Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) The workflow takes linear DNA library as input. ( b ) Ribosome display links genotype (RNAs transcribed from DNA input library that are stop codon free, and stall ribosome at the end of the transcript) and phenotype (folded VHH protein tethered to ribosomes due to the lack of stop codon in the RNA). ( c ) Selection cycle that enriches DNA encoding for VHHs that binds immobilized targets. ( d ) High throughput sequencing of full-length VHHs. ( e ) Sequences are grouped into clusters based on similarity of their CDRs, each cluster is distinct and represent a unique binding family. ( f ) The system outputs one representative sequence from each cluster to be synthesized and characterized for specific downstream applications. ( g ) Workflow for generating VHH library. VHH CDR randomization was introduced by PCR using a hairpin oligo (blocks DNA end from ligation) and an oligo with random 5’ sequence, followed by orientation-controlled ligation. Three successive PCR plus ligation cycles randomizes all three CDRs. ( h ) The final DNA library sequence structure. ( i ) One round of ribosome display and anti-Myc selection was performed after randomization of CDR1 and CDR2. The pie chart shows percentage of indicated sequence categories before and after anti-Myc selection. ( j ) Length distribution of DNA region encoding CDR1 of the VHH library before and after anti-Myc selection. Arrows indicate all correct-frame lengths showing increased percentage after anti-Myc selection.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Selection, Next-Generation Sequencing, Binding Assay, Sequencing, Synthesized, Ligation

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Position-wise amino acid profile of natural VHHs (298 VHHs, PDB) and ( b ) synthetic VHHs. Amino acids were color coded according to labels to the right, B indicates an empty position. Bar height is the relative percentage of each amino acids. The two most common amino acids were shown as patterned bars while others were shown as solid bars. ( c ) Plot of diversity index (as 1 – Gini index) for each amino acid position of natural VHHs and ( d ) synthetic VHHs.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques:

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Amino acid sequences encoded by frames that serve as templates for VHH library generation were aligned to the corresponding segments of the human IGHV3-23 (hIGHV3-23) or IGHJ4 (hIGHJ4). Positions in hIGHV3-23/hIGHJ4 that are identical to the corresponding position in at least one VHH frames are highlighted in orange. Positions in VHH frames that are identical to the corresponding position in hIGHV3-23/hIGHJ4 are highlighted in orange. hIGHV3-23 positions not identical to any VHH frames are numbered according to its position within the segment. Asterisks indicate VHH hallmark residues thought to be required for VHH’s independence of light chain. ( b ) Percent homology of VHH frames to the closest human gene. ( c ) List of VHH residues at positions numbered in (a) and representative human IGHV genes that encode the same VHH residue at the corresponding position. None: no human IGHV genes has the VHH residue at the corresponding position.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Residue

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Immobilization strategy for the target proteins: 3xFlag-tagged EGFP or RBD. ( b ) Pair-wise CDR match score (based on BLOSUM62 matrix) were calculated for 2000 randomly selected sequences from input library and output libraries after 3 rounds of selection. High match score populations appeared in the output libraries. Combining CDR1 and 2 match scores further separated high and low score population and a match score of 35 (black dashed line) was chosen as cut-off for downstream clustering analysis. ( c ) Percentage of indicated sequence categories in the input library and output libraries (EGFP, RBD). ( d ) Number of unique and shared clusters identified in EGFP and RBD output libraries. ( e ) Number of sequences for each size of RBD unique clusters. ( f ) ELISA assay revealed 3 strong binders (“s”) to RBD, 7 weak binders (“w”) and ( g ) 4 non-binders (“n”) among the 14 VHHs chosen for characterization. ( h ) SARS-CoV-2 S pseudotyped lentivirus neutralization assay showed 6 VHHs inhibiting infection >30% at 1μM on HEK293T expressing ACE2 and TMPRSS2. Data shown are two technical replicates, bars indicate the average of data, circles indicate values of each replicate.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Selection, Sequencing, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Expressing

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Amino acid profile of representative VHH sequence for each unique cluster identified from RBD and EGFP output libraries (“output binders”, 932 sequences). Plotted as described in . ( b ) Plot of diversity index (as 1 – Gini index) for each amino acid position of output binder VHHs.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Sequencing

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) r 2 values for the amino acid percentages in the indicated sequence group pairs at each CDR position. 298 natural VHHs (natural) and 298 randomly sampled sequences from input library (input) and output binders (output) were analyzed. Three random sampling trials were performed to generate three r 2 for each position. ( b ) Scatter plots of the percentage of each amino acid in the input library and the output binders and ( c ) that in the natural VHHs and the output binders at representative CDR positions. Circles are the mean and error bars are the standard deviation of data. ( d ) Root mean square error (RMSE, relative to Y = X line) values for the indicated sequence group pairs at each CDR position. Using the same randomly sampled sequences as (a). ( e ) Plot showing the similarity distances between the three sequence groups, with each connecting line length between two sequence groups indicating their RMSE. Vertical dashed lines indicate the middle point of the distance between output and natural sequence groups

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Sequencing, Sampling, Standard Deviation

Journal: bioRxiv

Article Title: A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

doi: 10.1101/2020.10.29.361287

Figure Lengend Snippet: ( a ) Affinity maturation workflow. ( b ) Two representative sections of position-wise post-minus pre-affinity maturation amino acid percent point change profile. White values indicate the original amino acid, yellow values indicate the beneficial mutation. Empty positions indicate amino acids not detected in either the pre-or post-selection libraries. ( c ) ELISA assay of VHH variants. ( d ) SARS-CoV-2 S pseudotyped lentivirus neutralization assay of VHHs on HEK293T expressing ACE2 and TMPRSS2. For (c) and (d), data shown are two technical replicates, bars indicate the average of data, circles indicate values of each replicate. ( e ) Scatter plot of ELISA assay absorbance versus pseudotyped lentivirus neutralization as percent infection inhibited. VHH concentration for both assays were 50 nM. Values are average of two technical replicates. Numbers on linear fitting lines were r 2 value for data within each family. ( f ) Dose-response curve for neutralization of pseudotyped lentiviral infection by VHHs. Markers are average of three technical replicates, error bars are standard deviation. ( g ) IC50 calculated from data in (f), presented as mean ± standard deviation.

Article Snippet: DNA encoding VHHs were obtained by gene synthesis (IDT) and cloned into pET vector in frame with a C-terminal 6XHis tag by Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix, New England Biolabs).

Techniques: Mutagenesis, Selection, Enzyme-linked Immunosorbent Assay, Neutralization, Expressing, Infection, Concentration Assay, Standard Deviation