Pro-Cys-Ile-Cys-Pro-Gly (SEQ ID NO.:27).
  • X is Val, Ala, Leu, He, Lys, Asp, Phe or Pro
  • Y is Glu, Val, Gin, Arg, Lys, or Pro
  • Z is Gly, Ala, He, Arg, Thr, or Lys
  • the sum of n and m is one to three, inclusive.
  • the DNA molecule of the invention may encode an immunoactive peptide consisting of 4-30 amino acid residues that conforms to the motif represented by Formula III:
  • each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
  • X is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Asp, Glu, Lys, Arg, His, Trp, Tyr, and Phe;
  • Y is selected from the group consisting of Ala, Val, Leu, He, 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; the formula III immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide.
  • a protein targeting sequence e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence
  • a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
  • the nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, inter leukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, inter leukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • 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
  • the sum of n and m is zero to eleven, inclusive.
  • 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.
  • Examples of such peptides of Formula III include the following:
  • Trp-Pro-Cys-Gly SEQ ID NO.:31
  • Gly-Pro-Cys-Ile-Leu-Asn SEQ ID NO.:32
  • the nucleic acid molecule of the invention may encode, for example, a peptide where X is Gly, Ala, He, Arg, Asp, T ⁇ , 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: A n -X p -Y-Cys-Z q -B m (Formula IV) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
  • 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, Phe, and Gin;
  • n, m, 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 immunoactive peptide of formula IV being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide.
  • a protein targeting sequence e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence
  • a signal peptide is linked to the N-terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
  • the nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • 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 4-10 amino acid residues). Most preferably, the peptide consists of 4-7 amino acid residues.
  • X s Gly, Y is Pro, and Z is Met, X s Ala, Y is Pro, and Z is T ⁇ , X s Ala, Y is Pro, and Z is Met, X s Glu, Y is Pro, and Z is T ⁇ , X s Ser, Y is Pro, and Z is T ⁇ , X s Leu, Y is Leu, and Z is Gly, X s Pro, Y is Arg, and Z is Arg, X s Gly, Y is Tyr, and Z is Pro, X s Val, Y is Val, and Z is Asn, X s Leu, Y is Ser, and Z is Gin, X s Ser, Y is Pro, and Z is Tyr, X s Ala, Y is Leu, and Z is Arg, X s Ala, Y is Pro, and Z is Tyr, X s Gly, Y is Ala, and Z is Pro, X s Lys, Y
  • 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
  • any amino acid residue of A adjacent to Y is not Ser, Gly, Ala, or Thr.
  • peptides of Formula IV include the following:
  • 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, T ⁇ , 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, T ⁇ , Glu, Leu, Met, and Lys;
  • 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 formula V immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a GPI attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide.
  • a protein targeting sequence e.g., a signal peptide, a GPI attachment signal, or a transmembrane sequence
  • a signal peptide is linked to the N-terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
  • the nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • a cytokine such as an interferon or interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
  • 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 immunoactive peptide conforms to the motif of Formula V, wherein W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, T ⁇ , 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. More preferably, at least one of X and Y is Met.
  • the immunoactive peptide conforms to the motif of Formula V, wherein W is selected from the group consisting of Gly, Asp, Arg, Ala, T ⁇ , 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 immunoactive peptide conforms to the motif of Formula V, wherein W is selected from the group consisting of Gly and Ser;
  • Z is selected from the group consisting of Phe, He, and Pro.
  • the immunoactive peptide may have Met and Cys or Met and Met aligned contiguously.
  • the peptide may conform in sequence to A-W-Met-Met-Z-B,
  • the peptide may contain Met and Cys, separated by no more than one amino acid.
  • the peptide may conform in sequence to A-Met-X-Cys-Z-B or
  • Lys-Met-Arg-Met-Lys (SEQ ED NO.:l 15), Phe-Met-Ile-Met-Lys (SEQ ID NO.:l 16),
  • Val-Ile-Met-Met-Leu-Thr (SEQ ID NO.: 125), Leu- Ala-Phe-Glu-Pro-Met-Met (SEQ ID NO. : 126) ,
  • Lys-Met-Arg-Met-Lys (SEQ ID NO.: 179).
  • Val-Gly-Glu-Ser-Phe-Thr (SEQ ID NO.: 149),
  • Asp-Pro-Cys-Ile-Ile (SEQ ID NO.: 163), Cys-Gly-Gly-Ile-Cys-Ile-Ala-Arg (SEQ ED NO.: 164), Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQID NO.: 165), Cys-His-Gly-Ser-Asp-Pro-Cys (SEQ ED NO.: 166), Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ED NO.: 167),
  • Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 168), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO. 168), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO. 168), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO. 168), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.
  • Tyr-Arg-Arg-Gly-Arg-Cys-Gly-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.: 170), Tyr-Arg-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 170), Tyr-Arg-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 170), Tyr-Arg-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly
  • 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 polypeptide linked to a protein targeting sequence. Of course, multiple coding sequences can be linked in tandem, separated by stop codons and potentially other noncoding sequence.
  • the polypeptides encoded by the nucleic acids of this invention may include a protein targeting sequence which targets the immunoactive peptide to a specific cellular compartment.
  • a signal peptide can be linked to the immunoactive 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.
  • 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 tissue- or cell-specific expression of one or more immunoactive peptides linked to a protein targeting sequence encoded on the nucleic acid molecule. 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 linked to a protein targeting sequence.
  • 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 vitro 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. If a GPI attachment signal is used, the polypeptide can be harvested from the medium only after cleavage with an enzyme such as phospholipase C.
  • an immunoactive peptide may be produced in a mammal (e.g., a human, non-human primate, 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 (e.g., a plasmid or virus) containing the nucleic acid molecule of the invention, or (3) a cell that contains and expresses the nucleic acid. In the latter case, a cell removed from the subject, or a descendant of such a cell, would be transduced with the nucleic acid ex vivo. Such cells (particularly mammalian cells such as human cells) are considered to be within the invention.
  • a mammal e.g., a human, non-human primate, mouse, rat, guinea pig, hamster, rabbit, dog, cat, cow, pig, goat, sheep or horse
  • Non-integrating viral vectors include he ⁇ es 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.
  • Other 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, "naked DNA,” 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 polypeptide 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 or expresses on its surface the immunoactive peptide.
  • cells of the patient could be transduced ex vivo by standard techniques, such as those described herein.
  • 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 clinical practitioners. 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.
  • the method for modulating the immune response in a patient further includes administration, including oral administration, of a cytokine such as an inteferon or an interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12 (see Rollwagen et al., Immunology Today 17:548-550 [1996] andHsieh et al., Science 260:547-549 [1993]).
  • a cytokine such as an inteferon or an interleukin: e.g., interferon- ⁇ , interferon- ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12 (see Rollwagen et al., Immunology Today 17:548-550 [1996] andHsieh et al., Science
  • 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, Sj ⁇ gren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome, orBehcet'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 hematopoietic system. Accordingly, a patient's cancer can be treated by any standard therapy known in the art, combined with administration to the patient of any of the above described nucleic acids.
  • Such standard therapies include surgery (e.g., removal of part or all of a solid tumor), exposure of the patient or a specific region or tissue of the patient to a therapeutic amount of ionizing radiation (e.g, ⁇ -particles, ⁇ -particles.
  • anticancer chemotherapy i.e., administration of one or more of the following cytotoxic chemotherapeutics: mechlorethamine, cyclophosphamide, ifosfamide, chlorambucil, melphalan, bursulfan, thiotepa, carmustine, lomustine, streptozocin, vincristine, vinblastine, paclitaxel, vinorelbine, docetaxel, methotrexate, mercaptopurine, thioguanine, fluorouracil, cytarabine, azacitidine, fludarabine, cladribine, and pentostatin).
  • Standard cancer therapies are described in Dale et al., Scientific American Medicine, Chapter 12: Oncology (1996).
  • nucleic acid molecule of the invention in the preparation of a medicament useful in treating any of the above conditions.
  • peptide 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 (Gin; Q), L-tyrosine (Tyr; Y), L-tryptophan (T
  • nucleic acid encompasses both RNA and DNA.
  • the nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand.
  • synthetic gene refers to a coding sequence that is not identical to any naturally-occurring, full length coding sequence. It can be operably linked to expression control elements. All publications, patents, and other references cited herein are inco ⁇ orated by reference in their entirety.
  • FIG. 1 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, IH, IV, or V, and (b) a protein targeting sequence that targets the peptide to a specific cellular compartment (i.e., the extracellular space) and a eukaryotic expression control sequence.
  • a signal peptide was used below as an example protein targeting sequence, other protein targeting sequences known in the art can be used, where targeting to an intracellular rather than extracellular compartment is desired. 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, BamHI, 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.
  • 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
  • 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. 1) 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-XhoI, (3) EcoRI-Hpal, (4) Hpal- BamHI, (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. Once the presence and orientation of the insert was confirmed by sequencing, large scale cultures were established in LB medium supplemented with ampicillin (50 ⁇ g/ml) and grown until the OD at 600 nm was 0.8.
  • PCR polymerase chain reaction
  • Chloramphenicol was then added to a final concentration of 180 ⁇ g/ml, and the flasks were incubated at 37°C, on a shaker, overnight.
  • the DNA constructs were isolated from the bacterial cultures using a QIAGEN aPlasimd kit (QIAGEN, GmbH, Hilden, Germany), according to the manufacturer's instructions.
  • 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 (Lindvall et al., 1991, Cancer Immunol. Immunother. 33:21-27). The tumors were kept in serial passage in syngeneic rats.
  • 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 1-glutamine, 1 mM pyruvate, 10 mM HEPES buffer, 10 mM NaHCO 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.
  • PCR polymerase chain reaction
  • an in vitro biological assay of the effect on proliferation of activated T lymphocytes of supernatant from SPMWl cells which had been transduced with a nucleic acid of the invention is consistent with the conclusion that the immunoactive peptide A (Met-Lys-Phe-Leu-Ser-Ala-Arg-Asp-Phe-His-Pro-Val-Ala-Phe-Leu-Gly-Leu-Met-Leu- Val-Thr-Thr-Thr-Thr-Ala-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg [SEQ ED NO:57]) is expressed and secreted by the transduced cells, and overcomes the suppressive effect of wild type SPMWl cells on T lymphocyte proliferation. Implantation of Transduced Cells
  • 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 ⁇ l 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.
  • V volume (in mm 3 )
  • a the largest diameter (in mm)
  • b the diameter pe ⁇ endicular to the largest diameter (in mm).
  • Example 1 Immunostimulation by Peptide A-expressing SPMWl Cells SPMWl mammary carcinoma cells in cell culture were infected with a retroviral vector containing an insert encoding the immunoactive peptide A. 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. As a control, 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.
  • Peptide A-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 peptide A-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 peptide A-expressing SPMWl cells involves a T cell-mediated immune response.
  • Ad9-101 mammary carcinoma cells in cell culture were infected with a retroviral vector containing an insert encoding the immunoactive peptide A. 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 peptide A-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 peptide A significantly impedes tumor growth in at least two model systems.
  • T lymphocytes are activated in response to in vivo expression of an immunoactive peptide
  • Rats were injected subcutaneously with either wild type Ad9- 101 cells or peptide A-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 10 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. 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 NaHCO 3 , 50 ⁇ M ⁇ -mercaptoethanol, and 10% FCS.
  • the cells On the fifth day in culture, the cells were exposed to [ 3 H]-thymidine (0.5 ⁇ Ci) for 6 hours. Although other cell types, such as monocytes, natural killer cells, and B lymphocytes are present, under the conditions of the culture, only T lymphocytes can respond with mitotic activity on day 5.
