Varying concentrations of P1A4(1O-3OO nM) were also tested to establish concentration dependencies of the assay (Fig. 4d). A series of concentrations of P1A4 spiked in PPE shows an increase in true Assoc-2 slopes as concentration approaches Kd (table 2). Where the Assoc- 1 step is not fully saturated, and the P1A4 sample concentrations of are at non-steady-state conditions, k_off predictions are less accurate. To address this, BATCH was developed to flag hits that show low expression or do not fully saturate in Assoc- 1 to indicated inaccuracies in predicted k_Oft values for the user’s discretion.

RAPID biopanning enables the identification o f rare antibodies against CHIP.

Carboxyl terminus of Hsp-70 interacting protein (CHIP) is an E3 ubiquitin ligase canonically known to interact with heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90), leading to the ubiquitination of misfolded clients as well as regulation of chaperone turnover. Recent work demonstrated that CHIP has substrate specificity extending beyond Hsp70/90 and predicted interactions suggest CHIP may have additional Hsp-independent roles in proteostasis and disease states¹⁵. To enable further biological studies, Abs against CHIP were developed beyond the available substrate binding inhibitors^{l l6}. CHIP is a challenging biopanning target due to its homodimeric structure and conformational flexibility, and therefore RAPID biopanning was employed.

Four rounds of standard biopanning were employed against CHIP with increased stringency each round. Subsequently, RAPID flow cytometry was performed by individually FITC labeling each output round. Flow cytometry data shows an increase in normalized MFI from round 2 through 3, and a significant drop is observed at round 4 (Fig 5a). The most enriched round (round 3) shows a normalized MFI of 2.11 which correlates to a reasonably high hit rate between 10 and 50 percent of a high affinity binder (~Kd =25 nM) according to previous control experiments with P1A4 (Fig 14c). As flow cytometry data suggests strong Fab-Ag binding enrichment had occurred, fluorescent activated sorting was deemed unnecessary, and BIAS was performed directly.

95 individual colonies were picked from round 3, and from PPE samples, 18 hits were identified by BIAS, and BATCH calculated k_OffS showing a wide range of values (9.40xl0⁶- 5.44xl0² s’¹) (Fig. 5b, Fig. 6, Table 3). Candidate hit Fabs that were recombinantly expressed and biochemically characterized exhibited similar trends of k_offS compared to those predicted by BIAS (R² =0.95), and their values were within ~2 fold of each other (Fig 5c). In parallel dot blots and ELIS As were performed to compare BIAS with standard screening methods. Dot blot experiments resulted in 76 hits (80% hit rate) in comparison to 18 hits identified from BIAS (Fig 5d). After exhaustive optimizations, no hits were identified using ELISAs. The biopanning campaign of CHIP demonstrates the power of RAPID biopanning as a method for accurately identifying the most enriched round, determining dominant factors of enrichment, and predicting hit rates of specific rounds of candidate hit screening. Notably, in this example, BIAS is shown to be a critical step in identifying true functional candidate hits and prioritizing potential binders for detailed biochemical characterizations.

Table 3. BIAS hit summary table for CHIP. Inaccurate k_off measurements are flagged (*- low expression #-unsaturuated Assoc- 1, !-poor exponential fit for Dissoc). Kd min and max are estimated based on general k_on of previously discovered Craik lab Fabs (5 x 10⁴ - 3 x 10⁵)

RAPID identifies rare binders against Gag.

Gaq is a ubiquitous heterotrimeric G protein subunit important in many G protein- coupled receptor signaling cascades. Like other G proteins, the conformation of Gaq is regulated by a guanine nucleotide. While Gaq is normally activated in diverse physiological states, several mutations lead to inappropriate activation of Gaq, leading to challenging diseases like uveal melanoma²⁵ or Sturge Weber syndrome²⁶. To understand how mutations in Gaq lead to activation, it was sought to develop antibody fragments that would enable structural studies. Gaq is a challenging target due to its known dynamic conformational flexibility. Six rounds of biopanning were employed against Gaq using the standard method, and

RAPID flow cytometry was used to monitor the enrichment progression (Fig 15a). A late enrichment is observed where the maximum NMFI (2.36) is reached in round 5. Interestingly, an increase in SD was also observed which would suggest that the high-affinity binders also exhibit superior expression/display propensities (Fig 15b, Fig 9). In line with the findings from the CHIP biopanning campaign, where significant enrichment was achieved, RAPID FACS was not necessary. BIAS was employed to 95 clones from round 5, where 27 hits were identified (28% hit rate) (Fig 6, Table 4). The clones occupying the top 12 ranks were determined to be identical. Additionally, independent of these, clones ranked 13 to 16 were also found to be identical (Fig 15c). Identical clones exhibited similar BIAS k_off values, and as demonstrated by the CHIP biopanning campaign, the candidate hit Fabs, which were biochemically characterized, displayed comparable koff values to those predicted by BIAS. Seven hits were predicted to be low expressors (flagged with*) and are ranked low on the BIAS rankings. This agrees with the RAPID flow cytometry results, where it was predicted that higher- affinity binders would also exhibit higher expression levels.

Table 4. BIAS hit summary table for Gotq. Inaccurate k_off measurements are flagged (*- low expression #-unsaturuated Assoc- 1, !-poor exponential fit for Dissoc). Kd min and max are estimated based on general k_ou of previously discovered Craik lab Fabs (5 x 10⁴ - 3 x 10⁵)

RAPID biopanning identifies rare binders against CS3D

Cyclic STAT3 decoy (CS3D) is a dsDNA decoy that targets STAT3 and inhibits expression of downstream STAT3 induced genes^{17 18}. To date, only a few recombinant Abs have been developed against nucleic acid targets, with most of them targeting unique structural motifs^5,6. The identification of Fabs against CS3D, a 15-mer dsDNA target with no distinct structural motifs, is presented as an example of a challenging target for which limited Ab discovery precedence exists. Both standard biopanning and RAPID biopanning were performed in parallel to provide a head-to-head comparison of the two methods.

