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Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries

ABSTRACT Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins β1 and β6 or αV and β1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.


Introduction
Therapeutic antibodies are entering clinical development and gaining approval in record numbers. 1 Bispecific antibodies, designed to engage two distinct targets or target epitopes, potentially represent the next generation of antibody therapeutics. There are over 90 bispecific antibodies in development, 2 and 2 currently approved: emicizumab-kxwh (Hemlibra®), co-targeting factor IX and factor X for hemophilia A; 3 and blinatumomab (Blincyto®), a bispecific T cell engager (BiTE) co-targeting CD3 and CD19 for acute lymphoblastic leukemia. 4,5 These are "obligate bispecific antibodies" where function is dependent upon the physical linkage of two distinct binding specificities, facilitating access to biological functions that cannot be achieved with either parental antibody alone or a mixture of the antibodies. The majority of obligate bispecific antibodies in development serve to redirect effector T cells or natural killer (NK) cells to tumorassociated antigens in a range of cancers, 6 but alternative potential mechanisms and obligate bispecific concepts in addition to bridging cells (in trans) are also being explored, such as receptor activation (in cis), receptor inhibition (in cis), cofactor mimetics and piggybacking. 7 Bispecific target selection to date has been largely hypothesis-driven; either by pre-selection of a defined antigen pair or a small number of functionally related targets, based on their known biological function. Although extensive functional screening of panels of bispecific antibodies has been reported to be necessary for optimal antibody selection for a given target pair, 8,9 a systematic, large-scale screening approach has not yet been fully exploited for the co-discovery of the optimal target antigen pairs for modulation of a given cellular response. The functional activity of bispecific antibodies, particularly those targeted to cell surface receptors, is difficult to predict from that of the individual parental antibodies. We propose high-throughput functional screening using diverse, combinatorial libraries of bispecific antibodies to find the optimum target pair and subsequently antibody pair, for modulation of any given disease-relevant cellular response. We have developed a bispecific screening format that enables simple generation and functional evaluation of hundreds of combinations of target specificities in high-throughput, disease-relevant human cell assays. Since antibodies that modulate receptor signaling are susceptible to geometric constraints, [10][11][12] this screening format, which we term Fab-K D -Fab, is monovalent and contains highly flexible linkages. The Fab-K D -Fab molecule is formed by in vitro mixing of two halves comprising Fab-fusion proteins, Fab-X and Fab-Y, where X and Y form a non-covalent heterodimeric association of sufficient affinity and stability to enable highthroughput screening in both short-and long-term complex human cell-based assays. Importantly, the Fab-K D -Fab is Fcfree to enable direct assessment of the effects of bispecific targeting, without interference from Fc-driven activity, particularly in assays using heterogenous immune cell populations.
We have evaluated our bispecific screening technology across various mechanism-based disease-relevant phenotypic in vitro models, and describe here three distinct applications using very different screening approaches utilizing human primary cells. Firstly, we describe the identification of a potent, obligate bispecific inhibitor of B cell receptor (BCR) function to target aberrant B cell activity characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). 13 This molecule was discovered in a high-throughput, short-term, human peripheral blood mononuclear cells (PBMC)-based flow cytometry signaling assay designed to mimic the B cell hyper-reactivity observed in SLE patients. By screening 1992 Fab-K D -Fab bispecific antibodies encompassing 23 targets and 300 different target combinations, we identified a bispecific antibody co-targeting CD79a/b and CD22, which was a potent inhibitor of a range of B cell functions. This functional activity was entirely dependent upon the molecule being a bispecific, as activity was not replicated by the single parental antibodies or a mixture of these antibodies.
Secondly, we describe the identification of potent bispecific inhibitors of extracellular matrix (ECM) accumulation, directed against target antigens in a coculture of human primary lung epithelial cells and lung fibroblasts. Excessive accumulation of ECM is a hallmark feature of fibrotic disease, which is recapitulated in the coculture screen mimicking the characteristics of fibrotic tissue remodeling and ECM accumulation in a 7 d, high-throughput, high-content imaging assay. 14,15 By screening 1671 Fab-K D -Fab bispecific antibodies encompassing 23 targets and 239 different target combinations, we identified a bispecific antibody co-targeting the β1 and β6 integrins, which showed obligate bispecific-dependent inhibition of fibronectin accumulation and additive bispecific-dependent inhibition of collagen I and III accumulation. In addition, we identified a bispecific antibody co-targeting the αV and β1 integrins, which showed considerably more potent inhibition of ECM accumulation compared to the αV integrin bivalent parental antibody.
In both of these screens, the primary hits were further confirmed and validated in secondary assays using a molecularly linked BYbe TM format 16 and a bispecific IgG format in the case of CD79a/b-CD22. The discovery of these new bispecific target pairs for different disease-relevant phenotypes demonstrates the utility of the Fab-K D -Fab screening format in assays modeling complex biology in human primary cells. Moreover, this approach shows the value of larger, less hypothesis-driven bispecific target discovery to exploit the unique therapeutic opportunities that bispecific antibody therapies can potentially deliver.
In this respect, our third screen demonstrates how larger screens of PBMC functional modulation can be executed to address broader mechanistic questions with data simultaneously captured for both the activation and inhibition of multiple end points from diverse cellular subsets under different stimulation conditions. The screen we describe focused on the activation of different T cell subsets and soluble mediators within a PBMC population, but this screen can easily be adapted by changing the stimuli, target cell subset(s), activation markers and soluble mediators measured to explore different mechanisms of interest. We assessed the effect of bispecific antibodies under three different stimulation conditions on four activation markers, covering seven unique cell subsets in addition to quantification of three soluble mediators. Although our immediate interest was in bispecifics that activated T cells, the screen was purposely designed to further capture bispecific T cell inhibitors for future potential interest with respect to autoimmune or inflammatory disease. We screened 6417 Fab-K D -Fab bispecific antibodies encompassing 49 targets and 969 different target combinations using PBMC from 2 independent donors. Here we illustrate a "snapshot" of the diversity of bispecific antibodies identified that either enhanced or inhibited T cell activity.

