Culture Method for Obtaining a Clonal Population of Antigen-Specific B Cells
The present invention relates to methods of isolating antigen-specific cells and producing antibodies therefrom.
1 - 77 . (canceled)
78 . A method of identifying a B cell that expresses an antigen-specific antibody, the method comprising:
(a) harvesting B cells from a rabbit that has been immunized against a desired human antigen;
(b) enriching the harvested rabbit B cells to increase the proportion of B cells that are specific for said antigen, thereby forming an enriched B cell population;
(c) culturing one or more sub-populations of cells from said enriched rabbit B cell population;
(d) determining whether said cultured sub-populations produce an antibody specific to said human antigen, thereby identifying one or more antigen-positive sub-populations; and
(e) determining whether an individual B cell produces an antibody specific to the antigen by a method comprising:
(i) isolating one or more individual B cells from one or more of said antigen-positive sub-populations of step (d);
(ii) providing an immobilized antigen comprising a matrix or solid support to which said antigen has been directly or indirectly attached;
(iii) incubating an individual B cell with said immobilized antigen;
(iv) detecting whether an antibody secreted by said individual B cell is bound to said immobilized antigen; and
(v) identifying a B cell that expresses an antibody specific to said antigen by its spatial proximity to said immobilized antigen bound to antibody secreted by said individual B cell.
79 . The method of claim 78 , wherein said immobilized antigen comprises antigen-loaded beads.
80 . The method of claim 78 , wherein step (e)(iv) comprises incubating said immobilized antigen with a secondary antibody that is coupled to a detectable label, wherein said secondary antibody is an anti-immunoglobulin antibody that binds antibodies of the host of step (a).
81 . The method of claim 80 , wherein said detectable label is a fluorophore.
82 . The method of claim 78 , wherein step (a) comprises harvesting rabbit B cells from at least one source selected from the spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells.
83 . The method of claim 78 , wherein step (a) comprises harvesting B cells from more than one source selected from the spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells and pooling said B cells from more than one source.
84 . The method of claim 78 , further comprising isolating or sequencing a nucleic acid encoding an antibody chain or fragment thereof from said individual B cell determined to produce an antibody specific to the antigen in step (e).
85 . The method of claim 84 , further comprising expressing a polypeptide encoded by said nucleic acid.
86 . The method of claim 85 , wherein said expression is performed in a recombinant cell.
87 . The method of claim 86 , wherein said recombinant cell is a yeast, bacterium, plant, insect, amphibian, or mammalian cell.
88 . The method of claim 87 , wherein said recombinant cell is a diploid yeast.
89 . The method of claim 88 , wherein the diploid yeast is a Pichia.
90 . The method of claim 78 , further comprising immunizing the host with the antigen prior to step (a).
91 . The method of claim 90 , wherein step (a) comprises harvesting B cells from the host at about 20 to about 90 days after said immunization.
92 . The method of claim 90 , wherein step (a) comprises harvesting B cells from the host at about 50 to about 60 days after said immunization.
93 . The method of claim 78 , wherein step (b) comprises affinity purification of antigen-specific B cells using an antigen directly or indirectly attached to a solid matrix or support.
94 . The method of claim 93 , wherein the solid matrix comprises magnetic beads.
95 . The method of claim 93 , wherein the solid matrix comprises a column.
96 . The method of claim 93 , wherein the antigen that is directly or indirectly attached to a solid matrix or support is biotinylated and is attached to the matrix or support via streptavidin, avidin, or neutravidin.
97 . The method of claim 78 , wherein the sub-populations of step (c) comprise no more than about 10,000 antigen-specific, antibody-secreting cells.
98 . The method of claim 78 , wherein the sub-populations of step (c) comprise about 50 to about 10,000 antigen-specific, antibody-secreting cells.
99 . The method of claim 78 , wherein the sub-populations of step (c) comprise about 50 to about 5,000 antigen-specific, antibody-secreting cells.
100 . The method of claim 78 , wherein the sub-populations of step (c) comprise about 50 to about 1,000 antigen-specific, antibody-secreting cells.
101 . The method of claim 78 , wherein the sub-populations of step (c) comprise about 50 to about 500 antigen-specific, antibody-secreting cells.
102 . The method of claim 78 , wherein the sub-populations of step (c) comprise about 50 to about 250 antigen-specific, antibody-secreting cells.
103 . The method of claim 78 , wherein the sub-populations of step (c) are cultured in a medium comprising feeder cells.
104 . The method of claim 103 , wherein the feeder cells are EL4B cells.
105 . The method of claim 78 , wherein the sub-populations of step (c) are cultured in a medium comprising activated T cell conditioned medium.
106 . The method of claim 78 , wherein the sub-populations of step (c) are cultured in a medium comprising between about 1% and about 5% activated rabbit T cell conditioned medium.
107 . The method of claim 78 , wherein the sub-populations of step (c) are cultured for at least 3 days.
108 . The method of claim 78 , wherein the sub-populations of step (c) are cultured for between about 3 days and about 5 days.
109 . The method of claim 78 , wherein the sub-populations of step (c) are cultured for at least one week.
110 . The method of claim 78 , further comprising determining whether said cultured sub-populations of step (c) produce an antibody that exhibits agonism or antagonism of antigen binding to a binding partner; induction or inhibition of the proliferation of a specific target cell type; induction or inhibition of lysis of a target cell; or induction or inhibition of a biological pathway involving the antigen.
