Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability

Report

Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability Graphical Abstract

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Laila Shehata, Daniel P. Maurer, Anna Z. Wec, ..., Xiaoyong Zhi, Yingda Xu, Laura M. Walker

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Description of the biophysical properties of 400 human B cellderived antibodies

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Human B cell-derived antibodies generally show ‘‘drug-like’’ developability profiles

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Affinity maturation leads to reduced antibody polyreactivity and hydrophobicity

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Somatic hypermutation is associated with decreased conformational stability

Shehata et al., 2019, Cell Reports 28, 3300–3308 September 24, 2019 ª 2019 The Author(s). https://doi.org/10.1016/j.celrep.2019.08.056

Shehata et al. analyze the biophysical properties of human antibodies derived from multiple B cell subsets and find that somatic hypermutation is associated with increased antibody specificity but diminished conformational stability. The results provide insight into the biophysical consequences of affinity maturation and have implications for antibody discovery and engineering.

Cell Reports

Report Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability Laila Shehata,1 Daniel P. Maurer,1 Anna Z. Wec,1 Asparouh Lilov,1 Elizabeth Champney,1 Tingwan Sun,1 Kimberly Archambault,1 Irina Burnina,1 Heather Lynaugh,1 Xiaoyong Zhi,1 Yingda Xu,1 and Laura M. Walker1,2,* 1Adimab,

LLC, Lebanon, NH 03766, USA Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2019.08.056 2Lead

SUMMARY

Monoclonal antibodies (mAbs) have recently emerged as one of the most promising classes of biotherapeutics. A potential advantage of B cell-derived mAbs as therapeutic agents is that they have been subjected to natural filtering mechanisms, which may enrich for B cell receptors (BCRs) with favorable biophysical properties. Here, we evaluated 400 human mAbs for polyreactivity, hydrophobicity, and thermal stability using high-throughput screening assays. Overall, mAbs derived from memory B cells and long-lived plasma cells (LLPCs) display reduced levels of polyreactivity, hydrophobicity, and thermal stability compared with naive B cell-derived mAbs. Somatic hypermutation (SHM) is inversely associated with all three biophysical properties, as well as BCR expression levels. Finally, the developability profiles of the human B cell-derived mAbs are comparable with those observed for clinical mAbs, suggesting their high therapeutic potential. The results provide insight into the biophysical consequences of affinity maturation and have implications for therapeutic antibody engineering and development. INTRODUCTION Monoclonal antibodies represent one of the most important classes of biotherapeutics and have created new treatment options for a wide range of disease indications. As of early 2019, there are more than 70 mAbs approved for therapeutic use and hundreds more in clinical trials. Although the majority of therapeutic mAbs on the market today originated from immunized mice or phage display technology, recent advances in single B cell technologies have fueled a burst in the discovery of human mAbs with native heavy- and light-chain pairing (Walker and Burton, 2018). Most of these mAbs target infectious disease agents, but recent studies have demonstrated that mAbs targeting therapeutically relevant self-antigens can also be recovered from certain patient populations (Bushey et al., 2016; Meyer et al., 2016; Sevigny et al., 2016). Successful mAb therapeutics must display functional binding activity as well as ‘‘drug-like’’ biophysical properties, such as low

aggregation propensity, high stability and solubility, low offtarget reactivity, and low viscosity. This suite of characteristics is often collectively termed ‘‘developability.’’ A multitude of early-stage screening assays have been established to identify lead molecules with favorable developability properties, therefore minimizing downstream development risks. Among these assays, some are designed to measure the propensity of mAbs to either self-associate or associate with a column matrix (Geng et al., 2014; Haverick et al., 2014). Antibody self-interaction can lead to aggregation, poor solubility, and high viscosity (Tessier et al., 2014). Complementary assays have also been developed to measure antibody cross-interaction with either a panel or heterogeneous mixture of non-cognate antigens. For example, ELISA- and flow cytometry-based assays have been developed that measure antibody polyreactivity to defined sets of structurally diverse antigens (e.g., single-stranded DNA [ssDNA], double-stranded DNA [dsDNA], flagellin, insulin, and lipopolysaccharide), baculovirus particles, mixtures of membrane and cytosolic proteins, or individual chaperone proteins (Ho¨tzel et al., 2012; Kelly et al., 2017; Wardemann et al., 2003; Xu et al., 2013). Finally, analytical hydrophobic interaction chromatography (HIC) is routinely used for measuring antibody hydrophobicity. High hydrophobicity in this assay has been shown to be associated with mAb aggregation and precipitation, which in turn can pose significant manufacturing and/or formulation hurdles (Chennamsetty et al., 2009; Tsai and Nussinov, 1997). Human B cells have been subjected to in vivo tolerance mechanisms, and therefore human B cell-derived mAbs would be expected to have a lower potential for immunogenicity, polyreactivity, and off-target reactivity. However, there are conflicting reports in the literature regarding the auto- and polyreactivity of human B cell-derived mAbs. Multiple studies have demonstrated that B cells can acquire poly- and autoreactivity by somatic hypermutation (SHM) (Diamond and Scharff, 1984; Shlomchik et al., 1987; Tiller et al., 2007), suggesting that tolerance within the germinal center (GC) is incomplete, whereas other studies have shown that autoreactive GC B cells are eliminated or ‘‘redeemed’’ following interaction with self-antigen (Ait-Azzouzene et al., 2010; Pulendran et al., 1995; Shokat and Goodnow, 1995). Furthermore, it is unclear if and how in vivo selection processes affect biophysical properties beyond specificity—such as stability, aggregation propensity, expression titer, and hydrophobicity—and how these properties may differ among mAbs derived from different B cell subpopulations.