  • the radioactivity inco ⁇ orated into acid-insoluble material which reflects the mitotic activity of the cells in culture, was measured with a scintillation counter. The amount of radioactivity inco ⁇ orated into homogeneously cultured lymph cells was subtracted from the amount of radioactivity inco ⁇ orated into heterogeneous cultures containing lymphatic and irradiated tumor cells.
  • rats were inoculated with either Ad9-101 wild type cells that had been irradiated with 10,000 rad (100 Gray), or clone 9 cells that had been similarly irradiated. (Irradiation prevents the cells from dividing more than once, but it does not immediately affect protein synthesis.) Animals received two subcutaneous injections of irradiated cells (1 x 10 6 cells/animal/dose), 14 days apart, while a group of control animals received comparable injections of phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Example 7 Immunomodulation by Peptide B-expressing. Peptide C-expressing, or
  • Ad9-101 Cells To compare the effects of three different immunoactive peptides on tumor growth, rats were inoculated with transduced Ad9-101 cells that expressed either peptide B (Met-T ⁇ - Phe-Leu-He-Leu-Phe-Leu-Ala-Leu-Ser-Leu-Gly-Gln-Ile- Asp-Ala- Ala-Pro-Gly-Cys-Cys- Pro-Gly [SEQ ID NO: 60]), peptide C (Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met- Leu-Leu-Val-Ala-Leu-Ser-Pro-Cys-Leu-Pro-Cys-Arg-Ala-Leu-Leu-Phe-Gly-Pro-Cys-Ile [SEQ ID NO:58]), or peptide D (Met-Arg-Leu-Arg-Leu-Leu-Val-Ser
  • the immunoactive peptide B (without signal sequence) has previously been shown to be immunostimulatory when administered directly in a delayed-type hypersensitivity (DTH) assay, while peptide C and peptide D (each without signal sequence) have shown activity consistent with immunosuppression.
  • DTH delayed-type hypersensitivity
  • peptide C and peptide D each without signal sequence
  • Approximately 20,000 transduced cells were injected subcutaneously into each rat (5 animals/group), and the tumors which developed were measured.
  • uncloned cells expressing peptide B formed tumors that grew somewhat more slowly than those which developed from wild type Ad9-101 cells, consistent with the immunostimulatory activity of this peptide observed in the DTH test.
  • the tumors that developed from uncloned cells expressing either peptide D or peptide C grew faster than the tumors that developed from wild type Ad9-101 cells (at least after day 22), consistent with the immunosuppressant activity of these peptides in the DTH test.
  • the delayed-type hypersensitivity assay described below is a useful predictor of the immunomodulatory effect of peptides expressed from the nucleic acid molecules of the invention.
  • Example 8 Inoculation of Various Clones of Peptide B- expressing Cells
  • the tumors that developed from uncloned peptide B-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).
  • the following assays can be used to identify immunostimulatory peptides linked to protein targeting sequences that can be delivered by gene therapy in accordance with the invention.
  • 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 asBalb/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 vanLoveren et al. (1984, J. Immunol. Methods 67:311), and expressed according to the formula: T ⁇ s - T t o (in ⁇ m 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 linked to a protein targeting sequence, and transplanted into a test animal.
  • Cells cultured in parallel, but untransduced or transduced only with the "empty" vector or a vector encoding a non-immunoactive peptide linked to the same protein targeting sequence, would be transplanted into control animals.
  • the encoded polypeptide would be deemed an effective immunostimulant.
  • the transduced cells could be irradiated and mixed with non-irradiated wild type cells prior to injection, so that the effect of the polypeptide on growth of wild type tumor cells in vivo would be measured.
  • Model systems developed from different sources of immortalized cells may provide the means to determine whether polypeptides are broadly effective or particularly effective in treating a certain type of cancer.
  • Genes encoding polypeptides 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 polypeptide 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 polypeptide-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 polypeptide-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 polypeptide-encoding vector or with polypeptide-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 linked to protein targeting sequences in the treatment of cancer.
  • Polypeptides that are ineffective in impeding tumor outgrowth, or that enhance tumor outgrowth, could be tested further, as described below, in order to determine whether they would be useful immunosuppressants.
  • polypeptide In order to determine whether a polypeptide 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 polypeptide, 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, B 10.BR, and B 10.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.
  • 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 polypeptide would be considered a successful immunosuppressant if it prolongs the time the grafted organ was tolerated by the host.
  • the effectiveness of an immunoactive peptide linked to a protein targeting sequence 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 polypeptides in vivo. These models are well known to skilled artisans and can be used to determine whether a given polypeptide 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 synthetic genes encoding immunoactive peptides linked to protein targeting sequences 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 10 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 polypeptide of interest, and the gene encoding it, function as an immunosuppressant potentially useful in treating autoimmune disease.
  • polypeptide-encoding vector e.g., anadenoviral vector
  • anadenoviral vector 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. These rats spontaneously develop autoantibodies against pancreatic islet cells and insulin, just as occurs in human Type I diabetes. Prior to development of full-scale diabetes in these animals, the vector of the invention could be targeted to their T lymphocytes, as discussed above, or to islet cells.
  • 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 polypeptide 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 linked to a protein tareting sequence.
  • the primary tumor could be treated by in situ 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.
  • 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.
  • 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. Both of these vectors are known to infect synovial cells in rabbits (Evans et al. Gene therapy for arthritis. In Wolff, J.A. Ed. Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser, Boston, MA, 1994 320-343).
  • Other embodiments are within the following claims. What is claimed is:

Abstract

Nucleic acid molecules encoding cysteine-containing or methionine-containing peptides that function to stimulate or suppress the immune response in a mammal are disclosed. Typically, these peptides contain no more than 30 amino acid residues and are linked to a protein targeting sequence at their amino terminal ends.

Description

SYNTHETIC GENES WITH IMMUNOMODULATORY EFFECTS
The field of the invention is non-antigen-specific immunomodulation, including both immunosuppression and immunostimulation.
Background of the Invention
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. Where global stimulation of the immune system is desired, this can sometimes be achieved with nonspecific agents such as adjuvants, interleukins, interferons, and colony-stimulating factors.
Occasionally, the immune system loses its ability to distinguish self from non-self. As a result, the individual may develop an autoimmune disease such as systemic lupus erythrematosis, Type I diabetes, or rheumatoid arthritis. These individuals would benefit greatly from suppression of the immune response, as would recipients of a transplanted organ or tissue.
The immune response may be generally suppressed by treatment with corticosteroids, azathioprine, cyclosporine, tacrolimus (FK506), rapamycin, or mycophenolate mofetil. In addition, 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. Summary of the Invention
It has been discovered that 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 protein targeting sequence.
In one embodiment, the DNA molecule of the invention encodes an immunoactive 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:
An-X-Cys-Cys-Y-Bm (Formula I) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Asn, Gin,
His, and Pro;
Y is selected from the group consisting Ala, Val, Leu, He, 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; where the formula I immunoactive peptide is linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide. In a preferred embodiment of the invention, a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell. By "protein targeting sequence" is meant any amino acid sequence which directs a polypeptide to which it is linked to a specific cellular compartment. Such compartments include, but are not limited to, the extracellular space, endoplasmic reticulum, mitochondria, nucleus, and plasma membrane (e.g., via a transmembrane sequence or a GPI linkage). Protein targeting sequences and the cellular compartments to which such sequences direct proteins are described in Alberts et al., 3rd ed., Molecular Biology of the Cell, Chapter 12, pages 551-599 (1995). GPI attachment sequences are described in Crise et al., J Virol 63:5328- 5333 (1989) and Salzwedel et al., J Virol 67:5279-5288 (1993). By a "glycosyl- phosphatidylinositol attachment signal" is meant an amino acid sequence within a polypeptide which leads to a covalent bond between the polypeptide and aglycolipid on the surface of a lipid bilayer: e.g., a cell membrane.
The nucleic acid can further encode a cytokine such as an interferon or interleukin; e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12. Preferably, X is Gly, Pro, He, Val, Asp, Leu, Glu, Gin, or Ala; Y is Gly, Pro, He, Val,
Asp, Leu, Glu, Ser, Phe, Tyr, or Thr; and the sum of n and m is zero to eleven, inclusive.
More preferably,
X is Gly and Y is Gly,
X is Pro and Y is Pro, X is Pro and Y is Val,
X is He and Y is Leu,
X is Pro and Y is Glu,
X is Glu and Y is Tyr,
X is Pro and Y is Phe, X is Glu and Y is Phe,
X is Ala and Y is Val,
X is Val and Y is He,
X is Gin and Y is Ser,
X is He and Y is Thr, X is Leu and Y is Asp, or X is Asp and Y is He; A and B vary according to the parameters above; and the sum of n and m is zero to eleven, inclusive.
Most preferably, the peptide represented by Formula I consists of 6-8 amino acid residues, the immunoactive peptide, and a protein targeting sequence. Examples of such immunoactive peptides of Formula I include the following:
Glu-Glu-Cys-Cys-Phe-Tyr (SEQ ID NO.:l),
Pro-Gly-Cys-Cys-Gly-Pro (SEQ ID NO.:2),
Pro-Gly-Cys-Cys-Pro-Gly (SEQ ID NO.:3),
Gly-Pro-Cys-Cys-Pro-Gly (SEQ ID NO.:4), Ala-Pro-Cys-Cys-Val-Pro (SEQ ID NO.:5),
Val-Ile-Cys-Cys-Leu-Thr (SEQ ID NO.:6),
Lys-Pro-Cys-Cys-Glu-Arg (SEQ ID NO.:7),
Lys-Glu-Cys-Cys-Tyr-Val (SEQ ID NO.: 8),
Thr-Pro-Cys-Cys-Phe-Ala (SEQ ID NO.:9), Leu-Ala-Cys-Cys-Val-Val (SEQ ID NO.: 10),
Pro-Val-Cys-Cys-Ile-Gly (SEQ ID NO.: 11),
Ser-Gln-Cys-Cys-Ser-Leu (SEQ ID NO.: 12),
Ser-Ile-Cys-Cys-Thr-Lys (SEQ ID NO.: 13),
Lys-Leu-Cys-Cys-Asp-Ile (SEQ ID NO.: 14), Pro-Ala-Cys-Cys-Gly-Pro (SEQ ID NO.: 15),
Pro-Asp-Cys-Cys-Ile-Pro (SEQ ID NO.: 16), and
Arg-Cys-Ser-Gly-Cys-Cys-Asn (SEQ ID NO.: 17).
Within formula I, A is preferably Gly, Lys, Arg, Cys, Ser, Val, Ala, Thr, Glu, Pro, Trp, Leu, Asp, Phe, or He; B is Leu, Arg, He, 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. Alternatively, A can be Pro, Gly, Glu, Ala, Val, Lys, Thr, Leu, or Ser; B can be Tyr, Pro, Gly, Thr, Arg, Val, Ala, Leu, Lys, or He; n is one; and m is one.
