Method for generating high affinity antibodies
The present disclosure relates to a method for identifying cells that express antigen-specific antibodies with a high binding affinity for a monomeric antigen. Using fluorescence activated cell sorting, cells expressing high affinity antigen-specific antibodies are selected from a population of immune cells isolated from a mammal that has been immunized with or otherwise exposed to the antigen. Nucleic acids encoding the high affinity antibodies can then be cloned into other lymphoid and non-lymphoid cells where the antibody can be expressed and from which the antibodies can be secreted.
1. A method for selecting primary antibody-producing cells that express antibodies exhibiting high affinity binding for an antigen of interest, the method comprising:
(a) contacting a population of primary antibody-producing cells that express antibody to the antigen of interest with a monomeric form of the antigen of interest for a time sufficient for the antigen of interest to bind to antibody on the surface of the cells, wherein the concentration of the monomeric antigen of interest is between 0.1-2 nM or between 5-10 nM;
(b) washing the cells for a period of time to remove unbound antigen;
(c) collecting cells bound to the antigen of interest; and
(d) sorting the cells collected in step (c) to obtain cells that express antibodies of high affinity, wherein the high affinity antibodies have a dissociation equilibrium constant (KD) of 0.1 pM to 25 nM.
2. The method of claim 1 , wherein the primary antibody-producing cells express antibody that is membrane-bound antibody.
3. The method of claim 1 , wherein the antigen comprises a detectable label.
4. The method of claim 3 , wherein the detectable label is conjugated directly to the antigen.
5. The method of claim 4 , wherein the detectable label is conjugated to antigen via a pair of binding partners.
6. The method of claim 5 , wherein one binding partner of the pair of binding partners is labeled with a detectable label.
7. The method of claim 5 , wherein the pair of binding partners is biotin/streptavidin.
8. The method of claim 3 , wherein the antigen is biotinylated and the biotinylated antigen is contacted with streptavidin comprising the detectable label.
9. The method of claim 8 , wherein cells that bind the biotinylated antigen are contacted with the streptavidin after step (b).
10. The method of claim 3 , wherein the detectable label is a fluorescent label.
11. The method of claim 10 , wherein step (c) comprises using fluorescence-activated cell sorting to collect the cells bound to the antigen of interest.
12. The method of claim 10 , wherein step (d) comprises sorting the cells by flow cytometry.
13. The method of claim 12 , wherein the cells are sorted into at least two populations of cells using fluorescence-activated cell sorting according to fluorescence intensity.
14. The method of claim 13 , wherein antibody affinity is determined by surface plasmon resonance or solution-affinity enzyme-linked immunosorbent assay (ELISA).
15. The method of claim 1 , wherein the cells sorted in step (d) are sorted as single cells.
16. The method of claim 1 , further comprising step (e), which comprises:
(i) isolating an antibody-encoding nucleic acid from the cells that express antibodies of high affinity of step (d);
(ii) transfecting a host cell with a vector comprising the antibody-encoding nucleic acid; and
(iii) growing the transfected host cell under conditions to support expression of antibody by the host cell.
17. The method of claim 16 , wherein the vector is an expression vector.
18. The method of claim 16 , wherein the host cell is Chinese hamster ovary (CHO) cell.
19. The method of claim 16 , wherein the antibody comprises a human heavy chain variable region and a human light chain variable region.
20. The method of claim 19 , wherein the antibody further comprises a human heavy chain constant region and a human light chain constant region.
21. The method of claim 19 , wherein the human heavy chain variable region is a human IgG heavy chain variable region.
22. The method of claim 19 , wherein the human light chain variable region is a human kappa light chain variable region.
23. The method of claim 19 , wherein the concentration of monomeric antigen is between 0.1 nM and 2.0 nM.
24. The method of claim 19 , wherein the concentration of monomeric antigen is between 5 nM and 10 nM.
25. The method of claim 1 , wherein the high-affinity antibodies comprise a human heavy chain variable region and human a light chain variable region.
26. The method of claim 25 , wherein the human heavy chain variable region is a human IgG heavy chain variable region.
27. The method of claim 25 , wherein the human light chain variable region is a human kappa light chain variable region.
28. The method of claim 25 , wherein the wherein the concentration of monomeric antigen is between 0.1 nM and 2.0 nM.
29. The method of claim 28 , wherein the concentration of monomeric antigen is 1 nM.
30. The method of claim 1 , wherein the high affinity antibodies have a K D of from 0.1 pM to 10 nM.
31. The method of claim 30 , wherein the high affinity antibodies have a K D of from 0.1 nM to 10 nM.
32. The method of claim 1 , wherein the population of primary antibody-producing cells is derived from antibody-producing cells obtained from a human that has been exposed to the antigen of interest.
33. The method of claim 32 , wherein the antibody-producing cells are obtained from peripheral blood of a human with humoral immunity to the antigen of interest.
34. The method of claim 33 , wherein the antibody-producing cells are peripheral blood mononuclear cells (PBMCs).
35. The method of claim 32 , wherein the population of human primary antibody-producing cells comprises lymphocytes selected from the group consisting of B-cells, memory B-cells and plasmablasts.
36. The method of claim 1 , wherein the population of primary antibody-producing cells is derived from antibody-producing cells obtained from a non-human mammal.
37. The method of claim 36 , wherein the antibody-producing cells are obtained from a non-human mammal with humoral immunity to the antigen of interest.
38. The method of claim 36 , wherein the antibody-producing cells are obtained from spleen, lymph node, bone marrow or peripheral blood of the non-human mammal.
39. The method of claim 36 , wherein the non-human mammal comprises a nucleic acid sequence encoding a human heavy chain variable region and a nucleic acid sequence encoding a human light chain variable region.
40. The method of claim 1 , wherein the period of time for washing the cells is 60 minutes or less.
41. The method of claim 1 , wherein the concentration of monomeric antigen is between 5 nM and 10 nM.
42. The method of claim 1 , wherein the concentration of monomeric antigen is 5 nM.
43. The method of claim 1 , wherein the concentration of monomeric antigen is between 0.1 and 2 nM.
44. The method of claim 43 , wherein the concentration of monomeric antigen is 1 nM.
45. The method of claim 44 , wherein the concentration of monomeric antigen is between 0.1 to 1 nM.
46. The method of claim 43 , wherein the concentration of monomeric antigen is 2 nM.
47. The method of claim 1 , wherein at least 90% of the cells sorted in step (d) express high-affinity antibodies.