3300 Cell Reports 28, 3300–3308, September 24, 2019 ª 2019 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Here, we analyzed and compared the polyreactivity, hydrophobicity, and thermal stability properties of 400 human mAbs derived from naive B cells, IgM and IgG memory B cells, and long-lived plasma cell (LLPC) subsets. These three assays were selected because previous studies have shown that they measure distinct biophysical characteristics (Jain et al., 2017). The results demonstrate that memory B cells and LLPCs encode antibodies with overall lower levels of polyreactivity and hydrophobicity, but also significantly reduced stability, compared with naive B cell-derived antibodies. SHM was inversely associated with all three of these properties, as well as B cell receptor (BCR) expression levels, suggesting that the process of affinity maturation leads to increased antibody specificity but impairs conformational stability. Importantly, the vast majority of B cellderived mAbs show similar thermal stability, polyreactivity, and hydrophobicity properties compared with clinically approved mAbs, suggesting that most B cell-derived mAbs have ‘‘druglike’’ biophysical properties. Altogether, the results provide insight into the trade-offs among antibody affinity, specificity, and stability and have implications for therapeutic antibody development. RESULTS Isolation of mAbs from Multiple Human B Cell Subsets To characterize the biophysical properties of human mAbs derived from different B cell subsets, we cloned and expressed 400 mAbs from LLPCs, IgG and IgM memory B cells, and naive B cells using a previously described single B cell isolation platform (Bornholdt et al., 2016). LLPCs (CD19
CD38+CD138+) were sorted from bone marrow mononuclear cell samples from four healthy donors (HDs), and naive (IgM+CD27
CD10
) and memory B cells (IgM+CD27+ or IgG+CD27+) were sorted from peripheral blood mononuclear cell (PBMC) samples from three HDs (Figures S1A and S1B). Antibody heavy- and light-chain variable (VH and VL) regions were amplified by single-cell PCR and subsequently cloned and expressed as full-length IgG1s in an engineered strain of Saccharomyces cerevisiae. All mAbs were cloned into an IgG1 isotype to allow the comparison of biophysical properties intrinsic to the antibody variable region. Notably, the mAbs cloned from each B cell subset showed the expected levels of SHM, with the order LLPCs > IgG+ memory B cells > IgM+ memory B cells > naive B cells (Scheid et al., 2011; Tsuiji et al., 2006; Weisel et al., 2016) (Figure S1C). Evaluation of Different Polyreactivity Assay Formats Previous studies have shown that polyreactive mAbs are cleared from circulation at a substantially faster rate than monospecific mAbs (Datta-Mannan et al., 2015; Ho¨tzel et al., 2012; Sigounas et al., 1994), and therefore we sought to assess the polyreactivity properties of the B cell-derived mAbs using an assay that could predict rapid non-target-mediated antibody clearance in vivo. To evaluate the relationship between human serum half-life and polyreactivity in different assay formats, we tested a panel of 16 anti-pathogen mAbs with pharmacokinetic data available from phase I clinical trials for polyreactivity in multiple previously described assays (Table S1). We used mAbs targeting infectious disease agents to ensure that the reported serum half-lives were

not affected by target-mediated clearance. The 16 anti-pathogen mAbs were cloned into an IgG1 expression vector, produced in HEK293 cells, and tested for non-specific binding to (1) a panel of non-cognate antigens (Tiller et al., 2007; Wardemann et al., 2003); (2) baculovirus particles (BVPs) (Ho¨tzel et al., 2012); (3) a mixture of membrane and cytosolic proteins (polyspecificity reagent [PSR]) (Xu et al., 2013); and (4) the HSP90 chaperone protein (Kelly et al., 2017) (Figure S2A). The correlation between the degree of polyreactivity and human serum half-life was the strongest for the PSR assay (R2 = 0.492) and BVP assay (R2 = 0.512) (Figure S2A). Because of the lower throughput of the BVP assay, we selected the PSR assay to measure the polyreactivity properties of the B cellderived mAbs. On the basis of the correlation shown in Figure S2A, mAbs with PSR scores <0.1, between 0.1 and 0.33, and >0.33 were defined as having no, low, and high polyreactivity, respectively. The median serum half-lives for the clinicalstage anti-pathogen mAbs in each of these groups were 24, 17.5, and 12.9 days, respectively (Figure S2B). Somatically Mutated mAbs Show Reduced Levels of Polyreactivity and Hydrophobicity Compared with Germline-Encoded mAbs On the basis of the results of the PSR assay described above, 12% to 18% of mAbs from each B cell subset exhibited some degree of polyreactivity (Figure 1A; Data S1). However, the naive B cell subset contained a higher proportion of polyreactive clones compared with the other B cell subsets (Figure 1A; Data S1). Approximately 6% of naive B cell-derived mAbs showed high polyreactivity in this assay, compared with 3% of mAbs cloned from IgM+ memory B cells, 1.4% of mAbs cloned from IgG+ memory B cells, 2% of mAbs cloned from LLPCs, and 4% of clinically approved mAbs (Figure 1A; Data S1 and S2). On the basis of this result, we hypothesized that SHM may be associated with the reduced levels of polyreactivity observed for the LLPC- and memory B cell-derived mAbs. Indeed, clustering of the mAbs on the basis of SHM load revealed that 8.5% of mAbs that lacked somatic mutations showed high polyreactivity, compared with only 2% of mAbs with 6–10 amino acid substitutions, 1.5% of mAbs with 11–20 amino acid substitutions, and 1% of mAbs with >20 amino acid substitutions (Figure 1B). Comparable results were observed when the analysis was performed on individual B cell subsets and a large panel of previously described anti-viral mAbs (Bornholdt et al., 2016; Rogers et al., 2017) (Figures 1C–1E). We next assessed the hydrophobicity of the B cell-derived mAbs using HIC, which is an assay that measures antibody binding to non-biological hydrophobic surfaces. Retention of mAbs on hydrophobic columns under salt-stress conditions has been shown to reflect overall hydrophobicity, with more hydrophobic mAbs showing increased retention times (Haverick et al., 2014; Kohli et al., 2015). On the basis of previous studies of clinicalstage mAbs (Jain et al., 2017), we defined mAbs with HIC retention times <10.5 min, between 10.5 and 11.5 min, and >11.5 min as having low, medium, and high hydrophobicity, respectively. Between 80% and 90% of mAbs from each B cell subset showed low hydrophobicity in this assay, which was similar to that observed for a panel of 42 clinically approved mAbs

Cell Reports 28, 3300–3308, September 24, 2019 3301

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(A) Percentages of mAbs isolated from each B cell subset that display high and low polyreactivity, as determined by the PSR assay. Forty-two clinically approved mAbs are also shown for comparison. (B) Percentages of B cell-derived mAbs with high and low polyreactivity, clustered according to the number of VH + VL amino acid substitutions. Naive, IgG and IgM memory, and LLPC-derived mAbs are pooled for this analysis. (C) Percentages of IgG memory B cell-derived mAbs with high and low polyreactivity, clustered according to the number of VH + VL amino acid substitutions. (D) Percentages of IgM memory B cell-derived mAbs with high and low polyreactivity, clustered according to the number of VH + VL amino acid substitutions. (E) Percentages of human B cell-derived anti-viral mAbs with high and low polyreactivity, clustered according to the number of VH + VL amino acid substitutions (n = 767) (Bornholdt et al., 2016; Rogers et al., 2017). aa, amino acid. Statistical comparisons were made using Fisher’s exact test (*p < 0.05, **p < 0.01, and ***p < 0.001). The results shown in (A)–(E) are representative of two independent experiments. See also Figures S1, S2, and S3 and Table S1.

