Method for cloning cognate antibodies
AssigneeSymphogen A/S
InventorsJesper Kastrup, Lars S. Nielsen, Per-Johan Meijer
Life of this patent
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Abstract
The invention relates to a procedure for linking cognate pairs of VH and VL encoding sequences from a population of cells enriched in particular surface antigen markers. The linking procedure involves a multiplex molecular amplification procedure capable of linking nucleotide sequences of interest in connection with the amplification, in particular polymerase chain reaction (multiplex PCR). The method is particularly advantageous for the generation of cognate pair libraries as well as combinatorial libraries of variable region encoding sequences from immunoglobulins. The invention also relates to methods for generation of chimeric human/non-human antibodies and expression libraries generated by such methods.
Claims36
A method of producing a library of cognate pairs comprising linked immunoglobulin variable region encoding sequences, said method comprising:
- (a) immunizing a rodent with a target antigen and providing a lymphocyte-comprising cell fraction from said rodent;
- (b) isolating from said lymphocyte-comprising cell fraction cells expressing CD43 and CD138 antigen or MHCII and B220 antigen;
- (c) distributing the isolated cells from step b) individually into a plurality of vessels to obtain a population of isolated single cells, wherein each cell expresses CD43 and CD138 antigen or MHCII and B220 antigen;
- and
- (d) amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest using templates derived from said isolated single cells;
- and effecting linkage of the amplified nucleotide sequences of interest to generate a library of cognate pairs of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence.
A method of producing a library of cognate pairs comprising linked immunoglobulin variable region encoding sequences, said method comprising:
- (a) immunizing a rodent with a target antigen and providing a lymphocyte-comprising cell fraction from said rodent;
- (b) isolating from said lymphocyte-comprising cell fraction cells that express CD43 and CD138 antigen;
- (c) distributing the isolated cells from step b) individually into a plurality of vessels to obtain a population of isolated single cells, wherein each cell expresses CD43 and CD138 antigen;
- and
- (d) amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest using templates derived from said isolated single cells;
- and effecting linkage of the amplified nucleotide sequences of interest to generate a library of cognate pairs of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence.
A method of producing a library of cognate pairs comprising linked immunoglobulin variable region encoding sequences, said method comprising:
- (a) immunizing a rodent with a target antigen and providing a lymphocyte-comprising cell fraction from said rodent;
- (b) enriching said lymphocyte-comprising cell fraction for a lymphocyte population expressing CD43 and CD138 antigen or MHCII and B220 antigen;
- (c) distributing cells from the enriched cell fraction obtained in step b) individually into a plurality of vessels to obtain a population of isolated single cells, wherein each cell expresses CD43 and CD138 antigen or MHCII and B220 antigen;
- and
- (d) amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest using templates derived from said isolated single cells;
- and effecting linkage of the amplified nucleotide sequences of interest to generate a library of cognate pairs of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence.
Dependencies are inferred from the claim text.