Beta2 microglobulin fusion proteins and high affinity variants

Abstract

β 2 -microglobulin fusion proteins and modified forms of β 2 -microglobulin are disclosed. The fusion proteins are shown to incorporate onto the surface of MHC I expressing mammalian cells and to cause an increased cytotoxic T-cell response to antigens presented by such cells. The fusion proteins are useful in methods of tumor therapy. Modified forms of human β 2 -microglobulin, particularly a form having a serine to valine transition at amino acid position 55 of the mature protein are shown to have an enhanced affinity for MHC I heavy chain, and are useful both in the disclosed fusion proteins and as a vaccine adjuvant where enhanced cytotoxic T-cell response is desired.

We claim:  
     
         1 . A fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is a β 2 -microglobulin.  
     
     
         2 . A fusion protein according to  claim 1  wherein the second amino acid sequence is a human β 2 -microglobulin.  
     
     
         3 . A fusion protein according to  claim 1  wherein the second amino acid sequence is hβ 2 m S55V.  
     
     
         4 . A fusion protein comprising first and second domains, wherein the second domain is β 2 m.  
     
     
         5 . A fusion protein according to  claim 4  wherein the first domain joined to the amino terminal of the second domain.  
     
     
         6 . A fusion protein according to  claim 4  wherein the second domain is hβ 2 m.  
     
     
         7 . A fusion protein according to  claim 4  wherein the first domain is a co-stimulatory protein.  
     
     
         8 . A fusion protein according to  claim 7  wherein the co-stimulatory protein is selected from the group consisting of B7.1 and B7.2.  
     
     
         9 . A fusion protein according to  claim 4  wherein the first domain is an integrin, a cytokine or a cell adhesion molecule.  
     
     
         10 . A fusion protein according to  claim 6  wherein the hβ 2 m is hβ 2 m S55V.  
     
     
         11 . A fusion protein according to  claim 4  wherein the first and second domains are linked by a peptide linker.  
     
     
         12 . A fusion protein according to  claim 4  wherein the fusion protein further comprises a signal peptide joined to the N terminus of the first domain.  
     
     
         13 . A fusion protein according to  claim 12  wherein the signal peptide is a β 2 m signal peptide.  
     
     
         14 . A fusion protein according to  claim 10  wherein the hβ 2 m S55V has an amino acid sequence as shown in Seq. I.D. No. 10.  
     
     
         15 . A fusion protein according to  claim 6  wherein the protein has an amino acid sequence selected from the group consisting of the sequences shown in Seq. I.D. Nos. 2 and 3.  
     
     
         16 . A recombinant nucleic acid molecule encoding a protein according to  claim 4 .  
     
     
         17 . A vector comprising a nucleic acid molecule according to  claim 16 .  
     
     
         18 . A transgenic cell comprising a nucleic acid molecule according to  claim 16 .  
     
     
         19 . A cell having a cell membrane comprising a fusion protein according to  claim 4 .  
     
     
         20 . A cell according to  claim 19  wherein the cell is a tumor cell.  
     
     
         21 . A protein comprising a structure X—Y wherein X is a protein domain and Y is a beta-2 microglobulin.  
     
     
         22 . A protein according to  claim 21  comprising a structure X-L-Y wherein L is a linker peptide.  
     
     
         23 . A protein according to  claim 22  comprising S—X-L-Y wherein S is a signal peptide.  
     
     
         24 . A protein according to  claim 23  wherein the signal peptide is a β 2 m signal peptide.  
     
     
         25 . A nucleic acid molecule encoding a protein according to  claim 21 .  
     
     
         26 . A protein according to  claim 21  wherein the protein has an amino acid sequence as shown in Seq. I.D. No. 2 or Seq. I.D. No. 3.  
     
     
         27 . A method of enhancing the immune response of a mammal to an antigen presented on the surface of a cell, the method comprising: 
 (a) contacting the cell with a fusion protein according to  claim 4  such that the fusion protein is presented on the surface of the cell; and    (b) administering the cell to a mammal.    
     
     
         28 . The method of  claim 27  wherein the first amino acid sequence is a co-stimulatory molecule.  
     
     
         29 . The method of  claim 28  wherein the co-stimulatory molecule is B7.1 orB7.2.  
     
     
         30 . The method of claim  29 .wherein the cell is a tumor cell.  
     
     
         31 . A method of enhancing the immune response of a mammal to an antigen presented on the surface of a cell, the method comprising: 
 (a) transforming the cell with a nucleic acid molecule according to  claim 16 , such that expression of the nucleic acid molecule results in expression of a fusion protein encoded by the nucleic acid molecule being presented on the surface of the cell; and    (b) administering the cell to a mammal.    
     
     
         32 . The method of  claim 31  wherein the first amino acid sequence is a co-stimulatory molecule.  
     
     
         33 . The method of  claim 32  wherein the co-stimulatory molecule is B7.1 or B7.2.  
     
     
         34 . The method of  claim 33  wherein the cell is a tumor cell.  
     
     
         35 . A human β 2 -microglobulin molecule having a valine residue at position 55.  
     
     
         36 . A human β 2 -microglobulin molecule according to  claim 35 , wherein the molecule comprises the amino acid sequence shown in Seq. I.D. No. 10.  
     
     
         37 . A vaccine preparation comprising at least one antigen and a molecule selected from the group consisting of 
 (a) a human β 2 -microglobulin molecule having a valine at position 55; and    (b) a fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is a β 2 -microglobulin.    
     
     
         38 . A vaccine preparation according to claim  37 (b) wherein the β 2 -microglobulin is hβ 2 m S55V.  
     
     
         39 . A vaccine preparation according to  claim 37  wherein the antigen is selected from the group consisting of bacterial, viral and tumor antigens.  
     
     
         40 . A method of vaccinating a mammal, comprising administering to the mammal a vaccine preparation according to  claim 37 .  
     
     
         41 . A method of vaccinating a mammal, comprising administering to the mammal an antigen and a microglobulin protein selected from the group consisting of: 
 (a) a human β 2 -microglobulin protein having a valine at position 55; and    (b) a fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is a β 2 -microglobulin.    
     
     
         42 . A method of stimulating a tumor-reactive cytotoxic T-cell response, comprising: 
 (a) isolating T-cells from a patient having a tumor;    (b) isolating tumor cells from the patient;    (c) incubating the tumor cells with a fusion protein according to  claim 4 , such that the fusion protein is presented on the surface of the tumor cells;    (d) incubating the T-cells in the presence of the fusion protein-presenting tumor cells to increase the number of tumor-reactive T-cells; and    (e) administering a therapeutically effective dose of the tumor-reactive T-cells to the patient.