Method for Linking Sequences of Interest

Abstract

Multiplex overlap-extension RT-PCR provides an efficient method of linking two or more nucleotide sequences encoding for domains or subunits of a heteromeric protein, in a single reaction. Especially, the linkage of variable region encoding sequences from e.g. immunoglobulins, T cell receptors or B cell receptors is eased with the method of the present invention. This allows for a more efficient way of generating libraries of variable region encoding sequences. The capability to perform the multiplex overlap-extension RT-PCR using template derived from an isolated single cell enables the generation of cognate pair libraries in a high-throughput format.

1 . A library of cognate pairs consisting of linked variable region encoding sequences, where each individual cognate pair is inserted into a vector capable of expressing said cognate pair. 
     
     
         2 . The library according to  claim 1 , wherein said cognate pairs of variable region encoding sequences are obtained by the method comprising:
 a) providing a lymphocyte-containing cell fraction from a donor;   b) optionally enriching for a particular lymphocyte population from said cell fraction;   c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels; and   d) amplifying and effecting linkage of the variable region encoding sequences contained in said population of isolated single cells.   
     
     
         3 . The library according to  claim 1 , wherein an individual member of said cognate pairs comprises an immunoglobulin light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence. 
     
     
         4 . The library according to  claim 3 , wherein said individual member encodes a full-length antibody selected from human immunoglobulin classes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. 
     
     
         5 . The library according to  claim 1 , wherein an individual member of said cognate pairs comprises a TcR alpha chain variable region encoding sequence associated with a beta chain variable region encoding sequence or a gamma chain variable region encoding sequence associated with delta chain variable region encoding sequence. 
     
     
         6 . The library according  claim 5 , wherein said individual member encodes a full-length TcR. 
     
     
         7 . The library of cognate pairs of linked variable region encoding sequences according to  claim 1 , wherein each individual member has been selected for its ability to encode for a protein exhibiting a desired binding specificity directed against a particular target. 
     
     
         8 . The library of cognate pairs according to  claim 3 , where the antibodies expressible from said library are capable of reacting with, or binding to Tetanus Toxin. 
     
     
         9 . A population of host cells comprising a library according to  claim 1 . 
     
     
         10 . The population of host cells according to  claim 9 , wherein the cells are mammalian cells. 
     
     
         11 . A recombinant polyclonal antibody expressed from host cells according to  claim 9 . 
     
     
         12 . The recombinant polyclonal antibody according to  claim 11 , wherein the individual antibody members are full-length, and the constant heavy chain region is chosen from the human immunoglobulin classes IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE, and the constant light chain region is chosen from human lambda or kappa chains. 
     
     
         13 . A recombinant polyclonal immunoglobulin or fragments thereof capable of reacting with or binding to Tetanus Toxin. 
     
     
         14 . The recombinant polyclonal immunoglobulin according to  claim 13 , wherein the individual members are cognate pairs. 
     
     
         15 . The recombinant polyclonal immunoglobulin according to  claim 13 , comprising at least two individual cognate pairs comprising an immunoglobulin heavy chain variable region encoding sequence associated with a light chain variable region encoding sequence with at least 90% identity to one individual SEQ ID pair, selected from the group consisting of SEQ ID pairs 229:276, 262:309, 240:287, 245:292, 267:314, 246:293, 258:305, 242:289, 231:278, 263:310, 232:279, 237:284, 255:302, 260:307, 251:298, 233:280, 228:275, 244:291, 250:297, 252:299, 264:311, 272:319, 235:282 or 238:285. 
     
     
         16 . A recombinant immunoglobulin or fragments thereof capable of reacting with or binding to Tetanus Toxin, wherein the complementarity determining regions (CDR1, CDR2 and CDR3) are derived from one or more of the SEQ ID pairs of claim  54 . 
     
     
         17 . A pharmaceutical composition comprising a recombinant polyclonal immunoglobulin according to  claim 11  as active ingredient, combined with at least one pharmaceutically acceptable excipient. 
     
     
         18 . A pharmaceutical composition comprising a recombinant polyclonal antibody capable of reacting with or binding to Tetanus Toxin as active ingredient, optionally combined with a pharmaceutically acceptable excipient. 
     
     
         19 . A pharmaceutical composition according to  claim 17  for use as a medicament. 
     
     
         20 . A method of preventing or treating a patient at risk of developing tetanus by administering to a patient in need thereof a composition comprising a recombinant polyclonal antibody capable of reacting with or binding to Tetanus Toxin. 
     