In a second embodiment, the DNA molecule of the invention may encode an immunoactive 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: An-X-Cys-Z-Cys-Y-Bm (Formula II) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids; X is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Asn, Gin,
Lys, Phe, His, and Pro;
Z is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Lys, His. Phe, Tyr, Arg, and Pro;
Y is selected from the group consisting of Ala, Val, Leu, He, 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; the formula II immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide. In a preferred embodiment of the invention, a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
The nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
Preferably, X is Gly, Pro, He, Val, Asp, Leu, Glu, Gin, or Ala; Y is selected from the group consisting of Gly, Glu, Val, Gin, Arg, Leu, Tyr, Phe, He, Ser, Thr, Asp, and Pro; Z is selected from the group consisting of He, Gly, Thr, Ala, Arg, and Lys; and the sum of n and m is zero to ten, inclusive. More preferably,
X is Gly and Y is Gly,
X is Pro and Y is Pro,
X is Pro and Y is Val,
X is He and Y is Leu, X is Pro and Y is Glu,
X is Glu and Y is Tyr, X is Pro and Y is Phe,
X is Glu and Y is Phe,
X is Ala and Y is Val,
X is Val and Y is He, X is Gin and Y is Ser,
X is He and Y is Thr,
X is Leu and Y is Asp, or
X is Asp and Y is He; Z is He, Gly, Thr, Ala, or Lys; A and B vary according to the parameters above; and the sum of n and m is zero to ten, inclusive. Examples of such peptides of Formula II include the following:
Val-Cys-Ile-Cys-Gln (SEQ ID NO.: 18),
Val-Cys-Gly-Cys-Arg (SEQ ID NO.: 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 NO.:23),
Leu-Cys-Ala-Cys-Val (SEQ ID NO.:24),
Phe-Cys-Ile-Cys-Lys (SEQ ID NO.:25),
Ala-Cys-Lys-Cys-Gln (SEQ ID NO.:26), and Gly-Pro-Cys-Ile-Cys-Pro-Gly (SEQ ID NO.:27).
In examples within formual II, X is Val, Ala, Leu, He, Lys, Asp, Phe or Pro; Y is Glu, Val, Gin, Arg, Lys, or Pro; Z is Gly, Ala, He, Arg, Thr, or Lys; and the sum of n and m is one to three, inclusive.
In a third embodiment, the DNA molecule of the invention may encode an immunoactive peptide consisting of 4-30 amino acid residues that conforms to the motif represented by Formula III:
An-X-Y-Cys-Z-Bm (Formula III) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids; X is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Asp, Glu, Lys, Arg, His, Trp, Tyr, and Phe;
Y is selected from the group consisting of Ala, Val, Leu, He, 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; the formula III immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide. In a preferred embodiment of the invention, a signal peptide is linked to the N- terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
The nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, inter leukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
Preferably, 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.
More preferably,
X s Gly, Y is Pro, and Z is He,
X s Gly, Y is Pro, and Z is Gly, X s Ala, Y is Pro, and Z is Ala, X s He, Y is Pro, and Z is Tyr, X s Ala, Y is Pro, and Z is He, X s Arg, Y is Pro, and Z is He, X s He, Y is Pro, and Z is He, X s Asp, Y is Pro, and Z is He, X s Trp, Y is Pro, and Z is He, X s Trp, Y is Pro, and Z is Gly, X s Gly, Y is He, and Z is He, X s Thr, Y is Pro, and Z is Tyr, X s Ala, Y is Pro, and 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. Examples of such peptides of Formula III include the following:
Gly-Pro-Cys-Gly (SEQ ID NO.:28),
Ala-Pro-Cys-Ala (SEQ ID NO.:29),
Ile-Pro-Cys-Tyr (SEQ ID NO.:30),
Trp-Pro-Cys-Gly (SEQ ID NO.:31 ), Gly-Pro-Cys-Ile-Leu-Asn (SEQ ID NO.:32),
Gly-Pro-Cys-Ile (SEQ ID NO.:33),
Leu-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:34),
Leu-Leu-Phe-Ala-Pro-Cys-Ile (SEQ ID NO.: 35),
Leu-Leu-Phe-Arg-Pro-Cys-Ile (SEQ ID NO.:36), Leu-Leu-Phe-Ile-Pro-Cys-Ile (SEQ ID NO.:37),
Leu-Leu-Phe-Asp-Pro-Cys-Ile (SEQ ID NO.:38),
Ala-Val-Trp-Thr-Pro-Cys-Tyr (SEQ ID NO.:39),
Phe-Val-Met-Ala-Pro-Cys-Phe (SEQ ID NO.:40),
Leu-Leu-Tyr-Ser-Pro-Cys-Phe (SEQ ID NO.:41), Ile-Ser-Gly-Pro-Cys-Pro-Lys (SEQ ID NO.:42),
Phe-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:43),
Leu-Phe-Gly-Pro-Cys-He-Leu (SEQ ID NO.:44),
Glu-Lys-Gly-Pro-Cys-Tyr-Arg (SEQ ID NO.:45),
Phe-Cys-Leu-Gly-Pro-Cys-Pro (SEQ ID NO.:46), Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:47),
Phe-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn (SEQ ID NO.:48),
Gly-Pro-Cys-Ile-Leu-Asn-Arg (SEQ D NO.:49),
Leu-Leu-Phe-T -Pro-Cys-Ile (SEQ D NO.:50),
Leu-Leu-Phe-Gly-Ile-Cys-Ile (SEQ ID NO.:51), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn
(SEQ ID NO.:52), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg (SEQ ED NO.:53),
Tφ-Cys-Gly-Pro-Cys-Lys-Met-Ile-Lys-Pro-Phe-Phe (SEQ ID NO.:54), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu
(SEQ ID NO.:55), and Phe-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu (SEQ ID NO.:56).
The nucleic acid molecule of the invention may encode, for example, a peptide where X is Gly, Ala, He, Arg, Asp, Tφ, 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.
In a fourth embodiment, 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: An-Xp-Y-Cys-Zq-Bm (Formula IV) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
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, Phe, and Gin;
Z is selected from the group consisting of Met, Tφ, Tyr, Phe, Gly, Pro, Arg, Asn, Gin, Ala, and Lys; n, m, 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 immunoactive peptide of formula IV being linked to a protein targeting sequence (e.g., a signal peptide, a glycosyl-phosphatidylinositol [GPI] attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide. In a preferred embodiment of the invention, a signal peptide is linked to the N-terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
The nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.
Preferably, 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 4-10 amino acid residues). Most preferably, the peptide consists of 4-7 amino acid residues.
More preferably,
X s Glu, Y is Pro, and Z is Met,
X s Gly, Y is Pro, and Z is Met, X s Ala, Y is Pro, and Z is Tφ, X s Ala, Y is Pro, and Z is Met, X s Glu, Y is Pro, and Z is Tφ, X s Ser, Y is Pro, and Z is Tφ, X s Leu, Y is Leu, and Z is Gly, X s Pro, Y is Arg, and Z is Arg, X s Gly, Y is Tyr, and Z is Pro, X s Val, Y is Val, and Z is Asn, X s Leu, Y is Ser, and Z is Gin, X s Ser, Y is Pro, and Z is Tyr, X s Ala, Y is Leu, and Z is Arg, X s Ala, Y is Pro, and Z is Tyr, X s Gly, Y is Ala, and Z is Pro, X s Lys, Y is Ser, and Z is Lys, X s Glu, Y is Pro, and Z is Phe, X s Glu, Y is Pro, and Z is Tyr, X s Ser, Y is Pro, and Z is Met, X s Ala, Y is Pro, and Z is Tyr, X s absent, Y is Leu, and Z is Phe, or X is Gly, Y is Pro, and Z is Tφ; 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.
Preferably, 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
(i) when p is 1, X is not Ser, Gly, Ala, or Thr, or
(ii) when p is 0, any amino acid residue of A adjacent to Y is not Ser, Gly, Ala, or Thr.
Examples of peptides of Formula IV include the following:
Gln-Cys-Ala-Leu-Cys-Arg (SEQ ID NO.:81), Val-Ala-Leu-Ser-Cys-Gln (SEQ ID NO.:82),
Ile-Val-Lys-Ser-Cys-Lys (SEQ ID NO.: 83),
Leu-Ala-Phe-Glu-Pro-Cys-Met (SEQ ED NO.: 84),
Leu-Leu-Pro-Gly-Pro-Cys-Met (SEQ ED NO.:85),
Met-Ala-Pro-Ala-Pro-Cys-Tφ (SEQ ID NO.:86), Ala-Leu-Tyr-Ala-Pro-Cys-Met (SEQ ID NO.:87),
Val-Leu-Tφ-Glu-Pro-Cys-Tφ (SEQ ID NO.:88), et-Leu-Phe-Ser-Pro-Cys-Tφ (SEQ ID NO.:89),
Leu-Leu-Cys-Gly-Pro-Ala-Ile (SEQ ID NO.:90),
Leu-Cys-Phe-Gly-Pro-Ala-Ile (SEQ ID NO.:91), Val-Met-Pro-Ser-Pro-Cys-Tyr (SEQ ID NO.:92),
Val-Val-Phe-Ala-Pro-Cys-Tyr (SEQ ID NO.:93),
Ala-Val-Pro-Glu-Pro-Cys-Phe (SEQ D NO.:94),
Met-Met-Tyr-Glu-Pro-Cys-Tyr (SEQ ID NO.:95),
Ala-Ala-Tφ-Ser-Pro-Cys-Met (SEQ ID NO.:96), Val-Ala-Tyr-Gly-Pro-Cys-Tφ (SEQ ID NO. :97)
Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile (SEQ ED NO.:98),
Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr (SEQ ID NO.:99),
Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg (SEQ ED NO.: 100),
Ala-Asn-Phe-Cys-Ala-Gly-Ala-Cys-Pro-Tyr-Leu-Tφ (SEQ ID NO.: 101),
Leu-Arg-Pro-Arg-Cys-Arg-Pro (SEQ ID NO.: 180), Pro-Gln-Val-Val-Cys-Asn-Tyr (SEQ ID NO.:181), Arg-Gly-Tyr-Cys-Pro-Tyr (SEQ ED NO.: 182), and Gln-Cys-Ala-Leu (SEQ ID NO.: 183).
Examples of peptides of Formula IV that contain a mammalian signal peptide sequence include the following:
Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met-Leu-Leu-Val-Ala-Leu-Ser-Pro-Cys- Leu-Pro-Cys-Arg-Ala-Leu-Ala-Phe-Glu-Pro-Cys-Met (SEQ ID NO.: 102),
Met-His-Leu-Ser-Leu-Ser-His-Gln-Tφ-Ser-Ser-Tφ-Thr-Val-Leu-Leu-Leu-Leu-Val-Ser- Asn-Leu-Leu-Leu-Tφ-Glu-Asn-Thr-Ala-Ser-Ala-Met-Ala-Pro-Ala-Pro-Cys-Tφ (SEQ ID NO.: 103),
Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-Leu-Leu-Leu-Tyr-Gln-Ala- Cys-Gly-Leu-Gln-Ala-Val-Leu-Tφ-Glu-Pro-Cys-Tφ (SEQ D NO.: 104),
Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-Leu-Leu-Leu-Tyr-Gln-Ala- Cys-Gly-Leu-Gln-Ala-Val-Met-Pro-Ser-Pro-Cys-Tyr (SEQ ID NO.: 105), Met-His-Leu-Ser-Leu-Ser-His-Gln-Tφ-Ser-Ser-Tφ-Thr-Val-Leu-Leu-Leu-Leu-Val-Ser-
Asn-Leu-Leu-Leu-Tφ-Glu-Asn-Thr-Ala-Ser-Ala-Met-Leu-Phe-Ser-Pro-Cys-Tφ (SEQ ID NO.: 106), and
Met-Gly-Phe-Leu-Lys-Phe-Ser-Pro-Phe-Leu-Val-Val-Ser-Ile-Leu-Leu-Leu-Tyr-Gln-Ala- Cys-Gly-Leu-Gln-Ala-Val-Val-Phe-Ala-Pro-Cys-Tyr (SEQ ID NO.: 107). In a fifth embodiment, 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:
An-W-X-Y-Zp-Bm (Formula V) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Tφ, 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, Tφ, Glu, Leu, Met, and Lys;
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 formula V immunoactive peptide being linked to a protein targeting sequence (e.g., a signal peptide, a GPI attachment signal, or a transmembrane sequence), e.g., at the amino terminus of the immunoactive peptide. In a preferred embodiment of the invention, a signal peptide is linked to the N-terminus and a transmembrane sequence is linked to the C-terminus of the immunoactive peptide, ensuring that the immunoactive peptide is displayed on the surface of a cell.
