Process for making human antibody producing B-lymphocytes

• US 4,444,887 A
• Filed: 03/12/1981
• Issued: 04/24/1984
• Est. Priority Date: 12/10/1979
• Status: Expired due to Fees

• Current Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Original Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Inventors: Hoffmann, Michael K.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/243,021
• Time in Patent Office: 1,139 Days
• Field of Search: 435/68, 435/240, 435/241
• US Class Current: 435/373
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2502/11 blood or immune system cells
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte

Abstract

Antibody producing human B-lymphocytes are produced by a process which comprises culturing human B-lymphocytes in a tissue culture medium in the presence of an antigen; helper signal producing agents comprising monocytes or monocyte conditioned medium containing Interleukin 1 (I1-1), and helper T-lymphocytes or helper T-lymphocyte replacing factor; and human serum; and thereafter recovering the antibody producing cells from the medium.

Claims

1. Process for making antibody producing human B-lymphocytes which comprises culturing a human lymphoid cell suspension containing human B-lymphocytes and a cell concentration of about 1×

10⁶ to 5×

10⁶ cells/ml in a tissue culture medium containing;

(a) 0.3 to 0.003% antigen;

(b) helper signal producing agents consisting essentially of;

(i) monocytes or conditioned medium containing interleukin 1 derived from said monocytes in an amount to result in a concentration of 0.1 to 30% and

(ii) helper T-lymphocytes or helper T-lymphocyte replacing factor obtained from activated T-cells; and

(c) about 1-10% human serum;

and thereafter recovering the antibody producing human B-Lymphocytes from the medium.

2. The process of claim 1 comprising the additional step of treating the lymphoid cell suspension to remove suppressor cells or their precursors prior to the culturing step.

3. The process of claim 1 wherein said tissue culture medium further comprises a B-lymphocyte mitogen at a concentration of about 0.3 to 0.00003%, as a proliferation promoter.

4. The process of claim 1 wherein said human lymphoid cell suspension contains in addition helper T-lymphocytes and said tissue culture medium comprises a T-lymphocyte mitogen as an activator for the helper T-lymphocytes.

5. The process of claim 1 comprising the additional step of treating the lymphoid cell suspension to remove T-lymphocytes prior to the culturing step.

6. Process of claim 5 wherein said removed T-lymphocytes are cultivated to produce T-lymphocyte helper factor which is isolated and added to the tissue culture medium.

7. Process of claim 1 wherein red blood cells comprise the antigen.

8. Process of claim 7 wherein the red blood cells are conjugated with a specific antigen.

9. Process of claim 1 wherein the antigen is concentration about 0.03%, by volume.

10. Process of claim 4 wherein the T-lymphocyte mitogen is Concanavalin A.

11. Process of claim 3 wherein the B-lymphocyte mitogen is the bacterial mitogen Staphylococcus aureus of concentration about 0.003%, by volume.

12. Process of claim 1 wherein said conditioned medium containing Interleukin 1 is in an amount to result in a concentration of about 10%, by volume.

13. Process of claim 1 wherein said human serum is a concentration about 5-10% by volume.

14. Process of claim 10 wherein said T-lymphocyte mitogen is Concanavalin A at a concentration of 0.3 to 10 μ

g/ml.

15. Process of claim 14 wherein said Concanavalin A is about 10 μ

g/ml.

16. Process of claim 1 wherein the culture medium is a Mishell-Dutton medium.

17. Process of claim 1 wherein the human lymphoid cell suspension contains in addition suppressor cells or their precursors and further comprises the steps of:

(a) delaying the addition of human serum to the medium until 16 to 24 hours after culturing has started; and

(b) adding to the culture medium initially 0.3 to 0.0003% of a B-lymphocyte mitogen as a proliferation promoter.

18. Process of claim 17 wherein said human lymphoid cell suspension contains in addition helper T-lymphocytes, further comprising the step of adding a T-lymphocyte mitogen as an activator for the helper T-lymphocytes.

19. Process of claim 17 comprising the additional step of removing T-lymphocytes from the cell suspension before culturing.

20. Process of claim 19 wherein the removed T-lymphocytes are cultivated to produce T-lymphocyte helper factor which is isolated and added to the B-lymphocyte culture medium.

21. Process of claim 17 wherein red blood cells comprise the antigen.

22. Process of claim 21 wherein the red blood cells are conjugated with a specific antigen.

23. Process of claim 17 wherein the antigen is added to the medium in concentrations of about 0.03% by volume.

24. Process of claim 17 wherein the mitogen is the bacterial mitogen, Staphylococcus aureus.

25. Process of claim 17 wherein the proliferation promoter is added to the medium in concentrations of about 0.003%, by volume.

26. Process of claim 17 wherein said conditioned medium containing Interleukin 1 is added to the culture medium in concentrations of about 10% by volume.

27. Process of claim 17 wherein the human serum is added to the culture medium in concentrations of about 5-10% by volume.

28. Process of claim 18 wherein 0.3 to 10 μ

g/ml Concanavalin A is added as a T-lymphocyte activator.

29. Process of claim 28 wherein the Concanavalin A is added to the medium in concentration of about 10 μ

g/ml.