Multiepitope polypeptides for cancer immunotherapy
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61K-039/00
G01N-033/574
C07H-021/04
C07H-021/00
C12P-021/06
C07K-014/82
출원번호
US-0388794
(2006-03-24)
공개번호
US-0246095
(2006-11-02)
우선권정보
IL-158140(2003-09-25)
발명자
/ 주소
Peretz,Tamar
Lotem,Michal
Frankenburg,Shoshana
Pitcovski,Jacob
Levi,Adva
Margalit,Hanah
Altuvia,Yael
출원인 / 주소
HADASIT MEDICAL RESEARCH SERVICES &
DEVELOPMENT LTD.
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM
GAVISH GALILEE BIO APPLICATIONS LTD.
대리인 / 주소
FLEIT KAIN GIBBONS GUTMAN BONGINI &
인용정보
피인용 횟수 :
0인용 특허 :
0
초록▼
Disclosed are recombinant multiple epitope polypeptides (MEPs) consisting of T cell epitopes derived from tumor-associated antigens capable of being presented by an antigen presenting cell (APC), the recombinant nucleic acid sequences and expression vectors encoding them, and host cells transfected
Disclosed are recombinant multiple epitope polypeptides (MEPs) consisting of T cell epitopes derived from tumor-associated antigens capable of being presented by an antigen presenting cell (APC), the recombinant nucleic acid sequences and expression vectors encoding them, and host cells transfected with said expression vectors. Further described are LTB-MEP fusion proteins comprising the E. Coli heat labile enterotoxin subunit B (LTB) peptide fused to a MEP by a synthetic linker, the recombinant nucleic acid sequences and expression vectors encoding them and host cell transfected with said expression vectors. Further included are compositions for inducing an immune response against malignancies, pharmaceutical compositions and a transdermal drug delivery system for the treatment of malignant disorders. Also disclosed are methods for conferring immunity against malignancies and for the treatment of malignant disorders.
대표청구항▼
1. A recombinant multiepitope polypeptide (MEP) wherein said MEP comprises at least two T cell epitopes, which may be identical or different, derived from a tumor associated antigen (TAA), and which epitopes are presented by an antigen presenting cell (APC) in the context of MHC Class I molecules a
1. A recombinant multiepitope polypeptide (MEP) wherein said MEP comprises at least two T cell epitopes, which may be identical or different, derived from a tumor associated antigen (TAA), and which epitopes are presented by an antigen presenting cell (APC) in the context of MHC Class I molecules and induce T cell activation, wherein each of said epitopes is operably linked to an adjacent epitope by a signal for proteasomal cleavage, which signals may be identical or different. 2. The recombinant polypeptide according to claim 1, further comprising adjuvants or carriers, wherein following proteasomal cleavage, cleavage products of said MEP are presented by an APC in the context of MHC Class I and Class II molecules, preferably, said carrier is any one of enterotoxin and Immunoglobulin Fc fragment, more preferably, said carrier is the E. Coli heat labile enterotoxin (LT), most preferably the B subunit of LT, designated LTB. 3. The recombinant polypeptide according to claim 1, wherein said TAAs are associated with any one of carcinomas, lymphomas, melanomas and sarcomas. 4. The recombinant polypeptide according to claim 3, wherein the signals for proteasomal cleavage, direct intracellular proteasomal excision of said MEP into epitope peptides and/or fragments, which are presented by an antigen presenting cell (APC) in the context of MHC Class I and Class II molecules, preferably, said signals for proteasomal cleavage are peptides selected from the group consisting of RKSY, RKSYL, ALL, SSL, AAY, AVHV, RVTIL and AASRY substantially as denoted by any one of SEQ ID NO: 19 to 23 and 41 to 43. 5. The recombinant polypeptide according to claim 4, wherein said epitopes are derived from any of the melanoma-associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1, preferably, said epitopes are selected from the group consisting of peptides 280-288 and 209-217 of gp100, peptide 369-377 of tyrosinase and peptide 27-35 of MART-1, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 15 to 18, respectively, or any functional homologue, variant, equivalent and derivative thereof. 6. A recombinant multiepitope polypeptide (MEP), wherein said MEP comprises at least two T cell epitopes, which may be identical or different, derived from melanoma-associated antigens selected from the group consisting of amino acids 280-288 of gp100, amino acids 209-217 of gp100, amino acids 369-377 of tyrosinase and amino acids 27-35 of MART-1, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 15 to 18, respectively, which epitopes are presented by an antigen presenting cell (APC) in the context of MHC Class I molecules and induce T cell activation, and wherein each of said epitopes is linked to an adjacent epitope by a signal for proteasomal cleavage, which signals may be identical or different, which signal is selected from the group consisting of RKSY, RKSYL, ALL, SSL AAY, substantially as denoted by SEQ ID NO: 19 to 23, respectively. 