The nucleic acid can further encode a cytokine such as an interferon or interleukin: e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12.. Preferably, 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.
More preferably, the immunoactive peptide conforms to the motif of Formula V, wherein W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Tφ, 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; and
Z is selected from the group consisting of Gly, Phe, Val, He, Pro, and Leu. More preferably, at least one of X and Y is Met.
More preferably, the immunoactive peptide conforms to the motif of Formula V, wherein W is selected from the group consisting of Gly, Asp, Arg, Ala, Tφ, and Ser;
X is selected from the group consisting of Pro and He;
Y is Met; and
Z is selected from the group consisting of Phe, He, and Pro.
Most preferably, the immunoactive peptide conforms to the motif of Formula V, 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 Pro.
The immunoactive peptide may have Met and Cys or Met and Met aligned contiguously. For example, the 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.
Alternatively, the peptide may contain Met and Cys, separated by no more than one amino acid. For example, the peptide may conform in sequence to A-Met-X-Cys-Z-B or
A-Cys-X-Met-Z-B.
Examples of such peptides of Formula V include the following:
Gly-Pro-Met-Ile (SEQ ID NO.: 114),
Lys-Met-Arg-Met-Lys (SEQ ED NO.:l 15), Phe-Met-Ile-Met-Lys (SEQ ID NO.:l 16),
Ile-Cys-Thr-Met-Glu (SEQ ED NO.:l 17),
I u-Met-Ala-Met-Val (SEQ ID NO.:l 18),
He-Met-Tyr-Met-Glu (SEQ ED NO.:l 19),
Ala-Pro-Met-Met-Val-Pro (SEQ ID NO.: 120), Gly-Pro-Met-Met-Pro-Gly (SEQ ED NO.: 121),
Gly-Pro-Cys-Met-Pro-Gly (SEQ ID NO.: 122),
Gly-Pro-Met-Cys-Pro-Gly (SEQ ID NO.: 123),
Pro-Gly-Met-Met-Gly-Pro (SEQ ID NO.: 124),
Val-Ile-Met-Met-Leu-Thr (SEQ ID NO.: 125), Leu- Ala-Phe-Glu-Pro-Met-Met (SEQ ID NO. : 126) ,
Met-Leu-Phe-Ser-Pro-Met-Tφ (SEQ ID NO.: 127),
Val-Val-Phe-Ala-Pro-Met-Tyr (SEQ ID NO.: 128),
Leu-Leu-Phe-Gly-Pro-Met-Ile (SEQ ID NO.: 129),
Leu-Leu-Tyr-Ser-Pro-Met-Phe (SEQ ID NO.: 130), Leu-Leu-Phe-Asp-Pro-Met-Ile (SEQ ID NO.: 131),
Leu-Leu-Phe-Tφ-Pro-Met-Ile (SEQ ID NO.: 132), Leu-Leu-Phe-Arg-Pro-Met-Ile (SEQ ID NO.: 133),
Leu-Leu-Phe-Ala-Pro-Met-Ile (SEQ D NO.: 134),
Leu-Leu-Phe-Gly-Ile-Met-Ile (SEQ ID NO.: 135),
Phe-Met-Leu-Gly-Pro-Met-Pro (SEQ D NO.: 136), Phe-Met-Ile-Met-Lys (SEQ ID NO.: 177),
Phe-Met-Leu-Gly-Pro-Met-Pro (SEQ ID NO.: 178), and
Lys-Met-Arg-Met-Lys (SEQ ID NO.: 179).
An example of a peptide of Formula V that contains a mammalian signal peptide is:
Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met-Leu-Leu-Val-Ala-Leu-Ser-Pro-Cys- Leu-Pro-Cys-Arg-Ala-Leu-Leu-Phe-Gly-Pro-Met-Ile (SEQ ID NO.: 137).
Preferably, the following peptides are excluded from Formulas I-V:
Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp-Val-Gln-Ala-Tφ-Ile-Arg-Gly-Cys-Arg-Leu (SEQ ID NO.: 139),
Ile-Asn-Thr-Lys-Cys-Tyr-Lys-Leu-Glu-His-Pro-Val-Thr-Gly-Cys-Gly (SEQ ID NO.: 140), Asp-Asn-Tyr-Arg-Gly-Tyr-Ser-Leu-Gly-Asn-Tφ-Val-Cys-Ala-Ala-Lys-Phe-Glu-Ser-Asn-
Phe-Thr-Gln (SEQ ED NO.: 141),
Ala-Pro-Ser-Pro-Leu-Pro-Glu-Thr-Thr-Glu-Asn-Val-Val-Cys-Ala-Leu-Gly (SEQ ID NO.: 176),
Ala-Pro-Ser-Pro-Leu-Pro-Glu-Thr-Thr-Glu-Asn-Val-Val-Cys-Ala-Leu-Gly-Leu-Thr-Val (SEQ ID NO.: 142),
Gly-Asp-Met-Tyr-Pro-Lys-Thr-Tφ-Ser-Gly-Met-Leu-Val-Gly-Ala-Leu-Cys-Ala-Leu-Ala- Gly-Val-Leu-Thr-Ile (SEQ D NO.: 143),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys (SEQ ID NO.: 144),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys-Gh-Glu-Leu-Leu (SEQ ID NO.: 145),
Val-Pro-Gly-Leu-Tyr-Ser-Pro-Cys-Arg-Ala-Phe-Phe-Asn-Lys (SEQ ED NO.: 146),
Glu-Ala-Ile-Tyr-Asp-Ile-Cys-Arg-Arg-Asn-Leu-Asp-Ile-Glu-Arg-Pro-Thr (SEQ E NO.: 147), Glu-Ala-Ile-Tyr-Asp-Ile-Cys-Arg-Arg-Asn-Leu-Asp-Ile (SEQ ED NO.: 148), Asp-Leu-Leu-Glu-Gln-Arg-Arg-Ala-Ala-Val-Asp-Thr-Tyr-Cys-Arg-His-Asn-Tyr-Gly-
Val-Gly-Glu-Ser-Phe-Thr (SEQ ID NO.: 149),
Thr-Ser-Ile-Leu-Cys-Tyr-Arg-Lys-Arg-Glu-Tφ-Ile-Lys (SEQ ID NO.: 150), Leu-Pro-Phe-Phe-Leu-Phe-Arg-Gln-Ala-Tyr-His-Pro-Asn-Asn-Ser-Ser-Pro-Val-Cys-Tyr (SEQ ID NO.: 151),
Gln-Ala-Lys-Phe-Phe-Ala-Cys-Ile-Lys-Arg-Ser-Asp-Gly-Ser-Cys-Ala-Tφ-Tyr-Arg-Gly-
Ala-Ala-Pro-Pro-Lys-Gln-Glu-Phe (SEQ ID NO.: 152),
Gln-Ala-Lys-Phe-Phe-Ala-Cys-Ile-Lys-Arg-Ser-Asp-Gly-Ser-Cys-Ala-Tφ-Tyr-Arg (SEQ
ID NO.:153), Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala-Ala-Ala-Met-Lys-Arg-His-Gly-Leu-Asp (SEQ ID
NO.: 154),
Ala-Glu-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Thr-Thr-Lys-Thr (SEQ ID NO.: 155), Lys-Asn-Ile-Phe-His-Phe-Lys-Val-Asn-Gln-Glu-Gly-Leu-Lys-Leu-Ser-Asn-Asp- t-Met
(SEQ ID NO.: 156), Leu-Glu-Cys-Gly-Pro-Cys-Phe-Leu (SEQ ED 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 (SEQ ID NO.: 159), Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-Ile (SEQ ID NO.: 160), Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ID NO.: 161), Thr-Pro-Pro-Thr-Pro-Cys-Pro-Ser (SEQ ID NO. : 162),
Asp-Pro-Cys-Ile-Ile (SEQ ID NO.: 163), Cys-Gly-Gly-Ile-Cys-Ile-Ala-Arg (SEQ ED NO.: 164), Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQID NO.: 165), Cys-His-Gly-Ser-Asp-Pro-Cys (SEQ ED NO.: 166), Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ED NO.: 167),
Tyr-Arg-Arg-Gly-Arg-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 168), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID
NO.: 169),
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-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 172), Tyr-Arg-Arg-Gly-Arg-Ala-Ala-Ala-Cys-Gly-Gly-Gly-Gly-Leu-Cys-Leu-Ala-Arg (SEQ ID NO.: 173),
Cys-Gly-Gly-Leu-Cys-Ala-Arg (SEQ ID NO.: 174), and 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 polypeptide linked to a protein targeting sequence. Of course, multiple coding sequences can be linked in tandem, separated by stop codons and potentially other noncoding sequence.
As stated above, the polypeptides encoded by the nucleic acids of this invention may include a protein targeting sequence which targets the immunoactive peptide to a specific cellular compartment. For example, a signal peptide can be linked to the immunoactive 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. 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 tissue- or cell-specific expression of one or more immunoactive peptides linked to a protein targeting sequence encoded on the nucleic acid molecule. 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 linked to a protein targeting sequence. These 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 vitro under conditions that permit expression of the immunoactive peptide, and harvesting the immunoactive peptide from the cell. If the vector encodes an immunoactive peptide linked to, for example, a signal peptide or GPI attachment signal, the immunoactive peptide may instead be harvested from the medium surrounding the cells. If a GPI attachment signal is used, the polypeptide can be harvested from the medium only after cleavage with an enzyme such as phospholipase C. Alternatively, an immunoactive peptide may be produced in a mammal (e.g., a human, non-human primate, 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 (e.g., a plasmid or virus) containing the nucleic acid molecule of the invention, or (3) a cell that contains and expresses the nucleic acid. In the latter case, a cell removed from the subject, or a descendant of such a cell, would be transduced with the nucleic acid ex vivo. Such cells (particularly mammalian cells such as human cells) are considered to be within the invention.
Non-integrating viral vectors include heφes 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. Other 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, "naked DNA," or biolistic methods.
The immunoactive peptides discussed herein were so named because of their ability to modulate an animal's immune response, as demonstrated by the biological assays described below. Thus, 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 polypeptide 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 or expresses on its surface the immunoactive peptide. In the latter case, 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 clinical practitioners. 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. The method for modulating the immune response in a patient further includes administration, including oral administration, of a cytokine such as an inteferon or an interleukin: e.g., interferon-α, interferon-β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, or interleukin- 12 (see Rollwagen et al., Immunology Today 17:548-550 [1996] andHsieh et al., Science 260:547-549 [1993]).
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. These 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, Sjόgren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome, orBehcet'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 hematopoietic system. Accordingly, a patient's cancer can be treated by any standard therapy known in the art, combined with administration to the patient of any of the above described nucleic acids. Such standard therapies include surgery (e.g., removal of part or all of a solid tumor), exposure of the patient or a specific region or tissue of the patient to a therapeutic amount of ionizing radiation (e.g, α-particles, β-particles. or gamma radiation), and anticancer chemotherapy (i.e., administration of one or more of the following cytotoxic chemotherapeutics: mechlorethamine, cyclophosphamide, ifosfamide, chlorambucil, melphalan, bursulfan, thiotepa, carmustine, lomustine, streptozocin, vincristine, vinblastine, paclitaxel, vinorelbine, docetaxel, methotrexate, mercaptopurine, thioguanine, fluorouracil, cytarabine, azacitidine, fludarabine, cladribine, and pentostatin). Standard cancer therapies are described in Dale et al., Scientific American Medicine, Chapter 12: Oncology (1996).
Also within the invention is use of the nucleic acid molecule of the invention in the preparation of a medicament useful in treating any of the above conditions.
By "peptide" is meant any chain of more than two amino acid residues, regardless of post-translational modification such as glycosylation or phosphorylation. As referred to herein, 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 (Gin; Q), L-tyrosine (Tyr; Y), L-tryptophan (Tφ; W), L-phenylalanine (Phe; F); and L-proline (Pro; P).
