Method and composition for altering a B cell mediated pathology
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 V H or V L region of a immunoglobulin molecule from particular B cells from a patient having a B cell mediated pathology, and an immunoglobulin constant region. The genes encoding V H and/or V L 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 an immunogenic carrier.
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 either a refractory low grade lymphoma or follicular B cell lymphoma;
(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 gone encoding a full length sequence of a human V L region isolated from a B cell clone of said patient having said refractory low grade lymphoma or follicular B cell lymphoma;
(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 first protein and/or said second protein is further conjugated to a carrier protein,
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 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.
5. 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.
6. 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.
7. The composition of claim 1 , wherein said proteins are used to treat non-Hodgkin's lymphoma.
8. 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.
9. 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.
10. 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 refractory low grade lymphoma or follicular B cell lymphoma;
(b) linking said gene encoding said entire V H region and a gene encodinig 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 refractory low grade lymphoma or follicular B cell lymphoma;
(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; 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.
11. The composition of claim 10 , wherein said expression vectors are baculovirus expression vectors.
12. The composition of claim 10 , wherein said expression vector is a bactilovirus 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.
13. The composition of claim 10 , wherein said proteins are conjugated to a keyhole-limpet hemocyanin (KLH).
14. The composition of claim 10 , wherein said composition is administered to said patient.
15. The composition of claim 14 , wherein said composition is administered to said patient with a monocyte chemotactic protein 3 (MCR 3) chemokine.
16. The composition of claim 15 , wherein said composition is administered to said patient with a granulocyte-macrophage-colony stimulating factor (GM-CSF) cytokine.
17. The composition of claim 10 , 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.
18. The composition of claim 10 , 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.