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(Figure 2A; Data S1 and S2). Interestingly, a larger fraction of naive B cell-derived mAbs displayed high hydrophobicity compared with memory B cell-derived mAbs, LLPC-derived mAbs, and clinically approved mAbs (Figure 2A; Data S1 and S2). Approximately 12.5% of mAbs derived from naive B cells showed high hydrophobicity, whereas only 2%–3% of mAbs derived from memory B cells and LLPCs showed such behavior (Figure 2A). Consistent with prior studies, we observed no correlation between hydrophobicity and polyreactivity (Jain et al., 2017) (Figure S3A). As observed for polyreactivity, somatically mutated mAbs showed significantly lower levels of hydrophobicity compared with germline-encoded mAbs (Figure 2B). Twentyfour percent of mAbs that lacked SHM displayed medium to high hydrophobicity compared with only 5%–7% of somatically mutated mAbs (Figure 2B). In summary, memory B cell- and LLPC-derived mAbs show overall lower polyreactivity and hydrophobicity compared with naive B cell-derived mAbs, and this difference is associated with the presence of SHM in the affinity matured B cell subsets. Somatically Mutated mAbs Show Overall Lower Thermal Stability Compared with Germline-Encoded mAbs We next assessed the apparent melting temperatures (TmApp), a predictor of thermodynamic stability and resistance to unfolding, of the B cell-derived mAbs, expressed as fragment antigen binding (Fab) domains, using differential scanning fluorimetry (DSF). Although the vast majority of mAbs cloned from each B cell sub-

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set had TmApp values that were comparable with those observed for clinically approved mAbs, the naive B cell-derived mAbs showed significantly higher TmApp values compared with memory B cell- and LLPC-derived mAbs (Figure 3A; Data S1 and S2). The median TmApp for the naive B cell-derived mAbs was 74 C, compared with 70 C for both the IgM memory B celland LLPC-derived mAbs and 68 C for the IgG memory B cellderived mAbs (Figure 3A). As expected, there was no correlation between thermal stability and polyreactivity or hydrophobicity (Figures S3B and S3C). Because one explanation for the above result is that SHM leads to decreased antibody stability, we analyzed the thermal stabilities of the mAbs clustered by their level of SHM. Interestingly, mAbs containing low levels of SHM (1–10 amino acid substitutions) showed significantly lower TmApp values compared with germline-encoded mAbs, but mAbs containing high SHM loads (>10 amino acid substitutions) did not show substantial further decreases in thermal stability (Figures 3B and 3C). The median TmApp for mAbs lacking SHM was 74 C, compared with 71 C for mAbs containing 1–5 amino acid substitutions, 69.5 C for mAbs containing 6–10 amino acid substitutions, 69.25 C for mAbs containing 11–20 amino acid substitutions, and 68.5 C for mAbs containing >20 amino acid substitutions. Furthermore, almost all of the B cell-derived mAbs (98%) displayed TmApp values > 60 C (Figures 3B and 3C). Thus, although SHM leads to an overall decrease in antibody thermal stability, there appears to be an in vivo BCR stability threshold that corresponds to a

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TmApp of approximately 60 C (Figures 3B and 3C). Notably, LLPCderived mAbs showed significantly higher thermostability compared with IgG+ memory B cell-derived mAbs, despite containing overall higher levels of SHM, suggesting that clones with increased BCR stability may be preferentially selected into this compartment. To further confirm that SHM leads to decreased thermal stability, we cloned and expressed the unmutated common ancestors (UCAs) of nine mAbs with varying levels of SHM and evaluated their thermal stabilities (Figure 3D; Data S3). In all cases, the UCA mAb exhibited an increase in TmApp (between 3 C and 12 C) compared with the mature antibody (Figure 3D), providing direct evidence that somatic mutations lead to reduced thermal stability. Somatically Mutated mAbs Show Overall Lower BCR Expression Levels Compared with Germline-Encoded mAbs Given the above results, coupled with previous studies showing a correlation between thermostability and expression titers in mammalian cells (Jain et al., 2017; Kowalski et al., 1998a, 1998b), we hypothesized that SHM may also be associated with decreased BCR expression levels. We therefore evaluated the SHM loads and thermal stabilities of mAbs derived from B cells with either high or low BCR density. Initial efforts using human B cells were unsuccessful because of the lack of secondary reagents that allowed clear separation of IgG1lo cells from other isotypes and IgG subclasses by flow cytometry. Because we observed significantly better separation on flow cytometry when using murine B cells and anti-mouse secondary reagents, we single-cell-sorted murine B220+IgM
IgD
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CD38+ IgG1+ CD79b+ B cells with either high or low levels of both IgG1 and the BCR component CD79b and amplified the corresponding VH and VL regions using single-cell PCR (Figure 4A; Figure S4A). Although plasmablasts, which express low levels of surface Ig, were not excluded from the sort, additional staining experiments using the plasmablast surface marker CD138 revealed that there were few to no plasmablasts in the sorted IgG1+ B cell populations (Figure S4B). Sequence analysis of mAbs isolated from either the top or bottom 5% of IgG1-expressing B cells revealed that B cells with low BCR expression levels encoded antibodies with significantly higher levels of SHM compared with B cells with high BCR expression levels (Figure 4B). The median level

(A) Percentages of mAbs isolated from each B cell subset that display high, medium, and low hydrophobicity. Polyreactivity data are also shown for 42 clinically approved mAbs for comparison. (B) Percentages of mAbs with high, medium, and low hydrophobicity, clustered according to the number of VH + VL amino acid substitutions. MAbs isolated from all B cell subsets were pooled for the analyses shown in (A) and (B). Statistical comparisons were made using Fisher’s exact test (**p < 0.01 and ***p < 0.001). The results shown in (A) and (B) are representative of two independent experiments. See also Figures S1 and S3.