7. The recombinant polypeptide according to claim 6, wherein said melanoma-associated multiepitope polypeptide, is designated MEP-Mel and has the amino acid sequence as denoted by SEQ ID NO: 2 or SEQ ID NO: 57, or any functional analogue, variant, equivalent and derivative thereof. 8. The recombinant polypeptide according to claim 4, wherein said epitopes are derived from any of the breast and ovarian carcinoma associated antigens Mucin-1 (MUC1) and Lactadherin (BA46), preferably, said epitopes are selected from the group consisting of peptides D6 (LLLTVLTVV) and A7 (NLTISDVSV) of MUC1, and peptides BA46-6 (NLFETPVEA) and BA46-7 (GLQHWVPEL) of Lactadherin, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 25 to 28, respectively, or any functional analogue, variant, equivalent and derivative thereof. 9. A recombinant multiepitope polypeptide (MEP) wherein said MEP comprises at least two T cell epitopes derived from epithelial carcinoma-associated antigens, which epitopes may be identical or different and are selected from the group consisting of peptides D6 (LLLTVLTVV) and A7 (NLTISDVSV) of MUC1, peptides BA46-6 (NLFETPVEA) and BA46-7 (GLQHWVPEL) of Lactadherin, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 25 to 28, respectively, which epitopes are presented by an antigen presenting cell (APC) in the context of MHC Class I molecules and induce T cell activation, and wherein each of said epitopes is linked to an adjacent epitope by a signal for proteasomal cleavage, which signals may be identical or different, which signal is selected from the group consisting of RKSYL, AAY, AVHV, RVTIL and AASRY substantially as denoted by SEQ ID NO: 20, 23 and 41 to 43, respectively. 10. The recombinant polypeptide according to claim 9, wherein said epithelial carcinoma derived multiepitope polypeptide, is designated MEP-Epi having the amino acid sequence as denoted by SEQ ID NO: 40, or any functional analogue, variant, equivalent and derivative thereof 11. A recombinant polypeptide according to claim 1, wherein said polypeptide is produced by a host cell transfected with an expression vector encoding the polypeptide. 12. A recombinant nucleic acid sequence encoding a fusion protein comprising: a) a multiepitope polypeptide (MEP) which sequence comprises at least two segments, wherein each of said segments, which may be identical or different, encodes a T cell epitope derived from a tumor associated antigen (TAA), and wherein each of said segments is operably linked to an adjacent segment by a spacer element, wherein each of said spacer elements, which may be identical or different, encodes a signal for proteasomal cleavage, operably linked via a suitable linking element, to b) a nucleic acid sequence encoding the B subunit of LT, LTB (SEQ ID NO: 52). wherein following proteasomal cleavage, cleavage products of said fusion protein are presented by an APC in the context of MHC Class I and Class II molecules. 13. The recombinant nucleic acid sequence encoding a fusion protein according to claim 12, wherein said nucleic acid is any one of DNA, RNA and any combination thereof. 14. The recombinant nucleic acid sequence encoding a fusion protein according to claim 13, wherein said TAAs are associated with any one of carcinomas, lymphomas, melanomas and sarcomas. 15. The recombinant nucleic acid sequence encoding a fusion protein according to claim 14, wherein said MEP epitopes are derived from any of the melanoma-associated antigens (MAA) tyrosinase, gp-100, MAGE-3 and MART-1. 16. The recombinant nucleic acid sequence encoding a fusion protein according to claim 15, wherein said MEP sequence comprises at least two segments, which may be identical or different, wherein each of said segments encodes T cell epitopes derived from melanoma associated antigens selected from the group consisting of amino acids 280-288 of gp100, amino acids 209-217 of gp100, amino acids 369-377 of tyrosinase and amino acids 27-35 of MART-1, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 15 to 18, respectively, and wherein each of said segments is operably linked to an adjacent segment by a spacer element, wherein each of said spacer elements, which may be identical or different, encodes a signal for proteasomal cleavage selected from the group consisting of RKSY, RKSYL, ALL, SSL and AAY, substantially as denoted by SEQ ID NO: 19 to 23, respectively, preferably, said sequence is denoted by SEQ ID NO: 54 and encodes a LTB-melanoma-derived multiepitope fusion protein, designated LTB-MEP-Mel having the amino acid sequence as denoted by SEQ ID NO: 56, or any functional analogue, variant, equivalent and derivative thereof. 17. The recombinant nucleic acid sequence encoding a fusion protein according to claim 14, wherein said MEP epitopes are derived from any of the breast and ovarian carcinoma associated antigens Mucin-1 (MUC1) and Lactadherin (BA46). 