The term "nucleic acid" encompasses both RNA and DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. The term "synthetic gene" refers to a coding sequence that is not identical to any naturally-occurring, full length coding sequence. It can be operably linked to expression control elements. All publications, patents, and other references cited herein are incoφorated by reference in their entirety.
The preferred methods, materials, and examples that will now be described are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, from the drawings, and from the claims.
Brief Description of the Drawings Fig. 1 is a schematic diagram of the Moloney murine sarcoma virus retroviral vector pLXSN.
Detailed Description
The experiments described herein utilize 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.
Vector Construction
The following procedures were carried out in order to construct vectors that could be used to transduce malignant cells in vitro. Each vector described below includes a sequence encoding (a) an immunoactive peptide that conforms to the motif represented in Formula I, II, IH, IV, or V, and (b) a protein targeting sequence that targets the peptide to a specific cellular compartment (i.e., the extracellular space) and a eukaryotic expression control sequence. Although a signal peptide was used below as an example protein targeting sequence, other protein targeting sequences known in the art can be used, where targeting to an intracellular rather than extracellular compartment is desired. 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, BamHI, Xhol, and Hpal.
In each case, the signal sequence was chosen on the basis of the first N-terminal amino acid of the immunoactive peptide. For example, 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. For experiments conducted with rat cells, signal sequences were chosen from those which occur naturally in rat cells. For use in other species, 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.
Similarly, 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. In order to anneal these two DNA molecules to one another, 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. In this case, the DNA was inserted into the Moloney murine sarcoma virus retroviral vector pLXSN (Fig. 1) 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-XhoI, (3) EcoRI-Hpal, (4) Hpal- BamHI, (5) Hpal-Xhol, or (6) XhoI-BamHI. To insert the DNA into the vector, 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 (DH5α 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. Once the presence and orientation of the insert was confirmed by sequencing, large scale cultures were established in LB medium supplemented with ampicillin (50 μg/ml) and grown until the OD at 600 nm was 0.8. Chloramphenicol was then added to a final concentration of 180 μg/ml, and the flasks were incubated at 37°C, on a shaker, overnight. The DNA constructs were isolated from the bacterial cultures using a QIAGEN aPlasimd kit (QIAGEN, GmbH, Hilden, Germany), according to the manufacturer's instructions.
Transduction of Cultured Cells
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 (Lindvall et al., 1991, Cancer Immunol. Immunother. 33:21-27). The tumors were kept in serial passage in syngeneic rats.
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 1-glutamine, 1 mM pyruvate, 10 mM HEPES buffer, 10 mM NaHCO3, and 5% fetal calf serum (FCS) in vessels obtained from NUNC (Roskilde, Denmark). To transduce the cells, DNA constructs were prepared as described above and used to transfect a retroviral packaging cell line GP+E or Psi2 with TRANSFECTAM™ (Promega, USA), according to the manufacturer's instructions. Transfectants were selected with G418 (300 μg/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.
An assay based on the polymerase chain reaction (PCR) was used to demonstrate that the DNA encoding an immunoactive peptide was transcribed into mRNA within the transduced cells.
In addition, an in vitro biological assay of the effect on proliferation of activated T lymphocytes of supernatant from SPMWl cells which had been transduced with a nucleic acid of the invention is consistent with the conclusion that the immunoactive peptide A (Met-Lys-Phe-Leu-Ser-Ala-Arg-Asp-Phe-His-Pro-Val-Ala-Phe-Leu-Gly-Leu-Met-Leu- Val-Thr-Thr-Thr-Ala-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg [SEQ ED NO:57]) is expressed and secreted by the transduced cells, and overcomes the suppressive effect of wild type SPMWl cells on T lymphocyte proliferation. Implantation of Transduced Cells
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. Each rat in the experimental group received a subcutaneous injection in the right hindlimb of approximately 200 μl of resuspended cells. As a control, 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. Assessment of Tumor Size in vivo
On the day the tumor cells were inoculated and at various intervals ranging up to 36 days afterward, the hindlimb was palpated at the site of injection. When a tumor could be felt, the largest diameter was measured with a caliper. A second measurement was taken peφendicular to the first. The volume of the tumor was calculated by using the formula V=0.4(a-b2), where V = volume (in mm3), a = the largest diameter (in mm), and b = the diameter peφendicular to the largest diameter (in mm).
Example 1 : Immunostimulation by Peptide A-expressing SPMWl Cells SPMWl mammary carcinoma cells in cell culture were infected with a retroviral vector containing an insert encoding the immunoactive peptide A. 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. As a control, 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. On any given day after injection, the mean size of the tumor that developed from peptide A-expressing SPMWl cells was less than one-tenth the size of the tumor that developed from wild type SPMWl cells (Table 1). This result demonstrates that a construct encoding and presumably expressing peptide A substantially impedes tumor growth in vivo.
Figure imgf000028_0001
Example 2: Lack of Immunostimulation by Peptide A- expressing SPMWl Cells in Nude Rats
Peptide A-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 peptide A-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 peptide A-expressing SPMWl cells involves a T cell-mediated immune response.
Figure imgf000029_0001
Example 3: Immunostimulation by Peptide A-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 A. 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 peptide A-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 peptide A significantly impedes tumor growth in at least two model systems.
Figure imgf000030_0001
Example 4: Immunostimulation bv two Peptide A- expressing Ad9-101 clones; clone 8 and clone 9
Cells from four different peptide A-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.
Figure imgf000031_0001
Example 5: Immunostimulation by Peptide A-expressing Ad9-101 Cells is Mediated by T Lymphocytes
In order to determine whether T lymphocytes are activated in response to in vivo expression of an immunoactive peptide, the following experiment, which extends from Example 4, was performed. Rats were injected subcutaneously with either wild type Ad9- 101 cells or peptide A-expressing Ad9-101 cells (clone 8 or clone 9). Each animal was killed when its tumor reached 50-100 mm3 in size, and approximately 2.5 x 105 cells were harvested from the lymph nodes associated with the tumor, i.e., the inguinal and para-aortal lymph nodes ipsilateral to the tumor. Cells were also harvested from the lymph nodes of normal rats (which were free of tumors). The lymphatic cells from each rat were placed in culture either as a homogeneous population, or in co-culture with approximately 7.5 x 103 lethally irradiated (8000 rad=80 Gray) wild type Ad9-101 cells. 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 NaHCO3, 50 μM β-mercaptoethanol, and 10% FCS. On the fifth day in culture, the cells were exposed to [3H]-thymidine (0.5 μCi) for 6 hours. Although other cell types, such as monocytes, natural killer cells, and B lymphocytes are present, under the conditions of the culture, only T lymphocytes can respond with mitotic activity on day 5. The radioactivity incoφorated into acid-insoluble material, which reflects the mitotic activity of the cells in culture, was measured with a scintillation counter. The amount of radioactivity incoφorated into homogeneously cultured lymph cells was subtracted from the amount of radioactivity incoφorated into heterogeneous cultures containing lymphatic and irradiated tumor cells. (The incoφorated 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. Table 5: 3H-th midine Inco oration b Cultured L m hatic Cells
Figure imgf000033_0001
These results demonstrate that, in the presence of lethally irradiated (but presumably still antigenic) wild type Ad9-101 cells, the mitotic activity of T lymphocytes harvested from animals that had been injected with peptide A-expressing Ad9-101 cells (clone 8 or clone 9) is substantially greater than the mitotic activity of T lymphocytes from either normal (non-injected) animals or animals that were injected with wild type Ad9-101 tumor cells. The data suggest that "vaccination" with wild type tumor cells actually decreases the ability of the animal's T lymphocytes to respond to a subsequent challenge with wild type tumor cells, while vaccination with peptide A-expressing tumor cells not only overcomes this inhibition, but renders the animal's T lymphocytes substantiallymore sensitive to challenge with wild type tumor cells than the T lymphocytes of an unimmunized animal.
Example 6: Immunization with Irradiated Peptide A- expressing Ad9-101 Cells
Impedes Subsequent Tumor formation bv Wild Type Ad9-101 Cells
In this experiment, rats were inoculated with either Ad9-101 wild type cells that had been irradiated with 10,000 rad (100 Gray), or clone 9 cells that had been similarly irradiated. (Irradiation prevents the cells from dividing more than once, but it does not immediately affect protein synthesis.) Animals received two subcutaneous injections of irradiated cells (1 x 106 cells/animal/dose), 14 days apart, while a group of control animals received comparable injections of phosphate buffered saline (PBS). Fourteen days following the second injection, by which time it is expected the irradiated cells had all been cleared from the injection site, the animals were challenged with an injection of 10,000 non-irradiated Ad9-101 wild type cells, and tumor growth was assessed. The tumors that developed in animals that had been injected with irradiated clone 9 cells were on average substantially smaller than the tumors that developed in rats that were injected with PBS or with irradiated wild type Ad9-101 cells (Table 6). Thus, the antitumor effect of peptide A persists even after the peptide A-expressing cells were presumably cleared from the test animals' bodies.
Figure imgf000035_0001
Example 7: Immunomodulation by Peptide B-expressing. Peptide C-expressing, or
Peptide D-expressing Ad9-101 Cells To compare the effects of three different immunoactive peptides on tumor growth, rats were inoculated with transduced Ad9-101 cells that expressed either peptide B (Met-Tφ- Phe-Leu-He-Leu-Phe-Leu-Ala-Leu-Ser-Leu-Gly-Gln-Ile- Asp-Ala- Ala-Pro-Gly-Cys-Cys- Pro-Gly [SEQ ID NO: 60]), peptide C (Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met- Leu-Leu-Val-Ala-Leu-Ser-Pro-Cys-Leu-Pro-Cys-Arg-Ala-Leu-Leu-Phe-Gly-Pro-Cys-Ile [SEQ ID NO:58]), or peptide D (Met-Arg-Leu-Arg-Leu-Leu-Val-Ser-Ala-Gly-Met-Leu- Leu-Val-Ala-Leu-Ser-Pro-Cys-Leu-Pro-Cys-Arg-Ala-Leu-Leu-Tyr-Ser-Pro-Cys-Phe [SEQ ID NO:59]). The immunoactive peptide B (without signal sequence) has previously been shown to be immunostimulatory when administered directly in a delayed-type hypersensitivity (DTH) assay, while peptide C and peptide D (each without signal sequence) have shown activity consistent with immunosuppression. Approximately 20,000 transduced cells were injected subcutaneously into each rat (5 animals/group), and the tumors which developed were measured. As shown in Table 7, uncloned cells expressing peptide B formed tumors that grew somewhat more slowly than those which developed from wild type Ad9-101 cells, consistent with the immunostimulatory activity of this peptide observed in the DTH test. In contrast, the tumors that developed from uncloned cells expressing either peptide D or peptide C grew faster than the tumors that developed from wild type Ad9-101 cells (at least after day 22), consistent with the immunosuppressant activity of these peptides in the DTH test. This suggests that the delayed-type hypersensitivity assay described below is a useful predictor of the immunomodulatory effect of peptides expressed from the nucleic acid molecules of the invention.
Figure imgf000037_0001
Example 8: Inoculation of Various Clones of Peptide B- expressing Cells
Several clones of Peptide B-expressing Ad9-101 cells were established. Each rat (n=5 animals/group) received an injection of either wild type AD9-101 cells, uncloned Peptide B-expressing Ad9-101 cells, or cells from one of the peptide B-expressing Ad9-101 clones designated 1, 2, 3, 5, 6, 7, and 9. Tumor growth was assessed as described above. The data gathered from animals injected with clone 6, clone 7, wild type, or uncloned peptide B-expressing cells are presented in Table 8. The tumors that developed from uncloned peptide B-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. In addition, clones 1, 2, 3, 5, and 9 showed essentially no tumor outgrowth through day 35 (data from clone 2 is shown in Table 8).
