key: cord-0898304-gm3k14b4 authors: Zupancic, Jennifer M.; Desai, Alec A.; Tessier, Peter M. title: Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis date: 2022-01-20 journal: STAR Protoc DOI: 10.1016/j.xpro.2021.101101 sha: a81201ed87eceea17503221bd65d691f26aa5358 doc_id: 898304 cord_uid: gm3k14b4 The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021). 1. Obtain the DNA sequence of a yeast surface display plasmid containing a representative nanobody. 2. Design forward (Forward primer #1) and reverse (Reverse primer #3) primers for amplifying the plasmid region encompassing the entire nanobody and additional plasmid DNA beyond the restriction sites that will be used for preparing the CDR-swapped library. Note: Amplified DNA should extend at least $30-50 base pairs beyond the restriction sites (i.e., N-and C-termini of the nanobody gene) that will be used for vector digestion. Overlap Step 1: A common framework nanobody library is prepared in a plasmid which enables nanobody display on yeast through a linker to the Aga2 protein. Magnetic-activated cell sorting (MACS) is first performed to enrich the naïve library for variants that bind the antigen. The enriched library is then sorted using fluorescence-activated cell sorting (FACS) to obtain a diverse population of cells that demonstrate antigen binding (see Figure 3 for details). Step 2: Nanobody plasmid DNA is isolated from yeast cells collected in the final sort performed in Step 1. PCRs are performed to amplify individual CDRs and overlapping DNA sequences from the nanobody framework and surrounding plasmid (see Figure 2 for details). DNA is then reassembled using overlap PCR to produce DNA sequences encoding entire nanobody genes composed of CDR sequences from one or more parental nanobodies. CDR-swapped nanobody library DNA is transformed into yeast cells to produce the CDR-swapped sub-library via homologous recombination in yeast. Step 3: The CDR-swapped library is displayed on the yeast surface and further sorted by FACS to select highaffinity variants (see Figure 3 for details). Plasmid DNA from yeast cells collected in the terminal sort is isolated, and the sequences of individual clones are determined. Individual nanobodies are cloned as Fc-fusion proteins and expressed via transient transfection in mammalian cells. The affinity and activity of nanobody-Fc fusion proteins is then analyzed. between this sequence and the digested vector will allow for the preparation of a CDR-swapped library via homologous recombination in yeast. Note: For the PCR conditions given below, the design of primers with melting temperatures of $58 C-62 C is recommended. (B) The DNA sequence of the nanobody and surrounding plasmid that are used for CDR-swapping mutagenesis. Gray sequences encode homologous sequences to the plasmid at the 5 0 and 3' ends. Nanobody framework regions are shown in black. Variable region DNA sequence corresponding to CDR1 (red), CDR2 (blue), and CDR3 (green) are shown as Xs. Individual PCRs amplify CDR1 (Forward and Reverse primer #1), CDR2 (Forward and Reverse primer #2), and CDR3 (Forward and Reverse primer #3). Regions recognized by primers that amplify individual CDRs are highlighted in yellow. Finally, the DNA segments are recombined using overlap extension PCR. Plasmid DNA at the 5' and 3 0 ends is amplified, including $50-60 base pairs that flank the restriction sites used for vector digest, in order to prepare plasmid DNA for homologous recombination and transformation into yeast. Restriction sites (NheI and XhoI) are highlighted in purple. 3. Design primers to amplify individual CDRs and overlapping DNA sequences a. Amplify CDR1 using Forward primer #1 and a reverse primer that binds in framework 2 of the nanobody library (Reverse primer #1). b. Amplify CDR2 using a forward primer that binds in framework 2 (Forward primer #2) and a reverse primer that binds in framework 3 (Reverse primer #2). c. Amplify CDR3 using a forward primer that binds in framework 3 (Forward primer #3) and Reverse primer #3. CRITICAL: The forward and reverse primers which bind in framework 2 and framework 3 should amplify enough overlapping DNA sequences (e.g., typically >15 base pairs) to perform overlap PCR using the forward and reverse primers designed in Step 3. It is recommended that the melting temperature of the primers designed in Step 3 and the overlapping regions of DNA amplified in framework 2 and framework 3 should not differ by >5 C. Timing: 2 days 4. Add volume corresponding to 1 3 10 7 streptavidin Dynabeads to a 1.5 mL tube, and place tube on DynaMag TM -2 Magnet. Note: For antigen that is not biotinylated, immobilization on the surface of magnetic beads with alternative surface chemistries like primary amine reactive tosyl group (tosyl activated Dynabeads, Invitrogen, Cat# 14-203) may be performed. Note: The volume of beads prepared may be scaled up or down depending upon the number of beads needed for analysis. 