Construction of linear expression cassettes

The plasmids of pTT5-mIgG1H, pTT5-mIg kappa, pTT5-HuIgG1H, pTT5-HuIg kappa, pTT5-HuIg lambda, and pTT5-HuIgG1Fab, constructed and kept by Sanofi US, were used as templates to prepare the DNA fragments for constructing heavy- and light-chain LEC (H_LECs/L_LECs) (Figure 2 and Table 7). The DNA fragment (P_CMV fragment) composed of CMV promoter and Ig leader sequence (human IgκV3 leader: MEAPAQLLFLLLLWLPDTTG) was amplified from the plasmid pTT5-mIgG1H with the primers F_CMV (5’-gaccgagcgcagcgagtc-3’), and R_CMV (5’-cggtggtatcagggagcc-3’) by using AccuPrime™ Pfx DNA Polymerase (Thermo Fisher Scientific, 12344032). The P_CMV fragment is common for both H-LEC and L-LEC. The DNA fragment containing mouse IgG1 constant region, stop codon and rabbit beta globin (rbGlob) poly (A) tail (muIgG1_C_H fragment) was amplified from the plasmid, pTT5-mIgG1H, using the primers, muIgHC_F (5’-ggatctgctgcccaaactaactc-3’), and IgHLC_R (5’- tctccgagggatctcgacc-3’). The DNA fragment of mouse Igκ constant region and rbGlob poly (A) tail (muIgC_κ fragment) was amplified from the plasmid of pTT5_mIg kappa using the primers muIgκC_F (5’-caactgtatccatcttcccaccatc-3’), and IgHLC_R (5’- tctccgagggatctcgacc-3’). The DNA fragment containing human IgG1 constant region or human IgG1 Fab constant region and rbGlob poly (A) tail (huIgG1_C_H fragment or huIgG1_F_ab fragment) were amplified from the plasmid of pTT5-huIgG1H or pTT5-huIgG1_Fab with the same primer pair, huIgHC_F (5’-cttcaaccaagggaccttctg-3’), and IgHLC_R (5’- tctccgagggatctcgacc-3’). The DNA fragment containing human Igκ constant region, stop codon and rbGlob poly (A) tail (huIgC_κ fragment) was amplified from the plasmid of pTT5_HuIg kappa with the primers, huIgκC_F (5’-gcactgtggcagccccttctg-3’), and IgHL(k)C_R (5’- tctccgagggatctcgacc-3’). The DNA fragment containing human Igλ constant region, stop codon and rbGlob poly (A) tail (huIgC_λ fragment) was amplified from the plasmid of pTT5_HuIg Lambda with the primers huIgλC_F (5’-ctgcaggccagcccaaagcag-3’), and IgHLC_R (5’- tctccgagggatctcgacc-3’). The PCR was carried out in a total volume of 50 µl with 1 unit of AccPrime Pfx DNA Polymerase, 5 µl of 10X Pfx AccuPrime PfxReaction mix, 1 ng of plasmid, 10 pmol of each primer at 95°C for 3 minutes followed by 27 cycles at 95°C for 17 seconds, 61°C for 30 seconds, 68°C for 80 seconds, and a final elongation step at 68°C for 2 minutes. The DNA fragments were purified by gel electrophoresis (ThermoFisher) and QIAquick Gel Extraction Kit (Qiagen). The primers F_CMV (5’-gaccgagcgcagcgagtc-3’) and IgHLC_R (5’- tctccgagggatctcgacc-3’) were also used for constructing H/L_LECs as described below.

To construct H/L_LECs by overlapping PCR, heavy and light chains from SBC and phage were amplified with tagged primers to allow addition of short DNA tag sequences to 5’ and 3’ ends of Ig V_H/V_L PCR products. These sequence tags were complementary to P_CMV fragment or Ig constant region DNA fragments (Table 7) for creation of human IgG1/κ or IgG1/λ whole or Fab fragments, mouse IgG1/κ constructs. The sequence tag at 5’ end of all V_H/V_L PCR products contained the same 24 nucleotides sequence (5’-ctctggctccctgataccaccggt-3’) that was complementary with the 3’ end of the P_CMV fragment. However, the sequence tag at the 3’ end of V_H/V_L PCR products was different and contained one of the following five sequence tags depending on whether it was a mouse or human heavy/light chain LEC: 1) a 22 nucleotide sequence (5’-gagttagtttgggcagcagatc-3’) at the 3’end of V_H which was complementary with 5’ end of muIgG1_C_H for creating H_LEC coding for mouse IgG1; 2) a 27 nucleotides sequence tag (5’- gaaagacagaaggtcccttggttgaag-3’) at the 3’ end of Ig V_H PCR fragment complementary with 5’ end of huIgG1_C_H/huIgG1_F_ab fragment for the construction of H_LEC coding either a full-length or Fab human IgG1 heavy chains; 3) a 21 nucleotide sequence (5’-ggatggtgggaagatggatac-3’) at the 3’end of VL PCR fragment complementary with 5' end of muIgC_κ.3’ for making L_LEC coding for C_κ chains; 4) a 29 nucleotides sequence (5’- gataaacacagaaggggctgccacagtgc-3’) at the 3’ end of Ig V_κ PCR fragment complementary with 5’ end of huIgC for L_LEC coding for human C_κ; 5) a 25 nucleotides sequence (5’-ggtgctgctttgggctggcctgcag-3’) at the 3’ end of Ig V_λ PCR fragment complementary with 5’ end of huIgC_λ for building L_LEC coding C_λ.

The cleaned amplicons of Ig V_H and V_L from single B cells or monoclonal phagemids were used to construct heavy chain and light chain LEC by overlapping PCR following the method previously described.²⁵ In brief, 5–20 ng of cleaned V_H or V_L PCR2 product was used in a 50 µl PCR reaction containing 1 unit of KOD Hot Start DNA polymerase (Millipore Sigma, 71086), 10 μmol of dNTP, 50 μmol of MgSO4, 10 pmol of each primer, 10 ng of P_CMV fragment, 10 ng of C_H fragment or C_L fragment (Figure 2). Relevant C_H fragments were used to assemble H_LECs for expression of mouse IgG1, huIgG1, or huIgG1_Fab (Figure 2). PCR cycles consisted of one cycle at 95°C for 2 minutes followed by 27 cycles of 95°C for 20 seconds, 62°C for 12 seconds, 70°C for 70 seconds, and a final elongation step at 70°C for 2 minutes. H-LEC and L-LEC amplicon generation was confirmed by running a sample of PCR products in agarose gels, and the LECs were purified using MinElute 96 UF PCR Purification Kit and eluted in 50 µl of ultrapure H₂O. DNA concentration was determining using nanodrop1000 (Thermo Fisher Scientific) and adjusted to 100 ng/μl