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 binding affinity 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 for a time sufficient for antigen to bind to antibody on the surface of the cells, wherein the concentration of the antigen of interest is 0.1 to 2 nM;
(b) washing the cells for a period of time to remove unbound antigen;
(c) collecting cells bound to the antigen of interest;
(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 (K D ) of 0.1 pM to 25 nM.
2 . The method of claim 1 , wherein the antigen is a monomeric protein.
3 . The method of claim 2 , 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 5 , wherein the antigen is biotinylated and the binding partner is streptavidin comprising a detectable label.
9 . The method of claim 8 , further comprising contacting cells that bind biotinylated antigen with streptavidin after step (b) and before step (c).
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 obtain cells that express antibody with high affinity for the antigen of interest.
12 . The method of claim 3 , further comprising collecting the cells that bind detectable label to isolate cells that express antibodies of high affinity for the antigen of interest.
13 . The method of claim 1 , wherein the concentration of antigen is about 1 nM.
14 . The method of claim 1 , wherein cells of the population of cells express antibody that is membrane-bound antibody.
15 . The method of claim 1 , wherein the period of time for washing the cells is between about 5 minutes to about 60 minutes.
16 . The method of claim 1 , wherein the washing the cells for a period of time comprises two (2) washes of about 5 minutes to about 30 minutes per wash.
17 . The method of claim 1 , further comprising isolating cells that express antibodies of high affinity from the collected cells bound to antigen.
18 . The method of claim 1 , wherein the primary antibody-producing cells are obtained from spleen, lymph node, peripheral blood and/or bone marrow.
19 . The method of claim 1 , wherein the affinity of the antibody is between about 0.1 pM to about 10 nM (K D ).
20 . 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);
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.