5. Allow sufficient time for beads to move to the side of the tube by the magnet and discard the liquid. 6. Wash the beads by resuspending in 1 mL of 13 PBS. Note: Another buffer may also be used for washing and storing the beads. Buffer should not contain any components that will interact with the surface chemistry of the beads (e.g., a buffer with a primary amine should not be used for washing tosyl beads). 7. Place beads in 13 PBS on magnet, allow beads to settle on the side of the tube by the magnet, and discard the liquid. 8. Repeat Steps 6 and 7. 9. Add 0.1 mg antigen to the beads and resuspend the bead solution to a total volume of 400 mL in same buffer (13 PBS) as used for washing the beads. 10. Mix the beads by end-over-end mixing for $12-48 h at room temperature (20 C-25 C). 11. Store beads at 4 C until ready to use. Note: Beads can be stored at 4 C for several months. Antigen immobilization time is variable depending on the type of beads used. For example, for streptavidin Dynabeads, 12-24 h at 4 C is sufficient whereas for tosyl activated Dynabeads, longer time is preferred (recommended by the manufacturer). Note: Autoclave tryptone, sodium chloride, yeast extract, and agar in 1 L DI H 2 O to dissolve. Add ampicillin once agar solution has cooled sufficiently to pour plates. Note: Ampicillin is used to select transformed bacteria containing a plasmid with an ampicillin resistance gene. Related antibiotics, including more stable ones (carbenicllin), are also appropriate. A suitable antibiotic and corresponding concentration should be chosen based on the plasmid being transformed. Tryptone 10 g/L 10 g Sodium chloride 10 g/L (171 mM) 10 g Note: Adjust to pH 3 with hydrochloric acid and make up the solution to 1 L. Recovering and inducing yeast libraries Yeast libraries are refreshed in growth media (SDCAA) prior to use in sorting. This growth step can be used to allow the library to recover after previous storage of the frozen library. Transferring the nanobody library to induction media (SDGCAA) then promotes nanobody expression on the surface of yeast cells for sorting. 1. Thaw a frozen aliquot of the nanobody yeast library on the benchtop at room temperature. a. The frozen library aliquot has >10-fold coverage of the diversity with >10 9 cells. 2. Add thawed yeast cells to 500 mL of SDCAA media in baffled flask. a. Supplement SDCAA media with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Penstrep. b. Incubate the flask at 30 C at 225 RPM for 18-24 h. 3. Calculate the cell density by measuring the optical density at 600 nm (OD 600 ). One OD 600 unit is equivalent to approximately 3 3 10 7 cells/mL. 4. Calculate the volume of cells required to seed 500 mL SDGCAA culture at a final OD 600 of 1. 5. Centrifuge the required number of cells at 25003g for 5 min. 6. Discard the supernatant, resuspend the cells in SDGCAA, and move the cells to a 500 mL SDGCAA culture in shake baffled flask. a. Supplement SDGCAA media with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Pen-strep. b. Incubate the flask at 20 C at 225 RPM for 36-48 h. Note: Yeast induction could be performed at 30 C for 18-22 h or at 20 C for 36-48 h. In certain cases, longer induction times help with higher surface expression. Magnetic-activated cell sorting, MACS (sort 1) Initial MACS selections allow for the processing of a large number of cells (10 8 -10 9 ) in early rounds of sorting. In order to ensure full processing of the entire library diversity, approximately 10-fold Elution buffer (0.1 M glycine) Final concentration Amount greater number of cells are processed than the expected library diversity. Performing a MACS selection enriches the library for clones that bind antigen and reduces library diversity to a level that can be processed via FACS. 7. Calculate the OD 600 of the SDGCAA culture. 8. Transfer volume corresponding to 10 9 cells to a 50 mL conical tube. 9. Centrifuge the cells at 25003g for 5 min at room temperature. 10. Discard the supernatant and wash the cells by resuspending in 25 mL PBSB. 11. Repeat Steps 9 and 10. 