of SHM in B cells with high or low BCR density was four and eight amino acid substitutions, respectively. Furthermore, the majority (65%) of B cells with high BCR expression levels contained fewer than six amino acid substitutions, whereas the majority (68%) of B cells with low BCR expression contained more than six amino acid substitutions (Figure 4C). Finally, mAbs derived from B cells with high BCR density showed overall higher TmApp values compared with mAbs derived from B cells with low BCR density, suggesting that there is an association between BCR expression levels and Fab stability (Figure 4D). Seventy-two percent of mAbs derived from the top 5% of IgG1-expressing B cells had TmApp values > 70 C compared with only 45% of mAbs derived from the bottom 5% of IgG1-expressing B cells (Figure 4E). Overall, the results suggest that SHM leads to decreased Fab thermal stability and support the existence of an in vivo BCR stability threshold. Sequence Features Associated with Favorable Biophysical Properties To evaluate if and how certain sequence features could be used to predict developability characteristics, we analyzed the biophysical properties of the B cell-derived mAbs clustered by light-chain (LC) class (Vk or Vl), VH germline gene family, theoretical isoelectric point (pI), and CDRH3 length. Overall, mAbs with Vl LCs showed similar levels of polyreactivity and hydrophobicity, but reduced thermal stability, compared with mAbs with Vk LCs (Figures 5A–5C). The median TmApp for mAbs with Vk LCs was 70 C compared with 68.5 C for mAbs with Vl LCs (Figure 5C). Furthermore, a higher proportion of mAbs with Vk LCs had TmApp values > 70 C compared with mAbs with Vl LCs (56% versus 35% for Vk and Vl, respectively) (Figure S5A). VH germline gene family had little to no effect on polyreactivity but had a significant effect on both hydrophobicity and thermal stability (Figures 5D–5F). A significantly higher proportion (18%) of mAbs belonging to the VH1 germline gene family showed medium to high hydrophobicity compared with mAbs belonging to the VH3 family (6%) (Figure 5E). Interestingly, this difference appeared to be due exclusively to the intrinsic hydrophobicity of the VH1–69 germline gene; 36% of mAbs that used VH1–69 showed medium to high hydrophobicity compared with only 12% of mAbs that used other VH1 germline genes

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pI did not have a substantial effect on hydrophobicity. Approximately 5% of mAbs with pIs > 9.3 showed medium to high hydrophobicity compared with 11% of mAbs with pIs < 9.3, but this difference did not reach statistical significance (Figure 5H). Finally, we evaluated whether the length of the heavy-chain complementarity region 3 (CDRH3) had a significant impact on polyreactivity and/or hydrophobicity. Overall, mAbs with long CDRH3s (>20 amino acids) showed significantly higher levels of polyreactivity compared with mAbs with short (7–14 amino acids) and medium-length (15–20 amino acids) CDRH3s (Figure 5I). Eleven percent of mAbs with long CDRH3s showed high polyreactivity compared with 1%–2% of mAbs with short or medium-length CDRH3s (Figure 5I). The median CDRH3 lengths of mAbs with no, low, and high levels of polyreactivity were 15, 16, and 20 amino acids, respectively (Figure S5C). MAbs with long CDRH3s also showed elevated hydrophobicity compared with mAbs with short CDRH3s. Approximately 20% of mAbs with long CDRH3s showed medium to high levels of hydrophobicity, compared with 13% of mAbs with medium-length CDRH3s and 4% of mAbs with short CDRH3s (Figure 5J). We conclude that LC class, VH germline gene use, pI, and CDRH3 length can have a substantial impact on antibody polyreactivity, hydrophobicity, and/or thermostability properties.

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(A) TmApp values of mAbs isolated from the indicated human B cell subsets. TmApp values of 42 clinically approved mAbs are shown for comparison. Black bars indicate medians. (B) TmApp values of human B cell-derived mAbs, clustered according to the number of VH + VL amino acid substitutions. MAbs isolated from all B cell subsets were pooled for this analysis. Black bars indicate medians. (C) TmApp values of human B cell-derived mAbs, plotted according to their level of SHM. MAbs isolated from all B cell subsets were pooled for this analysis. (D) TmApp values of nine affinity-matured mAbs (mature) and their corresponding unmutated common ancestors (UCAs). Mean values (bars) ± SEM of two independent experiments are shown. Statistical comparisons were made using the Mann-Whitney test (*p < 0.05, **p < 0.01, and ***p < 0.001). aa, amino acid. The results shown in (A)–(D) are representative of two or three independent experiments. See also Figures S1 and S3.

(Figure 5E). Notably, previous studies have shown that VH1–69 contains a germline-encoded hydrophobic motif (Ile53/Phe54 or Ile53/Leu54, depending on the allele) within the apex of the CDRH2 loop, which has been shown to be important for recognition of certain viral pathogens (Avnir et al., 2016; Chan et al., 2001; Gilman et al., 2016; Huang et al., 2004). We did not observe a bias toward either Phe54 or Leu54 in the subset of VH1–69 mAbs with elevated hydrophobicity (Data S4). Analysis of the TmApp data clustered by VH germline family revealed that mAbs belonging to the VH4 germline gene family showed overall higher thermostabilities relative to mAbs using other germlines (Figure 5F). The median TmApp for VH4-family mAbs was 73.5 C, compared with 70.5 C for VH2-family mAbs, 69 C for VH1- and VH3-family mAbs, and 65.5 C for VH5-family mAbs (Figure 5F). This difference did not appear to be due to a single highly stable VH4 germline gene, as all of the VH4 germlines showed median TmApp values > 70 C (Figure S5B). We next analyzed the impact of mAb pI on polyreactivity and hydrophobicity. As anticipated on the basis of previous studies (Datta-Mannan et al., 2015; Hinton et al., 2006; Igawa et al., 2010; Li et al., 2014), mAbs with high pIs (>9.3) were significantly more likely to display polyreactivity compared with mAbs with low pIs (<9.3). Thirty-three percent of mAbs with pIs > 9.3 displayed polyreactivity compared with 12.4% and 5.5% of mAbs with pIs between 9 and 9.3 and <9.3, respectively (Figure 5G). In contrast,

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DISCUSSION In this study, we used a high-throughput B cell cloning platform and multiple early-stage developability screening assays to define the biophysical properties of human mAbs derived from multiple B cell subsets. Our findings reveal that (1) human

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Figure 4. Sequencing and Thermal Stability Analysis of mAbs Isolated from B Cells with High or Low BCR Surface Density (A) Flow cytometric sorting of murine IgG1+ B cells with high or low BCR density. The plot shown is gated on B220+IgM
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CD38+ B cells. BCR expression levels were measured using a combination of IgG1 and immunoglobulin associated-beta (CD79b) surface staining intensities. The top and bottom 5% of B220+ IgM
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CD38+IgG1+CD79b+ B cells (shown within the gates) were defined as IgGhiCD79bhi and IgGloCD79blo, respectively, and these populations were single-cell-sorted for mAb cloning and sequencing. (B) Load of somatic mutations in mAbs isolated from IgGhiCD79bhi or IgGloCD79blo B cells.

