Brachyury protein, non-poxvirus non-yeast vectors encoding brachyury protein, and their use

Abstract

Brachyury protein can be used to induce Brachyury -specific CD4+ T cells in vivo and ex vivo. It is also disclosed that Brachyury protein can be used to stimulate the production of both Brachyury -specific CD4+ T cells and Brachyury -specific CD8+ T cells in a subject, such as a subject with cancer. In some embodiments, the methods include the administration of a Brachyury protein. In additional embodiments, the methods include the administration of a nucleic acid encoding the Brachyury protein, such as in a non-pox non-yeast vector. In further embodiments, the method include the administration of host cells expressing the Brachyury protein.

1 . A method for inducing an immune response to  Brachyury  comprising administering to a subject an effective amount of
 (a) a protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1;   (b) a polypeptide comprising at least 15 consecutive amino acids of the amino acid sequence set forth at SEQ ID NO: 1 that specifically binds a Major Histocompatibility Class (MHC class II) molecule, or through internalization and cross-presentation can bind to MHC Class I;   (c) a nucleic acid encoding the protein or the polypeptide;   (d) a bacterial host cell expressing the protein or the polypeptide; or   (e) a non-pox non-yeast vector encoding the protein or the polypeptide,   thereby inducing the immune response, wherein the immune response comprises a  Brachyury  specific CD4+ T cell response.   
     
     
         2 . The method of  claim 1 , wherein the immune response further comprises a  Brachyury  specific CD8+ T cell response. 
     
     
         3 . The method of  claim 1 , further comprising measuring the  Brachyury  specific CD4+ T cell response. 
     
     
         4 . A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of
 (a) a protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1;   (b) a polypeptide comprising at least 15 consecutive amino acids of the amino acid sequence set forth at SEQ ID NO: 1;   (c) a nucleic acid encoding the protein or the polypeptide;   (d) a host cell expressing the protein or the polypeptide; or   (e) a non-pox non-yeast vector encoding the protein or the polypeptide, thereby treating or preventing the cancer in the subject.   
     
     
         5 . The method of  claim 1 , wherein the subject is human. 
     
     
         6 . The method of  claim 1 , wherein the subject has cancer. 
     
     
         7 . The method of  claim 6 , wherein the cancer is a breast cancer, small intestine cancer, stomach cancer, kidney cancer, bladder cancer, uterus cancer, ovarian cancer, testes cancer, lung cancer, colon cancer, prostate cancer, chronic lymphocytic leukemia (CLL), a B cell lymphoma, a Burkitt's lymphoma or a Hodgkin's lymphoma. 
     
     
         8 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , comprising administering to the subject a liposome comprising the protein or polypeptide or chitosan. 
     
     
         14 . The method of  claim 1 , wherein the polypeptide comprises 15 to 435 consecutive amino acids of the amino acid sequence set forth as SEQ ID NO: 1. 
     
     
         15 . The method of  claim 1 , wherein the polypeptide comprises at least 20 consecutive amino acids of the amino acid sequence set forth as SEQ ID NO: 1. 
     
     
         16 . The method of  claim 1 , comprising administering to the subject the non-pox non-yeast vector encoding the protein, and wherein the non-pox non-yeast vector is a viral vector. 
     
     
         17 . The method of  claim 16 , wherein the viral vector is an adenovirus, an alphavirus, a lentivirus, a measles virus or a poliovirus vector. 
     
     
         18 . The method of  claim 1 , wherein the non-pox non-yeast vector encodes a costimulatory molecule. 
     
     
         19 . The method of  claim 18 , wherein the costimulatory molecule is one or more of B7-1, B7-2, LFA-3 or ICAM-1. 
     
     
         20 . The method of  claim 1 , comprising administering to the subject the non-pox non-yeast vector encoding the protein, wherein the vector comprises a DNA sequence encoding an immunostimulatory molecule, wherein the immunostimulatory molecule is selected from the group consisting of IL-2, ICAM-1, LFA-3, CD72, GM-CSF, TNF-α, IFN-γ, IL-12, and IL-6. 
     
     
         21 . The method of  claim 1 , comprising administering to the subject an effective amount of a  Listeria  or  Salmonella  host cell expressing the protein. 
     
     
         22 . The method of  claim 1 , further comprising administering to the subject an effective amount of an adjuvant. 
     
     
         23 . The method of  claim 22 , wherein the adjuvant is chitosan. 
     
     
         24 . The method of  claim 1 , further comprising administering to the subject a therapeutically effective amount of a second agent, wherein the second agent is a chemotherapeutic agent, radiation, or a small molecule targeted therapeutic, or monoclonal antibodies. 
     