12. Centrifuge the cells at 25003g for 5 min (room temperature) and discard the supernatant. Prepare 10% w/v milk solution in PBSB by dissolving 500 mg non-fat dry milk in 4.5 mL PBSB followed by end-over-end mixing at room temperature for 10-15 min. a. Spin down milk solution at 25003g for 5 min (room temperature). Move the supernatant to a new tube. Note: Centrifuging the milk solution prior to use removes insoluble aggregates and produces more consistent results. 13. Resuspend the cells in a final volume of 5 mL with 1% milk (dilute 10% milk solution in PBSB from Step 12) and 300 nM of biotinylated monovalent antigen (e.g., SARS-CoV-2 receptor-binding domain). a. Incubate cells with antigen for 3 h with end-over-end mixing at room temperature. 14. Post antigen incubation, centrifuge the cells at 2500xg for 5 min and wash the cells once by resuspending in 25 mL ice-cold PBSB. 15. Centrifuge the cells at 25003g for 5 min (room temperature). Note: LS columns are gravity columns. Before passing cells through them, they need to be placed on magnetic adapter (MidiMACS Separator and MACS MultiStand). Refer to manufacturer's instructions (https://www.miltenyibiotec.com/upload/assets/IM0001298.PDF) for more details. Optional: Pass cells through a 70 mm filter immediately prior to running cells through the LS columns. Filtering cells removes aggregates and allows cells to pass through the column without clogging. 22. Wash the captured beads once by passing 5 mL ice-cold PBSB through the LS column under the magnetic field. 23. Remove the LS column from the magnet and elute beads by passing 5 mL SDCAA through the column. 24. Transfer eluted beads and cells to a 50 mL SDCAA media. a. Supplement culture with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Pen-strep. 25. Plate dilutions (10-10003 from the 50 mL cultures) of the cell culture on yeast dropout plates to evaluate the numbers of cells retained during the selection. 26. Grow cells in SDCAA culture and on dropout plates at 30 C at 225 RPM for 2 d. The culture OD 600 is typically >8-10 after 2 d of growth. Pause point: Cells in SDCAA culture can be stored at 4 C for several weeks. After storing at 4 C, cells should be refreshed in fresh SDCAA for 18-24 h prior to induction in SDGCAA. Fluorescence-activated cell sorting, FACS (sorts prior to CDR shuffling) Timing: 5 h FACS selections performed prior to CDR shuffling enrich the sorted library to obtain a population of yeast cells from which binding signal can be easily distinguished from the background. This stage of sorting maintains or sequentially decreases the antigen concentration in order to selection for a binding population that displays modest affinity for the target antigen. 27. Induce the yeast cells collected in sort 1 in SDGCAA media as described above in Steps 3-6. Culture volume at this stage may be decreased to 5-8 mL. 28. Calculate the OD 600 of the SDGCAA culture. 29. Transfer volume corresponding to 10 7 -10 8 yeast cells (adjusted to sample 10-fold greater number of cells compared to the isolated library diversity in the previous sort) to a sterile 1.5 mL tube. Typically, at least 10 7 cells are prepared to enable collection of at least 3,000-5,000 cells. CRITICAL: It is recommended to sample at least 10-fold coverage of nanobody diversity in the early FACS selections (e.g., rounds 2-3) to sample full diversity obtained from the MACS selections (e.g., round 1). Note: Reducing the antigen concentration in progressive rounds of sorting allows for the separation and selection of nanobodies with higher affinities. Note: It is recommended to prepare samples at two different concentrations for sorting. In cases in which enrichment for clones that bind to the antigen is modest, it is recommended that the samples are prepared with the same antigen concentration and a three-fold dilution compared to the previous round. In cases in which enrichment for clones progresses rapidly between rounds, it recommended that the samples are prepared with three-fold and 10-fold reductions in antigen concentration compared to the previous round. Collection of cells from the lower of the two examined antigen concentrations that shows a distinct binding population typically allows for more efficient isolation of nanobodies with increased affinity. Optional: Add milk to achieve a final concentration of 1% milk. The presence of milk may help in reducing the collection of nanobodies with poor specificity. should be used to examine the log area of signal from the fluorophore used to detect nanobody expression on the x-axis and the log area of the signal for the fluorophore used to detect antigen binding on the y-axis. 48. Analyze a control sample to assess nanobody expression and secondary reagent binding. This sample should be labeled with primary and secondary antibodies for detecting nanobody expression as well as secondary reagent for detecting antigen binding. 