Ii-key/antigenic epitope hybrid peptide vaccines

• US 20040058881A1
• Filed: 09/24/2002
• Published: 03/25/2004
• Est. Priority Date: 09/24/2002
• Status: Active Grant

• Current Assignee: Nugenerex Immuno-Oncology Incorporated (Generex Biotechnology Corp.)
• Original Assignee: Antigen Express, Inc. (Generex Biotechnology Corp.)
• Inventors: Xu, Minzhen, Humphreys, Robert E.
• Granted Patent: US 7,179,645 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules

Abstract

Disclosed is a nucleic acid molecule comprising a first expressible sequence encoding a protein of interest or polypeptide of interest which contains an MHC Class II-presented epitope. In addition, the nucleic acid molecule comprises a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide. The antigen presentation enhancing hybrid polypeptide includes the following elements: i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: ______) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less.

Claims

1. A non-naturally occurring protein or polypeptide modified by recombinant DNA techniques, comprising:

a) a C-terminal element comprising an MHC Class II-presented epitope;

b) an N-terminal element comprising an Ii-key motif; and

c) an intervening element comprising a sequence from about 4 to about 11 amino acid residues;

the modification by recombinant DNA techniques taking place within elements b) and c).

2. The protein or polypeptide of claim 1 wherein the Ii-Key motif comprises a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM and at least one selected from the group consisting of HKR.

3. The method of claim 1 wherein the MHC Class II-presented epitope is a molecular feature in a therapeutically significant protein.

4. An expressible nucleic acid sequence encoding a non-naturally occurring protein or polypeptide, the protein or polypeptide comprising:

a) a C-terminal element comprising an MHC Class II-presented epitope;

b) an N-terminal element comprising an Ii-key motif, the Ii-Key motif comprising a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM and at least one selected from the group consisting of HKR; and

c) an intervening element comprising a sequence from about 4 to about 11 amino acid residues.

5. A method for suppressing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:

a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence encoding an Ii-Key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; and

b) modifying the Ii-Key motif to decrease its conformance to the archetypal Ii-Key regulatory motif, the archetype Ii-Key regulatory motif comprising a segment of 5 contiguous amino acids comprising at least two amino acids selected from the group consisting of leucine, isoleucine, valine, phenylalanine and methionine, and at lease one amino acid selected from the group consisting of histidine, lysine and arginine.

6. A method for enhancing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:

a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence lacking an Ii-key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; and

b) modifying the nucleic acid sequence to introduce an Ii-key motif appropriately spaced from the MHC Class II-presented epitope.

7. An isolated nucleic acid molecule comprising:

a) a first expressible sequence encoding a protein of interest or polypeptide of interest which contains an MHC Class II-presented epitope; and

b) a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide comprising;

i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO;

______) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;

ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and

iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less.

8. The isolated nucleic acid of claim 7 wherein the modifications of element a) are selected from the group consisting of:

a) deletion of amino acids from the C-terminus;

b) N-terminal extensions; and

c) amino acid substitutions.

9. The isolated nucleic acid of claim 7 wherein the amino acid substitutions to element a) exclude the substitution of amino acid residues aspartate or glutamate for an amino acid found in a wild-type mammalian Ii-Key sequence (SEQ ID NO ______).

10. The isolated nucleic acid of claim 7 wherein the C-terminal element further comprises an MHC Class I-presented epitope, or a portion thereof, the amino acid residues comprising the MHC Class I-presented epitope or portion thereof being constituent residues of the MHC Class II-presented epitope.

11. The isolated nucleic acid of claim 7 wherein the C-terminal element further comprises an antibody-recognized determinant, or a portion thereof, the amino acid residues comprising the antibody-recognized determinant or a portion thereof being constituent residues of the MHC Class II-presented epitope.

12. The isolated nucleic acid sequence of claim 7 wherein the protein or polypeptide of interest corresponds to a protein or polypeptide encoded by an infectious pathogen.

13. The isolated nucleic acid sequence of claim 12 wherein the infectious pathogen is selected from the group consisting of anthrax, EBOLA, HIV and influenza.

14. The isolated nucleic acid sequence of claim 7 wherein the infectious pathogen is vaccinia virus.

15. The isolated nucleic acid sequence of claim 7 wherein the protein of interest is gp42.

16. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:

a) providing a nucleic acid molecule comprising;

i) a first expressible sequence encoding a protein of interest, the protein of interest corresponding to a protein encoded by an infectious pathogen;

ii) a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide comprising;

1) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO;

______) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;

2) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and

3) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less; and

b) administering the nucleic acid molecule of step a) to a patient in an amount sufficient to stimulate an immune response.

17. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:

a) providing a first nucleic acid sequence comprising a first expressible sequence encoding a protein of interest, the protein of interest corresponding to a protein encoded by an infectious pathogen;

b) providing a second nucleic acid sequence comprising a second expressible sequence encoding an antigen presentation enhancing hybrid polypeptide comprising;

i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO;

______) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;

ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and

iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less; and

c) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; and

d) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.

18. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:

a) providing the genome of a vaccine pathogen of interest;

b) incorporating into the genome of step a) an expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide comprising;

i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO;

______) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;

ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being encoded by the genome of step a); and

iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less;

c) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; and

d) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.

19. A method for displaying a virus-specific antigenic epitope of interest on the surface of an MHC Class II molecule-positive cell in which Ii protein expression is suppressed, the method comprising:

a) providing an MHC Class II-positive cell; and

b) introducing into the cell of step a) a recombinant virus comprising;

i) an expressible nucleic acid sequence encoding the virus-specific antigenic epitope of interest; and

ii) an expressible reverse gene construct which encodes an RNA molecule which is complementary to an mRNA molecule which encodes human Ii protein, the RNA molecule having the ability to hybridize with the mRNA molecule thereby inhibiting translation of the mRNA molecule.

20. A method of claim 19 wherein the recombinant virus is a vaccinia virus.