B cell-derived mAbs generally show favorable biophysical properties, comparable with those observed for clinically approved mAbs, and (2) the process of antibody affinity maturation often leads to improved antibody specificity but impaired conformational stability. Previous studies investigating the polyreactivity of human B cell-derived mAbs have yielded conflicting conclusions (Dimitrov et al., 2013). For example, multiple studies have shown that germline-encoded mAbs display enhanced structural flexibility and binding promiscuity, and it has been suggested that these properties allow the naive B cell repertoire to recognize a virtually unlimited number of foreign molecules with a finite number of BCRs (Thorpe and Brooks, 2007; Willis et al., 2013). Correspondingly, affinity maturation has been shown to lead to paratope rigidification, and this reduction in conformational flexibility has been associated with a decrease in polyreactivity (Manivel et al., 2002; Schmidt et al., 2013). However, other studies have shown that a smaller fraction of naive B cells express polyreactive antibodies compared with IgG+ memory B cells, suggesting that polyreactivity is commonly introduced during GC selection (Tiller et al., 2007; Wardemann et al., 2003). Our results are consistent with the former studies concluding that affinity maturation leads to reduced antibody polyreactivity. We found that naive B cell-derived mAbs display overall higher polyreactivity and hydrophobicity compared with memory B cell- and LLPCderived mAbs. Furthermore, higher levels of SHM were associated with lower levels of polyreactivity, suggesting that increased residence time in the GC leads to enhanced antibody specificity. Potential explanations for the discrepancy between our results and those observed by Tiller et al. (2007) are differences in the thresholds used to define polyreactivity and/or the level of background binding observed in flow cytometry versus ELISA-based assays. Finally, although our findings are likely applicable to most B cell-derived mAbs, broadly neutralizing antibodies to HIV and influenza, which often contain high levels of SHM, have been shown to be frequently polyreactive (Andrews et al., 2015; Mouquet et al., 2010). Unexpectedly, we found that memory B cell- and LLPCderived mAbs showed significantly lower thermal stability relative to naive B cell-derived mAbs. Germline reversion studies and grouping of mAbs on the basis of SHM loads revealed that this difference was due to the presence of somatic mutations in the memory B cell- and LLPC-derived mAbs. Interestingly, however, the median and range of TmApp values for mAbs with both high and low levels of SHM were comparable, and almost all the mAbs in the panel displayed TmApp values > 60 C, pointing to the existence of in vivo selection mechanisms that dictate a minimum BCR stability threshold. Indeed, we found that B cells

(C) Percentage of mAbs isolated from IgGhiCD79bhi or IgGloCD79blo B cells with the indicated SHM loads. (D) TmApp values of mAbs isolated from IgGhiCD79bhi or IgGloCD79blo B cells. (E) Percentage of mAbs isolated from IgGhiCD79bhi or IgGloCD79blo B cells with the indicated TmApp values. Statistical comparisons were made using the Mann-Whitney test (**p < 0.01 and ****p < 0.0001). Red bars indicate medians. The results shown in (A)–(E) are representative of two independent experiments. See also Figure S4.

Cell Reports 28, 3300–3308, September 24, 2019 3305

G

3

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Figure 5. Sequence Characteristics Associated with Favorable Developability Properties (A) Percentages of Vk or Vl mAbs that display low and high levels of polyreactivity. (B) Percentages of Vk or Vl mAbs that show low, medium, and high hydrophobicity. (C) TmApp values of Vk and Vl mAbs. Red bars indicate median values. (D) Percentages of VH1, VH3, and VH4 antibodies that display low and high levels of polyreactivity. MAbs using VH2, VH6, and VH7 germline genes were excluded because of low sample numbers. (E) Percentages of VH1, VH3, VH4, and VH1–69 mAbs that display low, medium, and high hydrophobicity. MAbs using VH2, VH6, and VH7 germline genes were excluded because of low sample numbers. (F) TmApp values of mAbs using VH1, VH2, VH3, VH4, and VH5 germline gene families. Red bars indicate median values. (G) Percentages of mAbs with theoretical pIs <8.0, between 9.0 and 9.3, and >9.3 that display low and high levels of polyreactivity. (H) Percentages of mAbs with theoretical pIs <8.0, between 9.0 and 9.3, and >9.3 that display low, medium, and high hydrophobicity. (I) Percentages of mAbs with CDRH3 lengths of 7–14, 15–20, and >20 amino acids that display low and high levels of polyreactivity. (J) Percentages of mAbs with CDRH3 lengths of 7–14, 15–20, and >20 amino acids that display low, medium, and high hydrophobicity. Statistical comparisons shown in (E), (G), (I), and (J) were made using Fisher’s exact test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Statistical comparisons shown in (C) and (F) were made using the Mann-Whitney test (**p < 0.01, ***p < 0.001, and ****p < 0.0001). MAbs isolated from all B cell subsets were pooled for these analyses. aa, amino acid; VH1 (no 1–69), all VH1 germlines except VH1–69. The results shown in (A)–(F) are representative of at least two independent experiments. See also Figure S5.