With the standard method, a total of six rounds of biopanning was performed with increasing stringency of washes and decreasing amounts of Ag per round. 95 colonies were randomly picked each round 4-6 (285 clones total) for candidate hit screening using dot blots. Four unique Fabs (3B11, 3B12, 3C8, and 4E4) were identified from 38 randomly sequenced clones, where three of the Fabs (3B11, 3B12, and 3C8) differed by only one amino acid indicating that a strong enrichment had occurred for a specific family of related clones over others throughout the rounds. Purified Fabs exhibited weak binding against CS3D with k_off values ranging from 7.64 xlO'² - 2.77 xlO'¹ s'¹ at 2 mM Fab concentrations (Table 5).

Table 5. koff (measured at 2pM) and recombinant expression yields (E. coli) of Fabs from CS3D biopanning.

To thoroughly examine candidate clones that were picked from the standard method campaign, BIAS was employed to the same clones picked previously that were randomly sequenced. BIAS resulted in a hit rate of 230 out of 285 clones (81% hit rate) (Table 6). However, when the top 10 ranked candidates from each round where sequenced, they were revealed to be the same low-affinity clones that were identified previously (Table 7), with two additional weak binders 2C11 (koff, 4.19 xlO'¹ s'¹) and 2B12 (koff, 3.7 xlO'¹ s'¹) being identified. . It was hypothesized that strong growth/display propensity biases were a dominant factor during the campaign, leading primarily to the identification of low affinity binders.

Table 6. BIAS of randomly chosen clones from Round 4-6 of panning and Round 4 Ph AB sorting.

Table 7. Top 10 BIAS ranked candidate clones from standard biopanning Round 4-6 and their k_offS measured by BLI at 2 pM. Identical clones are italized or shown in bold.

RAPID biopanning was employed to identify higher affinity binders which was not achievable using the standard method. RAPID flow cytometry results showed that nearly no enrichment of binding had occurred during the campaign despite titers of output phage showing enrichment in round 3-5 (534-fold increase in output titer from round 5 relative to that of round 3), as shown with no significant changes of normalized MFI in any of the rounds (Fig 16a and Fig 7b). However, a higher fluorescent signal that does not significantly overlap with the control distribution (i.e., a minor shoulder distribution) emerged as biopanning proceeded, showing a maximum MFI at round 4. (Fig 16b and Fig. 7a). RAPID FACS was employed to isolate this population.

Beads from the round 4 output (top -4% fluorescent intensity) were sorted along with the beads from the majority population (bottom -96%) and both populations were used to infect TGI (E. coli) cells (Fig 16c). BIAS was performed separately against both populations where ~2-fold lower average BIAS k_offS were observed in the top -4 percent population hits compared to the bottom -96 percent population hits (P< 0.001), suggesting that the top -4 percent population contained ph-Fab that have more favorable kinetic properties (Fig 16d). BIAS hits of the top -4 percent sorted population were sequenced, and a new candidate binder, SP1-B3, was identified, along with identical clones of previously identified low-affinity binders. SP1-B3 exhibited 2.11 xlO'² s’¹ k_off which is ~3-fold lower than the previously identified Fab with the lowest k_Off, and over -20- fold lower than the Fab with the highest k_Off that was most frequently identified (Table 5). It is important to note that Fabs that were identified without RAPID biopanning exhibited significantly higher expression levels, as high as 27-fold (Table 5). As it has been well reported that the expression levels of displayed protein correlate with display propensities¹⁹ it is reasonable to conclude that display propensity bias was a strong factor during the biopanning campaign of CS3D, which led to the enrichment of weakly binding but high expressing ph-Fabs. The use of RAPID in this biopanning campaign enabled the direct isolation of a lower frequency, high affinity binder population, which resulted in the discovery of superior binding Abs, that otherwise would have not been discovered with the standard method.

Summary

The efficient identification of rare high-affinity Abs against challenging targets (i.e., low thermal stability, conformationally diverse, low expression of target, low antigenicity, low solubility, etc.) continues to be a major challenge in the Ab discovery field. For phage display based biopanning methods, challenging targets often exhibit weak Ab-Ag enrichment due to the low prevalence of high- affinity binders, and additional factors such as growth biases and inconsistent protein display propensities can affect, and even dominate the enrichment process. This ultimately leads to high-affinity binders never fully enriching and becoming difficult to identify, given the low probability of identification from common stochastic hit-picking screening methods.

The RAPID biopanning method was specifically designed as a solution for identifying rare high-affinity Abs against challenging targets using phage display. Where previous standard methods employ an approach where the total population of enriched displayed Abs are non- discriminatory screened, RAPID biopanning isolates/identifies a selective population of high affinity binders that are subsequently screened in a discriminatory manner. This was achieved by (1) accurately identifying the most enriched population of ph-Fab, (2) increasing the prevalence of low frequency high-affinity ph-Fabs by fluorescent activated sorting, and (3) rapidly screening candidate hits in a discriminatory matter to prioritize in-depth biochemical characterizations of promising binders. A method was developed for fluorescent labeling ph-Fab for quantitative measurements of ph-Fab bounding to Ag immobilized beads and a novel BLI method, BIAS, was developed for rapid real time analysis of candidate binders. Ultimately, RAPID biopanning follows a Label-Profile-Sort-Screen pipeline (Fig 1) and has been applied to two targets, CS3D and CHIP, where rare high-affinity binders were identified.

