IP Library Granted Patent US 8,962,315
Granted Patent B2
US 8,962,315 · App. 13/374,353 · Granted Feb 24, 2015

Compositions and methods including recombinant B lymphocyte cell line including at least one endogenous gene expressing at least one endogenous membrane immunoglobulin reactive to a first antigen and including at least one exogenously incorporated nucleic acid expressing at least one exogenous secreted immunoglobulin reactive to a second antigen

Inventors: Roderick A. Hyde (Redmond, WA); Wayne R. Kindsvogel (Seattle, WA)
Assignee: Elwha LLC
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Quick Facts
Patent No.
US 8,962,315
App. No.
13/374,353
Granted
Feb 24, 2015
Kind
B2
Abstract

Compositions and methods are disclosed herein for producing one or more immunoglobulins in an isolated B lymphocyte cell line. An isolated recombinant cell line includes an isolated B lymphocyte cell line capable of expressing at least one endogenous membrane immunoglobulin reactive to a first antigen and at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive to a second antigen.

Claims (71)

1. A method for producing a recombinant B lymphocyte cell line expressing at least two different immunoglobulins comprising:

isolating from a vertebrate subject exposed to at least one first antigen that is a B lymphocyte-activating antigen, a B lymphocyte cell expressing at least one first immunoglobulin that is an endogenous membrane immunoglobulin reactive to the at least one first antigen that is the B lymphocyte-activating antigen;

introducing into the isolated B lymphocyte cell at least one exogenous nucleic acid encoding at least one second immunoglobulin that is a secreted immunoglobulin reactive to at least one second antigen from a pathogenic agent to produce a recombinant B lymphocyte cell line; and

assaying for presence of the at least one exogenous secreted immunoglobulin reactive to the at least one second antigen from the pathogenic agent to select the recombinant B lymphocyte cell line.

2. The method of claim 1 comprising:

exposing the recombinant B lymphocyte cell line to the at least one first antigen; and

assaying production of the exogenous secreted immunoglobulin reactive to the at least one second antigen in the recombinant B lymphocyte cell line.

3. The method of claim 1 comprising:

selecting the recombinant B lymphocyte cell line expressing at least one endogenous secreted immunoglobulin reactive to the at least one first antigen.

4. The method of claim 1 comprising:

introducing into the recombinant B lymphocyte cell line at least one exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to the at least one second antigen to produce at least one second recombinant B lymphocyte cell line;

selecting the at least one second recombinant B lymphocyte cell line expressing at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin reactive to the at least one second antigen and the at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive to at least one second antigen.

5. The method of claim 1 , wherein the at least one first antigen and the at least one second antigen are different epitopes of a single antigenic polypeptide.

6. The method of claim 1 , wherein

the isolated B lymphocyte cell line is capable of disrupting expression of an endogenous secreted immunoglobulin reactive to the at least one first antigen.

7. The method of claim 1 comprising:

introducing into the recombinant B lymphocyte cell line at least one exogenous nucleic acid encoding at least one secreted immunoglobulin reactive to a third antigen to produce at least one second recombinant B lymphocyte cell line; and

selecting the at least one second recombinant B lymphocyte cell line expressing the at least one exogenous secreted immunoglobulin reactive to a third antigen from a pathogenic agent and expressing the at least one exogenous secreted immunoglobulin reactive to the at least one second antigen.

8. The method of claim 7 comprising:

exposing the at least one second recombinant B lymphocyte cell line to the at least one first antigen; and

assaying production of the exogenous secreted immunoglobulin reactive to third antigen in the at least one second recombinant B lymphocyte cell line.

9. The method of claim 1 comprising:

introducing into the isolated B lymphocyte cell line at least one exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to a third antigen that is a B lymphocyte-activating antigen;

selecting at least one second recombinant B lymphocyte cell line expressing the at least one membrane immunoglobulin reactive to the third antigen.

10. The method of claim 1 comprising:

exposing the recombinant B lymphocyte cell line to the at least one first antigen, and

assaying production of the exogenous secreted immunoglobulin reactive to the at least one second antigen in the recombinant B lymphocyte cell line.

11. The method of claim 10 comprising:

harvesting the exogenous secreted immunoglobulin reactive to the at least one second antigen from the recombinant B lymphocyte cell line or from a culture of the recombinant B lymphocyte cell line.

12. The method of claim 7 comprising:

exposing the at least one second recombinant B lymphocyte cell line to the at least one first antigen, and

assaying production of the exogenous secreted immunoglobulin reactive to the third antigen in the at least one second recombinant B lymphocyte cell line.

13. The method of claim 12 comprising:

harvesting the exogenous secreted immunoglobulin reactive to the third antigen from the recombinant B lymphocyte cell line or from a culture of the recombinant B lymphocyte cell line.

14. The method of claim 1 comprising:

assaying for the exogenous secreted immunoglobulin reactive to the at least one second antigen produced by the at least one recombinant B lymphocyte cell line in response to the membrane immunoglobulin binding to the at least one first antigen;

selecting the at least one recombinant B lymphocyte cell line;

culturing the at least one recombinant B lymphocyte cell line; and

isolating the exogenous secreted immunoglobulin reactive to the at least one second antigen.

