Method and composition for altering a b cell mediated pathology

Abstract

The present invention provides compositions for altering a B cell mediated pathology in a patient. The compositions may comprise at least one and/or two chimeric proteins. Each chimeric protein comprises at least a portion of either the VH or VL region of an immunoglobulin module from particular B cells from a patient having a B cell mediated pathology, and an immunoglobulin constant region. The genes encoding VH and/or VL regions and the genes encoding immunoglobulin constant regions are isolated and inserted in an expression vector. The chimeric proteins may be produced by introducing the expression vectors into insect cell lines. Proteins may be further purified and conjugated to a compound such as a immunogenic carrier.

What is claimed is: 
     
         1 . A composition comprising two different proteins produced by a process comprising the steps of:
 (a) isolating a gene encoding a full length sequence of a human V H  region isolated from a B cell clone of a human patient having a B cell mediated pathology;   (b) linking said gene encoding said entire V H  region and a gene encoding at least a portion of an immunoglobulin constant region in an expression vector to form a gene encoding the first of said different proteins;   (c) isolating a gene encoding a full length sequence of a human V L  region isolated from a B cell clone of said patient having said B cell mediated pathology;   (d) linking said gene encoding said entire V L  region and a gene encoding at least a portion of an immunoglobulin constant region into said expression vector to form a gene encoding the second of said different proteins;   (e) introducing said expression vector into insect cells, wherein said expression vector contains two separate expression cassettes each having a baculovirus promoter and a heterologous secretory signal sequence; and   (f) allowing the expression of said two different proteins having the same V H  and V L  amino acid sequences in said patient, wherein said composition further comprises a cytokine or chemokine, and wherein said composition elicits an active immune response.   
     
     
         2 . The composition of  claim 1  wherein said carrier protein is a keyhole-limpet hemocyanin (KLH). 
     
     
         3 . The composition of  claim 1 , wherein said composition is administered to said patient with a granulocyte-macrophage-colony stimulating factor (GM-CSF) cytokine. 
     
     
         4 . The composition of  claim 1 , wherein said first protein and/or said second protein is further conjugated to a carrier protein 
     
     
         5 . The composition of  claim 1 , wherein said gene encoding said first of said proteins comprising said entire V H  region and a first immunoglobulin constant region is controlled by a p10 promoter in an expression vector, and said gene encoding said second of said proteins comprising said entire V L  region and a second first immunoglobulin constant region is controlled by a polyhedrin promoter in an expression vector. 
     
     
         6 . The composition of  claim 1 , wherein one of said genes encoding said immunoglobulin constant regions is a gene encoding at least a portion of a human κ or λ constant region. 
     
     
         7 . The composition of  claim 1 , wherein said gene encoding one of said immunoglobulin constant regions is a gene encoding at least a portion of an immunoglobulin constant region selected from the group consisting of a human IgGγ1 constant region, a human IgGγ2 constant region, a human IgGγ3 constant region, a human IgGγ4 constant region, a human IgA1 constant region, a human IgA2 constant region, a human IgM constant region, a human IgD constant region, a human IgE constant region, a human κ, chain constant region, and a human λ chain constant region. 
     
     
         8 . The composition of  claim 1 , wherein said proteins are used to treat refractory low grade lymphoma or follicular B cell lymphoma. 
     
     
         9 . The composition of  claim 1 , wherein said proteins are produced in insect cells selected from the group consisting of the  Trichoplusia ni  cell line and the  Spodoptera frugiperda  cell line, and wherein said expression vector is a baculovirus expression vector that comprises a honey bee melittin secretory signal sequence and a human placental alkaline phosphatase secretory signal sequence. 
     
     
         10 . The composition of  claim 1 , wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a human placental alkaline phosphatase secretory signal sequence. 
     
     
         11 . A composition comprising two separate and distinct proteins produced by a process comprising the steps of:
 (a) isolating a gene encoding a full length sequence of a human V H  region isolated from a B cell clone of a human patient having a B cell mediated pathology;   (b) linking said gene encoding said entire V H  region and a gene encoding at least a portion of an immunoglobulin constant region in an expression vector to form a gene encoding the first of said separate and distinct proteins;   (c) isolating a gene encoding a full length sequence of a human V L  region isolated from a B cell clone of said patient having said B cell mediated pathology;   (d) linking said gene encoding said entire V L  region and a gene encoding at least a portion of an immunoglobulin constant region into an expression vector to form a gene encoding the second of said separate and distinct proteins;   (e) introducing said expression vectors into insect cells, wherein said expression vectors contain two separate expression cassettes each having a baculovirus promoter and a heterologous secretory signal sequence, wherein (i) said promoters are selected from the group consisting of p10 promoter and polyhedrin promoter, and (ii) said secretory signal sequences are selected from the group consisting of human placental alkaline phosphatase secretory signal sequence, honey bee melittin secretory signal sequence, and the endogenous secretory signal sequence associated with the immunoglobulin genes isolated from said patient; and   (f) allowing the expression of said two separate and distinct proteins having the same V H  and V L  amino acid sequences in said patient.   
     
     
         12 . The composition of  claim 11 , wherein said expression vectors are baculovirus expression vectors. 
     
     
         13 . The composition of  claim 11 , wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a human placental alkaline phosphatase secretory signal sequence. 
     
     
         14 . The composition of  claim 11 , wherein said first protein and/or said second protein is further conjugated to a carrier protein and is administered to said patient together with a cytokine or chemokine, and wherein said composition elicits an active immune response. 
     
     
         15 . The composition of  claim 11 , wherein said proteins are conjugated to a keyhole-limpet hemocyanin (KLH). 
     
     
         16 . The composition of  claim 11 , wherein said composition is administered to said patient. 
     
     
         17 . The composition of  claim 16 , wherein said composition is administered to said patient with a monocyte chemotactic protein 3 (MCR 3) chemokine. 
     
     
         18 . The composition of  claim 17 , wherein said composition is administered to said patient with a granulocyte-macrophage-colony stimulating factor (GM-CSF) cytokine. 
     
     
         19 . The composition of  claim 11 , wherein said gene encoding said first of the proteins comprising a full length V H  region and a first immunoglobulin constant region is controlled by a p10 promoter, and said gene encoding said second of the proteins comprising a full length V L  region and a second first immunoglobulin constant region is controlled by a polyhedrin promoter. 
     
     
         20 . The composition of  claim 11 , wherein one of said genes encoding at least a portion of said immunoglobulin constant regions is a gene encoding at least a portion of a human κ or λ constant region, and wherein said gene encoding at least a portion of said immunoglobulin constant regions is a gene encoding at least a portion of an immunoglobulin constant region selected from the group consisting of a human IgGγ1 constant region, a human IgGγ2 constant region, a human IgGγ3 constant region, a human IgGγ4 constant region, a human IgA1 constant region, a human IgA2 constant region, a human IgM constant region, a human IgD constant region, a human IgE constant region, a human k chain constant region, and a human 1 chain constant region.