Robust labeling of ph-Fab was achieved using NHS-FITC. The labeling reaction is simple (only NHS-FITC and borate buffer required), fast (total reaction time 1 h in RT), reliable, and effective for the quantitative measurement of ph-Fab bound to Ag-Beads in flow cytometry. Most importantly, FITC labeling does not disrupt the binding of displayed Fab to Ag, nor does it exhibit any significant variabilities in labeling between different ph-Fabs.

By individually FITC labeling ph-Fab libraries from a single biopanning campaign and analyzing bound ph-Fab on Ag-Beads on flow cytometry, the enrichment progression of the campaign can be profiled. The progression of the global distribution of bound ph-Fab from subsequent rounds of biopanning can indicate dominant factors that affected enrichment (i.e., Fab-Ag binding, growth rates of phagemid containing E. coli. Fab display propensities). This is superior to phage ELIS As and calculations of phage titers of output ph-Fabs as these methods yield a single measurement of bound phage as opposed to a global distribution.

For campaigns that struggle to identify higher- affinity binders, fluorescent activated sorting of higher fluorescent populations can be an effective solution, as beads that exhibit higher fluorescent intensities contain greater enriched populations of higher- affinity binders. The frequency and variation in affinity of the higher- affinity binder can result in either a broader fluorescence distribution or a discernible population in FACS, as observed in the CS3D biopanning campaign. As shown with the CS3D biopanning campaign, a rare higher affinity binder, SP1-B3, was identified by isolating the ph-Fab-Ag-bead complexes exhibiting higher fluorescent signal by FACS. It is worth noting that previous efforts of standard magnetic bead biopanning followed by random clone picking or even applying the more stringent BIAS screening to these clones failed to identify new higher-affinity binders, indicating that the pool of ph-Fab examined itself had severely low frequencies of higher- affinity ph-Fabs. In the case for the CS3D campaign, the significantly lower expression levels of SP1-B3 (as much as ~20-fold less) compared to the weaker binders suggests that, at least in part, Fab display propensities played a role in the enrichment process. The example of the CS3D biopanning campaign highlights the use of fluorescent labeled ph-Fab libraries coupled with FACS, where rare high- affinity binders can be retrieved from a large pool of weaker binders, previously enriched for factors other than Fab-Ag binding.

It is crucial to the outcome of the antibody discovery campaign to correctly prioritize candidate binders for in-depth biochemical characterization as time and resources are practically limited in a lab setting. To this point a discriminatory hit screening method, BIAS, was developed to use Abs from crude extracts to measure real-time binding to immobilized Ags using BLI. The raw data is analyzed with an in-house developed script, BATCH, and clones are categorized and rank ordered based on their kinetic off-rates. The discriminatory identification of clones allows for the prioritization of more promising candidates and further investigation and characterizations can be done in line with the BIAS rankings. As shown with the CHIP biopanning campaign, BIAS is more discriminatory compared to conventional screening methods such as dot blots and provides more in-depth details of candidate clones compared to ELISAs (i.e., Fab expression levels, k_Off values etc.). This is demonstrated by the most promising candidate binder, Fl, showing no obvious indication of being a promising candidate from dot blot results (Fig 5d). BIAS hits from the CHIP campaign show similar kinetic off-rate trends compared to the BIAS rankings, and of the five Fabs characterized, four show good agreement (~2-fold) of predicted koff values compared to biochemically characterized koffs.

RAPID biopanning, particularly where stringent fluorescent activated sorting was needed, can on occasion yield lower expression levels as shown with SP1-B3 (Table 5) or be poorer growers. Expression of these binders in full-length IgG formats in mammalian cells can help overcome low expression yields in bacteria, which is a widely employed solution in improving Ab expression. As an example, SP1-B3 expressed in an IgG format shows a 220-fold increase in expression (33 mg/L culture) compared to its Fab counterpail.

While the RAPID biopanning pipeline synergizes high quality ph-Fab population isolation with rapid discriminatory screening of candidate clones, each step is highly modular and can be utilized independently or in combination with other existing protocols. For example, as is common with YSD coupled with FACS, iterative rounds of fluorescent activated sorting could be performed for continuous enrichment of high quality ph-Fab populations. Also, where functional assays are available for hit screening, functional screens can be utilized in place of BIAS, post flow cytometry profiling, and/or fluorescent activated sorting. Finally, BIAS can be utilized with different Ab formats (i.e., scFv, nanobodies etc.) and different anti-tag secondary IgGs as well.

In conclusion, it is demonstrated that RAPID biopanning couples efficiency with precision to allow for a more selective biopanning campaign to identify rare high-affinity candidate clones, particularly for challenging targets.