111 . The method of claim 78 , further comprising determining the antigen binding affinity of an antibody produced by said cultured sub-populations of step (c).
112 . A method of identifying a rabbit B cell that expresses an antigen-specific antibody, the method comprising:
(a) harvesting B cells from a rabbi that has been immunized with a desired human antigen;
(b) enriching the harvested B cells to increase the proportion of B cells that are specific for said antigen by affinity purification of antigen-specific B cells using an antigen directly or indirectly attached to a solid matrix or support, thereby forming an enriched B cell population;
(c) culturing one or more sub-populations of cells from said enriched B cell population;
(d) determining whether said cultured sub-populations produce an antibody specific to said antigen, thereby identifying one or more antigen-positive sub-populations; and
(e) determining whether an individual B cell produces an antibody specific to the antigen by a method comprising:
(i) isolating one or more individual B cells from one or more of said antigen-positive sub-populations of step (d);
(ii) providing an immobilized antigen comprising a matrix or solid support to which said antigen has been directly or indirectly attached;
(iii) incubating an individual B cell with said immobilized antigen;
(iv) incubating said immobilized antigen with a secondary antibody that is coupled to a detectable label, wherein said secondary antibody is a host-specific anti-immunoglobulin antibody, wherein said secondary antibody is an anti-immunoglobulin antibody that binds antibodies of the host of step (a), thereby detecting whether an antibody secreted by said individual B cell is bound to said immobilized antigen; and
(v) identifying a B cell that expresses an antibody specific to said antigen by its spatial proximity to said immobilized antigen bound to antibody secreted by said individual B cell. (i)
113 . The method of claim 112 , wherein said detectable label in step (e)(iv) is a fluorophore.
114 . The method of claim 112 , wherein said immobilized antigen comprises antigen-loaded beads.
115 . The method of claim 112 , wherein step (a) comprises harvesting B cells from at least one source selected from the spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells.
116 . The method of claim 112 , wherein step (a) comprises harvesting B cells from more than one source selected from the spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells and pooling said B cells from more than one source.
117 . The method of claim 112 , further comprising isolating or sequencing a nucleic acid encoding an antibody chain or fragment thereof from said individual B cell determined to produce an antibody specific to the antigen in step (e).
118 . The method of claim 117 , further comprising expressing a polypeptide encoded by said nucleic acid.
119 . The method of claim 118 , wherein said expression is performed in a recombinant cell.
120 . The method of claim 119 , wherein said recombinant cell is a yeast, bacterium, plant, insect, amphibian, or mammalian cell.
121 . The method of claim 119 , wherein said recombinant cell is a diploid yeast.
122 . The method of claim 121 , wherein the diploid yeast is a Pichia.
123 . The method of claim 112 , wherein step (a) comprises harvesting B cells from the host at about 20 to about 90 days after said immunization.
124 . The method of claim 123 , wherein step (a) comprises harvesting B cells from the host at about 50 to about 60 days after said immunization.
125 . The method of claim 112 , wherein in step (b) the solid matrix comprises magnetic beads.
126 . The method of claim 112 , wherein in step (b) the solid matrix comprises a column.
127 . The method of claim 112 , wherein in step (b) the antigen that is directly or indirectly attached to a solid matrix or support is biotinylated and is attached to the matrix or support via streptavidin, avidin, or neutravidin.
128 . The method of claim 112 , wherein the sub-populations of step (c) comprise no more than about 10,000 antigen-specific, antibody-secreting cells.
129 . The method of claim 112 , wherein the sub-populations of step (c) comprise about 50 to about 10,000 antigen-specific, antibody-secreting cells.
130 . The method of claim 112 , wherein the sub-populations of step (c) comprise about 50 to about 5,000 antigen-specific, antibody-secreting cells.
131 . The method of claim 112 , wherein the sub-populations of step (c) comprise about 50 to about 1,000 antigen-specific, antibody-secreting cells.
132 . The method of claim 112 , wherein the sub-populations of step (c) comprise about 50 to about 500 antigen-specific, antibody-secreting cells.
133 . The method of claim 112 , wherein the sub-populations of step (c) comprise about 50 to about 250 antigen-specific, antibody-secreting cells.
134 . The method of claim 112 , wherein the sub-populations of step (c) are cultured in a medium comprising feeder cells.
135 . The method of claim 134 , wherein the feeder cells are EL4B cells.
136 . The method of claim 117 , wherein the sub-populations of step (c) are cultured in a medium comprising activated T cell conditioned medium.
137 . The method of claim 112 , wherein the sub-populations of step (c) are cultured in a medium comprising between about 1% and about 5% activated rabbit T cell conditioned medium.
138 . The method of claim 137 , wherein the sub-populations of step (c) are cultured for at least 3 days.
139 . The method of claim 117 , wherein the sub-populations of step (c) are cultured for between about 3 days and about 5 days.
140 . The method of claim 137 , wherein the sub-populations of step (c) are cultured for at least one week.
141 . The method of claim 112 , further comprising determining whether said cultured sub-populations of step (c) produce an antibody that exhibits agonism or antagonism of antigen binding to a binding partner; induction or inhibition of the proliferation of a specific target cell type; induction or inhibition of lysis of a target cell; or induction or inhibition of a biological pathway involving the antigen.
142 . The method of claim 112 , further comprising determining the antigen binding affinity of an antibody produced by said cultured sub-populations of step (c).