A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Figures & data

Figure 1. Schematic representation of batch reformatting of scFvs to IgGs. (A) Upper panel: Amplification of the phage display vector with 5′ primers annealing to VL FW1 and 3′ primers annealing to VH FW4. This results in the amplification of the whole plasmid except the linker between the VH and VL (dotted line). The primers carry complementary overhangs corresponding to the 5′ and 3′ ends of a donor fragment (B). This donor fragment represents the hinge and IgG1 constant domains followed by translation stop and polyA sites (in green) and the light chain control elements consisting of the LC promoter and signal sequence (in blue). This intermediate fragment is then fused in frame with VH and VL by means of In-Fusion technology. The resulting pool of plasmids is shown in the bottom panel (C, IgG intermediate pool). The IgG cassette from the intermediate vector pool is PCR amplified using 5′ primers that anneal to the FW1 of VH and 3′ primers that anneal to the FW4 of VL. The primers have overhangs to anneal to the signal sequence of HC and 5′ end of the Cκ or Cλ. The PCR products are then cloned into the IgG vector (D). The result is a pool of plasmid that codes for the entire HC and LC and can express IgG in mammalian cells. HSS, heavy chain signal sequence.

Figure 2. Maintenance of VH and VL pairing during batch reformatting of a mini-library of 31 scFvs with known sequences to full-length IgG. Antibody amplification steps of the reformatting process were done using either standard aqueous PCR conditions (A) or using emulsion PCR where DNA templates are separated within water-in-oil droplets (B). scFv DNA sequences from each pool were identified by their VH and VL CDR3 sequences. Numbers in the table show IgG sequences observed with the certain VH and VL combinations indicated. IgG having the original VH-VL pairing are indicated by shading. Values in square brackets indicate the % correct pairing rate in each population from 86 or 87 scFv sequences analyzed.

Figure 3. Representation of VH CDR3 sequences in scFv populations before and after batch reformatting for SiPF. Randomly chosen individual colonies were given an arbitrary identity based on their VH CDR3 sequence. Sequence representation is shown as a percentage frequency in scFv (129 sequences) or SiPF (176 sequences) populations. The 3 most frequent VH CDR3 sequences, and the representation of other sequences, is broadly the same for both populations. 16% (scFv) and 23% (SiPF) of the sequences in each population do not overlap, most likely due to sample size and the low probability of re-sampling relatively rare sequences.

Table 1. Comparison of variable domain sequences of phagemid scFv from Round 2 of selection and as VH and VL in IgG after batch reformatting to IgG.

	PRE SiPF (phagemid)						SiPF Vector
	Library 1			Library 2			Library 1				Library 2
							λ				λ		κ
	λ	352	κ	λ	264	κ	VH	VL	VH	κ	VH	VL	VH	VL
Total reads	229		5	136		27	176	176	176	176	176	176	176	176
Full length, in-frame scFv		66%			62%		157	166	148	167	161	165	158	164
							89%	94%	84%	95%	91%	94%	90%	93%

Figure 4. (A) Expression of 1496 IgG in Expi293F cells. Cells were transfected with mini-prep DNA, harvested after 6 d and IgG expression levels was determined using octet protein A. The mean titer for this set of antibodies was 70 µg/ml. (B) Reproducibility of the high-throughput mammalian expression system. A set of 352 antibodies were expressed in 4 96-well blocks and the titer determined after 6-days (week 1). Two weeks later the same antibodies were re-expressed under the same conditions (week 2). The expression of each antibody in each of the separate instance is highly comparable.

Figure 5. More potent anti-cytokine antibodies are identified by SiPF than by scFv screening. Comparison of potency of the 2 leading antibodies identified from scFv screening (Ab048) or SiPF screening (Ab049) of phage display selections that were performed to identify an antagonistic antibody for a soluble cytokine. Neutralization of 1nM cytokine-induced receptor activation by Ab048 and Ab009 are shown in a cell-based receptor reporter enzyme complementation assay (PathHunter, DiscoverRx).

Figure 6. Flow diagrams comparing typical routes from phage display selection to the identification of functional antibodies, highlighting the utility of the SiPF platform in reducing surrogate steps (e.g., scFv format, binding based / biochemical) assays. Rapid conversion of phage scFv to IgG allows earlier analysis of larger numbers of IgG in more relevant, functional cell-based assays.

Figure 7. FMAT analysis of cell-binding of anti FPR1 antibodies as IgG supernatant generated using the SiPF platform. Four different FPR1 binding antibodies identified from cell-based phage display selections are shown. Hy38-1 SiPF is a positive control antibody known to recognize FPR1³³ that has been expressed using the SiPF platform.

Table 2. Primers for intermediate pool generation. Sequences in red are complementary to the 5′ end of VL FW1 (5′ primers) or the 3′ end of VH FW4 (3′ primers). Sequences in blue are overhangs in 5′ primers that are complementary to the LC control elements in the donor fragment. Sequences in green are overhangs in 3′ primers that are complementary to the hinge region of IgG1 in the donor fragment.