Materials and Methods

Standard biopanning

Standard biopanning with magnetic beads were performed as previously described^20,21. Briefly, a human naive B-cell phage displayed Fab library (diversity 4.1 xlO¹⁰) was used against CHIP and CS3D. Biotinylated Ags (EZ-link biotinylation Pierce) were immobilized to magnetic streptavidin beads (Dynabeads M-270 Streptavidin), and subsequently the ph-Fab library was added. For the CHIP campaign, 25 mM HEPES, 50 mM KC1 was used for the binding, and 25 mM HEPES, 50 mM KC1, 0.05% Tween-20 was used for washing. For the CS3D campaign, 20 mM Tris-HCl, 150 mM NaCl, 0.05% NP-40, 1 mM EDTA was used for the binding, and both PBS and PBS-T (0.05% Tween-20) was used for washing. Both campaigns introduced negative selections against magnetic beads with no Ag starting at round 2. Individual clones were selected and screened with either Dot blots, ELISAs, and BIAS and subsequent hits were sequenced. Plasmids containing unique sequences of Fabs were used to transform BL21(DE3) E. coli cells for further biochemical analysis of Fab. Phage preparation

Fab-display cd phage were first amplified and prepared using standard methods. Briefly, 50 ml cultures (2xYT, 2% glucose, 100 ug/ml Ampicillin) of phagemid containing E. coli cells were incubated at 37°C with 200 rpm shaking until OD600 reached ~0.5. Subsequently, 10 ml of this culture was infected with M13KO7 helper phage at 10:1 helper phage to cell ratio. Culture was incubated at 37°C for 30 min without shaking followed by 20 min with shaking at 200 rpm. Infected cells were collected by centrifugation and cells were resuspended with fresh media (2xYT 100 mg/ml Ampicillin, 50 mg/ml Kanamycin). Cultures were grown overnight, and amplified phage were isolated by adding PEG 6000/2.5M NaCl phage to the supernatant of the overnighted culture. Phage yield was analyzed by taking OD268 measurements.

Phage labeling with NHS-FITC

NHS-Fluorescein (Thermo Scientific) was prepared in DMSO (1 mg/ml final concentration). For the standard protocol labeling reaction consisted of the following: 50 pl of NHS-FITC (Img/ ml) stock 500 pl of Phage (OD268 = 1, final), and 40 pl of Borate buffer (0.67M). Previous studies have shown that NHS-esters, including NHS-FITC, can efficiently label M13 phage via the N-termini and lysine residues of the pVIII coat proteins^22,2 . Labelling reaction was incubated in RT for 1 hr in the dark. PEG 6000/2.5M NaCl was added to the reaction to precipitate phage and incubated on ice for 15 min. Precipitated phage were collected by centrifugation (max speed for 5 min) and supernatant was removed. Pellets were resuspended in PBS and process was repeated. Phage were washed and precipitated three times total. Samples were immediately used in subsequent experiments. A plate reader (BioTek Synergy H4 plate reader) was used for fluorescent measurements and optical density of phage and FITC were analyzed using nanodrop. Phage labeling was aimed to maximize fluorescent signal per phage while maintaining solubility to achieve maximum dynamic range of fluorescent distribution. Labeling conditions were optimized using M13 helper phage. Normalized fluorescence shows an increase in relation to the number of FITC conjugated per phage, (Fig 12b). Further labeling caused precipitation and was not further investigated. The washing and the reaction incubation time was also optimized (Fig 12a, 12c) where a final reaction time of 1 hour and a total of three washes determined to be optimal. Flow cytometry and fluorescent activated sorting

All experiments of flow cytometry biopanning profiling and fluorescent activated sorting were done with biotinylated Ags immobilized with streptavidin coated beads. SPHEROTM Streptavidin Polystyrene particles, (3.0 - 3.9 pm) were used to immobilize Ag. First, the beads were blocked with 2% BSA- buffer for 1 hour. Beads were then washed by resuspending beads in 1 % BSA-buffer and subsequently centrifuging the beads (7k rpm for 2 min) to remove supernatant. This process was repeated three times. Biotinylated Ag was then added to beads at 1% BSA final concentration and incubated for one hour. Meanwhile, labeled phage were blocked in 1% BSA PBS for one hour. After Ag-immobilized beads were washed, blocked phage was added to the beads and incubated for 1-1.5 hours. After the binding phase, beads were washed 1-3 times with l%BSA-buffer and passed through a 40 pm cell strainer. Subsequently, flow cytometry analysis or fluorescent activated sorting was performed. All sorting was performed on a BDFACS Aria II and all flow cytometry analysis were performed on a benchtop Beckman Cytoflex Analyzer or BDFACSCaliber machine. Bead populations were gated with FSC and SSC parameters and later only singlet populations were analyzed by gating linear FSA and FSW. Of singlet population of beads, histogram analysis was done with FITC. For fluorescent activated sorting, correct percentage of events were sorted. Sorted beads were used to infect fresh TGI cells (OD600-0.7) and cells were plate for picking individual clones.

96-well Periylasmic extract preparation

Single colony clones were picked and inoculated into 2xYT media containing 2% glucose and 100 mg/ml Ampicillin in round bottom 96 well plates (150 ml of media per well). Cultures were grown overnight at 37°C with 200 rpm shaking. Following day 96 well cultures were inoculated (12 pl per well) into 96 well deep plates containing 2xYT media with 0.1% glucose and 100 pg/ml Ampicillin (1200 pl of media per well) and grown for 4-6 hours until culture is turbid. Fab expression was induced with 300 pl of 2xYT, ampicillin mg/ml and 5 mM IPTG and were left overnight at 30°C with 200 rpm shaking. Periplasmic extracts were collected by osmotic shock. Briefly, cells from overnight cultures were collected by centrifugation at 2000 g for 25 min and 375 pl of ice-cold TES buffer (200 mM Tris-HCl, 0.5 mM EDTA, 500 mM Sucrose, pH = 8) was added directly into each well and incubated with shaking at RT. Subsequently 1125 l of ice-cold water was added in each well and mixed thoroughly. The periplasmic fraction (supernatant) was collected by centrifugation at 2000 g for 25 min and stored at -20 °C for future experiments.

Dot blots

From 96-well periplasmic extracts, 2-3 pl was applied to nitrocellulose membranes. After ~10 min, the membrane was blocked with 2%-TBS-milk and gently rocked at RT. After 1 hr, membrane was gently rinsed with TBS-T (0.05% Tween-20) and 1%TBS-Milk with anti-myc HRP(9E10) (1:5000 dilution) was added and incubated for 1 hr. Washes were performed with TBS-T (x2) and TBS (x2), and 1 ml of Immobilon Forte Western HRP substrate was added for imaging on a ChmiDoc MP imaging system.

Biolayer interferometry (BLI)

All buffers were filter sterilized with 0.22 mm filters prior to preparing samples. Black 384 well microplates were used to set up BLI plates and streptavidin tips were purchased by Sartorius. Kinetic constants of Fabs were determined by Octet RED384 system with continuous shaking at 1000 rpm in RT. Prior to the experiment, biological tips (model name) were presoaked in buffer to allow for equilibration for 1 hour. Data were analyzed using 1 : 1 interaction model on the ForteBio data analysis software 12.0.

Biolayer interferometry antibody screen (BIAS)

Instrument and reagent set up was identical to that of standard BLI. Anti-myc Ab (9E10) (Merk) was used for the Assoc-2. For the control experiments, BLI was run in following method: Baseline (1 min), Load, Baseline (1 min), Assoc-1 (10 min), Assoc-2 (10 min), Dissoc (10 min), and for CHIP and CS3D BIAS runs, Assoc-1 (3 min), Assoc-2 (2 min 30 sec), and Dissoc (3 min) was used as the final protocol. Dependent on the Ag that was used, loading step was adjusted where loading was immediately terminated once rate of loading changed to allow for equal distribution of Ag on tip. Raw data files of the run(s) were then used in the BATCH to rank order candidate hits according to their predicted dissociation rates. The true Assoc-2 slope of the PPE spiked sample without 9E10 calculated by BATCH was further used as the threshold value for determining hits (Table 1). BIAS Algorithm Triaging Confirmed Hits (BATCH) development.

A BIAS function was developed to effectively process raw BLI sensogram data, to categorize and rank order candidate hits according to their kinetic properties. A brief schematic of the categorization, ranking system, and outputs is listed in Fig. 10. BATCH will process all raw BLI data files, a user-created methods file, and a user-created thresholds file. Due to buffer mismatches occurring during the BIAS runs, each step is trimmed at the beginning and end to avoid erroneous noise. Briefly, the Assoc- 1 and Dissoc steps are fitted as a one phase exponential association and one phase exponential decay respectively. To account for continued Fab binding during the Assoc-2 step, the one phase exponential association fit of the Assoc- 1 step is extrapolated through Assoc-2 as the “extrapolated association- 1 curve”. The “true Assoc- 2 slope” only accounts for the anti-myc IgG signal contribution and is calculated by subtracting the extrapolated association curve by the raw Assoc-2 curve. The “true Assoc-2 slope” is fitted with a linear regression to quantify a significant signal shift. Importantly the “true Assoc-2 slope” distinguishes “Hits” versus “False positives”. Threshold values were determined by control experiments (Fig 4b-4d, Table 1). BATCH outputs R2 values for all fits in each step which determines which category each clone is classified into based on a threshold value (figure 11). BATCH assumes a pseudo first-order kinetic model for predicting koffs. By using the calculated koffs from the Dissoc step, BATCH will predict a range of Kd based on previous Craik lab Fab kons and rank clones classified as “Hits”. BATCH will generate two tables to provide a summary of results and a more detailed view of all processed data. To give the user more information on the reason behind classification of clones, the comment section provides more details (i.e., no dissociation, no association, R2 values too low etc.). Threshold and kon values can be changed according to the user’s experimental set up where secondary IgG or Ab format is different.

Exceptions to the BIAS rankings

Clones that are flagged in the BIAS rankings indicate inaccuracies in koff predictions. For these clones, the “true association-2 slope” can be a better predictor of binding, as exchange rates of Fab to Fab-9E10 complex during Assoc-2 are directly correlated to koff of the Fab. Specifically, rapid exchange of Fab to Fab-9E10 indicate a high koff, which translate to a high “true association-2 slope”. Therefore, koff is inversely proportional to the true association-2 slope. A simple guide to further distinguishing flagged BIAS results is shown in Fig 13. Fab expression

Freshly transformed BL21(DE3) E. coli single colony clones were picked and inoculated into 50 ml of 2xYT media containing 2% glucose and 100 mg/ml Ampicillin. Starter cultures were grown overnight at 37oC with 200 rpm shaking. The next day, the starter culture was inoculated to 1 L of 2xYT media with 0.1% glucose and 100 mg/ml Ampicillin (OD600 0.05 final) and after incubation (37°C with 200 rpm shaking), protein expression was induced with IPTG (1 mM final) at OD600 of 0.6 and continued to grow overnight (20°C with 200 rpm shaking). Periplasmic extracts containing Fabs were collected by osmotic shock. Briefly, cells from overnight cultures were collected by centrifugation at 9000 g for 15 min and -15 ml of ice-cold TES buffer was added and resuspended thoroughly to achieve a homogenous mixture. The mixture was then incubated with gentle shaking at 4oC for 1 hour. Subsequently -25 ml of ice-cold water was added and incubated with gentle shaking for 45 min. The periplasmic fraction (supernatant) was collected by centrifugation at 10,000 g for 30 min and loaded to Ni- NTA resin for affinity purification. Purified Fabs were dialyzed against PBS (lOkDa MWCO) and size exclusion chromatography was performed to further purify the Fabs and remove any aggregates (AKTA autopurification system with a Superdex 200 10/300GL column). Fractions exhibiting correct size were pooled and Fab concentrations were determined by absorbance at 280 nm.