3306 Cell Reports 28, 3300–3308, September 24, 2019

with low BCR density contained significantly higher SHM loads compared with B cells with high BCR density, suggesting that SHM is also associated with decreased BCR expression levels. Presumably, low BCR density leads to reduced B cell competitiveness in GCs because of an impaired ability to extract and present antigen to Tfh cells (Victora et al., 2010). The above results also suggest that germline reversion of bystander somatic mutations in therapeutic antibody candidates may yield improvements in stability. In summary, our experiments reveal that mAbs derived from human B cells generally show favorable developability properties, comparable with those observed for clinically approved mAbs, but that the process of affinity maturation often leads to enhanced antibody specificity at the cost of conformational stability. Future studies should investigate the underlying biological selection processes that imbue human B cell-derived antibodies with favorable biophysical properties, which may in turn inform antibody engineering strategies. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Human Subjects METHOD DETAILS B Single B cell sorting B Amplification and cloning of antibody variable genes B Expression and purification of IgGs and Fab fragments B PSR and chaperone binding assays B Polyreactivity ELISA B Thermostability analysis B Hydrophobicity analysis B BVP binding assay B Mouse immunizations QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY B Data Availability

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.08.056. ACKNOWLEDGMENTS We thank M. Va´squez and E. Krauland for helpful discussions and comments on the manuscript. We also thank C. Williams for assistance with figure preparation. All the IgGs were sequenced by Adimab’s molecular core and produced by the high-throughput expression group. Biolayer interferometry binding experiments were performed by Adimab’s protein analytics group. AUTHOR CONTRIBUTIONS L.M.W. and L.S. designed research. L.S., D.P.M., A.Z.W., A.L., E.C., T.S., K.A., I.B., H.L., X.Z., and Y.X. performed research. L.M.W., L.S., D.M., X.Z., Y.X., T.S., and A.Z.W. analyzed data. L.M.W. wrote the paper.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Mouse anti-human CD19 PECy7 (clone H1B19)

BioLegend

Cat#302218; RRID:AB_314248

Mouse anti-human CD3 PerCP-Cy5.5 (Clone UCHT1)

BioLegend

Cat#300430; RRID:AB_893299

Mouse anti-human CD8 PerCP-Cy5.5 (Clone SK1)

BioLegend

Cat#344710; RRID:AB_2044010

Mouse anti-human CD14 PerCP-Cy5.5 (Clone 61D3)

Thermo Fisher Scientific

Cat#45-0149-42; RRID:AB_1518736

Mouse anti-human CD16 PerCP-Cy5.5 (Clone 3GB)

BioLegend

Cat#302028; RRID:AB_893262

Antibodies

Mouse anti-human IgG BV605 (Clone G18-145)

BD Biosciences

Cat#563246; RRID:AB_2738092

Mouse anti-human CD27 BV421

BioLegend

Cat#356418; RRID:AB_2562599

Mouse anti-human IgM APC (clone MHM-88)

BioLegend

Cat#314510; RRID:AB_493011

Mouse anti-human CD10 BV605 (clone HI10a)

BioLegend

Cat#312222; RRID:AB_2562157

Mouse anti-human CD138 FITC (clone MI15)

BioLegend

Cat#356508; RRID:AB_2561882

Mouse anti-human CD38 APC (clone HIT2)

BioLegend

Cat#303510; RRID:AB_314362

Rat anti-mouse CD3 PerCP-Cy5.5 (clone 17A2)

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Cat#100218; RRID:AB_1595492

Rat anti-mouse TER-119 PerCP-Cy5.5 (clone TER-119)

BD Biosciences

Cat#560512; RRID:AB_10561844

Rat anti-mouse Ly-6G/Ly-6C PerCP-Cy5.5 (clone Rb6-8C5)

BD Biosciences

Cat#552093; RRID:AB_394334

Armenian Hamster anti-mouse CD11c PerCP-Cy5.5 (clone HL3)

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Cat#560584; RRID:AB_1727422

Rat anti-mouse B220 PECy7 (clone RA3-6B2)

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Cat#103222; RRID:AB_313005

Rat anti-mouse IgD APC-Cy7 (clone 11-26c.2a)

BioLegend

Cat#405715; RRID:AB_10660304

Rat anti-mouse IgM BV605 (clone RMM-1)

BioLegend

Cat#406523; RRID:AB_2563358

Rat anti-mouse GL7 BV421 (clone GL7)

BD Biosciences

Cat#562967; RRID:AB_2737922

Rat anti-mouse CD38 APC (clone 90)

eBioscience

Cat#17-0381-81; RRID:AB_469381

Rat anti-mouse IgG1 PE (clone RMG1-1)

BioLegend

Cat#406607; RRID:AB_10551439

Armenian Hamster anti-mouse CD79b FITC (clone HM79-12)

BioLegend

Cat#132805; RRID:AB_2244530

Rat anti-mouse CD138 BV510 (clone 281-2)

Biolegend

Cat#142521; RRID:AB_2562727

Goat F(ab’)2-anti-human kappa FITC

Southern Biotech

Cat#2062-02; RRID:AB_2795737

Healthy donor bone marrow mononuclear cells

AllCells

https://www.allcells.com/

Healthy human peripheral blood mononuclear cells

Dartmouth Hitchcock Medical Center

N/A

Extravadin-R-PE

Sigma Aldrich

Cat#E4011-1ML

Propidium Iodide

Sigma Aldrich

Cat#P4170-25MG

NP-40

Thermo Scientific

Cat#85124

RNaseOUT

Invitrogen

Cat#10777019

NHS-LC-Biotin

ThermoFisher

Cat#21336

Recombinant HSP90

Kelly et al., 2017

Addgene plasmid #22487

Baculovirus particles

BlueSky Biotech

Cat#25690

Invitrogen

Cat#18080044

This paper

Data S1

Taconic

N/A

Tiller et al., 2008

N/A

Biological Samples

Chemicals, Peptides, and Recombinant Proteins

Critical Commercial Assays SuperScript III Reverse Transcriptase Kit Deposited Data Human monoclonal antibody sequences Experimental Models: Organisms/Strains BALB/c mice Oligonucleotides For primers used for RT-PCR and nested PCRs

