METHODS AND COMPOSITIONS FOR INDUCING AN IMMUNE RESPONSE TO EGFRVIII

• US 20140037662A1
• Filed: 11/17/2011
• Published: 02/06/2014
• Est. Priority Date: 11/17/2010
• Status: Active Grant

• Current Assignee: Aduro Biotech, Inc. (Novartis Ag), Providence Health & Services - Oregon (Providence St. Joseph Health)
• Original Assignee: Aduro Biotech, Inc. (Novartis Ag), Providence Health & Services - Oregon (Providence St. Joseph Health)
• Inventors: Lauer, Peter M., Bahjat, Keith
• Granted Patent: US 9,200,057 B2
• Time in Patent Office: Days
• US Class Current: 424/185.1
• CPC Class Codes: A61K 2039/522 avirulent or attenuated
• CPC Class Codes: A61K 2039/523 expressing foreign proteins
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/001104 Epidermal growth factor receptors [EGFR]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators

Abstract

The present invention relates to methods of inducing a T-cell response against a EGFRvIII in a subject. These method comprise administering to a subject a composition which expresses at least one immunogenic polypeptide, the amino acid sequence of which comprise a plurality of EGFRvIII polypeptide sequences, the sequence of which each comprise EEKKGNYV (SEQ ID NO: 3), and/or administering the polypeptide itself.

Claims

1. A method of inducing a T-cell response to EGFRvIII in a subject, said method comprising:

recombinantly expressing at least one immunogenic polypeptide, the amino acid sequence of which comprise a plurality of EGFRvIII polypeptide sequences, the sequence of which each comprise EEKKGNYV (SEQ ID NO;

     3), in said subject under conditions selected to induce said T cell response in said subject.

2. The method of claim 1 wherein said plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of LEEKKGNYV (SEQ ID NO:

4), LEEKKGNYVVTDH (SEQ ID NO;

     2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO;

     5).

3. The method of claim 2 wherein said plurality of EGFRvIII polypeptide sequences comprise at least three copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

4. The method of claim 2 wherein said plurality of EGFRvIII polypeptide sequences comprise at least five copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

5. The method of claim 1, wherein each EGFRvIII polypeptide sequence is flanked by a sequence which is configured to be cleaved by the proteasome.

6. The method of claim 5, wherein each EGFRvIII polypeptide sequence is flanked by a sequence selected from the group consisting of ASKVL↓

ADGSVK;

ASKVA↓

GDGSIK;

LSKVL↓

ADGSVK;

LAKSL↓

ADLAVK;

ASVVA↓

GIGSIA;

GVEKI↓

NAANKG; and

DGSKKA↓

GDGNKK (SEQ ID NOS;

6-12), wherein each arrow represents an EGFRvIII polypeptide sequence.

7. The method of claim 1, wherein the immunogenic polypeptide is expressed in said subject by administering a Listeria monocytogenes bacterium comprising a nucleic acid sequence encoding said immunogenic polypeptide integrated into the genome of said bacterium operably linked to control sequences which cause the expression of the immunogenic polypeptide in the subject.

8. The method of claim 7, wherein the bacterium is an actA deletion mutant or an actA insertion mutant, an inlB deletion mutant or an inlB insertion mutant or a Δ

actA/Δ

inlB mutant comprising both an actA deletion or an actA insertion and an inlB deletion or an inlB insertion.

9. The method of claim 8, wherein said nucleic acid sequence has been integrated into a virulence gene of said bacterium, and the integration of said nucleic acid sequence disrupts expression of the virulence gene or disrupts a coding sequence of the virulence gene.

10. The method of claim 9, wherein the virulence gene is actA or inlB.

11. The method of claim 1, wherein the bacterium is an attenuated Listeria monocytogenes.

12. The method of claim 11, wherein the bacterium is Lm Δ

actA/Δ

inlB.

13. The method of claim 12, wherein the bacterium further comprises a genetic mutation that attentuates the ability of the bacterium to repair nucleic acid.

14. The method of claim 13, wherein the genetic mutation is in one or more genes selected from phrB, uvrA, uvrB, uvrC, uvrD and recA.