Conversion of Fab into Full-Length IgG

The methods used here were described in detail in a previous study 24. Briefly, the heavy and light chain regions of Fabs were cloned out from their respective phagemids and then individually cloned into a pTT5-SP-Hl mammalian expression vector through Gibson Assembly® (NEB #E2611) methods. The Gibson assembly product was transformed into NEB® 5a Competent E. Coli cells (NEB #C2987H) and plasmids were isolated using a ZymoPurell Plasmid Maxiprep Kit (Zymo Research #D4203) and confirmed through sequencing.

Small-scale Expression and Purification of Full-Length IgG in Mammalian Cells

The expression protocol used here was based on the Expi293TM Expression System (ThermoFisher #A14635). Expi293 suspension cells were seeded at a final density of 2.5 x 106 cells/mL in a 6-well plate. The next day cells were diluted to a final density of 3 x 106 cells/mL and a total of 1 .0 pg/mL of plasmid DNA was transfected using ExpiFectamineTM 293 Transfection Kit. Transfected cells were then incubated on an orbital shaker at 37°C and 8% CO2 overnight and ExpiFectamine 293 Transfection Enhancer 1 and 2 were added to the cells. Secreted IgG was harvested 6 days post-transfection and the supernatant was incubated with PierceTM Protein G Plus Agarose (ThermoScientific #22851) resin at room temperature for 1 hour. After washing with 10 column volumes of PBS, the IgG was eluted with 2 column volumes of 100 mM glycine, pH 2.5 and neutralized by 1 M Tris, pH 8.5. The eluted fractions were determined by SDS-PAGE gel and fractions containing pure IgG were collected and dialyzed against PBS buffer.

Statistics and Reproducibility

NHS-FITC phage labeling experiments were performed in triplicates and data were presented as mean ± SD. Flow cytometry and FACS data was analyzed by FlowJo 10.9.0 from at least 10,000 events. The statistical significance between the BIAS off-rates of sorted and unsorted populations were determined by A two-way ANOVA test on Prism (Graphpad).

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18. Johnson, D. E., O’Keefe, R. A. & Grandis, J. R. Targeting the 1L-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol. 15, 234-248 (2018).

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23. Li, K. et al. Chemical Modification of M13 Bacteriophage and Its Application in Cancer Cell Imaging. Bioconjug. Chem. 21, 1369-1377 (2010).

24. Duriseti, S. et al. Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration. J. Biol. Chem. 285, 26878-26888 (2010).

25. Van Raamsdonk, C. D. et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599-602 (2009).

26. Shirley, M. D. et al. Sturge-Weber Syndrome and Port-Wine Stains Caused by Somatic Mutation in GNAQ. N. Engl. J. Med. 368, 1971-1979 (2013). In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

It will be understood by those within the ail that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (c.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or“B” or “A and B.”

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein arc principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the ail, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. § 112(6) is not invoked.

Claims

CLAIMS WHAT IS CLAIMED IS:

2. The method according to Claim 1, wherein the method further comprises contacting the sensor tip with a buffer and measuring a dissociation rate of the first antibody from the target of interest.

3. The method according to Claims 1 or 2, wherein the method is performed simultaneously or sequentially on a plurality of solutions comprising different antibodies.

4. The method according to any one of Claims 1 to 3, wherein the antibodies are produced from a phage display library.

5. The method according to any one of Claims 1 to 4, wherein the antibodies are Fab, Fv, scFv, VH domain antibodies, or nanobodies.

6. The method according to any one of Claims 1-5, wherein the solution comprising multiple copies of the first antibody is generated from a bacteria infected with a phage displaying the first antibody, wherein the phage is identified by a method comprising: fluorescently labeling a phage display library comprising a multitude of different antibodies displayed on phages; contacting the labeled phage display library with the target of interest immobilized on beads to produce bead-phage complexes; and analyzing the bead-phage complexes using flow cytometry to generate a profile of the complexes; using the profile to identify high-affinity antibodies.

7. The method according to Claim 6, wherein the profile comprises a histogram representing the frequency of complexes having different fluorescence intensities.

8. The method according to Claim 7, wherein a median fluorescence intensity (MFI) and/or a standard deviation (SD) are calculated for one or more components of the profile.

9. The method according to Claim 8, wherein the high affinity antibodies correspond to a component of the profile having a high MFI.

10. The method according to Claims 8 or 9, wherein the high affinity antibodies correspond to a component of the profile having a high SD.

11 . The method of according to any one of Claims 6 to 10, further comprising: sorting the bcad-phagc complexes based on a level of fluorescent phages bound to the beads, thereby separating complexes exhibiting a threshold level of fluorescence from complexes exhibiting fluorescence below the threshold level.

12. The method according to any one of Claims 6 to 11, wherein the phage display library comprises phages from two or more rounds of preselection for phages that bind to the target of interest.

13. The method according to Claim 12, wherein the phage display library comprises phages from four or more rounds of preselection for phages that bind to the target of interest.

14. The method according to Claims 12 or 13, wherein phages from each round are separately contacted with the target of interest immobilized on beads and analyzed using flow cytometry to generate a profile for phages from each round of preselection.

15. The method according to Claim 14, comprising using the generated profiles to identify high-affinity antibodies.

16. The method according to Claim 15, wherein the high-affinity antibodies have the profile with the highest MFI.

17. The method according to any one of Claims 6 to 16, wherein the phage display library is labeled with fluorescein isothiocyanate (FITC) or an Alexa Fluor (AF) dye.