5′ kappa_1	GCTCCCCGGGGCGCGCTGT GAC ATC CAG WTG ACC CAG TCT
5′ Kappa_2	GCTCCCCGGGGCGCGCTGT GAT ATT GTG ATG ACC CAS ACT
5′ Kappa_3	GCTCCCCGGGGCGCGCTGT GAA ATT GTG WTG ACR CAG TCT
5′ Kappa_4	GCTCCCCGGGGCGCGCTGT GAT RTT GTG ATG ACW CAG TCT
5′ Kappa_5	GCTCCCCGGGGCGCGCTGT GAA ACG ACA CTC ACG CAG TCT
5′ Kappa_6	GCTCCCCGGGGCGCGCTGT GAA ATT GTG CTG ACT CAG TCT
5′ lambda_1	GCTCCCCGGGGCGCGCTGT CAG TCT GTG YTG ACK CAG CCR
5′ lambda_2	GCTCCCCGGGGCGCGCTGT CAG TCT GCC CTG ACT CAG CCT
5′ lambda_3	GCTCCCCGGGGCGCGCTGT TCC TAT GWG CTG ACT CAG CYA
5′ lambda_4	GCTCCCCGGGGCGCGCTGT CWG CCT GTG CTG ACT CAG CCM
5′ lambda_5	GCTCCCCGGGGCGCGCTGT TCT TCT GAG CTG ACT CAG GAC
5′ lambda_6	GCTCCCCGGGGCGCGCTGT AAT TTT ATG CTG ACT CAG CCC
5′ lambda_7	GCTCCCCGGGGCGCGCTGT CAG YCT GTA CTG ACT CAA CCG
5′ lambda_8	GCTCCCCGGGGCGCGCTGT CAG RCT GTG GTG ACY CAG GAG
5′ lambda_9	GCTCCCCGGGGCGCGCTGT CAG CCT GTG CTG ACT CAA TC
5′ lambda_10	GCTCCCCGGGGCGCGCTGT CAG GCA GGG CTG ACT CAG CCA
3′ heavy_1	TGGGCCCTTGGTCGACGC TGA GGA GAC RGT GAC CAG GGT KCC
3′ heavy_2	TGGGCCCTTGGTCGACGC TGA AGA GAC GGT GAC CAT TGT CCC
3′ heavy_3	TGGGCCCTTGGTCGACGC TGA GGA GAC GGT GAC CGT GGT CCC

Table 3. Primers for final IgG pool generation. Sequences in red are complementary to 5′ end of VH FW1 (5′ primers) or the 3′ end of VL FW4 (3′ primers). Sequences in blue are overhangs in 5′ primers that are complementary to the HC signal sequence region of the IgG acceptor vector. Sequences in black are overhangs in 3′ primers that are complementary to the 5′ end of either the kappa or lambda CL region of the IgG acceptor vector.

5′ heavy_1	TCTCCACAGGTGTACACTCC CAG RTG CAG CTG GTG CAR T
5′ heavy_2	TCTCCACAGGTGTACACTCC SAG GTC CAG CTG GTR CAG T
5′ heavy_3	TCTCCACAGGTGTACACTCC CAG RTC ACC TTG AAG GAG T
5′ heavy_4	TCTCCACAGGTGTACACTCC SAG GTGCAG CTG KTG GAG
5′ heavy_5	TCTCCACAGGTGTACACTCC CAG GTG CAG CTA CAG CAG T
5′ heavy_6	TCTCCACAGGTGTACACTCC CAG GTA CAG CTG CAG CAG TC
5′ heavy_7	TCTCCACAGGTGTACACTCC GAR GTG CAG CTG GTG CAG
5′ heavy_8	TCTCCACAGGTGTACACTCC CAG STG CAG CTG CAG GAG TC
5′ heavy_9	TCTCCACAGGTGTACACTCC GAG GTG CAG CTG TTG GAG TCT
3′ kappa_1	GTGCAGCCACCGTACGTTT GAT HTC CAC YTT GGT CCC
3′ kappa_2	GTGCAGCCACCGTACGTTT GAT CTC CAG CTT GGT CCC
3′ kappa_3	GTGCAGCCACCGTACGTTT AAT CTC CAG TCG TGT CCC
3′ lambda_1	AGTGACCGAGGGCGCCGCCTTGGGCTGACC TAG GAC GGT GAC CTT GGT CCC
3′ lambda_2	AGTGACCGAGGGCGCCGCCTTGGGCTGACC TAG GAC GGT CAG CTT GGT CCC
3′ lambda_3	AGTGACCGAGGGCGCCGCCTTGGGCTGACC GAG GAC GGT CAG CTG GGT