Generation of the Fab-K D -Fab bispecific antibody screening format
To facilitate the generation of large bispecific antibody libraries, two Fab-fusion proteins, Fab-X (Fab-scFv) and Fab-Y (Fab-peptide), were designed based on the reported strong-affinity antibodies to a peptide derived from the yeast transcription factor GCN4. 17 This antibody (52SR4) and target (GCN4 peptide) combination was selected based on three key criteria relating to the target: (1) it is not expressed in human cells, (2) the epitope is linear, and (3) it has a very slow dissociation rate from the antibody. The Fab is fused to either 52SR4 scFv (X) or GCN4 peptide (Y) (Figure 1a). Fab-X and Fab-Y proteins with specificities toward a range of target antigens were expressed transiently as C-terminally His-tagged proteins. The expression level of the Fab-X in the cell culture supernatant typically ranged from 50 to 250 µg/ mL, whereas the expression level of the Fab-Y typically ranged from 100 to 400 µg/mL. Proteins were immobilized metal affinity chromatography (IMAC) purified, and the purified proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion ultra-performance liquid chromatography (SE-UPLC); representative data are shown in Figure S1 and Figure 1. The reducing SDS-PAGE gel ( Figure S1) shows banding patterns that indicated the constructs were being expressed correctly, with bands at approximately 50 kDa and 25 kDa (Fab-X) or a doublet at approximately 25 kDa (Fab-Y). Fab-X and Fab-Y proteins were highly monomeric following a one-step affinity purification, typically displaying monomer levels of approximately 90% and over 95%, respectively (Figure 1b).
Exploiting the reported strong-affinity interaction between 52SR4 scFv (X) and the GCN4 peptide (Y), 17 the bispecific screening format was made by mixing the Fab-X and Fab-Y in vitro in a 1:1 molar ratio to allow complex formation ( Figure 1a). The resulting Fab-K D -Fab complexes were highly pure after mixing ( Figure 1b); therefore, no further purification or processing steps were required. SE-UPLC analysis showed that the Fab-X and Fab-Y complexed within minutes of mixing, and were stable at 37°C, with Fab-K D -Fab integrity remaining unchanged over 7 d (Figure 1c). The Fab-K D -Fab bispecific antibody format is therefore highly pure and stable, and thus amenable to highthroughput screening in both short-and long-term functional assays.

Bispecific screen for inhibitors of BCR signaling
Cross-linking the BCR on mature B cells leads to rapid induction of downstream signaling events. The rapid coordinated action of Src family kinases on CD79a and CD79b intracellular domains leads to their phosphorylation and activates Syk, which in turn activates multiple downstream partners such as PLCγ2 and BTK. 18 BCR signals then integrate with signals from associated regulatory molecules (e.g., CD19 and CD22) leading to modulation of Akt, calcium flux and NF-κB, 19 which Fab-X and Fab-Y, where X is a scFv with strong binding affinity for peptide Y. (b) Representative SE-UPLC data of a Fab-X, Fab-Y and Fab-K D -Fab following mixing of the Fab-X and Fab-Y in a 1:1 molar ratio in PBS, pH7.4. Peak heights are normalized to 100%. (c) SE-UPLC analysis of the Fab-K D -Fab complex following incubation at 37°C in PBS, pH7.4 for 5 min, 1 h, 24 h, 48 h and 7 d. Peak heights are normalized to 100%. Gel filtration protein standards were loaded for molecular weight estimation. (d) Generation of bispecific-monovalent Fab-K D -Fabs (Fab1-X + Fab2-Y; or Fab2-X + Fab1-Y); (e) Fab mixture controls (Fab1-X + Fab2-X; or Fab1-Y + Fab2-Y); (f) Generation of monospecific-monovalent Fab-K D -Fab controls (Negative Fab-X + Fab1-Y; or Fab2-X + negative Fab-Y); (g) Generation of monospecific-bivalent Fab-K D -Fab controls (Fab1-X + Fab1-Y; or Fab2-X + Fab2-Y).
activate or inhibit B cell activity in a context-dependent fashion. These responses are subsequently additionally fine-tuned by members of the MAP kinase family such as p38 and Erk1 and 2. 20 In autoimmunity, many of these signaling pathways are altered and can contribute directly and indirectly to the disease state. 21 Aberrant BCR signaling has been implicated in the selection and survival of B cells, B cell tolerance, germinal center reactions and the generation of short-lived plasmablasts, all of which can contribute to autoimmunity. 21 To assess the contribution of BCR signaling in the context of an autoimmune disease, we measured signaling readouts downstream of the BCR in SLE patients and compared these with healthy individuals. In order, to encompass the whole of the BCR signaling pathway we chose to measure the key signaling nodes Syk, Akt, NF-κB and Erk1/2. When compared to healthy volunteers, SLE patient samples showed a greater activation of all parts of the BCR pathway measured, seen as increased protein phosphorylation by flow cytometry (Figure 2). Using this information, we sought to develop a high-throughput human B cell signaling assay that mimicked the elevated signaling in SLE patients. To do this, we activated healthy naïve B cells within PBMC using an anti-IgM polyclonal antibody, which activated the same signaling proteins measured in SLE patients. We then screened bispecific antibodies to B cell surface antigens to identify novel bispecific target combinations that inhibited BCR activation. Key readouts of the upstream BCR pathway (phosphorylated Syk, PLCγ2 and Akt) were chosen for primary screening as they gave robust assay signals at the same activation time-point. Additional readouts such as phosphorylated NF-κB and p38 were used for follow-on studies to give further pathway coverage.
To generate Fab-X and Fab-Y libraries, target antigens were selected based on their expression on B cells or other immune cells that are likely to contribute to B cell function. Antibodies were made to a total of 23 different antigens and between 1 and 8 unique V-regions to each antigen were selected for screening from different V-region sequence families, based purely on their ability to bind PBMC as a Fab-Y. We deliberately did not pre-select antibodies of defined affinity, activity, or epitope to enable unbiased bispecific discovery, driven entirely by the functional cellular activity of the V-region combination. Fab-X and Fab-Y antibodies covering these 23 unique B cell antigens were purified and mixed to generate 1992 Fab-K D -Fab molecules for screening in the B cell signaling assay. In total, bispecific antibodies encompassing 300 antigen combinations were screened. A Ward's clustered 22 heat map of the average percentage inhibition of intracellular phosphorylated Akt, PLCγ2 and Syk (pAkt, pPLCγ2 and pSyk) in gated B cells was generated ( Figure 3a). Each line in the clustered heat map represents a different Fab-K D -Fab molecule. The cluster analysis was pruned to 30 clusters. In the most inhibitory cluster depicted at the bottom of the dendrogram and detailed in cluster 30 ( Figure 3b), over 80% of the bispecific antibodies had specificity for CD79a/b (herein referred to as CD79) and CD22. A total of 24 different CD79-CD22 bispecifics were tested, comprising 3 different V-regions to CD79 with 8 different V-regions to CD22, which all showed greater than 50% inhibition of at least two different readouts. Although our interest in the context of SLE was to identify inhibitors of B cell signaling, the screen was designed in such a way to also capture agonists of B cell signaling to cover additional future interest in oncology or infectious disease. These agonists are depicted at the top of the heat map in Figure 3a and detailed in the first few clusters in Figure 3b, with the strongest agonists being in clusters 1-3.