49. Adjust voltages for lasers exciting the fluorophores for detecting nanobody expression and antigen binding. a. Adjust the voltage for the laser used for detecting nanobody expression such that two distinct cell populations are observed (See Figure 3) . i. One population (left) is cells that do not express nanobodies on their surface. ii. The other population (right) is cells that express nanobodies. iii. Quadrants may be drawn to guide distinguishing the nanobody non-expressing and expressing cells. b. Adjust the voltage for the laser used for detecting antigen binding such that signal from this laser is minimal in the control sample. i. Voltage should be adjusted such that the signal is spaced away from the cytogram axis. ii. Voltage and quadrants should be adjusted such that minimal signal is seen in the upper two quadrants. 50. Analyze positive samples to assess both nanobody expression and antigen binding. Positive samples should include samples in which cells have been incubated with antigen at desired concentrations and labeled with the secondary reagent for detecting bound antigen. These samples should also be labeled with primary and secondary reagents for detecting antibody expression. a. Draw a gate for cell collection in the upper right-hand quadrant of the cytogram. i. Gate should be drawn sufficiently above the horizontal line dividing the quadrants such that a minimal number of cells fall within this gate in the control sample. ii. Gate should be drawn to have a diagonal slope on the right side. The nanobody library is sorted first using MACS (e.g., one MACS sort against monovalent SARS-CoV-2 receptor binding domain, RBD). Next, the enriched library is sorted by FACS against bivalent antigen (e.g., RBD-Fc) at relatively high antigen concentrations, and then it is further sorted by FACS against monovalent antigens (e.g., monovalent RBD) and/or at reduced antigen concentrations. A diagonal gate is drawn during FACS selections to collect yeast cells that bind antigen in a manner that is proportional to nanobody expression to enrich for high-affinity clones. The gates are drawn to minimize the percentage of cells appearing within the gates in the control sample to avoid enrichment of nanobodies that bind the secondary reagents. After several rounds of enriching the library via FACS, yeast plasmid DNA is isolated and CDR-swapping mutagenesis is performed. Finally, additional FACS sorts are performed following CDR-swapping mutagenesis to isolate high-affinity nanobodies. Pause point: Cells in SDCAA culture can be stored at 4 C for several weeks. After storing at 4 C, cells should be refreshed in fresh SDCAA for 18-24 h prior to induction in SDGCAA. Timing: 1.5 h This step isolates the plasmid DNA from the enriched library that will be used as a template for CDR shuffling. 53. Obtain 1 mL of the 8 mL SDCAA culture collected in final FACS sort prior to CDR-swapping mutagenesis. Note: Remaining cell culture can be stored at 4 C for several weeks. For long-term storage, cells can be frozen at À80 C in a solution of 30% glycerol and 0.67% yeast nitrogen base (without amino acids). 54. Recover nanobody library plasmids from 1 mL of sorted cells using a Zymo Yeast Miniprep Kit according to the manufacturer's protocol (https://files.zymoresearch.com/protocols/ _D2004_Zymoprep_Yeast_Plasmid_Miniprep_II.pdf). Timing: 2 days DNA segments encoding individual CDRs and overlapping constant regions of the nanobodies are amplified. DNA-encoding nanobodies, which consist of combinations of the individual CDRs, are then reassembled via overlap PCR. Step 54 with Q5 DNA polymerase and associated reagents (Table 1) . a. Amplify DNA segment including CDR1 (N-terminal homology-framework 1 to framework 2) using Forward primer #1 and Reverse primer #1. Pause point: Isolated DNA can be stored at À20 C. 59. Mix amplified PCR products corresponding to each CDR gene in equal mass ratio along with terminal primers, Q5 polymerase, and reagents described below (Table 2 ) in a PCR tube to perform overlap PCR. 60. Place PCR tube in a PCR block with a heated lid and run PCR at the same conditions as Step 56. Note: Sufficient replicates (16-32 of the above described 50 mL PCR reactions is usually sufficient) of this overlap PCR reaction should be performed in order to generate 12 mg of insert DNA. 61. Purify the PCR product by separation using a 1% agarose gel followed by cutting the DNA band at the expected size ($520 base pairs for the given primers). 