18. The recombinant nucleic acid sequence encoding a fusion protein according to claim 17, wherein said MEP sequence comprises at least two segments, which may be identical or different, wherein each of said segments encodes T cell epitopes derived from breast and ovarian carcinoma-associated antigens selected from the group consisting of peptides D6 (LLLTVLTVV) and A7 (NLTISDVSV) of MUC1, peptides BA46-6 (NLFETPVEA) and BA46-7 (GLQHVWVPEL) of Lactadherin, substantially as denoted by the amino acid sequences as denoted by SEQ ID NO: 25 to 28, respectively, and wherein each of said segments is operably linked to an adjacent segment by a spacer element, wherein each of said spacer elements, which may be identical or different, encodes a signal for proteasomal cleavage selected from the group consisting of: RKSYL, AAY, AVHV, RVTIL and AASRY, substantially as denoted by any one of SEQ ID NO: 20, 23 and 41 to 43, respectively, preferably, said sequence encodes a LTB-breast cancer derived multiepitope fusion protein composed of SEQ ID NOs: 52, 53 and 39, or any functional analogue, variant, equivalent and derivative thereof designated LTB-MEP-Epi. 19. An expression vector encoding a multiepitope fusion protein comprising: a) a nucleic acid sequences encoding for a multiepitope polypeptide (MEP) which comprises at least two segments, wherein each of said segments, which may be identical or different, encodes a T cell epitope derived from a tumor associated antigen (TAA), and wherein each of said segments is operably linked to an adjacent segment by a spacer element, wherein each of said spacer elements, which may be identical or different, encodes a signal for proteasomal cleavage, operably linked to b) a nucleic acid sequence encoding LTB (SEQ ID NO: 52), and operably linked to c) control, promoting and/or regulatory elements; wherein following proteasomal cleavage, cleavage products of said fusion protein are presented by an APC in the context of MHC Class I and Class II molecules. 20. The expression vector as described in claim 19, wherein the fusion protein encoding sequence is defined in claim 12. 21. An expression system consisting of a host cell transfected with any one of the expression vectors defined in claim 20, wherein said host cell is any one of eukaryotic and prokaryotic cell, preferably a mammalian antigen presenting cell (APC). 22. A fusion protein comprising the LTB peptide sequence fused to a MEP polypeptide, wherein following proteasomal cleavage, cleavage products of said fusion protein are presented by an APC in the context of MHC Class I and Class II molecules and induce B and T cell activation. 23. The fusion protein according to claim 22, wherein said fusion protein comprises a melanoma-associated multiepitope polypeptide fused to LTB, which fusion protein is designated LTB-MEP-Mel and has the amino acid sequence as denoted by SEQ ID NO: 56, or any functional analogue, variant, equivalent and derivative thereof 24. The fusion protein according to claim 22, wherein said fusion protein comprises an epithelial carcinoma derived multiepitope polypeptide fused to LTB, which fusion protein is designated LTB-MEP-Epi composed by the amino acid of sequences SEQ ID NO: 55 and 40, or any functional analogue, variant, equivalent and derivative thereof. 25. A composition for inducing an immune response directed against malignancy in a mammalian subject, comprising as an active ingredient a multiepitope polypeptide (MEP) as defined in claim 1. 26. A composition for inducing an immune response directed against malignancy in a mammalian subject, comprising as an active ingredient at least one of a LTB-MEP fusion protein as defined in claim 22, a recombinant nucleic acid sequence encoding said LTB-MEP fusion protein as defined in claim 12, an expression vector encoding said LTB-MEP fusion protein as defined in claim 19, and an expression system consisting of a host cell transfected with said vectors as defined in claim 21. 27. A pharmaceutical composition for the treatment of a malignant disorder in a mammalian subject, comprising as an active ingredient a multiepitope polypeptide (MEP) as defined in claim 1, and optionally further comprising pharmaceutically acceptable carrier, diluent, excipient, adjuvant and additive. 28. A pharmaceutical composition for the treatment of a malignant disorder in a mammalian subject, comprising as an active ingredient any one of a LTB-MEP fusion protein as defined in claim 22, a recombinant nucleic acid sequence encoding said LTB-MEP fusion protein as defined in claim 12, an expression vector encoding said LTB-MEP fusion protein as defined in claim 19, and a host cell transfected with said vectors as defined in claim 21, optionally further comprising pharmaceutically acceptable carrier, diluent, excipient, adjuvant and additive. 