15. The method of claim 8 comprising:

assaying for the exogenous secreted immunoglobulin reactive to the third antigen produced by the at least one second recombinant B lymphocyte cell line in response to the membrane immunoglobulin binding to the first antigen;

selecting the at least one recombinant B lymphocyte cell line;

culturing the at least one recombinant B lymphocyte cell line; and

isolating the exogenous secreted immunoglobulin reactive to the third antigen.

16. The method of claim 1 , wherein activating the at least one endogenous membrane immunoglobulin with the first antigen is capable of controlling expression of the at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive to the second antigen.

17. The method of claim 1 , wherein

the isolated B lymphocyte cell line comprises at least one of naïve B lymphocytes, immature B lymphocytes, transitional B lymphocytes, mature B lymphocytes, follicular B lymphocytes, memory B lymphocytes, plasmablasts, or plasma cells.

18. The method of claim 1 , wherein the B lymphocyte-activating antigen is one or more of a model antigen, dinitrophenol, or keyhole limpet hemocyanin.

19. The method of claim 1 , wherein the B lymphocyte-activating antigen is one or more of influenza antigen or S. aureus antigen.

20. A method for producing a recombinant B lymphocyte cell line expressing at least two different immunoglobulins comprising:

isolating from a vertebrate subject exposed to at least one first antigen from a pathogenic agent, a B lymphocyte cell expressing at least one first immunoglobulin that is an endogenous membrane immunoglobulin reactive to the at least one first antigen from the pathogenic agent;

introducing into the isolated B lymphocyte cell at least one exogenous nucleic acid encoding at least one second immunoglobulin that is a secreted immunoglobulin reactive to at least one second antigen from the pathogenic agent to produce a recombinant B lymphocyte cell line; and

assaying for presence of the at least one exogenous secreted immunoglobulin reactive to the at least one second antigen from the pathogenic agent to select the recombinant B lymphocyte cell line.

21. The method of claim 20 comprising:

exposing the recombinant B lymphocyte cell line to the at least one first antigen; and

assaying production of the exogenous secreted immunoglobulin reactive to the at least one second antigen in the recombinant B lymphocyte cell line.

22. The method of claim 20 comprising:

selecting the recombinant B lymphocyte cell line expressing at least one endogenous secreted immunoglobulin reactive to the at least one first antigen.

23. The method of claim 20 , wherein the at least one first antigen and the at least one second antigen are two different epitopes of a single antigenic polypeptide.

24. The method of claim 20 , comprising:

introducing into the recombinant B lymphocyte cell line at least one exogenous nucleic acid encoding at least one membrane immunoglobulin reactive to the at least one second antigen to produce at least one second recombinant B lymphocyte cell line;

selecting the at least one second recombinant B lymphocyte cell line expressing at least one exogenously incorporated nucleic acid encoding at least one membrane immunoglobulin reactive to the at least one second antigen and the at least one exogenously incorporated nucleic acid encoding at least one secreted immunoglobulin reactive to at least one second antigen.

25. The method of claim 20 , wherein the at least one first antigen and the at least one second antigen are different epitopes of a single antigenic polypeptide.

26. The method of claim 20 , wherein

the isolated B lymphocyte cell line is capable of disrupting expression of an endogenous secreted immunoglobulin reactive to the at least one first antigen.

27. The method of claim 20 , comprising:

introducing into the recombinant B lymphocyte cell line at least one exogenous nucleic acid encoding at least one secreted immunoglobulin reactive to a third antigen to produce at least one second recombinant B lymphocyte cell line; and

selecting the at least one second recombinant B lymphocyte cell line expressing the at least one exogenous secreted immunoglobulin reactive to a third antigen from a pathogenic agent and expressing the at least one exogenous secreted immunoglobulin reactive to the at least one second antigen.

28. The method of claim 27 , comprising:

exposing the at least one second recombinant B lymphocyte cell line to the at least one first antigen; and

assaying production of the exogenous secreted immunoglobulin reactive to third antigen in the at least one second recombinant B lymphocyte cell line.

Assignments (4)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 6, 2021
From: KOTA BIOTHERAPEUTICS, LLC
To: THE INVENTION SCIENCE FUND II, LLC
Reel/Frame 056159/0064 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 13, 2019
From: THE INVENTION SCIENCE FUND II, LLC
To: KOTA BIOTHERAPEUTICS, LLC
Reel/Frame 050995/0470 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 29, 2019
From: ELWHA LLC
To: THE INVENTION SCIENCE FUND II, LLC
Reel/Frame 050216/0254 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 13, 2012
From: HYDE, RODERICK A.; KINDSVOGEL, WAYNE R.
To: ELWHA LLC
Reel/Frame 027855/0763 →
Continuity (2)
Continuation In Part 13374351 · Dec 22, 2011
Related Publication 20130164277A1 · Jun 27, 2013