Methods and compositions for generating human monoclonal antibodies
The present invention provides a method for generating human monoclonal antibodies, especially those that are specific for surface antigens representative of a particular cell type. The present invention also includes populations of monoclonal antibodies produced by the invention methods, populations of polynucleotides comprising sequences encoding the immunoglobulins or fragments thereof, which are capable of binding to antigens representative of a cell type of interest.
1. An isolated population of non-transformed human B cell clones that produce human monoclonal antibodies immunoreactive with cell surface antigens, wherein said isolated population of non-transformed human B cell clones is generated by:
(a) (1) culturing in vitro in an immunization medium, an isolated population of human lymphocytes comprising a plurality of non-transformed human B lymphocytes and cells that natively express said cell surface antigens that have been immobilized to a solid substrate, and thereby contacting said isolated population of human lymphocytes comprising said plurality of non-transformed human B lymphocytes with said cells that natively express said cell surface antigens that have been immobilized to the solid substrate, and
(2) allowing one or more non-transformed human B lymphocytes to specifically bind to the immobilized cells bearing said cell surface antigens,
wherein the surfaces of the non-transformed human B lymphocytes are free of serum, and
(b) removing unbound B lymphocytes thereby selecting the non-transformed human B lymphocytes that specifically bind to the cells bearing the cell surface antigens, and
(c) culturing the selected B lymphocytes of (b) under conditions favorable for B cell proliferation to yield a plurality of non-transformed B cell clones that produce human monoclonal antibodies immunoreactive with the cell surface antigens.
2. The isolated population of non-transformed human B cell clones of claim 1 , said human B cell clones produce a population of human monoclonal antibodies exhibiting binding specificity to the cell surface antigens representative of the specific cell type.
3. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cells that natively express said cell surface antigens have been grown in serum-free medium.
4. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cells that natively express said cell surface antigens are grown in the form of a monolayer.
5. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cells that natively express said cell surface antigens are of embryonic or adult origin.
6. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cells that natively express said cell surface antigens are human cells.
7. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cells that natively express said cell surface antigens are eukaryotic cells.
8. The isolated population of non-transformed human B cell clones of claim 1 , wherein the cell surface antigens are presented on the surface of an intact cell.