15. The method of claim 11, wherein the bacterium is a Listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein which is constitutively active.

16. The method of claim 11, wherein the bacterium is a killed but metabolically active Listeria monocytogenes.

17. The method of claim 16, wherein the bacterium is a Listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein which is constitutively active.

18. The method of claim 7, wherein the nucleic acid sequence is codon optimized for expression by Listeria monocytogenes.

19. The method of claim 7, wherein said conditions selected to induce said T cell response in said subject comprise administering said Listeria monocytogenes by one or more routes of administration selected from the group consisting of orally, intramuscularly, intravenously, intradermally, and subcutaneously to said subject.

20. The method of claim 1, wherein said immunogenic polypeptide is expressed as a fusion protein comprising a secretory signal sequence.

21. The method of claim 20, wherein the secretory signal sequence is a Listeria monocytogenes ActA signal sequence.

22. The method of claim 20, wherein said immunogenic polypeptide is expressed as a fusion protein comprising an in frame ActA-N100 sequence selected from the group consisting of SEQ ID NO:

28, SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31, or an amino acid sequence having at least 90% sequence identity to said ActA-N100 sequence.

23. The method of claim 7, wherein said method comprises administering a Listeria monocytogenes expressing a fusion protein comprising:

an ActA-N100 sequence selected from the group consisting of SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31 or an amino acid sequence having at least 90% sequence identity to said ActA-N100 sequence; and

a plurality of amino acid sequences having the sequence PASRALEEKKGNYVVTDHGSC (SEQ ID NO;

     5), each of said plurality of amino acid sequences being flanked by a sequence which is configured to be cleaved by the proteasome,

wherein said fusion protein is expressed from a nucleic acid sequence operably linked to a Listeria monocytogenes ActA promoter.

24. The method of claim 1, wherein said subject has an EGFRvIII-expressing tumor.

25. The method of claim 24, wherein said subject has a glioma.

26. The method of claim 1, wherein said composition, when delivered to said subject, induces an increase in the serum concentration of one or more proteins selected from the group consisting of IL-12p70, IFN-γ

, IL-6, TNF α

, and MCP-1 at 24 hours following said delivery; and

induces a CD4+ and/or CD8+ antigen-specific T cell response against EGFRvIII.

27. A composition comprising:

a bacterium or virus which comprises a nucleic acid encoding at least one immunogenic polypeptide, the amino acid sequence of which comprise a plurality of EGFRvIII polypeptide sequences, the sequence of which each comprise EEKKGNYV (SEQ ID NO;

     3).

28. The composition of claim 27 wherein said plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of LEEKKGNYV (SEQ ID NO:

4), LEEKKGNYVVTDH (SEQ ID NO;

     2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO;

     5).

29. The composition of claim 28 wherein said plurality of EGFRvIII polypeptide sequences comprise at least three copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

30. The composition of claim 28 wherein said plurality of EGFRvIII polypeptide sequences comprise at least five copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

31. The composition of claim 27, wherein each EGFRvIII polypeptide sequence is flanked by a sequence which is configured to be cleaved by the proteasome.

50. A pharmaceutical composition comprising:

the composition of claim 27; and

a pharmaceutically acceptable excipient.

32-49. -49. (canceled)

51. An isolated nucleic acid molecule encoding at least one immunogenic polypeptide, the amino acid sequence of which comprise a plurality of EGFRvIII polypeptide sequences, the sequence of which each comprise EEKKGNYV (SEQ ID NO:

3).

52. The isolated nucleic acid molecule of claim 51 wherein said plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of LEEKKGNYV (SEQ ID NO:

4), LEEKKGNYVVTDH (SEQ ID NO;

     2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO;

     5).

53. The isolated nucleic acid molecule of claim 51 wherein said plurality of EGFRvIII polypeptide sequences comprise at least three copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

54. The isolated nucleic acid molecule of claim 51 wherein said plurality of EGFRvIII polypeptide sequences comprise at least five copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO:

5).

55. The isolated nucleic acid molecule of claim 51, wherein each EGFRvIII polypeptide sequence is flanked by a sequence which is configured to be cleaved by the proteasome.

56-66. -66. (canceled)