Claims for Patent: 6,673,573

Summary for Patent: 6,673,573

Title:	Expression of immunoglobulin-cytokine fusion proteins in malignant B cells
Abstract:	According to the invention, there is provided a vector for the expression of immunoglobulin-cytokine fusion proteins in malignant B cells at least containing operably linked to each other (a) a region of at least 1.5 kb which is homologous to a region of the .mu. intron or the .kappa. intron and which lacks a functional C.sub..mu. or C.sub..kappa. enhancer or contains a non-functional C.sub.82 or C.sub..kappa. enhancer; (b) at least one DNA sequence encoding a domain of an immunoglobulin or a part thereof; (c) a DNA sequence encoding a cytokine; and (d) a marker gene selectable in eukaryotic B cells and lacking a functional enhancer region wherein the expression of said marker following integration is controlled by the cellular C.sub..mu. or C.sub..kappa. enhancer.
Inventor(s):	Mocikat; Ralph (Munich, DE)
Assignee:	GSF-Forschungszentrum fur Umwelt und Gesundheit (Oberschleissheim, DE)
Application Number:	09/064,026
Patent Claims:	1. Method for the expression of an immunoglobulin-cytokine fusion protein in malignant B cells in vitro, said method comprising: (a) introducing into a malignant B cell a vector comprising the following components operably linked together: (i) a region of at least 1.5 kb which is homologous to a region of the .mu. intron or the .kappa. intron, (ii) at least one DNA sequence encoding a constant region of an immunoglobulin or a part of the constant region, (iii) a DNA sequence encoding a cytokine, and (iv) a marker gene which is selectable in eukaryotic B cells and contains a functional enhancer, whereby after homologous recombination said immunoglobulin-cytokine fusion protein is expressed, comprising the variable region of the endogenous immunoglobulin of said malignant B cell fused with said constant region of an immunoglobulin or a part of said constant region and said cytokine; and (b) treating the cells to render them replication-incompetent. 2. Method according to claim 1, comprising an additional step of selecting and identifying cells stably expressing the fusion protein between steps (a) and (b). 3. Method according to claim 2, wherein the selection is carried out in a medium containing mycophenolic acid, G418, or hygromycin as a selective agent. 4. Method according to claim 1, wherein step (a) is performed by means of transfection. 5. Method according to claim 4, wherein said transfection is performed by electroporation, calcium phosphate co-precipitation, lipofection, the DEAE dextran technique or by retroviral gene transfer. 6. Method according to claim 1, wherein following introduction of a vector comprising the following components operably linked together: (i) a region of at least 1.5 kb which is homologous to a region of the .mu. intron or the .kappa. intron, (ii) at least one DNA sequence encoding a constant region of an immunoglobulin or a part of the constant region, (iii) a DNA sequence encoding a cytokine, and (iv) a marker gene which is selectable in eukaryotic B cells and contains a functional enhancer, a site-specific integration of said vector at the immunoglobulin heavy chain locus 3' of the heavy chain V gene of the malignant B cell occurs by homologous recombination. 7. Method according to claim 1, wherein the expression is controlled by the endogenous V.sub.H promoter.

Details for Patent 6,673,573

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2017-04-22
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2017-04-22
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2017-04-22