CD79-CD22 bispecific validation in the BYbe TM format
To demonstrate that the target pair biology identified in the Fab-K D -Fab screening format is reproduced in a therapeutically relevant bispecific format, it was important to test the activity of the CD79-CD22 bispecific in a molecularly linked construct that has no potential to dissociate. To test this, we used the BYbe TM format, 16 which comprises a Fab (with specificity toward antigen 1) fused to a disulfide-stabilized scFv (with specificity toward antigen 2) via a G4S linker ( Figure  S2a). Bispecific-monovalent BYbe TM proteins were tested alongside monospecific-monovalent BYbe TM controls (comprising negative binding scFv), monospecific-bivalent BYbe TM controls and a negative BYbe TM control (comprising negative binding Fab and scFv) in the B cell signaling assay. BYbe TM schematics and SE-UPLC data for purified BYbe TM proteins are shown in Figure S2a. Similar concentration-response curves and IC 50 values were obtained with the bispecific Fab-K D -Fab and BYbe TM molecules, with the bispecific BYbe TM proteins consistently displaying a modest increase in potency compared to the bispecific Fab-K D -Fab proteins ( Figure 5, Table S1). In both bispecific BYbe TM orientations (CD79 Fab-CD22 scFv and CD22 Fab-CD79 scFv), robust inhibition of pAkt (82-86%), pp38 (88-91%), pPLCγ2 (58--74%) and pNF-κB (92-94%) signaling was observed, which was equal to or moderately increased compared to the Fab-K D -Fab. This increased inhibition is likely a result of physically linking the two antigen-binding specificities in the BYbe TM molecule. All other BYbe TM proteins showed minimal inhibition of these activation readouts, with the exception of the CD79-CD79 bivalent BYbe TM molecule. Analogous to the Fab-K D -Fab format, the CD79-CD79 bivalent BYbe TM showed inhibition of pp38 and pNF-κB, but with reduced efficacy (50-54%) compared to the CD79-CD22 bispecific molecules (88-94%) ( Figure 5, Table S1). The CD79-CD79 + CD22-CD22 bivalent BYbe TM mixture showed inhibition of activation readouts that were comparable to the CD79-CD79 bivalent molecule; thus, the activity observed with this mixture is likely attributed to the CD79-CD79 bivalent molecule. To further understand the differences in inhibition of B cell signaling by the CD79-CD22 bispecific and CD79-CD79 bivalent molecules, we tested their effects on B cell signaling in the absence of anti-IgM stimulation. Of all the BYbe TM proteins tested, only the CD79-CD79 bivalent BYbe TM protein and not the CD79-CD22 bispecific BYbe TM protein resulted in a concentration-dependent increase in pAkt ( Figure S3), with no concomitant activation of PLCγ2, p38 or NF-κB (unpublished data). We hypothesize that the lack of bivalency for CD79 coupled to the specificity for Percentage inhibition of pAkt, pPLCγ2 and pSyk relative to anti-IgM and untreated controls are depicted. Red denotes inhibition of BCR activity (max 100% inhibition), blue denotes enhancement of BCR activity (max minus 180% inhibition) and gray denotes baseline (no effect on BCR activity). Each horizontal line represents a different Fab-K D -Fab molecule. The cluster analysis was pruned to 30 clusters depicted in alternating blue and green on the dendrogram and the CD79-CD22 target pair selected from the most inhibitory bottom cluster (cluster 30). (b) Average percentage inhibition of pAkt, pPLCγ2 and pSyk for all Fab-K D -Fab molecules in each of the 30 clusters defined in the dendrogram. Scale on Y-axis is defined for each cluster range covering 100% inhibition to minus 180% inhibition (activation). The cluster from which the CD79-CD22 target pair was selected (where greater than 50% inhibition was observed for all 3 parameters) is highlighted. Each horizontal line represents a different Fab-K D -Fab molecule, and each color a different target combination.
CD22 in the bispecific BYbe TM molecule overrides the capacity for CD79 targeting to induce B cell activation. 23

Inhibition of B cell responses with a CD22-CD79 BYbe TM and CD22-CD79 bispecific IgG
Following activation through the BCR, and with subsequent costimulation, B cells proliferate and differentiate, producing cytokines and antibodies that contribute to an ongoing immune response. After validation of CD79-CD22 bispecific BYbe TM activity in the B cell signaling assay, we therefore evaluated the outcome of targeting these two antigens on additional B cell functional responses. Since the CD79-CD22 and CD22-CD79 BYbe TM proteins previously gave comparable results, we selected one orientation, the CD22-CD79 BYbe TM , and assessed inhibition of B cell proliferation, cell surface marker activation and cytokine production. Control CD79-CD79 and CD22-CD22 bivalent BYbe TM proteins, or a mixture of the two, were also tested, as well as a negative control BYbe TM . All molecules were tested at a final concentration of 100 nM, as this concentration gave maximum inhibition in all readouts of the B cell signaling assay ( Figure 5). To mimic a T cell-dependent antigen response, B cells were costimulated with anti-IgM and anti-CD40 antibodies. Proliferation was measured after 5 d and the percentage inhibition of a given response was calculated for each molecule. B cell proliferation was inhibited by approximately 90% by the CD22-CD79 BYbe TM (Figure 6a) whilst little or no inhibition was observed with the negative or bivalent control BYbe TM molecules (<20%). The bivalent mixture of CD79-CD79 + CD22-CD22 BYbe TM proteins also failed to inhibit B cell proliferation, confirming that the CD22-CD79 molecule is an obligate bispecific, as the inhibitory function cannot be replicated by antibodies targeting either of the antigens alone or mixtures of these antibodies.
CD86 is upregulated on activated B cells and plays a crucial role in B-T cell cross-talk through interactions with CD28 and CTLA-4. 24 We therefore examined CD86 expression by flow cytometry. In parallel to inhibition of proliferation, CD86 expression was inhibited when cells were treated with the CD22-CD79 BYbe TM protein (>70%), but not with any of the bivalent BYbe TM proteins or the bivalent BYbe TM mixture ( Figure 6b).
Inflammatory cytokines such as Lymphotoxin-α can be released by activated B cells. We therefore analyzed if the CD22-CD79 BYbe TM could also inhibit the release of such cytokines in comparison to bivalent BYbe TM proteins or the bivalent BYbe TM mixture. Purified B cells were stimulated with anti-IgM/G (to activate naïve and memory B cells) and anti-CD40 antibodies, and Lymphotoxin-α levels were measured after 3 d. We observed considerable inhibition of Lymphotoxin-α (>95%) with only the CD22-CD79 BYbe TM and none of the control molecules (Figure 6c). In addition, we confirmed that conversion of the CD22-CD79 Fab-K D -Fab to a bispecific IgG antibody format still retained inhibition of cytokine release (>80%) compared to a negative control IgG (~10%) (Figure 6d). SE-UPLC data for purified IgG proteins are shown in Figure S2b. Bispecific BYbe TM proteins were tested alongside monospecific-monovalent BYbe TM proteins, monospecific-bivalent BYbe TM proteins and bivalent BYbe TM mixtures. For each molecule or mixture, 8-point concentration-response assays were performed in triplicate and pAkt, pp38, pPLCγ2 and pNF-κB levels were measured in B cells from the PBMC populations treated with anti-IgM. Percentage inhibition values relative to maximum and minimum control well data were calculated. Mean ± standard deviation of 4 donors are plotted.