     
         21 . A method for producing a library of cognate pairs comprising linked variable region encoding nucleotide sequences, the method comprising:
 a) providing a lymphocyte-containing cell fraction from a donor;   b) distributing cells from said cell fraction individually into a plurality of vessels to obtain a population of isolated single cells;   c) amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest comprising variable region encoding sequences using templates derived from said isolated single cells; and   d) effecting linkage of the amplified nucleotide sequences of interest.   
     
     
         22 . The method of  claim 21 , wherein the cell fraction is enriched for a particular lymphocyte population. 
     
     
         23 . The method of  claim 21 , wherein individual isolated single cells in the population of single cells are expanded to populations of isogenic cells prior to performing amplification and linkage. 
     
     
         24 . The method of  claim 21 , wherein the lymphocyte-containing cell fraction comprises whole blood, bone marrow, mononuclear cells or white blood cells. 
     
     
         25 . The method of  claim 22 , wherein the lymphocyte-containing cell fraction is enriched for B lymphocytes. 
     
     
         26 . The method of  claim 22 , wherein the lymphocyte-containing cell fraction is enriched for plasma cells. 
     
     
         27 . The method of  claim 22 , wherein cells from the lymphocyte-containing cell fraction are enriched for antigen specificity. 
     
     
         28 . The method of  claim 22 , wherein the lymphocyte-containing cell fraction is enriched for T lymphocytes. 
     
     
         29 . The method of  claim 21 , wherein the nucleotide sequences of interest comprise immunoglobulin variable region encoding sequences and the linkage generates cognate pairs of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence. 
     
     
         30 . The method of  claim 21 , wherein the multiplex molecular amplification procedure is a multiplex RT-PCR amplification. 
     
     
         31 . The method of  claim 30 , wherein the multiplex RT-PCR amplification is a two step process comprising a separate reverse transcription (RT) step prior to the multiplex PCR amplification. 
     
     
         32 . The method of  claim 31 , wherein said linkage of the nucleotide sequences of interest is effected in association with the multiplex PCR amplification, utilizing a multiplex overlap-extension primer mix. 
     
     
         33 . The method of  claim 30 , wherein the 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. 
     
     
         34 . The method of  claim 21 , wherein said linkage of the nucleotide sequences of interest is performed in the same vessel as the multiplex molecular amplification. 
     
     
         35 . The method of  claim 21 , further comprising creating a sub-library by selecting a subset of cognate pairs of linked variable region sequences that encode binding proteins with a desired target specificity, thereby generating a library of target-specific cognate pairs of variable region encoding sequences. 
     
     
         36 . The method of  claim 35 , further comprising transferring said library of target-specific cognate pairs of variable region encoding sequences to a mammalian expression vector. 
     
     
         37 . The method of  claim 36 , wherein the nucleotide sequences of interest comprise immunoglobulin variable region encoding sequences and the linkage generates a cognate pair of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence. 
     
     
         38 . The method of  claim 37 , wherein the mammalian expression vector encodes at least one constant domain selected from the group consisting of human immunoglobulin classes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, kappa light chain and lambda light chain. 
     
     
         39 . The method of  claim 21 , further comprising inserting the library of cognate pairs into a vector. 
     
     
         40 . The method of  claim 39 , wherein said vector is selected from the group consisting of cloning vectors, shuttle vectors, display vectors and expression vectors. 
     
     
         41 . The method of  claim 40 , wherein individual members of the library of cognate pairs comprise an immunoglobulin heavy chain variable region encoding sequence associated with a light chain variable region encoding sequence, and said sequences are inserted in-frame into a vector containing sequences encoding at least one immunoglobulin constant domain or a fragment thereof. 
     
     
         42 . The method of  claim 41 , wherein said vector is a mammalian expression vector. 
     
     
         43 . The method of  claim 39 , further comprising the steps:
 a) introducing a vector comprising linked variable region encoding nucleotide sequences into a host cell;   b) cultivating said host cell under conditions suitable for expression of the linked variable region sequences; and   c) obtaining the protein product expressed from the vector inserted into said host cell.   
     
     
         44 . The method of  claim 43 , wherein said protein product is a monoclonal antibody. 
     
     
         45 . A library of cognate pairs of linked variable region encoding nucleotide sequences obtained by the method of  claim 21 . 
     
     
         46 . The library of  claim 45 , wherein individual members of the library comprise a cognate pair of linked immunoglobulin variable region encoding sequences, each cognate pair comprising a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence. 
     
     
         47 . The library of  claim 46 , wherein individual members of the library encode a full-length antibody selected from the group consisting of human immunoglobulin classes IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM.