Expression vector production and high-throughput cell screening

Abstract

The present invention relates inter alia to expression vector production as well as application to the production of host cells for protein repertoire expression and high-throughput screening. The invention also relates to primers useful for PCR amplification of nucleotide sequences encoding human antibody variable domains.

What is claimed herein is: 
     
         1 . A kit comprising one or more mixtures selected from
 a) two or more PCR primers, each primer comprising at least 15 contiguous nucleotides of a human antibody variable gene segment UTR sequence for hybridising to the 5′UTR sequence of an antibody variable gene segment selected from: IGHV1-8; IGHV1-2; IGHV1-3*01; IGHV1-18; IGHV1-24; IGHV2-5 and/or IGHV2-26; IGHV3-7; IGHV4-4; IGHV6-1; IGHV7-4-1; IGHV3-9; IGHV3-11; IGHV3-13; IGHV3-15; IGHV3-20; IGHV3-21; IGHV3-23; one, more or all of IGKV1-5, 1-12, 1-8, ID-8, ID-43, ID-16, ID-9; one, more or all of IGKV1-6, 1-13, ID-12, ID-13; IGKV1-17 and/or ID-17; one, more or all of IGKV1-27, 1-33, ID-39; one, more or all of IGKV2-28, 2-30, 2D-40; IqKV2-24; IGKV3 Family; IGKV4-1; IGKV5-2; IGLV1-40; IGLV1-47; IGLV10-54; IGLV2-23; IGLV3-1; IGLV3-10; IGLV3-12; IGLV3-19; IGLV3-21; IGLV3-22; IGLV3-25; IGLV3-16; IGLV3-9; IGLV4-3; IGLV3-2; IGLV5-45; IGLV7-43; IGLV9-49; IGLV1-40; IGLV1-47 and IGLV10-54, for performing PCR to copy the gene segment;   b) a PCR primer mixture comprising at least a first isolated PCR primer and a second isolated PCR primer, wherein the PCR primers are different and each comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-47; and   c) a PCR primer mixture comprising at least a first isolated PCR primer and a second isolated PCR primer, wherein the first PCR primer is capable of hybridising to a human antibody variable region gene segment 5′UTR sequence of a gene comprised by a target nucleic acid;
 wherein the variable region gene segment encodes a human V region; and the second PCR primer is capable of hybridising to a second sequence; 
 wherein the second sequence is comprised by the target nucleic acid and is 3′ to the UTR sequence; 
 wherein the first isolated PCR primer comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-47; and wherein the second isolated PCR primer comprises an antibody constant region sequence. 
   
     
     
         2 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 1-17; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy VH gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-17; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy VH gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-17.   
     
     
         3 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy Vκ gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 18-26; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy Vκ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 18-26; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy Vκ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 18-26.   
     
     
         4 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy Vλ gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 27-47; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 27-47; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 27-47.   
     
     
         5 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vκ gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 1-26; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy VH gene segments and Vκ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-26; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy VH gene segments and Vκ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-26.   
     
     
         6 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of at least 3 PCR primers which copy VH gene segments and 3 PCR primers which copy Vκ gene segments, wherein each PCR primer comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-26; or   b. wherein the mixture comprises at least 3 PCR primers which copy VH gene segments and at least 3 PCR primers which copy Vκ gene segments and wherein each PCR primer comprises a sequence which is at least 90% identical to a sequence selected from the SEQ ID NOs: 1-26; or   c. wherein the mixture comprises at least 3 PCR primers which copy VH gene segments and at least 3 PCR primers which copy Vκ gene segments and wherein each PCR primer comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-26.   
     
     
         7 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vκ gene segments and comprise at least 15 contiguous nucleotides of each of SEQ ID NOs: 1-26; or   b. a first group of at least 26 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments and Vκ gene segments and wherein each of the 26 PCR primers consists of a sequence which is at least 90% identical to a sequence selected from SEQ ID Nos: 1-26, such that a sequence at least 90% identical to each of the sequences of SEQ ID NOs: 1-26 is present in the first group; or   c. a first group of at least 26 different PCR primers comprising the first isolated PCR primer, wherein each of the PCR primers in the first group copy VH gene segments and Vκ gene segments and wherein all of the 26 PCR primers consists of a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-26, such that a sequence at least 90% identical to each of the sequences of SEQ ID NOs: 1-26 is present in the first group.   
     
     
         8 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vκ gene segments and comprise at least 20 contiguous nucleotides of each of SEQ ID NOs: 1-26; or   b. a first group of at least 26 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments and Vκ gene segments and wherein each of the 26 PCR primers consists of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID Nos: 1-26, such that a sequence at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-26 is present in the first group; or   c. a first group of at least 26 different PCR primers comprising the first isolated PCR primer, wherein all of the PCR primers in the first group copy VH gene segments and Vκ gene segments and wherein each of the 26 PCR primers consists of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 1-26, such that a sequence at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-26 is present in the first group.   
     
     
         9 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vλ gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 1-17 and 27-47; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-17 and 27-47; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-17 and 27-47.   
     
     
         10 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of at least at least 3 PCR primers which copy VH gene segments and at least 3 PCR primers which copy Vλ gene segments and each PCR primer comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-17 and 27-47; or   b. wherein the mixture comprises at least 3 PCR primers copy VH gene segments and at least 3 PCR primers which copy Vλ gene segments and wherein each PCR primer sequence comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47; or   c. wherein the mixture comprises least 3 PCR primers which copy VH gene segments and at least 3 PCR primers which copy Vλ gene segments and wherein each PCR primer comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47.   
     
     
         11 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vλ gene segments and comprise at least 15 contiguous nucleotides of each of SEQ ID NOs: 1-17 and 27-47; or   b. a first group of at least 38 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and wherein each of the 38 different PCR primers consist of a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47 such that a sequence at least 90% identical to each of the sequences of SEQ ID NOs: 1-17 and 27-47 is present in the first group; or   c. a first group of at least 38 different PCR primers comprising the first isolated PCR primer, wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and wherein each of the 38 different PCR primers consist of a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47 such that a sequence at least 90% identical to each of the sequences of SEQ ID NOs: 1-17 and 27-47 is present in the first group.   
     
     
         12 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments and Vλ gene segments and comprise at least 20 contiguous nucleotides of each of SEQ ID NOs: 1-17 and 27-47; or   b. a first group of at least 38 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and wherein each of the 38 different PCR primers consist of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47 such that a sequence at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-17 and 27-47 is present in the first group; or   c. a first group of at least 38 different PCR primers comprising the first isolated PCR primer, wherein all of the PCR primers in the first group copy VH gene segments and Vλ gene segments and wherein each of the 38 different PCR primers consist of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 1-17 and 27-47 such that a sequence at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-17 and 27-47 is present in the first group.   
     
     
         13 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments, Vκ gene segments and Vλ gene segments and comprise at least 15 contiguous nucleotides of SEQ ID NOs: 1-47; or   b. a first group of PCR primers comprising the first and second isolated PCR primers, and wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-47; or   c. a first group of PCR primers comprising the first isolated PCR primer, and wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and the PCR primer sequences comprise a sequence which is at least 90% identical to the sequences of SEQ ID NOs: 1-47.   
     
     
         14 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of at least at least 3 PCR primers which copy VH gene segments, at least 3 PCR primers which copy Vκ gene segments and at least 3 PCR primers which copy Vλ gene segments and wherein each PCR primer comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-47; or   b. wherein the mixture comprises at least 3 PCR primers which copy VH gene segments, at least 3 PCR primers which copy Vκ gene segments and at least 3 PCR primers which copy Vλ gene segments and wherein each PCR primer comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-47; or   c. wherein the mixture comprises at least 3 PCR primers which copy VH gene segments, at least 3 PCR primers which copy Vκ gene segments and at least 3 PCR primers which copy Vλ gene segments and wherein each PCR primer comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-47.   
     
     
         15 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the PCR primers copy VH gene segments, Vκ gene segments and Vλ gene segments and comprise at least 15 contiguous nucleotides of each of SEQ ID NOs: 1-47; or   b. a first group of at least 48 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and wherein each of the 48 different PCR primers consist of a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-47, such that a sequence at least 90% identical to each of the sequences of SEQ ID NOs: 1-47 is present in the first group; or   c. a first group of at least 48 different PCR primers comprising the first isolated PCR primer, wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and wherein each of the 48 different PCR primers consist of a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 1-47, such that a sequence which is at least 90% identical to each of the sequences of SEQ ID NOs: 1-47 is present in the first group.   
     
     
         16 . The kit according to  claim 1 , wherein the kit comprises
 a. a mixture of two or more PCR primers, wherein all of the primers copy VH gene segments, Vκ gene segments and Vλ gene segments and comprise at least 20 contiguous nucleotides of each of SEQ ID NOs: 1-47; or   b. a first group of at least 48 different PCR primers comprising the first and second isolated PCR primers, wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and wherein each of the 48 different PCR primers consist of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 1-47, such that a sequence at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-47 is present in the first group; or   c. a first group of at least 48 different PCR primers comprising the first isolated PCR primer, wherein all of the PCR primers in the first group copy VH gene segments, Vκ gene segments and Vλ gene segments and wherein each of the 48 different PCR primers consist of a sequence which is at least 95% or 100% identical to a sequence selected from SEQ ID NOs: 1-47, such that a sequence which is at least 95% or 100% identical to each of the sequences of SEQ ID NOs: 1-47 is present in the first group.   
     
     
         17 . The kit according to claim  1 b, wherein the first PCR primer is capable of hybridising to a human antibody variable region gene segment 5′UTR sequence of a gene comprised by a target nucleic acid;
 wherein the gene encodes a human V region and/or the second PCR primer is capable of hybridising to a second sequence; and 
 wherein the second sequence is comprised by the target nucleic acid and is 3′ to the UTR sequence. 
 
     
     
         18 . The kit according to  claim 1 , wherein the mixture further comprises at least one further isolated PCR primer, wherein the further PCR primer comprises at least one of:
 a. an antibody heavy chain constant region sequence;   b. an antibody kappa chain constant region sequence; and   c. an antibody lambda chain constant region sequence.   
     
     
         19 . The kit according to  claim 18 , wherein the further PCR primer comprises at least one of:
 a. an antibody heavy chain constant region sequence at least 90% identical to SEQ ID NO 48 or 49;   b. an antibody kappa chain constant region sequence at least 90% identical to SEQ ID NO: 50 or 51; and   c. an antibody lambda chain constant region sequence at least 90% identical to SEQ ID NO: 52 or 53.   
     
     
         20 . The kit according to  claim 18 , wherein the further PCR primer comprises at least one of:
 a. an antibody heavy chain constant region sequence at least 95% or 100% identical to SEQ ID NO 48 or 49;   b. an antibody kappa chain constant region sequence at least 95% or 100% identical to SEQ ID NO: 50 or 51; and   c. an antibody lambda chain constant region sequence at least 95% or 100% identical to SEQ ID NO: 52 or 53.   
     
     
         21 . The kit according to claim  1 c, wherein the first PCR primer comprises a promoter nucleotide sequence 5′ of the UTR sequence which is a human cytomegalovirus promoter fragment.