Bispecific screen for inhibitors of ECM accumulation
In our second example, we sought to identify novel target combinations with potential anti-fibrotic activity. Fibrotic disease is thought to initiate with a chronic organ insult resulting in an inflammatory infiltrate of macrophages that release cytokines such as transforming growth factor-β; this induces the resident fibroblast population to proliferate and differentiate into myofibroblasts, which release an excess of ECM into the surrounding tissue. 25 This ECM protein accumulation is a hallmark of fibrosis and has been used to measure fibrotic disease in many different organ systems. We therefore developed an assay that mimicked this tissue remodeling, by assessment of ECM accumulation in the absence or presence of Fab-K D -Fab proteins. The previously described high-content, high-throughput in vitro ECM kidney coculture assay 14,26 was modified to model ECM accumulation by lung-derived cells.
ECM accumulation was measured in a 7 d coculture system of human primary lung fibroblasts and epithelial cells, whereby coculturing the two cell types resulted in spontaneous ECM accumulation. Three specific ECM components, fibronectin, collagen I and collagen III, were quantified.
To generate Fab-X and Fab-Y libraries, we selected 23 target antigens based on their known involvement in fibrotic mechanisms and expression level in the cells used in the coculture assay. Antibodies were made to a total of 23 different antigens, and between 1 and 5 unique V-regions were selected for screening based purely on their ability to bind the target antigen as a Fab-Y and originating from different V-region sequence families. Fab-X and Fab-Y antibodies covering these 23 unique antigens were purified and mixed to generate 1671 Fab-K D -Fab molecules for screening. In total, bispecific antibodies encompassing 239 antigen combinations were screened. Two independent screens were performed with Fab-K D -Fab Inhibition of CD86 expression on B cells from CellTrace TM violet-labeled PBMC stimulated with anti-IgM and anti-CD40 antibodies ± test molecules (100 nM). In (a) and (b), inhibition was calculated relative to maximum (anti-IgM stimulation only) and minimum (no stimulation) controls. For each test condition, each dot represents the mean of triplicate wells from a single donor and a total of 3 donors are plotted. (c) BYbe TM protein activity in a cytokine release assay. (d) Bispecific IgG activity in a cytokine release assay. In (c) and (d), Lymphotoxin-α was measured following treatment of purified B cells stimulated with anti-IgM/G and anti-CD40 antibodies ± test molecules (100 nM). Percentage inhibition values relative to maximum and minimum controls were calculated. For each test condition each dot represents the mean of triplicate wells from a single donor and a total of either 4 (c) or 6 (d) donors are plotted. The solid horizontal lines represent the mean ± standard deviation.  molecules tested at a final concentration of 10 nM. A heat map of the screen data was created using unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering, 27 which resulted in three discrete clusters of antigen combinations (Figure 7a). Primary screen hits were identified as those that induced more than or equal to 50% inhibition of fibronectin and collagen I and III in both screens; these were located in cluster 3 of the dendrogram (Figure 7b). This resulted in 28 hits, from which two lead antigen pairs that induced maximal inhibition of ECM accumulation were selected: β1-β6 (bispecific co-targeting integrins β1 and β6) and αV-β1 (bispecific co-targeting integrins αV and β1). From a total of 14 integrin β1-β6 bispecifics tested, comprising 4 different V-regions to integrin β1 and 4 different V-regions to integrin β6, only 3 resulted in more than 50% inhibition of both fibronectin and collagens I and III. A total of 23 integrin αV-β1 bispecifics were tested, comprising 6 different V-regions to integrin αV and 5 different V-regions to integrin β1, of which 12 resulted in greater than 50% inhibition of both fibronectin and collagens I and III.

β1-β6 and αV-β1 bispecific validation in the Fab-K D -Fab format
To validate the primary hits identified in the ECM lung coculture assay, the integrin β1-β6 and αV-β1 Fab-K D -Fab screen hits were assayed at a single concentration of 10 nM alongside monospecific-bivalent Fab-K D -Fab controls, monospecificmonovalent Fab-K D -Fab controls and Fab-Y mixtures. Accumulation of fibronectin and collagen I and III was quantified; representative images are shown in Figure 8a.
Integrin β1-β6 bispecific Fab-K D -Fab molecules in both orientations, β1-β6 (β1 Fab-X-β6 Fab-Y) and β6-β1 (β6 Fab-X-β1 Fab-Y), induced considerable inhibition of fibronectin (96-97%) and collagen I and III (~85-88%) accumulation. The monospecific-bivalent Fab-K D -Fab controls and monospecificmonovalent Fab-K D -Fab and Fab-Y mixture controls showed limited inhibition of fibronectin deposition (<38%) in comparison (Figure 8b), demonstrating the obligate bispecific effect of the integrin β1-β6 pair with respect to inhibition of fibronectin accumulation. The effects of the control molecules were less clear-cut with respect to collagen I and III accumulation. The integrin β6-β6 bivalent Fab-K D -Fab control and β6 monospecific-monovalent Fab-K D -Fab control showed limited inhibition of collagen I and III accumulation (<30%). However, the equivalent controls containing only integrin β1 molecules showed up to 62% inhibition, with the β1 + β6 Fab-Y mixture showing approximately 73% inhibition of collagen I and III accumulation. Thus, with respect to inhibition of collagen I and III accumulation, this apparent additive effect was enhanced when the integrin β1 and β6 Fabs were physically linked in the bispecific Fab-K D -Fab molecules.
Overall, both the integrin β1-β6 and αV-β1 bispecific antibodies showed enhanced bispecific-dependent effects on ECM accumulation that ranged from obligate to additive activity depending on the target combination and on the respective ECM protein. Cell viability at the end of the assay was similar across all Fab-K D -Fab treatment conditions compared to untreated coculture controls ( Figure S4), indicating that the inhibition of ECM accumulation induced by the bispecific molecules was not due to direct effects on cell viability.