18. The method according to Claim 17, wherein the phage display library is labeled with FITC.

19. The method according to Claim 17, wherein the phage display library is labeled with AF647.

20. The method according to any one of Claims 6 to 19, wherein the phages are M 13 phages.

21. The method according to Claim 20, wherein the M13 pages are labeled via N-termini and/or lysine residues of the pVIII coat proteins.

22. The method according to any one of Claims 6 to 21, wherein the phage display library is generated from bacteria infected with a phage.

23. The method according to Claim 22, wherein the bacteria are Escherichia coli.

24. The method according to Claims 22 or 23, wherein the solution comprising multiple copies of a first antibody is crude periplasmic extract (PPE) from bacteria infected with a phage.

25. The method according to Claim 24, wherein the first antibody comprises a tag and the second antibody binds to the tag.

26. The method according to any one of Claims 2-25, wherein the buffer comprises no detectable copies of the first antibody or the second antibody.

27. The method according to any one of Claims 2-26, wherein the method further comprises determining a binding kinetic parameter of first antibody to the target of interest using tip measurements.

28. The method according to Claim 27, wherein the binding kinetic parameter is a dissociation rate constant.

29. The method according to Claim 28, wherein the dissociation rate constant is determined using measurements generated while the tip is contacting the buffer.

30. The method according to any one of the preceding Claims, wherein the method further comprises determining false positives using tip measurements.

31. The method according to Claim 30, wherein the false positives arc determined using measurements generated while the tip is contacting the second aliquot of the solution comprising multiple copies of the first antibody and wherein the second aliquot further comprises the second antibody that binds to the first antibody, wherein presence of (a) and absence of (b) identifies the first antibody as a false positive antibody.

32. The method according to any one of the preceding Claims, wherein the method further comprises determining if the sensor tip is saturated by antibody copies using tip measurements.

33. The method according to Claim 32, wherein the saturation is determined using measurements generated while the tip is in the first aliquot of the solution.

34. A system configured to perform the method according to any one of Claims 1 to 33, optionally wherein the system comprises a biolayer interferometry device and a cell sorter.

35. A biolayer interferometry device configured to perform the screening according to any one of Claims 1 to 33.

36. A method for identifying antibodies with affinity to a target of interest, the method comprising: conducting a first round of biopanning comprising isolating phages displaying antibodies that bind to the target of interest and fluorescently labeling the phages to generate a first labeled population of phages; conducting a second round of biopanning comprising isolating phages displaying antibodies that bind to the target of interest and fluorescently labeling the phages to generate a second labeled population of phages; separately contacting the first and the second labeled population of phages with the target of interest immobilized on beads to produce bead-phage complexes; analyzing the complexes using flow cytometry to generate a first fluorescence profile of the complexes produced from the first labeled population of phages and a second fluorescence profile of second labeled population of phages; and using the first and second profiles to identify antibodies with affinity to the target of interest.

37. The method according to Claim 36, wherein the profile comprises a histogram representing the frequency of complexes having different fluorescence intensities.

38. The method according to Claim 37, wherein a median fluorescence intensity (MFI) and/or a standard deviation (SD) are calculated for one or more components of the profiles.

39. The method according to Claim 38, wherein the antibodies associated with the profile having the highest MFI are identified as high affinity antibodies.

40. The method according to Claim 38, wherein when the MFIs of each profile are similar, the antibodies associated with the profile with the highest SD are identified as high affinity antibodies.

41. The method according to any one of Claims 38 to 40, wherein the antibodies associated with the profile with the highest MFI and SD are identified as high affinity antibodies.

42. The method of according to any one of Claims 36 to 41, wherein the isolating further comprises: sorting the complexes based on a level of fluorescent phages bound to the beads, thereby separating complexes exhibiting a threshold level of fluorescence from complexes exhibiting fluorescence below the threshold level; wherein the high affinity antibodies are present in the complexes exhibiting the threshold level of fluorescence.

43. The method according to any one of Claims 36 to 42, wherein the method comprises conducting three or more rounds of biopanning to generate three or more labeled populations of phages.

44. The method according to Claim 43, wherein the method comprises conducting four or more rounds of biopanning to generate four or more labeled populations of phages.

45. The method according to any one of Claims 36 to 44, wherein the method further comprises screening the high affinity antibodies individually to identify antibodies with a high affinity to the target of interest using bio-layer interferometry (BLI).

46. The method according to Claim 45, wherein the screening comprises: infecting bacteria with phages expressing the high affinity antibodies; separating the bacteria into single cells and growing bacterial colonies that each express a single type of antibody; preparing an extract from the colonies to provide a plurality of solutions, each solution comprising multiple copies of single type of antibody; contacting in parallel, a first aliquot of each of the plurality of solutions with a sensor tip comprising the target of interest immobilized thereon; contacting in parallel, a second aliquot of each of the plurality of solutions with the sensor tip, wherein the second aliquot further comprises a second antibody that binds to each of the single type of antibodies; wherein binding between the target of interest, the antibodies expressed by the phages, and the second antibody are determined by irradiating the target of interest with light and measuring wavelength shift over time using the sensor tip; and wherein a difference in the rate of change of wavelength shift over time between when the sensor tip is in contact with first aliquot and when the sensor tip is in contact with the second aliquot is used to identify whether the antibody expressed by the phages has affinity to the target of interest.

47. The method according to Claim 46, wherein the method further comprises contacting a third buffer with the sensor tip.

48. The method according to Claims 46 or 47, wherein the phages produce an antibody comprising a tag and the second antibody binds to the tag.

49. The method according to any one of Claims 46 to 48, wherein the screening is performed for phages produced from at least 10, at least 30, at least 100, at least 300, or more individual bacterial colonies.