62. Isolate insert DNA using a Qiagen Gel Extraction kit according to the manufacturer's protocol (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/ dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/?catno=28706). 63. Incubate vector with NheI-HF and XhoI restriction enzymes at 37 C overnight ($12-18 h) according to the manufacturer's protocol (https://www.neb.com/protocols/2012/12/07/ optimizing-restriction-endonuclease-reactions). Note: Sufficient DNA should be digested in order to generate 4 mg of digested vector DNA (pCTCON2). 64. Add Quick CIP (calf intestinal alkaline phosphatase) to restriction digest reaction and incubate at 37 C for at least 15 min. 65. Purify the digested vector by separation using a 1% agarose. 66. Isolate digested vector DNA using a Qiagen Gel Extraction kit according to the manufacturer's protocol (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rnapurification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/?catno=28706). Note: Digested vector DNA should be stored frozen at À20 C until use. Timing: 5 days Plasmid DNA encoding the CDR-shuffled nanobodies is transformed into yeast cells to generate a library for subsequent sorting. 67. Mix 12 mg of insert DNA (from Step 62) with 4 mg linearized plasmid (from Step 66). 68. Ethanol precipitate DNA with Pellet Paint NF Co-precipitant according to the manufacturer's protocol (https://www.emdmillipore.com/US/en/product/Pellet-Paint-NF-Co-Precipitant,EMD_ BIO-70748#anchor_USP). 69. Cover ethanol precipitated DNA with aluminum foil and allow to dry completely overnight in a vacuum or chemical fume hood at room temperature. 70. Transform ethanol precipitated DNA into EBY100 yeast cells. 71. Two days before transforming the library, start a 5 mL culture of EBY100 yeast cells and grow cells overnight ($12-18 h) at 30 C with agitation at 225 RPM. a. Supplement culture with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Pen-strep. 72. One day before transforming the library, seed a 50 mL culture of EBY100 yeast cells from the 5 mL culture started the previous day and grow cells overnight ($12-24 h) at 30 C with agitation at 225 RPM. a. Supplement culture with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Pen-strep. 73. The day that the library will be transformed, inoculate a culture of EBY100 yeast cells from the 50 mL culture started the previous day to achieve a final OD 600 of 0.3. a. Start 50 mL of culture per library to be transformed. b. Supplement culture with 100 mg/mL ampicillin, 100 mg/mL kanamycin, and 0.013 Pen-strep. c. Grow cells at 30 C with agitation at 225 RPM. Pause point: A library in SDCAA culture can be stored at 4 C for several weeks. After storing at 4 C, the library should be refreshed in SDCAA for 18-24 h prior to induction in SDGCAA. For long-term storage, the library may be frozen at À80 C in solution of 30% glycerol and 0.67% yeast nitrogen base (without amino acids). Note: For the sort immediately following generation of the CDR-swapped library, antigen concentration should be maintained at the value used in the last selection before library generation. Note: Subsequent rounds of sorting should further decrease the antigen concentration in order to select for high-affinity nanobodies. Preparation of samples with antigen at two distinct concentrations is recommended (as described above) because round-to-round enrichment is difficult to predict. Collection of cells from the lower of the two examined antigen concentrations typically allows for more efficient collection nanobodies with increased affinity. This protocol for CDR shuffling is expected to lead to the isolation of nanobody variants with increased affinity relative to those generated without the incorporation of CDR-swapping mutagenesis into the selection process. Isolation of high-affinity lead nanobodies from a synthetic library using CDR shuffling is expected to reduce the need for further affinity maturation of selected lead nanobodies using methods such as error-prone PCR to generate sub-libraries for further screening. This mutagenesis strategy has the potential to generate high-quality lead nanobodies that may be directly used in biological studies, such as potently neutralizing SARS-CoV-2 nanobodies . A limitation of this protocol is that it requires a common framework library, and it has yet to be evaluated if this method may be generalized to synthetic libraries that contain framework diversity or more complex libraries such as non-immune and