β1-β6 and αV-β1 bispecific validation in the BYbe TM format
The integrin β1-β6 and integrin αV-β1 target combinations identified in the single concentration ECM lung coculture bispecific screen were further validated in a concentrationresponse assay, measuring inhibition of fibronectin, collagen I and III accumulation after 7 d. The activity of the Fab-K D -Fab bispecific was analyzed alongside the equivalent molecularly linked bispecific BYbe TM format, which contained either the αV or β6 specificity in the Fab position and β1 specificity in the scFv position. Bivalent BYbe TM antibodies that contained the same V-regions in both Fab and scFv positions were also included; BYbe TM schematics and SE-UPLC data for purified BYbe TM proteins are shown in Figure S2c. Similar concentrationresponse curves and IC 50 values were obtained with Fab-K D -Fab and BYbe TM bispecifics for both integrin β1-β6 ( Figure 9a) and integrin αV-β1 bispecifics (Figure 9b), with the BYbe TM consistently demonstrating a moderate increase in potency compared to the Fab-K D -Fab (Table S2). Moreover, the concentration-response data confirmed that both the integrin β1-β6 and integrin αV-β1 bispecific BYbe TM antibodies more potently inhibited both fibronectin and collagen I and III accumulation than the corresponding bivalent BYbe TM antibodies (integrin αV-αV, integrin β1-β1 and integrin β6-β6).

Bispecific screen for activators of T cell subsets
PBMC represent and cover the major leukocyte classes involved in both innate and adaptive immunity, apart from granulocytes. PBMC comprise a heterogenous population of cells that when manipulated in vitro provide a more physiologically relevant cellular environment compared to isolated component cell types, such as T cells, B cells and monocytes, that are no longer capable of responding to paracrine and autocrine signals provided by other cells. As such, identification of molecules modulating specific cell subsets within the wider PBMC population has increased translational potential to more complex biological systems. Screens can be adapted e1859049-10 specifically for different PBMC subsets of interest; however, here, we focused on identification of modulators of T cell activation.
In recent years, immunotherapy has become an established treatment option for an increasing number of cancer patients, exemplified by the increased use of therapeutic antibody-based immune checkpoint inhibitors (CPIs), including the now wellestablished CTLA-4 and PD-1-targeted biologics, ipilimumab, pembrolizumab and nivolumab. 29 Nevertheless, differences in efficacy across patient groups, ranging from complete responses to treatment relapse and even failure to respond, 30 imply that these mechanisms alone are insufficient. Hence, there is potential to identify novel modulators of T cell activation based on unbiased biology and novel bispecific target pairs to enhance T cell activation as additional or alternative treatments of cancer and infectious diseases.
We generated Fab-X and Fab-Y libraries targeting antigens that were selected based on their expression on T cells or other immune cells known to interact with T cells. Antibodies were made to a total of 49 different antigens and between 1 and 4 unique V-regions to each antigen were selected for screening from different V-region sequence families, based purely on their ability to bind PBMC as a Fab-Y. We deliberately did not pre-select antibodies of defined affinity, activity, or epitope to enable unbiased bispecific discovery, driven entirely by the functional cellular activity of the V-region combination. Fab-X and Fab-Y antibodies covering these 49 unique antigens were purified and mixed to generate 6417 Fab-K D -Fab molecules for screening in a PBMC assay. In total, bispecific antibodies encompassing 969 antigen combinations were screened at 100 nM using PBMC from two independent donors. Cell activation was evaluated in unstimulated, anti-CD3-stimulated and Staphylococcal enterotoxin B (SEB)stimulated culture conditions. The activation markers CD69, CD71, CD25 and CD137 were measured on seven different cell subsets in addition to quantification of the soluble mediators granzyme B, interferon (IFN)-γ and interleukin (IL)-2 in the conditioned medium. The cell subsets evaluated comprised both naïve and memory CD4 and CD8 T cells, monocytes, NK cells and B cells. This highly multiplexed screen format generated a large multidimensional dataset that we are currently evaluating using a range of analytical and informatics approaches. Examples of the diverse range of bispecific antibody activities identified are shown in Figure 10 using an interactive Spotfire® data visualization and mining tool that can be interrogated to query different phenotypic screen outputs, either one at a time or providing a global view of all bispecific antibodies in the screen. Example screening output heat maps for CD8 and CD4 T cell activation and a soluble mediator granzyme B are shown in Figure 10a-c; each square represents a different Fab-K D -Fab molecule. Figure 10a shows expression of the activation marker CD137 on anti-CD3stimulated CD8 T cells (both naïve and memory). Figure 10b shows expression of the activation marker CD25 on CD4 T cells (naïve and memory) stimulated with anti-CD3. Figure  10c shows modulation of granzyme B in SEB-stimulated PBMC. Activators of T cell activity are depicted in blue, whereas inhibitors of T cell activity are depicted in red. Bispecifics comprising a Fab-X targeting the costimulatory molecule CD28 or the canonical CPIs, PD-1 or CTLA-4, are highlighted to illustrate the diversity of activities observed with different Fab-K D -Fab molecules and target combinations, though multiple novel T cell-activating target combinations beyond the PD-1 and CTLA-4 axis were also identified. bispecific BYbe TM proteins were tested alongside monospecific-bivalent BYbe TM controls in a concentration-response assay measuring fibronectin and collagens I and III. Five biological replicates for each data point were tested and plotted as mean ± standard deviation.