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Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by Laura Walker (laura.walker@ adimab.com). Antibodies generated in this study will be made available on request, but we may require a completed Materials Transfer Agreement if there is potential for commercial application. EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjects Blood samples (50-100 cc) were obtained from healthy donors aged 25-55 years and processed in the Immune Monitoring and Flow Cytometry Core Laboratory at the Geisel School of Medicine at Dartmouth to isolate peripheral blood-derived B cells. Isolated cells and plasma were stored frozen in aliquots. This study complies with all relevant ethical regulations for work with human participants and was approved by the Committee for the Protection of Human Subjects, Dartmouth-Hitchcock Medical Center and Dartmouth College. Human bone marrow samples were purchased from AllCells (https://www.allcells.com/). Bone marrow samples were collected from the posterior iliac crest of healthy adult donors of unknown age and gender. Aspirate (50 ml) was drawn from each hip from a maximum of 2 sites using multiple syringes. The aspirate was filtered to remove clots, bone chips, spicules, and then pooled to normalize cell concentration between draws. METHOD DETAILS Single B cell sorting For human B cell sorting experiments, PBMCs were stained using anti-human CD19 (PECy7), CD3 (PerCP-Cy5.5), CD8 (PerCPCy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), CD27 (BV421), IgM (APC), CD10 (BV605), and IgG (BV605). Bone marrow samples were stained using anti-human CD19 (PECy7), CD3 (PerCP-Cy5.5), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), CD138 (FITC), and CD38 (APC). Single cells were sorted on a BD FACS Aria II (BD Biosciences) into 96-well PCR plates (BioRAD) containing 20 ul/well of lysis buffer [5 ml of 5X first strand cDNA buffer (Invitrogen), 0.625 ml of 10% NP-40 (Thermo Scientific), 0.25 ml RNaseOUT (Invitrogen), 1.25 ml dithiothreitol (DTT) (Invitrogen), and 12.8 ml dH2O]. Plates were immediately stored at
80 C. For murine B cell sorting experiments, splenocytes from six mice previously immunized with hen egg lysozyme were pooled and enriched for B cells using a mouse B cell isolation kit (Miltenyi Biotec, 130-090-862). The single cell suspension was subsequently labeled with propidium iodide (Sigma Aldrich), anti-mouse CD3 (PerCP-Cy5.5), TER-119 (PerCP-Cy5.5), Ly-6G/Ly-6C (PerCPCy5.5), CD11c (PerCP-Cy5.5), B220 (PE-Cy7), IgD (APC-Cy7), IgM (BV605), GL7 (BV421), CD38 (APC), IgG1 (PE), CD79b (FITC), and CD138 (BV510) for 30 min on ice. Single cells were sorted on a BD FACS Aria II (BD Biosciences) into 96-well cell culture plates (Corning, 3799) containing 20 ml per well of lysis buffer [5 ml of 5X first strand cDNA buffer (Invitrogen), 0.625 ml of 10% NP-40 (Thermo Scientific, 85124), 0.25 ml RNaseOUT (Invitrogen), 1.25 ml of 0.1M DTT (Invitrogen), and 12.875 ml dH2O. Plates were immediately stored at
80 C. Amplification and cloning of antibody variable genes Human antibody variable genes (VH, VK, and VL) were amplified by reverse transcription PCR and nested PCRs using cocktails of IgG- and IgM-specific primers, as described previously (Tiller et al., 2008). Murine antibody variable genes (VH and VK) were amplified using IgG-specific primers, as described previously (Tiller et al., 2009). The primers used in the second round of PCR contained 40 base pairs of 50 and 30 homology to the digested expression vectors, allowing for cloning by homologous recombination into S. cerevisiae. The PCR products were cloned into S. cerevisiae using the lithium acetate method for chemical transformation(Gietz and Woods, 2002) using 10 ml of unpurified VH and VL PCR product and 200 ng of the digested expression vectors. Following the transformation, individual yeast colonies were picked for sequencing and characterization. Approximately 50, 150, 65, and 145 mAbs were cloned from naive, LLPCs, IgM memory B cells, and IgG memory B cells, respectively. Somatic mutations were determined based on alignment of VH and VL sequences to human or murine reference germlines using the IMGT gene database. For germline reversion studies, VH and VL sequences of each antibody were aligned to human reference germlines, and the CDR and framework regions were reverted to the nearest inferred germline precursor. gBlocks Gene Fragments (Integrated DNA

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Technologies) with 40 base pairs of 50 and 30 homology to digested expression vectors were used to clone the antibody genes into S. cerevisiae using the lithium acetate method for chemical transformation, as described above. Expression and purification of IgGs and Fab fragments B cell-derived IgGs were expressed in S. cerevisiae cultures grown in 24-well plates, as described previously(Bornholdt et al., 2016). Briefly, Fab fragments were produced by digesting the IgGs with papain for 2 hours at 30 C. The reaction was terminated using iodacetamide, and the Fab and Fc mixtures were passed over Protein A agarose to remove undigested IgG and Fc fragments. The resulting flow-through was passed over CaptureSelect IgG-CH1 affinity resin (Thermo Fisher Scientific). The Fab fragments were then eluted with 200 mM acetic acid and 50 mM NaCl (pH 3.5) into 1/8th volume of 2 M HEPES (pH 8.0), and buffer-exchanged into phosphate-buffered saline (PBS; pH 7.0). The 16 anti-infectious disease IgGs were expressed by transient co-transfection of heavy and light chain plasmids into HEK293 cells. One day prior to transfection, HEK293 cells were passaged at 2.0 - 2.5 X 106 cells/ml. On the day of transfection, cells were pelleted by centrifuging at 400 g for 5 min, and cell pellets were resuspended in fresh FreeStyle F17 medium at a density of 4 X 106 cells/ml and returned to the incubator. A transfection mixture was prepared by first diluting the plasmid DNA preparations in FreeStyle F17 medium (1.33 mg total plasmid DNA per ml of culture). Transfection agent, PEIpro (Polyplus Transfection, Illkirch, France), was then added to the diluted DNA at a DNA-to-PEI ratio of 1:2, and the mixture was incubated at room temperature for 10 min. The transfection mixture was then added to the culture. Cultures were harvested six days post transfection by two rounds of centrifugation, each at 2000xg for 5 min, and the clarified conditioned medium subject to antibody purification. Cell supernatants were purified by passing over Protein A agarose (MabSelect SuRe from GE Healthcare Life Sciences). The bound antibodies were washed with PBS, eluted with 200 mM acetic acid / 50 mM NaCl pH 3.5 into 1/8th volume 2M HEPES (pH 8.0), and buffer-exchanged into PBS (pH 7.0). Fab fragments were generated by digesting the IgGs with papain for 2 hours at 30 C. The digestion was terminated by the addition of iodoacetamide, and the Fab and Fc mixtures were passed over Protein A agarose to remove Fc fragments and undigested IgG. The flow-through of the Protein A resin was then passed over CaptureSelect IgG-CH1 affinity resin (Thermo Fisher Scientific) and eluted with 200 mM acetic acid and 50 mM NaCl pH 3.5 into 1/8th volume 2M HEPES pH 8.0. Fab fragments then were buffer-exchanged into PBS pH 7.0. PSR and chaperone binding assays Polyspecificity reagent- and chaperone-binding were assessed as described previously(Kelly et al., 2017; Xu et al., 2013). Briefly, soluble membrane protein (SMP) and soluble cytosolic protein (SCP) fractions were prepared from Chinese hamster ovary cells and biotinylated with NHS-LC-Biotin reagent (Thermo Fisher Scientific). Recombinant HSP90 was biotinylated using NHS-LC-Biotin reagent. Two million IgG-presenting yeast were transferred to a 96-well assay plate and pelleted to remove the supernatant. The yeast pellets were resuspended in either 50 ml of 1 mM biotinylated HSP90 or a 1:10 diluted stock of biotinylated SCPs and SMPs and incubated on ice for 20 minutes. Cells were washed twice with ice-cold PBSF, and the samples were incubated in 50 ml of secondary labeling mix (Extravadin-R-PE, goat F(ab’) 2-anti human LC-FITC, and propidium iodide) on ice for 20 minutes. The samples were analyzed for both IgG expression (LC-FITC) and chaperone- or polyspecificity reagent (PSR)-binding using a FACS Canto II (BD Biosciences) with an HTS sample injector. Mean fluorescence intensity (MFI) values of each were recorded and the PSR binding value normalized as follows. To normalize the PSR assay MFI values between 0 and 1, every run included three control mAbs with low, medium and high MFI values: mAb1(low), mAb2(medium), and mAb3(high). The identities and sequences of the control mAbs have been described previously(Kelly et al., 2017). The normalization scheme consists of two data transformations. First, Equation 1 revises the MFI of a mAb (indexed as x) to the observed light chain fluorescence intensity (LC_FITC) of mAb2, to obtain a standardized value, MFIx . MFIx = MFIx