immune libraries. The translation of this method to libraries with framework diversity would require more complex primer design, such as mixtures of primers or degenerate primers, to account for the diversity in the framework regions used for primer annealing in CDR-swapping mutagenesis. The library may not have been sufficiently enriched for clones that bind the antigen. Further enrichment of the library using a second round of MACS (as described in Steps 7-26) may lead to a clear binding population by FACS. Alternatively, if a bivalent antigen (e.g., antigen-Fc fusion protein) is available, FACS sorting may be performed with this antigen to detect a binding population through higher avidity. Library shows enrichment of nanobodies binding to secondary labeling reagents by FACS (Step 48). Selections have likely resulted in the enrichment of nanobodies that bind to secondary labeling reagents. For an antigen that is available with various detection tags (e.g., biotin, epitope tag, Fc region), the use of antigen with different tags in alternating rounds of sorting is encouraged. If antigen with multiple tags is not available or enrichment for nanobodies that bind to secondary reagents can otherwise not be avoided by gating on FACS, a negative selection to remove secondary binders should be performed using control samples without antigen, as described in Step 48. This selection ll OPEN ACCESS should collect cells that express nanobodies but do not show interaction with the secondary reagent used to detect antigen binding. Problem 3 PCR product is not visible on agarose gel used to analyze the overlap PCR product (Step 61). Ensure that PCR conditions for overlap PCR reactions (set up in Steps 59-60) agree with those described. An equal mass of each CDR product [e.g., 1-10 ng of each CDR (CDR1, CDR2, and CDR3)] should be used. The melting temperature of the primers and the overlapping DNA segments in frameworks 2 and 3 should be within 5 C of each other and greater than the PCR annealing temperature. Expression of nanobody-Fc fusion proteins results in low yield (Step 158). The expression conditions may be altered to increase nanobody yield. Nanobody-Fc fusion plasmids should be prepared using codon frequencies for the species of cell which will be utilized for protein expression (e.g., human codon frequencies should be used for HEK293-6E cells). Nanobody-Fc fusion protein expression may also be performed using gene dosing (10% nanobody-Fc plasmid and 90% salmon sperm DNA) for clones which do not express well under standard conditions. Gene dosing may improve the expression of some nanobody-Fc fusion proteins. Binding curves for analyzing affinity of isolated nanobody-Fc fusion proteins do not saturate (Step 174). Ensure that appropriate concentrations of both nanobody-Fc fusion protein and antigen are used. A range of nanobody-Fc fusion concentrations should be examined to obtain a sigmoidal binding curve from the signals measured in Step 174. If a sigmoidal binding curve cannot be obtained by adjusting the concentrations of the evaluated nanobody-Fc fusion protein, then antigen-coated beads with either higher or lower loading of antigen may be examined. Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peter Tessier (ptessier@umich.edu). This study generated a new Aga2 display format of the common framework nanobody library reported previously (McMahon et al., 2018) . The original library was obtained from Andrew Kruse's lab and requires a Material Transfer Agreement (MTA). This study did not generate or analyze any unique datasets or codes. We thank Andrew Kruse for providing the nanobody library used in this work. We also thank members of the Tessier lab for their helpful suggestions. This work was supported by the National Institutes of Health (RF1AG059723 and R35GM136300 to P.M.T.), National Science Foundation (CBET Prepare nanobody Fc-fusion plasmids in pTT5 vector or other equivalent vectors (e.g., pBIO-CAM5) Cut bands corresponding to nanobody coding sequences. b. Purify DNA using a Qiagen Gel Extraction kit according to the manufacturer's protocol For the vector, add Quick CIP calf alkaline phosphatase to the reaction according to the manufacturer's protocol Purify the digested insert using a Qiagen PCR Purification kit according to the manufacturer's protocol Purify DNA using a Qiagen Gel Extraction kit according to the manufacturer's protocol Ligate digested insert and linearized backbone with T4 ligase according to the manufacturer's protocol Transform the ligated plasmids into competent DH5a cells Plate transformed