Discussion
Bispecific antibodies can elicit novel biological functions that cannot be achieved with conventional monospecific antibodies, though novel bispecific target discovery remains a challenge. The functional activity of bispecific antibodies is not necessarily predicted from that of the parental antibodies. We thus propose that identification of the optimum target pair for modulation of a given disease phenotype is best achieved by high-throughput functional screening using diverse, combinatorial bispecific antibody libraries covering multiple targets. We describe here a novel in vitro combinatorial bispecific antibody format that enables rapid and efficient generation of large, high-quality bispecific antibody panels. We used these bispecific antibody panels in three high-throughput dual target discovery campaigns that incorporated distinct phenotypic screens to identify novel drug target pairs and bispecific antibodies for diverse disease indications.
Firstly, we developed a flow cytometry signaling assay using anti-IgM-stimulated PBMC to mimic the elevated B cell activation observed in SLE patients. Through screening 1992 bispecific antibodies encompassing 23 targets and 300 different antigen combinations, we identified a novel obligate bispecific BCR inhibitor co-targeting CD79a/b and CD22, which potently inhibited B cell activation. Activity of the identified CD79-CD22 target combination was entirely bispecificdependent, with minimal activity being observed with the parental antibodies alone or mixtures of the two antibodies. CD22 is an inhibitory regulatory molecule recruited to the BCR following activation to limit the immune response and prevent over-activation. 31 We speculate that the simultaneous physical co-engagement of CD22 with the positive signaling component of the BCR, CD79, by the bispecific antibody favors negative inhibitory signaling via CD22, thus overriding BCR activation via CD79. 32,33 Secondly, we identified bispecifics co-targeting integrins β1 and β6 or αV and β1 as potent inhibitors of ECM accumulation from a longer-term high-content imaging assay from a screen containing 1671 bispecific antibodies encompassing 23 targets and 239 antigen combinations. These two novel bispecific target pairs displayed unique characteristics in the modulation of the ECM response over a 7 d assay period: the β1-β6 bispecific showed obligate inhibitory activity with respect to fibronectin accumulation and additive inhibitory activity with respect to collagen I and III accumulation; the bivalent αV-αV molecules were substantial inhibitors of ECM accumulation that could be enhanced further when paired with anti-β1 in the αV-β1 bispecific formats.
Thirdly, we introduced the concept of evaluating the modulation of multiple signaling pathways in diverse PBMC subsets in a single screen, combined with the ability to include different activating stimuli and markers for multiple cellular phenotypes. We demonstrated the ability to identify both activating and inhibitory bispecifics; in this case, identifying multiple activators as well as inhibitors of different T cell subsets. Validation of this unbiased screening approach was exemplified by the identification of several new target combinations with the reference costimulator CD28 and known CPIs such as PD-1 and CTLA-4, in addition to many additional novel nonpredicted target pairs. Across the three examples described, we screened thousands of bispecific antibodies to hundreds of antigen combinations to identify these novel bispecifics. High-throughput combinatorial target discovery on this scale has not been described to date, although unbiased bispecific functional screening has been reported to be necessary for identification of the optimal V-region pair. 8,9 We were able to generate sufficient numbers of bispecific antibodies at high purity for screening owing to the unique way in which our bispecific Fab-K D -Fab antibody format is formed. Following in vitro mixing of the two component Fab-X and Fab-Y halves, the resulting bispecific complex is of sufficiently high purity and thus requires no further purification or processing prior to screening. Chemical conjugation techniques have historically been used to couple Fabs, but these methods still suffer from low yield and product heterogeneity. 34 Homogenous Fab conjugates have been generated by chemical conjugation of proteolytically cleaved IgGs, though reduction and oxidation in addition to multiple purification steps were required. 9 More recently, split protein reconstitution methods such as the SpyTag/SpyCatcher system have been used to covalently couple antibody fragments; however, SpyCatcher fusions can suffer from low expression 35 and multiple polishing steps are required to isolate homogeneous-ligated product. 36,37 Sortasemediated covalent antibody conjugates have been successfully screened in biological assays without further purification of the conjugated product, 38 but certain assays may require removal of the sortase prior to screening. Combinatorial bispecific IgG libraries have also been generated by controlled Fab arm exchange, though removal of reducing agent is required following bispecific formation, and IgG bispecifics may be further hampered by restricted Fab arm geometry. The binding geometries of different IgG isotypes can vary dramatically, owing to differences in hinge sequences and flexibilities of Fab arms, 39 and this can manifest in vastly altered activities. 40 More importantly, activity from the Fc domain in IgG-based bispecifics may complicate the analysis of the effects of bispecific target engagement in cell assays utilizing immune cells. The Fab-K D -Fab is therefore designed to be Fc-free for direct assessment of the effects of bispecific targeting, without interference from Fc-driven activity. Additional Fc function can be analyzed subsequently with selection of a wide choice of therapeutic bispecific antibody formats. 2 Importantly, in several examples, we showed that the functional activity of the Fab-K D -Fab screening hits is verified in a molecularly linked therapeutically relevant antibody format. Fab-K D -Fab activity was retained in a BYbe TM format, 16 which can subsequently be converted to a TrYbe TM therapeutic format 41 by the addition of an albumin-binding domain. We also demonstrated that the obligate CD79-CD22 bispecific activity was replicated in a full-length bispecific IgG format in a B cell cytokine release assay. The Fab-K D -Fab target discovery format can thus reliably predict the effects of bispecific targeting in both Fab and scFv-based therapeutic antibody formats.
In summary, we have designed a unique bispecific screening format, that allows rapid generation of large numbers of highquality bispecific antibodies to enable unbiased dual target identification by high-throughput functional screening in biologically relevant assays. Our in vitro combinatorial format technology can also be applied to smaller hypothesized screens for selection of optimal target pairs or V-region pairs for bispecific or biparatopic antibody development. V-regions suitable for expression as a Fab can be derived from any commonly used source, including immunizations or display libraries. Bispecific activity can also be assessed using Fab-K D -Fab molecules generated from in vitro mixing of Fab-X and Fab-Y transiently expressing cell culture supernatants, without the need for purification, enabling even faster bispecific functional evaluation (data not shown). Our antibody library currently comprises approximately 1,500 purified Fab-X and Fab-Y molecules covering 150 different target specificities, with the potential to make approximately 450,000 unique Fab-K D -Fab molecules by in vitro Fab-X and Fab-Y mixing. As well as bispecific Fab-K D -Fab molecules, all the associated bivalent and single antigen-binding controls can also be easily made by combining the relevant component halves in vitro.
As the search for validated drug targets becomes increasingly challenging, phenotypic screening strategies are gaining momentum. Studies show that phenotypic drug discovery has been more successful in identifying small molecule drugs than target-based screening 42 and this target-agnostic approach is now being applied to monoclonal antibody drug discovery. 43 However, phenotypic screening with bispecific antibodies has been challenging owing to the difficulties in isolating the thousands of bispecific antibodies required at sufficient purity for screening. Our Fab-K D -Fab format overcomes this hurdle, and our bispecific screening platform can be applied to any disease area using translational human cell assays amenable to high-throughput screening. Our technology offers access to new or enhanced biology that cannot be attained with conventional antibodies, and thus provides a means to identify potentially firstin-class bispecific antibody therapeutics toward novel drug target pairings.

