Method for Cloning Cognate Antibodies

Abstract

The invention relates to a procedure for linking cognate pairs of V H and V L 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.

1 . A method for generating a vector encoding a chimeric antibody with human constant regions and rodent variable regions, said method comprising:
 (a) providing a lymphocyte-comprising cell fraction from a rodent;   (b) obtaining a population of isolated single cells by distributing cells from said cell fraction individually into a plurality of vessels;   (c) amplifying and effecting linkage of the variable region encoding nucleic acids contained in said population of isolated single cells by amplifying, in a multiplex molecular amplification procedure, said nucleic acids using a template derived from an isolated single cell or a population of isogenic cells; and effecting linkage of the amplified nucleic acids encoding variable regions of heavy and light chains;   (d) effecting linkage of the amplified variable regions to human constant regions; and   (e) inserting the obtained nucleic acid into a vector.   
     
     
         2 . The method of  claim 1 , wherein said multiplex molecular amplification procedure is a multiplex RT'-PCR amplification. 
     
     
         3 . The method according to  claim 2 , wherein said multiplex RT-PCR amplification is a two step process comprising a separate reverse transcription (RT) step prior to the multiplex PCR amplification. 
     
     
         4 . The method according to  claim 2 , wherein said multiplex RT-PCR amplification is performed in a single step comprising initially adding all the components necessary to perform both reverse transcription (RT) and multiplex PCR amplification into a single vessel. 
     
     
         5 . The method according to  claim 1 , wherein said linkage of the nucleotide sequences of interest is performed in the same vessel as the multiplex molecular amplification. 
     
     
         6 . The method according, to  claim 2 , wherein said linkage of the nucleotide sequences of interest is effected in association with the multiplex RT-PCR amplification, utilizing a multiplex overlap-extension primer mix. 
     
     
         7 . The method according to  claim 1 , wherein said linkage of the nucleotide sequences of interest is effected by ligation. 
     
     
         8 . The method according to  claim 1 , wherein an additional molecular amplification is performed utilizing a primer mix adapted for amplifying the linked nucleic acid sequences of interest. 
     
     
         9 . The method according to  claim 6 , wherein the multiplex overlap-extension primer mix comprises primer sets wherein at least one primer set member of each primer set comprises an overlap-extension tail capable of hybridizing to the overlap-extension tail of a primer set member of a second primer set. 
     
     
         10 . The method according to  claim 6 , wherein the multiplex overlap-extension primer mix comprises:
 (a) at least one mKappar1 or hmJK primer complementary to the sense strand of an immunoglobulin light chain region encoding sequence;   (b) at least one mVK primer complementary to the antisense strand of an immunoglobulin light chain variable region encoding sequence or light chain variable region leader sequence, and capable of forming a primer set with the primer(s) in (a);   (c) at least one mCHrev1, mHCrev1-ext, or mJH primer complementary to the sense strand of an immunoglobulin heavy chain domain encoding sequence; and   (d) at least one mVH primer complementary to the antisense strand of an immunoglobulin heavy chain variable region encoding sequence or heavy chain variable region leader sequence, and capable of forming a primer set with the primer(s) in (c).   
     
     
         11 . The method of  claim 2 , wherein the PCR product is inserted into an expression vector. 
     
     
         12 . The method of  claim 11 , wherein a dual promoter cassette is inserted into the expression construct, the dual promoter cassette being capable of directing the simultaneous expression of heavy and light chains, preferably wherein the dual promoter cassette is bidirectional. 
     
     
         13 . The method of  claim 12 , wherein the dual promoter cassette further includes a nucleic acid sequence coding for dual signal peptides. 
     
     
         14 . The method of  claim 11 , wherein the expression vector comprises a human constant light chain encoding sequence or a fragment thereof and/or a human constant heavy chain encoding sequence or a fragment thereof. 
     
     
         15 . The method according to  claim 1 , comprising a further amplification step, wherein a polynucleotide encoding a human constant light chain or a fragment thereof with an overlap capable of providing linkage to the variable light chain, is added to the PCR mixture together with a primer set capable of amplification of a construct comprising in order: a marine VH chain, a linker a murine VL chain, and a human constant light chain. 
     
     
         16 . The method according to  claim 1 , comprising a further amplification step, wherein a polynucleotide encoding human constant heavy chain or a fragment thereof with an overlap capable of providing linkage to the variable heavy chain, is added to the PCR mixture together with a primer set capable of amplification of a construct comprising in order: a human constant heavy chain, a marine VH chain, a linker, and a murine VL chain. 
     
     
         17 . A library of vectors encoding chimeric antibodies, each antibody member consisting of rodent immunoglobulin variable region encoding sequences, and human immunoglobulin heavy and light chain constant regions. 
     
     
         18 . The library according to  claim 17 . wherein said vectors are obtained by the method of:
 (a) providing a lymphocyte-comprising cell fraction from a rodent donor;   (b) obtaining a population of isolated single cells by distributing cells from said cell fraction individually into a plurality of vessels, wherein at least a subpopulation of the cells express CD43 and CD138 antigen or and B220 antigen; and   (c) amplifying and effecting linkage of the variable region encoding sequences contained in said population of isolated single cells by amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest using a template derived from an isolated single cell or a population of isogenic cells; and effecting linkage of the nucleotide sequences of interest amplified; and   (d) inserting the linked nucleotide sequences of (c) into a vector.   
     
     
         19 . The library according to  claim 17 , wherein said vectors are obtained by the method of:
 (a) providing a lymphocyte-comprising cell fraction from a rodent;   (b) obtaining a population of isolated single cells by distributing cells from said cell fraction individually into a plurality of vessels;   (c) amplifying and effecting linkage of the variable region encoding nucleic acids contained in said population of isolated single cells by amplifying, in a multiplex molecular amplification procedure, said nucleic acids using a template derived from an isolated single cell or a population of isogenic cells; and effecting linkage of the amplified nucleic acids encoding, variable regions of heavy and light chains;   (d) effecting linkage of the amplified variable regions to human constant regions; and   (e) inserting the obtained nucleic acid of (d) into a vector.   
     
     
         20 . The library of  claim 17 , wherein the light chain constant region is a kappa constant region. 
     
     
         21 . A sub-library which codes for antibodies exhibiting desired binding specificities directed against a particular target, selected from a library according to  claims 17 .