LC FITCmab2 LC FITCx

1

Next, the standardized MFI values for the mAbs are converted to normalized scores by piecewise linear interpolation using Equations 2a-c. By definition, score values of 0.1, 0.33, and 0.66 on the normalization correspond to 1:5 MFImab1 , MFImab2 , and MFImab3 , respectively. A sigmoid-like hyperbolic tangent asymptoting to 1 (Equation 2d) is used for MFIx values exceeding MFImab3 .   MFIx
MFImab1 Scorex = max 0; 0:2 MFIx %1:5 MFImab1 2a MFImab1  Scorex = 0:1 + 0:23

MFIx
1:5 MFImab1 MFImab2
1:5 MFImab1



  MFIx
MFImab2 Scorex = 0:33 1 + MFImab3
MFImab2

1:5 MFImab1 < MFIx %MFImab2

MFImab2 < MFIx %MFImab3

2b

2c

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   MFIx
MFImab3 Scorex = 0:33 2 + tanh MFImab3

MFImab3 < MFIx

2d

Polyreactivity ELISA Antibody polyreactivity was determined by ELISA as described previously(Wardemann et al., 2003). Briefly, high-protein binding ELISA plates (Costar) were incubated overnight with 50 ml/well of antigen in carbonate binding buffer. Calf thymus dsDNA was used at 10 mg/ml, human insulin at 5 mg/ml, and LPS from E. coli at 10 mg/ml. Plates were blocked with 200 ml/well of blocking buffer (0.5 M EDTA pH 8.0, 10X PBS, Tween-20) for 1 hour at 37 C. IgGs were diluted 4-fold in PBS, starting at 1 mg/mL, and then added to the plates (50 ml/well) and incubated for 1 hour at 37 C. Plates were incubated for 1 hour at 37 C with 75 ml/well of HRP-conjugated goat-anti human IgG, diluted 1:2000 in blocking buffer. Plates were developed with AquaBlue ELISA substrate, and the area under the curve (AUC) was calculated using GraphPad Prism. Thermostability analysis Apparent melting temperatures were determined using a CFX96 Real-Time System (BioRad), based on a previously described protocol(He et al., 2011). Briefly, 20 mL of a 1 mg/ml sample was mixed with 10 mL of 20 3 SYPRO orange. The plate was scanned from 40 C to 95 C at a rate of 0.5 C/2 min. The Fab TmApp was assigned using the first derivative of the raw data obtained from the BioRad analysis software. Hydrophobicity analysis The methodology for this assay was described previously(Estep et al., 2015). In brief, 5 mg IgG (1 mg/mL) were spiked into a mobile phase A solution (1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5) to achieve a final ammonium sulfate concentration of 1 M before analysis. A Sepax Proteomix HIC butyl-NP5 column was used with a liner gradient of mobile phase A and mobile phase B solution (0.1 M sodium phosphate, pH 6.5) over 20 min at a flow rate of 1 ml/min with UV absorbance monitoring at 280 nm. BVP binding assay Antibody binding to baculovirus particles was measured as described previously(Ho¨tzel et al., 2012; Jain et al., 2017). Briefly, ELISA plates (Corning) were incubated at 4 C overnight with 50 ml/well BVP stock (BlueSky Biotech) diluted with 50 ml/well of 50 mM sodium carbonate (pH 9.6). Unbound BVPs were aspirated from the wells, and the plates were blocked with 100 ml/well of blocking buffer (PBS with 0.5% BSA) for 1 hour at room temperature. Plates were washed 3 times with PBS and then mAbs (pre-diluted to 1 mM in blocking buffer) were added. After 1 hour, plates were washed 6 times with PBS. HRP-conjugated anti-human IgG was added (50 ml/well) and incubated for 1 hour, followed by 6 washes with PBS. Plates were developed with 50 ml/well of TMB substrate for 10-15 minutes. Reactions were terminated with the addition of 50 ml/well of 2 M sulfuric acid. The BVP score was calculated by normalizing the absorbance at 450 nM to control wells containing no test antibody. Mouse immunizations Six BALB/c mice (Taconic Biosciences) were immunized via intraperitoneal injection with 10 mg recombinant hen egg lysozyme in Sigma Adjuvant System, followed by two additional immunizations, 4 and 5 weeks apart. Splenocytes were harvested and pooled 4 days after the last immunization. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical details of experiments can be found in the figure legends, figures, and results. Statistical comparisons in Figures 1 and 2 were performed using Fisher’s exact test. Statistical comparisons in Figures 3 and 4 were performed using the Mann-Whitney test. Statistical comparisons in Figure 5 were performed using Fisher’s exact test and the Mann-Whitney test. All analyses were carried out in GraphPad Prism. DATA AND CODE AVAILABILITY Data Availability The published article includes all datasets generated during this study. Antibody sequences are available in Data S1.

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