     
         25 . The method of  claim 24 , wherein the second agent is an epithelial growth factor receptor inhibitor, a transforming growth factor (TGF)-β inhibitor, or a tyrosine kinase inhibitor. 
     
     
         26 . The method of  claim 1 , wherein the subject has cancer, and wherein the cancer is a chemotherapy resistant cancer or a radiation resistant cancer. 
     
     
         27 . A non-pox non-yeast vector comprising a polynucleotide encoding:
 (a) an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1; or   (b) a polypeptide comprising 15 to 435 consecutive amino acids of the amino acid sequence set forth as SEQ ID NO: 1 that specifically binds a Major Histocompatibility Class (MHC class II) molecule, or through internalization and cross-presentation can bind to MHC Class I.   
     
     
         28 . The non-pox non-yeast vector of  claim 27 , wherein the vector is expressed in  Salmonella  or  Listeria.   
     
     
         29 . The non-pox non-yeast vector of  claim 27 , wherein the vector is a viral vector. 
     
     
         30 . The non-pox non-yeast vector of  claim 29 , wherein the vector is an alphavirus, a lentiviurs, an adenovirus, a measles virus or a poliovirus vector. 
     
     
         31 . The non-pox non-yeast vector of  claim 27 , comprising a polynucleotide encoding a polypeptide at least 15 and at most 400 consecutive amino acids of the amino acid sequence set forth at SEQ ID NO: 1 that specifically binds a Major Histocompatibility Class (MHC class II) molecule. 
     
     
         32 . The non-pox non-yeast vector of  claim 27 , wherein the vector encodes a costimulatory molecule. 
     
     
         33 . The non-pox non-yeast vector of  claim 32 , wherein the costimulatory molecule is one or more of B7-1, B7-2, LFA or ICAM-1. 
     
     
         34 . The non-pox non-yeast vector of  claim 27 , wherein the vector comprises an exogenous DNA sequence encoding an immunostimulatory molecule, wherein the immunostimulatory molecule is selected from the group consisting of IL-2, ICAM-1, LFA-3, CD72, GM-CSF, TNF-α, IFN-γ, IL-12, and IL-6. 
     
     
         35 . An isolated host cell comprising the vector of  claim 27 . 
     
     
         36 . An isolated host cell transformed with the vector of  claim 28 , wherein the host cell is a  Listeria  or a  Salmonella  host cell. 
     
     
         37 . A composition comprising an effective amount of (a) the non-pox non-yeast vector of  claim 27  or a host cell comprising the vector and (b) a pharmaceutically acceptable carrier. 
     
     
         38 . The composition of  claim 37 , further comprising an effective amount of an adjuvant. 
     
     
         39 . The composition of  claim 38 , wherein the adjuvant is chitosan or a liposome. 
     
     
         40 . The composition of  claim 37 , further comprising an effective amount of at least one of a cytokine selected from the group consisting of IL-2, GM-CSF, TNF-α, IL-12, IL-6, IL-15 and IL-15/IL-15 receptor complex. 
     
     
         41 . A method for inhibiting the growth of a cancer cell in a subject, the method comprising,
 contacting a dendritic cell with
 (a) a protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1; 
 (b) a polypeptide comprising at least 15 consecutive amino acids of the amino acid sequence set forth at SEQ ID NO: 1; or 
 (c) a  Listeria  or  Salmonella  host cell expressing the protein or the polypeptide; to produce a specific antigen presenting cell; and 
   administering the specific antigen presenting cell to the subject, thereby inducing an immune response and inhibiting the growth of the cancer cell.   
     
     
         42 . The method of  claim 41 , wherein the subject is human. 
     
     
         43 . The method of  claim 41 , wherein the cancer is a breast cancer, small intestine cancer, stomach cancer, kidney cancer, bladder cancer, uterus cancer, ovarian cancer, testes cancer, lung cancer, colon cancer, prostate cancer, chronic lymphocytic leukemia (CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma. 
     
     
         44 . The method of  claim 41 , wherein the protein comprises the amino acid sequence set forth as SEQ ID NO: 1. 
     
     
         45 . The method of  claim 41 , further comprising administering to the subject a non-pox non-yeast vector encoding the protein or polypeptide, and wherein the non-pox non-yeast vector is a viral vector. 
     
     
         46 . The method of  claim 45 , wherein the viral vector is an adenovirus, an alphavirus, a lentivirus, a measles virus or a poliovirus vector. 
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 4 , wherein the subject is at risk of developing cancer. 
     
     
         49 . The method of  claim 4 , wherein the subject has high grade prostatic intraepithelial neoplasia, familial adenomatous polyposis, or atypia of the breast.