Reagents
All materials, reagents and cell lines were sourced from Thermo Fisher Scientific unless otherwise stated.

Plasmid construction
The Fab-X and Fab-Y light chains were constructed as signal peptide-VL1-MsCκ for mouse-and human-derived V-regions, and signal peptide-VL1-MsCκ S171C for rabbit-derived V-regions, where VL1 is the variable light domain 1 and MsCκ is the mouse kappa light chain constant domain. The Fab-X heavy chain was constructed as signal peptide-VH1-MsCH1-Ala(Gly 4 )-VL2-(Gly 4 Ser) 4 -VH2-10xHis-Myc, where VH1 is the variable heavy domain 1, MsCH1 is the mouse gamma-1 heavy chain CH1 constant domain, VL2 is the 52SR4 variable light domain and VH2 is the 52SR4 variable heavy domain. 17 10xHis and Myc are C-terminal epitope tags. The Fab-Y heavy chain was constructed as signal peptide-VH1-MsCH1-Ala,Ser(Gly 4 )-GCN4 peptide 17 -10xHis. The BYbe TM light chain was constructed as signal peptide-VL1-RbCκ, where VL1 is the variable light domain 1 and RbCκ is the rabbit kappa light chain constant domain. The BYbe TM heavy chain was constructed as signal peptide-VH1-RbCH1-Ser(Gly 4 Ser) 2 -VH2-(Gly 4 Ser) 4 -VL2 ± 6xHis, where VH1 is the variable heavy domain 1, RbCH1 is the rabbit gamma-1 heavy chain CH1 constant domain, VH2 is the variable heavy domain 2, VL2 is the variable light domain 2 and 6xHis is a C-terminal epitope tag. VL2 and VH2 in both Fab-X and BYbe TM heavy chains were disulfide stabilized, containing cysteines at VH44-VL100 (Kabat numbering), which is a disulfide bond location that is well tolerated among different scFv [44][45][46] and serves to prevent multimerization of Fab-X and BYbe TM proteins post-purification. 44 For full-length human IgG, light chain constructs comprised VL-hCκ S171C and heavy chain constructs comprised VH-hIgG4 containing S228P 47 and knobsinto-holes mutations. 48 Additional Fc null mutations, F234A and L235A, were incorporated to exclude the contributions of FcγR engagement in the cell assays. 49 All proteins were expressed from one of two closely related CMV-containing UCB-modified mammalian expression plasmids; pNAFL and pNAFH for light and heavy chains, respectively.

Antibody production
Antibody V-regions were derived from commonly used sources, such as rabbit and mouse immunization and subsequent B cell isolation, 50,51 bio-panning of naïve human antibody phage display libraries (reviewed by Hammers et al. 52 ) or publicly available sequences. V-regions were ligated into Fab-X, Fab-Y, BYbe TM16 or knobs-into-holes IgG 48 expression plasmids using standard cloning methods. Expression plasmids were cotransfected into Expi293F TM cells using ExpiFectamine TM transfection kits or CHO-SXE cells 53 using ExpiCHO TM transfection kits according to the manufacturer's instructions. Cell culture supernatants were harvested by centrifugation and clarified 7-14 d post-transfection by passage through a 0.22-µm filter. Product concentration was determined by a protein G high-performance liquid chromatography (HPLC) assay by comparison of the A 280 signal to a Fab standard. Fab-X and Fab-Y proteins were purified by one-step IMAC under endotoxin-free conditions using a vacuum-based purification system. Briefly, Ni 2+sepharose resin was mixed with cell culture supernatant, packed into a column, washed with 50 mM Na 2 PHO 4 , 500 mM NaCl, pH 6.2 followed by 500 mM NaCl and eluted with 50 mM sodium acetate, 1 M NaCl, pH 4.0. Eluates were concentrated, buffer exchanged into phosphate-buffered saline (PBS), pH 7.4 and sterile filtered. BYbe TM proteins were purified from clarified cell culture supernatant by affinity chromatography followed by a preparative size exclusion polishing step. BYbe TM proteins with a 6xHis tag were IMAC purified, whereas untagged BYbe TM proteins were purified using Protein G. Briefly, tagged BYbe TM proteins were loaded onto a 5 mL Nickel HisTrap TM Excel column (GE Healthcare), then washed with 10 mM Na 2 PHO 4 , 500 mM NaCl, pH 7.4 followed by 10 mM Na 2 PHO 4 , 500 mM NaCl, 25 mM imidazole. Bound material was eluted with a 10 mM Na 2 PHO 4 , 500 mM NaCl, 250 mM imidazole, pH 7.4 step elution. Untagged BYbe TM proteins were loaded onto a Gammabind Plus Sepharose® XK26/20 column (GE Healthcare), washed with PBS, pH 7.4, eluted with a 100 mM glycine, pH 2.7 step elution and neutralized with 2 M Tris/HCl pH 8.5. Fractions containing BYbe TM proteins from the affinity capture step were pooled and concentrated using Amicon® Ultra centrifugal spin columns (Merck Millipore). Monomeric BYbe TM proteins were isolated by application onto a HiLoad® 16/60 Superdex® 200 column (GE Healthcare) equilibrated with PBS, pH 7.4. Fractions containing monomeric protein were pooled, sterile filtered and stored at 4°C. Parental IgG proteins were purified by flowing clarified supernatant over a 5 mL MabSelect SuRe TM column (GE Healthcare), which was washed with PBS, pH 7.4 prior to a 0.1 M sodium citrate, pH 3.6 step elution and neutralization with 2 M Tris/HCl, pH 8.5. Affinity purification was followed by a preparative gel filtration step using a Superdex® 200 10/300 GL column (GE Healthcare). Bispecific IgGs were generated by mixing relevant parental IgG molecules at a 1:1 molar ratio in the presence of 50 mM βmercaptoethylamine overnight at 20°C. The mixtures were subjected to gel filtration and appropriate fractions were collected to isolate the final product in PBS, pH 7.4.

