Method of producing human monoclonal antibodies and kit therefor

• US 5,567,610 A
• Filed: 10/17/1994
• Issued: 10/22/1996
• Est. Priority Date: 09/04/1986
• Status: Expired due to Term

• Current Assignee: BioInvent International AB
• Original Assignee: BioInvent International AB
• Inventors: Danielsson, Lena, Moller, Susanna, Borrebaeck, Carl
• Primary Examiner(s): Ziska, Suzanne E.
• Application Number:
  US08/323,593
• Time in Patent Office: 736 Days
• Field of Search: 435/172.3, 435/172.2, 435/240.2
• US Class Current: 435/451
• CPC Class Codes: A61K 39/0005 Vertebrate antigens from snakes A61K39/38
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 5/0087 Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte

Abstract

A method for the removal of, for in vitro immunization, undesired cells from human lymphocyte containing cell populations is disclosed. For the method, use is made of lysosomotropic agents which are caused to act in vitro on human lymphocyte containing cell populations.

Also disclosed is a method in the in vitro immunization of lymphocytes for use in the production of human monoclonal antibodies. In this method, lysosomotropic agents are caused to act in vitro on human lymphocyte containing cell populations for removing cell populations having a negative effect on the in vitro immunization, whereupon the lymphocytes are in vitro immunized antigen-specifically and immortalized.

Finally, a kit for in vitro immunization of lymphocytes for use in the production of human monoclonal antibodies is disclosed. The kit comprises as active constituents a container containing lymphokines and a container containing lysosomotropic agents.

Claims

1. In an improved method for in vitro immunization of lymphocytes for use in production of human monoclonal antibodies, the improvement comprising:

exposing human peripheral blood lymphocytes to a lysomotrophic agent selected from the group consisting of L-leucine-O-methyl ester, L-glutamic acid dimethyl ester and L-leucyl-L-leucine-O-methyl ester to produce treated human lymphocytes; and

exposing said treated human lymphocytes to an antigen to activate lymphocytes wherein said treated, activated lymphocytes produce antibody which specifically binds said antigen.

2. The method of claim 1, wherein said lysosomotropic agent is L-leucine-O-methyl ester.

3. The method of claim 1, wherein said lysosomotropic agent is L-glutamic acid dimethyl ester.

4. The method of claim 1, wherein said lysosomotropic agent is L-leucyl-L-leucine-O-methyl ester.

5. The method of claim 2, which further comprises the step of immortalizing said activated lymphocytes, wherein said step of immortalizing is selected from the group consisting of fusion of activated lymphocytes with human or murine myelomas, infections of said lymphocytes with Epstein-Barr virus, fusion with a lymphoblastoid cell line and fusion with lymphoma cells.

6. The method of claim 3, which further comprises the step of immortalizing said activated lymphocytes, wherein said step of immortalizing is selected from the group consisting of fusion of activated lymphocytes with human or murine myelomas, infections of said lymphocytes with Epstein-Barr virus, fusion with a lymphoblastoid cell line and fusion with lymphoma cells.

7. The method of claim 4, which further comprises the step of immortalizing said activated lymphocytes,wherein said step of immortalizing is selected from the group consisting of fusion of activated lymphocytes with human or murine myelomas, infections of said lymphocytes with Epstein-Barr virus, fusion with a lymphoblastoid cell line and fusion with lymphoma cells.

8. The method of claim 5, which further comprises culturing said immortalized activated lymphocytes and recovering monoclonal antibodies produced by said immortalized activated lymphocytes.

9. The method of claim 6, which further comprises culturing said immortalized activated lymphocytes and recovering monoclonal antibodies produced by said immortalized activated lymphocytes.

10. The method of claim 7, which further comprises culturing said immortalized activated lymphocytes and recovering monoclonal antibodies produced by said immortalized activated lymphocytes.

11. The method of claim 1 wherein said lysosomotropic agent is a peptide having a C-terminal L-leucine-O-methyl ester.