Figure imgf000038_0001
Example 9: Expression of IL-7 has No Effect on Tumor Growth
In order to demonstrate that the immunomodulatory effect observed in the experimental paradigms described above is due to the expression of nucleic acid molecules that encode peptides conforming to Formula I, π, III, IV, or V, a nucleic acid molecule encoding a peptide that does not conform to any of these formulas was studied according to the paradigm described in Example 1. Approximately 10,000 SPMWl cells transduced with EL-7 (interleukin 7) were subcutaneously injected into the hindlimbs of each of 7 rats. As a control, an equivalent group of rats was similarly injected with approximately 10,000 wild type (i.e., non-transduced) SPMWl cells. The size of the tumor that developed in vivo was estimated in both groups for up to seventeen days following injection. As shown in Table 9, expression of IL-7 had no effect on tumor growth, and therefore presumably no effect on the animals' immune response.
Figure imgf000039_0001
Additional Assays for Identifying Immunostimulants
The following assays can be used to identify immunostimulatory peptides linked to protein targeting sequences that can be delivered by gene therapy in accordance with the invention.
Delayed Type Hypersensitivity (DTH) Test
The ability of a DNA molecule to encode a polypeptide that modulates the immune response can be determined using the delayed type hypersensitivity (DTH) test in 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 asBalb/c mice, are sensitized by exposure to 4-ethoxymethylene-2-phenyloxazolin-5-one (OXA; Sigma Chemical Co.). On Day 0, 150 μl of an absolute ethanol-acetone (3: 1) solution containing 3% OXA is applied to the animal's shaved abdomen. Treatment with the immunoactive peptide itself (e.g., by topical or IV administration), or with gene therapy using a nucleic acid encoding the peptide, is then begun (methods of administration are discussed below). Approximately one week after sensitization, the thickness of the animal's ears is measured with an Oditest spring caliper before both ears are challenged by topical application of 20 μl 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 vanLoveren et al. (1984, J. Immunol. Methods 67:311), and expressed according to the formula: T^^s - Tto (in μm 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. Alternatively, a spontaneous or induced tumor from an animal (e.g., a spontaneous tumor from a human) could be used. Cells from any of these sources could be placed in culture, transduced with a nucleic acid encoding a candidate immunoactive peptide linked to a protein targeting sequence, and transplanted into a test animal. Cells cultured in parallel, but untransduced or transduced only with the "empty" vector or a vector encoding a non-immunoactive peptide linked to the same protein targeting sequence, would be transplanted into control animals. If the transplanted cells that expressed the candidate polypeptide failed to produce a tumor, or produced a tumor that was significantly smaller than that found in control animals, the encoded polypeptide would be deemed an effective immunostimulant. In a variation on this assay, the transduced cells could be irradiated and mixed with non-irradiated wild type cells prior to injection, so that the effect of the polypeptide on growth of wild type tumor cells in vivo would be measured. Model systems developed from different sources of immortalized cells may provide the means to determine whether polypeptides are broadly effective or particularly effective in treating a certain type of cancer. Immunization Assay
Genes encoding polypeptides 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. As a control, animals could be injected with irradiated wild type tumor cells. Subsequently, both groups of animals would be challenged with wild type tumor cells and examined for tumor formation. If the polypeptide 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 polypeptide-expressing cells. By performing the analysis of a given gene in model systems that employ immortalized cells from an array of tumors, it should be possible to determine whether the gene encodes a polypeptide that could serve as a broad- based vaccine, or whether it is primarily effective against a particular form of cancer. In a variation on this method, one could use as test animals transgenic animals (e.g., mice) that spontaneously develop tumors. 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. Assay for Tumor Regression
Another way to assay a polypeptide-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. When a tumor of a given size has formed in a test animal, the animal is injected with the polypeptide-encoding vector or with polypeptide-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 linked to protein targeting sequences in the treatment of cancer. In each of the assays described above, it would be possible to show that the immune system was suppressed by the gene product in question by performing the experiment presented in Example 5. This experiment would demonstrate that the gene product exerted its influence by stimulating the activity of T lymphocytes, rather than by a mechanism that is independent of the immune system.
Polypeptides that are ineffective in impeding tumor outgrowth, or that enhance tumor outgrowth, could be tested further, as described below, in order to determine whether they would be useful immunosuppressants.
Assays for Identifying Immunosuppressants
The following procedures could be employed in order to determine whether a given polypeptide is an immunosuppressant that could be usefully delivered by genetic therapy, Transplantation Paradigms
In order to determine whether a polypeptide 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 polypeptide, 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.
Alternatively, the graft itself could be transduced with the nucleic acid of the invention.
Strains of mice such as C57B1-10, B 10.BR, and B 10.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.
A method for performing cardiac grafts by anastomosis of the donor heart to the great vessels in the abdomen of the host was first published by Ono et al. (1969, J. Thorac.
Cardiovasc. Surg. 57:225; see also Corry et al., 1973, Transplantation 16:343). According to this surgical procedure, 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. Once 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. With regard to the current invention, a given polypeptide would be considered a successful immunosuppressant if it prolongs the time the grafted organ was tolerated by the host.
The effectiveness of an immunoactive peptide linked to a protein targeting sequence can also be assessed following a skin graft. To perform skin grafts on a rodent, 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. Generally, 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
Models of autoimmune disease provide another means to assess polypeptides in vivo. These models are well known to skilled artisans and can be used to determine whether a given polypeptide is an immunosuppressant that would be therapeutically useful in treating a specific autoimmune disease when delivered via genetic therapy.
Autoimmune diseases that have been modeled in animals include rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosis (SLE), type I diabetes, and autoimmune diseases of the thyroid and central nervous system. For example, 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. 37:9; see also Fundamental Immunology, 1989, Paul, Ed., Raven Press, New York, NY). Similarly, a shift to lethal nephritis is seen in the progeny of NBZ X SWR matings (Data et al., 1976, Nature 263:412). The histological appearance of renal lesions in SNF1 mice has been well characterized (Eastcott et al., 1983, J. Immunol. 131:2232; Paul, supra). Therefore, the general health of the animal as well as the histological appearance of renal tissue can be used to determine whether the administration of genes encoding immunoactive peptides linked to protein targeting sequences can effectively suppress the immune response in an animal model of SLE. 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). Alternatively, 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 μl total) into the base of the tail. Arthritis develops 2-3 weeks after immunization. The ability of synthetic genes encoding immunoactive peptides linked to protein targeting sequences 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 μg/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 107 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 polypeptide of interest, and the gene encoding it, function as an immunosuppressant potentially useful in treating autoimmune disease.
Alternatively, one could introduce the polypeptide-encoding vector, e.g., anadenoviral vector, directly into the cells of the joint synovium. 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. These rats spontaneously develop autoantibodies against pancreatic islet cells and insulin, just as occurs in human Type I diabetes. Prior to development of full-scale diabetes in these animals, the vector of the invention could be targeted to their T lymphocytes, as discussed above, or to islet cells.
Human Therapy The application of 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.
Ex vfvo-based Human Therapies
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.
For treatment of rheumatoid arthritis, 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 polypeptide at the site of the inflammation. A retroviral or adeno-associated viral vector would be appropriate for this ex vivo infection procedure.
In vz'vo-based Human Therapies
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. For example, after surgical removal of a primary tumor, residual cells may be targeted by treating the vicinity of the tumor with a composition containing a retroviral vector encoding an immunostimulatory peptide linked to a protein tareting sequence. Instead of surgery, the primary tumor could be treated by in situ 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. Alternatively, one could utilize an engineered cell attachment ligand on the vector or the viral particle to accomplish preferential targeting of specific cells in accordance with standard methods.
An example of a 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, α-fetoprotein promoter for hepatocellular carcinoma (Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043) or the tyrosinase promoter for melanoma (Hart et al., 1995, Br. Med. Bull. 51(3):647-655). Alternatively, a constitutively active promoter could be used, e.g., the SV40 promoter or CMV promoter. One could treat rheumatoid arthritis via direct inoculation of the vector into the affected joint in order to infect the synovial cells in situ. Adeno-associated viral vectors may be used if long-term expression is desired, or an adenoviral vector for shorter-term expression. Both of these vectors are known to infect synovial cells in rabbits (Evans et al. Gene therapy for arthritis. In Wolff, J.A. Ed. Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser, Boston, MA, 1994 320-343). Other embodiments are within the following claims. What is claimed is:

Claims

Claims
1. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula I: An-X-Cys-Cys-Y-Bm (I) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Asn, Gin, His, and Pro;
Y is selected from the group consisting Ala, Val, Leu, He, Gly, Ser, Thr, Asp, Glu, Asn, 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; the coding sequence further encoding a protein targeting sequence.
2. The nucleic acid of claim 1 , wherein the protein targeting sequence is a signal peptide.
3. The nucleic acid of claim 1 , wherein the protein targeting sequence is a glycosyl- phosphatidylinositol attachment signal.
4. The nucleic acid of claim 1 , wherein the protein targeting sequence is a transmembrane sequence.
5. The nucleic acid of claim 1, wherein the protein targeting sequence is linked to the amino terminus of the immunoactive peptide.
6. The nucleic acid of claim 1 , wherein said nucleic acid further comprises a second coding sequence encoding a cytokine.
7. The nucleic acid of claim 6, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
8. The nucleic acid molecule of claim 1 , wherein X is selected from the group consisting of Gly, Pro, He, Val, Asp, Leu, Glu, Gin, and Ala; Y is selected from the group consisting of Gly, Pro, He, Val, Asp, Leu, Glu, Ser, Phe, Tyr, and Thr; and the sum of n and m is zero to eleven, inclusive.
9. The nucleic acid molecule of claim 1, wherein
X is Gly and Y is Pro,
X is Gly and Y is Gly,
X is Pro and Y is Pro,
X is Pro and Y is Val, X is He and Y is Leu,
X is Pro and Y is Glu,
X is Glu and Y is Tyr,
X is Pro and Y is Phe,
X is Glu and Y is Phe, X is Ala and Y is Val,
X is Val and Y is He,
X is Gin and Y is Ser,
X is He and Y is Thr,
X is Leu and Y is Asp, or X is Asp and Y is He; and the sum of n and m is zero to eleven, inclusive.
10. The nucleic acid molecule of claim 1, wherein said immunoactive peptide is selected from the group consisting of Glu-Glu-Cys-Cys-Phe-Tyr (SEQ ID NO.: 1 ),
Pro-Gly-Cys-Cys-Gly-Pro (SEQ ID NO.:2), Pro-Gly-Cys-Cys-Pro-Gly (SEQ ID NO.. -3),
Gly-Pro-Cys-Cys-Pro-Gly (SEQ ID NO.:4),
Ala-Pro-Cys-Cys-Val-Pro (SEQ ID NO.:5),
Val-Ile-Cys-Cys-Leu-Thr (SEQ ID NO.:6), Lys-Pro-Cys-Cys-Glu-Arg (SEQ ID NO.:7),
Lys-Glu-Cys-Cys-Tyr-Val (SEQ ID NO.:8),
Thr-Pro-Cys-Cys-Phe-Ala (SEQ ID NO.:9),
Leu-Ala-Cys-Cys-Val-Val (SEQ ID NO.: 10),
Pro-Val-Cys-Cys-Ile-Gly (SEQ D NO.:l 1), Ser-Gln-Cys-Cys-Ser-Leu (SEQ ID NO.: 12),
Ser-Ile-Cys-Cys-Thr-Lys (SEQ ID NO.: 13),
Lys-Leu-Cys-Cys-Asp-Ile (SEQ ID NO.: 14),
Pro-Ala-Cys-Cys-Gly-Pro (SEQ ED NO.: 15),
Pro-Asp-Cys-Cys-He-Pro (SEQ ID NO.: 16), and Arg-Cys-Ser-Gly-Cys-Cys-Asn (SEQ ID NO. : 17).
11. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula II:
An-X-Cys-Z-Cys-Y-Bm (Jl) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Asn, Gin, His, Lys, Phe, and Pro; Z is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Lys, and
Arg;
Y is selected from the group consisting of Ala, Val, Leu, He, Gly, Asp, Glu, Lys, Arg, Gin, Tyr, Phe, Ser, Thr, and Pro; and n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-five, inclusive; the coding sequence further encoding a protein targeting sequence.
12. The nucleic acid of claim 1 1, wherein SEQ ID NOs. 139 through 156 are excluded from Formula π.
13. The nucleic acid of claim 1 1 , wherein the protein targeting sequence is a signal peptide.
14. The nucleic acid of claim 1 1, wherein the protein targeting sequence is a glycosyl- phosphatidylinositol attachment signal.
15. The nucleic acid of claim 11, wherein the protein targeting sequence is a transmembrane sequence.
16. The nucleic acid of claim 11, wherein the protein targeting sequence is linked to the amino terminus of the immunoactive peptide.
17. The nucleic acid of claim 11, wherein said nucleic acid further comprises a second coding sequence encoding a cytokine.
18. The nucleic acid of claim 17, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
19. The nucleic acid molecule of claim 1 1, wherein X is selected from the group consisting of Gly, Pro, He, Val, Asp, Leu, Glu, Gin, and Ala; Y is selected from the group consisting of Gly, Glu, Val, Gin, Arg, Leu, Tyr, Phe, He, Ser, Thr, Asp, and Pro; Z is selected from the group consisting of He, Gly, Thr, Ala, Arg, and Lys; and the sum of n and m is zero to ten, inclusive.
20. The nucleic acid molecule of claim 11 , wherein
X is Gly and Y is Gly, X is Pro and Y is Pro,
X is Pro and Y is Val,
X is He and Y is Leu,
X is Pro and Y is Glu, X is Glu and Y is Tyr,
X is Pro and Y is Phe,
X is Glu and Y is Phe,
X is Ala and Y is Val,
X is Val and Y is He, X is Gin and Y is Ser,
X is He and Y is Thr,
X is Leu and Y is Asp, or
X is Asp and Y is He; Z is He, Gly, Thr, Ala, or Lys; and the sum of n and m is zero to ten, inclusive.
21. The nucleic acid molecule of claim 11, wherein said immunoactive peptide is selected from the group consisting of
Val-Cys-Ile-Cys-Gln (SEQ ED NO.: 18),
Val-Cys-Gly-Cys-Arg (SEQ ID NO.: 19), Lys-Cys-Arg-Cys-Lys (SEQ ID NO.:20),
Asp-Cys-He-Cys-Gln (SEQ ED NO.:21),
He-Cys-Thr-Cys-Glu (SEQ ID NO.:22),
He-Cys-Thr-Cys-Arg (SEQ ID NO.:23),
Leu-Cys-Ala-Cys-Val (SEQ ID NO.:24), Phe-Cys-He-Cys-Lys (SEQ ID NO.:25),
Ala-Cys-Lys-Cys-Gln (SEQ ID NO.:26), and
Gly-Pro-Cys-He-Cys-Pro-Gly (SEQ ID NO.:27).
22. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula HI:
An-X-Y-Cys-Z-Bm (HI) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
X is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Asp, Glu, Lys, Arg, His, Tφ, Tyr, and Phe;
Y is selected from the group consisting of Ala, Val, Leu, He, Gly, Pro, and Gin;
Z is selected from the group consisting of Ala, Val, Leu, He, Gly, Ser, Thr, Lys, His, Phe, Tyr, Arg, and Pro; and n and m are whole integers chosen with the proviso that the sum of n and m is zero to twenty-six, inclusive; the coding sequence further encoding a protein targeting sequence.
23. The nucleic acid of claim 22, wherein SEQ ED NOs. 139 through 156 are excluded from Formula EOT..
24. The nucleic acid of claim 22, wherein the protein targeting sequence is a signal peptide.
25. The nucleic acid of claim 22, wherein the protein targeting sequence is a glycosyl- phosphatidylinositol attachment signal.
26. The nucleic acid of claim 22, wherein the protein targeting sequence is a transmembrane sequence.
27. The nucleic acid of claim 22, wherein the protein targeting sequence is linked to the amino terminus of the immunoactive peptide.
28. The nucleic acid of claim 22, wherein said nucleic acid further comprises a second coding sequence encoding a cytokine.
29. The nucleic acid of claim 28, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
30. The nucleic acid molecule of claim 22, wherein X is selected from the group consisting of Gly, Ala, He, Asp, Thr, Ser, Arg, and Tφ; Y is selected from the group consisting of He, Gly, and Pro; Z is selected from the group consisting of Lys, He, Phe, Pro, Ala, Tyr, and Gly; and the sum of n and m is zero to eleven, inclusive.
31 The nucleic acid molecule of claim 22, wherein X s Gly, Y is Pro and Z is He, X s Gly, Y is Pro and Z is Gly, X s Ala, Y is Pro and Z is Ala, X s He, Y is Pro and Z is Tyr, X s Ala, Y is Pro and Z is He, X s Arg, Y is Pro and Z is He, X s He, Y is Pro and Z is He, X s Asp, Y is Pro and Z is He, X s Tφ, Y is Pro and Z is He, X s Tφ, Y is Pro and Z is Gly, X s Gly, Y is He and Z is He, X s Thr, Y is Pro and Z is Tyr, X s Ala, Y is Pro and Z is Phe, X s Ser, Y is Pro and Z is Phe, X s Gly, Y is Pro and Z is Pro, X s Gly, Y is Pro and Z is Tyr.
32. The nucleic acid molecule of claim 22, wherein said immunoactive peptide is selected from the group consisting of Gly-Pro-Cys-Gly (SEQ ID NO.:28), Ala-Pro-Cys-Ala (SEQ ID NO.:29), Ile-Pro-Cys-Tyr (SEQ ID NO.:30), Tφ-Pro-Cys-Gly (SEQ ID NO.:31), Gly-Pro-Cys-Ile-Leu-Asn (SEQ ID NO.:32), Gly-Pro-Cys-Ile (SEQ ID NO.:33), Leu-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:34), Leu-Leu-Phe-Ala-Pro-Cys-Ile (SEQ ID NO.:35), Leu-Leu-Phe-Arg-Pro-Cys-Ile (SEQ ID NO.:36), Leu-Leu-Phe-He-Pro-Cys-Ile (SEQ D NO.:37), Leu-Leu-Phe-Asp-Pro-Cys-Ile (SEQ ID NO.:38), Ala-Val-Tφ-Thr-Pro-Cys-Tyr (SEQ ID NO.:39), Phe-Val-Met-Ala-Pro-Cys-Phe (SEQ ED NO.:40), Leu-Leu-Tyr-Ser-Pro-Cys-Phe (SEQ ID NO.:41), He-Ser-Gly-Pro-Cys-Pro-Lys (SEQ ID NO.:42), Phe-Leu-Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:43), Leu-Phe-Gly-Pro-Cys-Ile-Leu (SEQ ED NO.:44), Glu-Lys-Gly-Pro-Cys-Tyr-Arg (SEQ ID NO.:45), Phe-Cys-Leu-Gly-Pro-Cys-Pro (SEQ ID NO.:46), Phe-Gly-Pro-Cys-Ile (SEQ ID NO.:47), Phe-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn (SEQ ID NO.:48), Gly-Pro-Cys-πe-Leu-Asn-Arg (SEQ ED NO.:49), Leu-Leu-Phe-Tφ-Pro-Cys-Ile (SEQ ED NO.:50), Leu-Leu-Phe-Gly-Ile-Cys-He (SEQ ID NO.:51), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn
(SEQ ID NO.:52), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg
(SEQ ID NO.:53), Tφ-Cys-Gly-Pro-Cys-Lys-Met-Ile-Lys-Pro-Phe-Phe
(SEQ ID NO.:54), Leu-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu
(SEQ ID NO.:55), Phe-Leu-Phe-Gly-Pro-Cys-Ile-Leu-Asn-Arg-Leu-Met-Glu (SEQ ID NO.:56),
33. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula IV:
Aπ-Xp-Y-Cys-Zq-B, (IV) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
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, Phe, and Gin;
Z is selected from the group consisting of Met, Tφ, Tyr, Phe, Gly, Pro, Arg, Asn, Gin, Ala, and Lys; n, m, 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 coding sequence further encoding a protein targeting sequence.
34. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula IV:
AΓÇ₧-Xp-Y-Cys-Z^Bm (IV) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids; X is selected from the group consisting of Ser, Glu, Gly, Ala, Leu, Pro, Thr, Val, Asn, and Lys; Y is Gin;
Z is selected from the group consisting of Met, Tφ, Tyr, Phe, Gly, Pro, Arg, Asn, Gin, Ala, and Lys; n, m, 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; provided that SEQ ED NOs. 139 through 156 are excluded from Formula IV.
35. The nucleic acid of claim 33, wherein the protein targeting sequence is a signal peptide.
36. The nucleic acid of claim 33, wherein the protein targeting sequence is a glycosyl- phosphatidylinositol attachment signal.
37. The nucleic acid of claim 33, wherein the protein targeting sequence is a transmembrane sequence.
38. The nucleic acid of claim 33, wherein the protein targeting sequence is linked to the amino terminus of the immunoactive peptide.
39. The nucleic acid of claim 33, wherein said nucleic acid further comprises a second coding sequence encoding a cytokine.
40. The nucleic acid of claim 39, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
41. The nucleic acid molecule of claim 33, wherein both p and q are 1, and the sum of n and m is zero to 16, inclusive.
42. The nucleic acid molecule of claim 33, wherein both p and q are 1 , and the sum of n and m is zero to 11 , inclusive.
43. The nucleic acid molecule of claim 33, wherein both p and q are 1, and the sum of n and m is zero to 3, inclusive.
44. The nucleic acid molecule of claim 33, wherein X is Glu, Y is Pro, and Z is Met,
X is Gly, Y is Pro, and Z is Met,
X is Ala, Y is Pro, and Z is Tφ,
X is Ala, Y is Pro, and Z is Met,
X is Glu, Y is Pro, and Z is Tφ, X is Ser, Y is Pro, and Z is Tφ,
X is Leu, Y is Leu, and 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, and 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, and Z is Lys,
X is Glu, Y is Pro, and 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 Tφ.
45. The nucleic acid molecule of claim 33, wherein said immunoactive peptide is selected from the group consisting of Gln-Cys-Ala-Leu-Cys-Arg (SEQ ED NO.:81),
Val-Ala-Leu-Ser-Cys-Gln (SEQ ID NO.:82), Ile-Val-Lys-Ser-Cys-Lys (SEQ ID NO.: 83), Leu-Ala-Phe-Glu-Pro-Cys-Met (SEQ ID NO.:84), Leu-Leu-Pro-Gly-Pro-Cys-Met (SEQ ID NO.: 85), Met- Ala-Pro- Ala-Pro-Cys-Tφ (SEQ ID NO.: 86), Ala-Leu-Tyr-Ala-Pro-Cys-Met (SEQ ED NO.: 87),
Val-Leu-Tφ-Glu-Pro-Cys-Tφ (SEQ ID NO.:88), Met-Leu-Phe-Ser-Pro-Cys-Tφ (SEQ ID NO.: 89), Leu-Leu-Cys-Gly-Pro-Ala-Ile (SEQ D NO.:90), Leu-Cys-Phe-Gly-Pro- Ala-He (SEQ ED NO.:91), Val-Met-Pro-Ser-Pro-Cys-Tyr (SEQ ID NO.:92),
Val-Val-Phe-Ala-Pro-Cys-Tyr (SEQ ID NO.:93), Ala-Val-Pro-Glu-Pro-Cys-Phe (SEQ ED NO.:94), Met-Met-Tyr-Glu-Pro-Cys-Tyr (SEQ ID NO.:95), Ala-Ala-Tφ-Ser-Pro-Cys-Met (SEQ ED NO.:96), Val-Ala-Tyr-Gly-Pro-Cys-Tφ (SEQ ID NO.:97)
Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile (SEQ ID NO.:98), Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr (SEQ ED NO.:99), Pro-Gin- Val-Val-Cys-Asn-Tyr-Arg (SEQ ID NO.: 100), or Ala-Asn-Phe-Cys-Ala-Gly-Ala-Cys-Pro-Tyr-Leu-Tφ (SEQ ID NO.: 101)
Leu-Arg-Pro-Arg-Cys-Arg-Pro (SEQ ID NO.: 180), Pro-Gin- Val-Val-Cys-Asn-Tyr (SEQ ID NO.: 181), Arg-Gly-Tyr-Cys-Pro-Tyr (SEQ ID NO.: 182), and Gln-Cys-Ala-Leu (SEQ ID NO.: 183).
46. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula V:
Figure imgf000058_0001
where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Tφ, 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, Tφ, Glu, Leu, Met, and Lys;
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 coding sequence further encoding a protein targeting sequence.
47. A nucleic acid molecule comprising a coding sequence encoding an immunoactive peptide of Formula V: An-W-X-Y-Zp-Bm (V) where each A and each B is independently selected from any of the 20 common, naturally occurring amino acids;
W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Tφ, 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 Lys;
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; provided that SEQ ED NOs. 139 through 156 are excluded from Formula V.
48. The nucleic acid of claim 46, wherein the protein targeting sequence is a signal peptide.
49. The nucleic acid of claim 46, wherein the protein targeting sequence is a glycosyl- phosphatidylinositol attachment signal.
50. The nucleic acid of claim 46, wherein the protein targeting sequence is a transmembrane sequence.
51. The nucleic acid of claim 46, wherein the protein targeting sequence is linked to the amino terminus of the immunoactive peptide.
52. The nucleic acid of claim 46, wherein said nucleic acid further comprises a second coding sequence encoding a cytokine.
53. The nucleic acid of claim 52, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
54. The nucleic acid molecule of claim 46, wherein p is one and the sum of n and m is zero to 16, inclusive.
55. The nucleic acid molecule of claim 46, wherein p is one and the sum of n and m is zero to 11, inclusive.
56. The nucleic acid molecule of claim 46, wherein p is one and the sum of n and m is zero to 3, inclusive.
57. The nucleic acid molecule of claim 46, wherein
W is selected from the group consisting of Gly, Pro, Asp, Arg, Ala, He, Tφ, 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; and Z is selected from the group consisting of Gly, Phe, Val, He, Pro, and Leu.
58. The nucleic acid molecule of claim 46, wherein at least one of X and Y is Met.
59. The nucleic acid molecule of claim 46, wherein W is selected from the group consisting of Gly, Asp, Arg, Ala, Tφ, and Ser;
X is selected from the group consisting of Pro and He;
Y is Met; and
Z is selected from the group consisting of Phe, He, and Pro.
60. The nucleic acid molecule of claim 46, 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 Pro.
61. The nucleic acid molecule of claim 46, said immunoactive peptide having a Met and a Cys aligned contiguously.
62. The nucleic acid molecule of claim 46, said immunoactive peptide having a Met and a Cys separated by no more than one amino acid.
63. The nucleic acid molecule of claim 46, wherein said immunoactive peptide is selected from the group consisting of
Gly-Pro-Met-Ile (SEQ ID NO.: 114), Lys-Met-Arg-Met-Lys (SEQ ID NO.: 1 15)
Phe-Met-Ile-Met-Lys (SEQ ED NO.: l 16), Ile-Cys-Thr-Met-Glu (SEQ ED NO.:l 17),
Leu-Met- Ala-Met-Val (SEQ ID NO.: 118),
Ile-Met-Tyr-Met-Glu (SEQ ID NO.:l 19),
Ala-Pro-Met-Met-Val-Pro (SEQ ED NO.: 120), Gly-Pro-Met-Met-Pro-Gly (SEQ ID NO. : 121 ),
Gly-Pro-Cys-Met-Pro-Gly (SEQ ID NO.: 122),
Gly-Pro-Met-Cys-Pro-Gly (SEQ ID NO.: 123),
Pro-Gly-Met-Met-Gly-Pro (SEQ ID NO.: 124),
Val-Ile-Met-Met-Leu-Thr (SEQ ID NO.: 125), Leu- Ala-Phe-Glu-Pro-Met-Met (SEQ ED NO. : 126),
Met-Leu-Phe-Ser-Pro-Met-Tφ (SEQ ID NO.: 127),
Val-Val-Phe-Ala-Pro-Met-Tyr (SEQ ID NO.: 128),
Leu-Leu-Phe-Gly-Pro-Met-Ile (SEQ ID NO.: 129),
Leu-Leu-Tyr-Ser-Pro-Met-Phe (SEQ D NO.: 130), Leu-Leu-Phe-Asp-Pro-Met-He (SEQ ID NO.: 131),
Leu-Leu-Phe-Tφ-Pro-Met-Ile (SEQ ID NO.: 132),
Leu-Leu-Phe-Arg-Pro-Met-Ile (SEQ ID NO.: 133),
Leu-Leu-Phe-Ala-Pro-Met-Ile (SEQ ID NO.: 134),
Leu-Leu-Phe-Gly-Ile-Met-Ile (SEQ ED NO.: 135), Phe-Met-Leu-Gly-Pro-Met-Pro (SEQ ID NO. : 136),
Phe-Met-Ile-Met-Lys (SEQ ID NO.: 177),
Phe-Met-Leu-Gly-Pro-Met-Pro (SEQ ID NO.: 178), and
Lys-Met-Arg-Met-Lys (SEQ ID NO.: 179).
64. The nucleic acid molecule of claim 1, claim 11, claim 22, claim 33, or claim 46, wherein said molecule further comprises a mammalian expression control sequence operatively linked to the coding sequence.
65. A mammalian expression vector comprising the nucleic acid molecule of claim 1, claim 11, claim 22, claim 33, or claim 46.
66. The expression vector of claim 65, wherein said vector is a viral vector.
67. A viral particle capable of infecting a mammalian cell, said viral particle comprising the expression vector of claim 66.
68. A mammalian cell containing the nucleic acid molecule of claim 64.
69. A method of producing an immunoactive peptide, said method comprising introducing into a mammalian cell the nucleic acid molecule of claim 64, culturing said cell under conditions that permit expression of said immunoactive peptide, and harvesting said immunoactive peptide from said cell or from the medium surrounding said cell.
70. A method of producing an immunoactive peptide in a mammal, said method comprising introducing into a cell of the mammal the nucleic acid molecule of claim 64, under conditions permitting expression of the nucleic acid molecule in the cell.
71. A method of producing an immunoactive peptide in a mammal, said method comprising introducing into the mammal the cell of claim 68, wherein the nucleic acid molecule is expressed in the cell to produce the immunoactive peptide in the mammal.
72. A method for modulating the immune response in a patient, said method comprising administering to the patient the nucleic acid molecule of claim 64, wherein expression of the nucleic acid molecule in a cell of the patient modulates the immune response of the patient.
73. The method of claim 72, wherein said method further comprises administration of a cytokine to the patient.
74; The method of claim 73, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin- 10, and interleukin- 12.
75. The method of claim 73, wherein the cytokine is administered orally.
76. The method of claim 75, wherein the cytokine is selected from the group consisting of interferon-╬▒, interferon-╬▓, interleukin-2, interleukin-5, and interleukin-6.
77. A method of treating cancer in a patient, comprising the steps of:
(a) performing surgery on the patient to remove at least a portion of the cancer; and
(b) administering to the patient the nucleic acid molecule of claim 64.
78. A method of treating cancer in a patient, comprising the steps of:
(a) exposing the patient to a therapeutic level of ionizing radiation; and
(b) before, during or after the exposure to radiation, administering to the patient the nucleic acid molecule of claim 64.
79. The method of claim 78, wherein the radiation is localized to a specific region or tissue of the patient's body.
80. A method of treating cancer in a patient comprising administering to the patient (a) the nucleic acid molecule of claim 64, and (b) a chemotherapeutic compound cytotoxic to cells of the cancer.
81. The method of claim 72, wherein said administration is carried out by introducing said nucleic acid molecule into the patient's bloodstream.
82. The method of claim 72, wherein said administration is carried out by introducing said nucleic acid molecule into the synovial fluid of the patient.
83. The method of claim 72, wherein said administration is carried out by introducing said nucleic acid molecule into the vicinity of a tumor of the patient.
84. A method for modulating the immune response in a patient, said method comprising administering to said patient the cell of claim 68.
85. The method of claim 84, wherein said cell is a descendant of a cell of said patient, said nucleic acid molecule having been introduced into said descendant cell, or a precursor of said descendant cell, ex vivo.
86. The method of claim 85, wherein said administration is carried out by introducing said cell into the patient's bloodstream.
87. The method of claim 85, wherein said administration is carried out by introducing said cell into the synovial fluid of the patient.
88. The method of claim 85, wherein said administration is carried out by introducing said cell into the vicinity of a tumor of the patient.
89. The nucleic acid molecule of claim 1, claim 11, claim 22, claim 33, or claim 46, wherein said immunoactive peptide is immunosuppressive in a mammal.
90. The nucleic acid molecule of claim 1, claim 11, claim 22, claim 33, or claim 46, wherein said immunoactive peptide is immunostimulatory in a mammal.
91. The method of claim 72, wherein the immunoactive peptide is immunosuppressive in the patient.
92. The method of claim 91, wherein said patient is suspected of having an autoimmune disease.
93. The method of claim 72, wherein the immunoactive peptide is immunostimulatory in the patient.
94. The method of claim 93, wherein the patient is suspected of having cancer.
95. The nucleic acid molecule of claim 2, claim 13, claim 24, claim 35, or claim 48, wherein said signal peptide is a human signal peptide.
96. The nucleic acid molecule of claim 8, wherein A is Gly, Lys, Arg, Cys, Ser, Val, Ala, Thr, Glu, Pro, Tφ, Leu, Asp, Phe, or He; B is Leu, Arg, He, Val, Pro, Ala, Tyr, Gly, Tφ, Thr, Lys, Met, Asp, Glu, or Phe; and the sum of n and m is two to four, inclusive.
97. The nucleic acid molecule of claim 8, wherein A is Pro, Gly, Glu, Ala, Val, Lys,
Thr, Leu, or Ser; B is Tyr, Pro, Gly, Thr, Arg, Val, Ala, Leu, Lys, or He; n is one; and m is one.
98. The nucleic acid molecule of claim 19, wherein X is Val, Ala, Leu, He, Lys, Asp, Phe or Pro; Y is Glu, Val, Gin, Arg, Lys, or Pro; Z is Gly, Ala, He, Arg, Thr, or Lys; and the sum of n and m is one to three, inclusive.
99. The nucleic acid molecule of claim 43 or 44, wherein X is Gly, Ala, He, Arg, Asp, Tφ, 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.
100. A therapeutic composition comprising the nucleic acid molecule of claim 1, claim 11, claim 22, claim 33, or claim 46, and a pharmaceutically acceptable carrier.
101. The nucleic acid molecule of claim 33, wherein said immunoactive peptide is 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
(i) when p is 1, X is not Ser, Gly, Ala, or Thr, or
(ii) when p is 0, any amino acid residue of A adjacent to Y is not Ser, Gly, Ala, or Thr.
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