cells on LB ampicillin plates and grow cells at 37 C for 18-20 h. 144. Pick individual colonies and grow in LB media supplemented with 100 mg/mL ampicillin overnight ($18-24 h) at 37 C Isolate the plasmids from DH5a using a Qiagen Miniprep kit according to the manufacturer's protocol Confirm plasmid sequence via Sanger sequencing Nanobody-Fc fusion protein expression Timing: 1 week Nanobody-Fc fusion proteins are expressed via transient transfection in a mammalian cell line Maintain and passage at a density of $1.8-2 million cells/mL. b. Add prepared plasmid mixture to cells at a density of $1.8-2 million cells/mL in culture tubes containing $25 mL cell culture Add 750 mL of 20% w/v yeastolate to culture 24-48 h post transfection Grow cells for another 3-5 d at 37 C with 5% CO 2 and mild agitation Incubate the supernatant and beads at 4 C overnight ($12-24 h) with mild agitation. 154. Capture beads in filter columns under vacuum and wash with $50 mL of PBS M glycine (pH 3.0) for 15 min to elute the protein solution and collect it by centrifugation Buffer exchange the proteins into 20 mM acetate (pH 5.0) using Zeba desalting columns as per manufacturer's protocol Filter nanobody Fc-fusions through a 0.22 mm filter, aliquot, and store at À80 C until use Determine the protein concentrations by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer and the purity using SDS-PAGE Wash the beads once by resuspending them in 200 mL ice-cold PBSB Centrifuge the plate containing the beads at 25003g for 5 min (4 C) and discard supernatant Resuspend the beads in 200 mL ice-cold secondary labeling reagents and incubate on ice for 4 min. a. Prepare secondary labeling solution in ice-cold PBSB b. Add 1:300 dilution (final concentration of $5 mg/mL) of goat anti-human IgG AlexaFluor 647 for detection of antigen binding Centrifuge the plate containing the beads at 25003g for 5 min (4 C) and discard supernatant. 171. Wash the beads once by resuspending them in 200 mL ice-cold PBSB Centrifuge the plate containing the beads at 25003g for 5 min (4 C) and discard supernatant Resuspend the beads in ice-cold PBSB and immediately analyze by flow cytometry. 174. Analyze the antigen-binding signal of the singlet population of beads Biointerfaces Institute (to P.M.T.), and Albert M. Mattocks Chair Rational affinity maturation of antiamyloid antibodies with high conformational and sequence specificity Transient gene expression in suspension HEK293-EBNA1 Cells Yeast surface display platform for rapid discovery of conformationally selective nanobodies Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDRswapping mutagenesis Timing: 5 h Apparent affinities of bivalent nanobodies are determined for individual nanobodies formatted as Fc-fusion proteins. A range of soluble nanobody-Fc fusions are incubated with antigen immobilized on the surface of magnetic beads, and binding signals are measured using flow cytometry. Binding curves can then be fit to the data.159. Prepare 1 3 10 5 antigen coated beads per sample to be examined. a. Transfer beads to a 1.5 mL tube and place tube on DynaMag TM -2 Magnet. b. Discard the liquid. c. Wash the beads by resuspending in 1 mL of PBSB. d. Place tube containing beads on the magnet for 2-3 min and discard the liquid. e. Repeat sub steps c and d. f. Resuspend the beads in 1 mL of 10% milk and mix for 1 h with end-over-end mixing to block the beads. g. Place tube containing beads on the magnet and discard the liquid. h. Wash the beads by resuspending in 1 mL of PBSB. i. Place tube containing beads on the magnet for 2-3 min and discard the liquid. j. Resuspend beads in PBSB (typically 0.1-1 mL of PBSB). 160. Thaw an aliquot of nanobody-Fc fusion protein. 161. Centrifuge nanobody-Fc fusion in a tabletop centrifuge at max speed for 5 min. Transfer supernatant to a fresh tube.Note: Centrifuging nanobody-Fc fusion proteins helps to remove any aggregates generated during each freeze/thaw cycle and produces more consistent results.162. Measure the A 280 signal for each nanobody using a Nanodrop to determine the protein concentration. 163. Prepare dilutions of nanobody-Fc fusion proteins in PBSB supplemented with 1% milk at concentrations (e.g., 0.1-1000 nM) desired for analysis. 164. Add 1 3 10 5 antigen-coated beads to antibody dilutions in each well of a 96-well plate. 165. Incubate soluble nanobody-Fc fusions and beads at 350 RPM and room temperature for 3 h in a final volume of 200 mL. 166. Centrifuge the plate containing the beads at 25003g for 5 min (4 C) and discard supernatant. The authors declare no competing interests.