50. The method according to any one of Claims 36 to 49, wherein the phage display library is labeled with fluorescein isothiocyanate (FITC) or an Alexa Fluor (AF) dye.

51. The method according to Claim 50, wherein the phage display library is labeled with FITC.

52. The method according to Claim 50, wherein the phage display library is labeled with AF647.

53. The method according to any one of Claims 36 to 52, wherein the phages are M13 phages.

54. The method according to Claim 53, wherein the M13 pages are labeled via N-termini and/or lysine residues of the pVIII coat proteins.

55. The method according to any one of Claims 46 to 54, wherein the phage display library is generated using bacteria of the genus Escherichia.

56. The method according to Claim 55, wherein the bacteria are Escherichia coli bacteria.

57. The method according to any one of Claims 44 to 56, wherein the solution comprises crude pcriplasmic extract (PPE) produced from bacteria.

58. The method according to any one of Claims 47 to 56, wherein the buffer does not include a chaotropic agent.

59. The method according to any one of Claims 47 to 58, wherein the buffer comprises a chaotropic agent.

60. The method according to any one of Claims 47 to 59, wherein the method further comprises determining a binding kinetic parameter of the antibodies to the target of interest using tip measurements.

61. The method according to Claim 60, wherein the binding kinetic parameter is a dissociation rate constant.

62. The method according to Claim 61, wherein the dissociation rate constant is determined using measurements generated while the tip is in the buffer.

63. The method according to any one of Claims 46 to 62, wherein the method further comprises determining false positives using tip measurements.

64. The method according to Claim 63, wherein the false positives are determined using measurements generated while the tip is in the second aliquot.

65. The method according to any one of Claims 46 to 64, wherein the method further comprises determining if the sensor tip is saturated by antibody copies using tip measurements.

66. The method according to Claim 65, wherein the saturation is determined using measurements generated while the tip is in the first aliquot the solution.

67. A system configured to perform the method according to any one of Claims 36 to 66.

68. A biolayer interferometry device configured to perform the screening according to any one of Claims 35 to 66.

70. The system according to Claim 69, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to refine raw data from the second segment by extrapolating the association curve fit to the first segment through the second segment and subtracting the extrapolated association curve from the raw data of the second segment, wherein the line is fit to the refined second segment data.

71 . The system according to Claims 69 or 70, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to section the interferometry data into three consecutive segments, wherein a one phase exponential decay curve is fit to raw data from the third segment.

72. The system according to Claim 71, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to determine a goodness of fit metric for each fitted curve.

73. The system according to Claim 72, wherein the goodness of fit metric is an r-squared value.

74. The system according to Claim 73, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to detect negatives of the antibody copies not binding to the target of interest, false positives of non-specific binding proteins binding to the target of interest, and/or hits of the antibody copies binding to the target of interest.

75. The system according to Claim 74, wherein negatives and false positives are detected by determining if the slope of the line fit to the second segment is below a predetermined threshold value.

76. The system according to Claim 74 or 75, wherein negatives and false positives are detected by determining if the r-squared value of the line fit to the second segment is below a predetermined threshold value.

77. The system according to any one of Claim 74 to 76, wherein false positives are detected by determining if the r-squared value of the association curve fit to the first segment meets or exceeds a predetermined threshold value.

78. The system according to any one of Claim 74 to 76, wherein negatives are detected by determining if the r-squarcd value of the association curve fit to the first segment is below a predetermined threshold value.

79. The system according to any one of Claim 74 to 78, wherein the hits are detected by determining if the slope of the line fit to the second segment meets of exceeds a predetermined threshold value and if the r-squared value of the line fit to the second segment meets or exceeds a predetermined threshold value.

80. The system according to Claim 79, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to determine if the sensor tip is saturated by antibody copies by calculating a slope of the extrapolated association curve.

81. The system according to Claim 80, wherein the sensor tip is determined to be saturated if the slope of the extrapolated association curve meets or exceeds a predetermined threshold value.

82. The system according to any one of the Claims 79 to 81, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to determine antibody clone expression by calculating the total wavelength shift of the first segment.

83. The system according to Claim 82, wherein the antibody copies arc determined to have low expression if the total wavelength shift of the first segment is below a predetermined threshold value.

84. The system according to any one of the Claims 79 to 83, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to predict a dissociation rate for the antibody copies and the target of interest.

85. The system according to Claim 84, wherein the dissociation rate is predicted using the exponential decay curve fit to the third segment.

86. The system according to Claims 84 or 85, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to determine if the predicted dissociation rate is inaccurate.

87. The system according to Claim 86, wherein the dissociation rate is determined to be inaccurate if the r-squared value of the exponential decay curve fit to the third segment is below a predetermined threshold value.

88. The system according to any one of the Claims 69 to 87, wherein the system is configured to identify antibodies with affinity to the target of interest for a plurality of inputs each comprising sequential wavelength shift measurements resulting from interactions between a sensor tip comprising a target of interest, copies of an antibody, and tag binding antibodies.

89. The system according to Claim 88, wherein the copies for each input are of a different antibody.

90. The system according to Claims 88 or 89, wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to rank the inputs based on a binding kinetic parameter predicted for each of the different antibodies to the target of interest.

91. The system according to Claim 90, wherein the binding kinetic parameter is a dissociation rate constant.

92. The system according to any one of the Claims 69 to 91, wherein the system further comprises a bio-layer interferometry sensor tip configured to generate the raw bio-layer interferometry data and transmit it to the processor, the sensor tip comprising: a surface comprising the target of interest; a light source configured to irradiate the surface; and a detector configured to detect an interaction between antibodies and the target of interest.