29. The composition according to claim 25, wherein said adjuvant is IgG Fc fragment and/or an enterotoxin, preferably LTB. 30. The composition according to claim 25, wherein said malignancy or malignant disorder is any one of carcinomas, lymphomas, melanomas and sarcomas, preferably, said malignancy or malignant disorder is melanoma. 31. The composition according to claim 30, wherein said mammalian subject is human. 32. The composition according to claim 31, wherein said APC are autologous dendritic cells (DC). 33. A method for conferring immunity against a malignancy in a mammalian subject, comprising the step of administering to said subject a multiepitope polypeptide (MEP) as defined in claim 1, an autologous APC loaded with said MEP or a composition comprising the same, in an amount sufficient to induce in said subject an immune response against said malignancy. 34. A method for conferring immunity against a malignancy in a mammalian subject, comprising the step of administering to said subject a LTB-MEP fusion protein as defined in claim 22, a recombinant nucleic acid sequence encoding said LTB-MEP fusion protein as defined in claim 12, an expression vector encoding said LTB-MEP fusion protein as defined in claim 19, an expression system consisting of a host cell transfected with said vector as defined in claim 21, an autologous APC loaded with said LTB-MEP fusion protein or a composition comprising the same, in an amount sufficient to induce in said subject an immune response against said malignancy. 35. A method for the treatment of a malignant disorder in a mammalian subject in need, comprising the step of administering to said subject a multiepitope polypeptide (MEP) as defined in claim 1, an autologous APC loaded with said MEP or a composition comprising the same, in an amount sufficient to induce in said subject an immune response against said malignancy. 36. A method for the treatment of a malignant disorder in a mammalian subject in need, comprising the step of administering to said subject a LTB-MEP fusion protein as defined in claim 22, a recombinant nucleic acid sequence encoding said LTB-MEP fusion protein as defined in claim 12, an expression vector encoding said LTB-MEP fusion protein as defined in claim 19, an expression system consisting of a host cell transfected with said vector as defined in claim 21, an autologous APC loaded with said LTB-MEP fusion protein or a composition comprising the same, in an amount sufficient to induce in said subject an immune response against said malignancy. 37. The method according to claim 33, wherein said malignancy is selected from the group consisting of carcinomas, lymphomas, melanomas and sarcomas, preferably, said malignancy or a malignant disorder is melanoma. 38. The method according to claim 37, wherein said mammal is human. 39. The method according to claim 38, wherein said host cell transfected with said vector is an autologous APC, preferably, said APC is autologous dendritic cell (DC). 40. A transdermal drug delivery system for the treatment of a malignant disorder by inducing a systemic antigen specific immune response in a mammalian subject in need, comprising as an active ingredient at least the B subunit of the LT protein (LTB) conjugated to or mixed with a multiepitope polypeptide, MEP, as defined in claim 1 which drug delivery system optionally further comprises pharmaceutically acceptable carrier, diluent, excipient, adjuvant and/or additive. 41. The transdermal drug delivery system according to claim 40, wherein said LTB is recombinantly fused to MEP, creating the LTB-MEP fusion protein as defined in claim 22. 42. The transdermal drug delivery system according to claim 40, in the form of an ointment, cream, spray, patches, sustained-release patches or any other suitable transdermal delivery vehicle. 43. The transdermal drug delivery system according to claim 40, wherein said malignancy is selected from the group consisting of carcinomas, lymphomas, melanomas and sarcomas, preferably, said malignancy or a malignant disorder is melanoma. 44. The transdermal drug delivery system according to claim 40, wherein said immune response results in the production of antibodies, helper and cytotoxic T lymphocytes, specific for different antigens associated with said malignancy, comprised in said LTB-MEP fusion protein. 45. A method for the treatment of a malignant disorder in a mammalian subject in need, comprising the step of applying to said subject the transdermal drug delivery system as defined in claim 40, in an amount sufficient to induce in said subject a systemic immune response against said malignancy. 46. The method according to claim 45, wherein said malignancy is selected from the group consisting of carcinomas, lymphomas, melanomas and sarcomas, preferably, said malignancy or a malignant disorder is melanoma. 47. The method according to claim 46, wherein said mammal is human.
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