Antibody quality control
Purified antibodies were quantified by UV spectrophotometry using the Dropsense 96 (Trinean) or Cary 50 UV-Vis spectrophotometer (Agilent Technologies). Molecular weights were estimated by SDS-PAGE or capillary electrophoresis using the Labchip GXII Touch HT (Perkin Elmer) under reducing and non-reducing conditions, according to the manufacturer's instructions. NuPAGE® was performed on a Novex TM 4-20% Tris-glycine gel in Tris-Glycine SDS buffer according to the manufacturer's instructions. Purified protein samples (5 µg) contained 1× NuPAGE® LDS sample buffer and either 1× NuPAGE® sample reducing agent (reduced samples) or 10 mM N-Ethylmaleimide (non-reduced samples). SeeBlue TM Plus2 pre-stained protein standard was used as a molecular weight marker and protein bands were detected using InstantBlue® gel stain (Expedeon). Monomer level was determined by size exclusion ultra-performance liquid chromatography (SE-UPLC) or size exclusion HPLC. For SE-UPLC, 1 µg purified protein sample was injected onto an ACQUITY BEH200 column (Waters) and developed with an isocratic gradient of 200 mM sodium phosphate, pH 7.0 at 0.35 mL/ min. Signal detection was by absorbance at 280 nm and multichannel fluorescence. For SE-HPLC, 20 µg purified protein sample was injected onto a TSK Gel G3000SWXL, 7.8 × 300 mm column (Tosoh Bioscience) and developed with an isocratic gradient of 200 mM sodium phosphate, pH 7.0 at 1 mL/min. Signal detection was by absorbance at 280 nm. Gel filtration protein standards (BioRad) were loaded for molecular weight estimation. Since all purified Fab-X and BYbe TM proteins contained disulfide stabilized scFv, the observed monomer level is independent of protein concentration and unaffected by on-column dilution effects. Endotoxin was measured using the Endosafe® Portable Test System (Charles River) or the Limulus Amebocyte Lysate (LAL) chromogenic endotoxin quantification kit (Pierce), according to the manufacturer's instructions to ensure endotoxin levels of all antibodies were <1 EU/mg.

PBMC preparation
SLE donor PBMC were obtained from Tissue Solutions. Healthy volunteer PBMC were isolated from leukocyte cone blood (NHS Blood and Transplant Service) by density-gradient centrifugation in Leucosep TM tubes (Greiner-Bio-One). PBMC were frozen and stored in liquid nitrogen in accordance with UCB Celltech UK HTA License Number 12,504. Frozen PBMC were thawed, washed and resuspended in cell culture medium (RPMI 1640 medium supplemented with 10% (v/v) heatinactivated fetal bovine serum, 100 U/mL Penicillin, 100 µg/ mL Streptomycin and 2 mM Glutamax TM ) to the following densities: 3.33 × 10 6 cells/mL for SLE and healthy volunteer comparison assays, 1.25 × 10 6 cells/mL for primary screening and 5.00 × 10 6 cells/mL for concentration-response assays. Cell suspension (60 µL) was added to a v-bottomed 96-well polystyrene microtiter plate (VWR), which was sealed with a gasporous adhesive cover and incubated at 37°C, 5% CO 2 for 60 min to allow the cells to acclimatize. For the B cell cytokine release assay, B cells were purified from PBMC by negative selection using the B cell Isolation Kit II (Miltenyi Biotec) to >95% purity by flow cytometry.

Intracellular B cell signaling analysis by flow cytometry
To generate Fab-K D -Fabs, Fab-X and Fab-Y molecules were mixed in cell culture medium at an equimolar ratio and incubated for 1 h at 37°C. BYbe TM proteins were diluted in cell culture medium. Prepared Fab-K D -Fab or BYbe TM proteins were incubated with acclimatized PBMC for 1 h. The cells were then stimulated via the BCR by addition of 25 µg/ mL (final assay concentration) goat anti-human IgM antibody (Southern Biotech). Medium alone was added to nonstimulated cells. Cells were incubated at 37°C, 5% CO 2 for 15 min and then fixed with 100 µL/well BD Cytofix TM fixation buffer (BD Biosciences) at room temperature for 15 min. Cells were centrifuged at 500 × g for 5 min and the supernatant was removed. Plates were then vortexed and 100 µL/well ice-cold BD Permeabilization Buffer III (BD Biosciences) was added for 30 min. Cells were washed twice in flow buffer (PBS, pH 7.4 supplemented with 1% (w/v) BSA, 0.1% (v/v) NaN 3 , 2 mM EDTA) using an ELX automated plate washer (BioTek). Cells were resuspended by vortexing before Phosflow TM antibody staining. For assays comparing healthy volunteer with SLE patient B cells, two different antibody panels (BD Biosciences) were used containing antibodies to B cell surface markers and phosphorylated NF-κB (pNF-κB), Akt (pAkt), p38 (pp38), Syk (pSyk) and Erk1/2 (pErk1/2). Panels consisted of either: CD20-Alexa Fluor® 647 (

B cell cytokine release assay
Isolated B cells were resuspended at 2.00 × 10 6 cells/mL in cell culture medium. The isolated cells (100 µL/well) were added to round-bottomed 96-well plates and BYbe TM proteins or bispecific IgG molecules (100 nM final concentration) were added across triplicate wells. B cells were stimulated with 12 µg/mL anti-IgM/G (Jackson ImmunoResearch) and 1 µg/mL anti-CD40 antibody (Biotechne) at 37°C, 5% CO 2 for 3 d. Conditioned medium was harvested from each well and Lymphotoxin-α was measured using a Lymphotoxin-α/TNFβ DuoSet® ELISA (Biotechne). Absorbance readings at 450 nm were measured on a Synergy Neo2 multi-mode microplate reader (BioTek) and data analyzed using GraphPad Prism software (version 8.1.1, GraphPad Software, Inc.).

Fibrosis assay cell preparation
Human small airway epithelial cells (SAEpC) (ATCC, PCS-301-010) were maintained in airway epithelial cell basal medium (ATCC) supplemented with bronchial epithelial cell growth kit (ATCC). Human primary lung fibroblasts derived from a patient with idiopathic pulmonary fibrosis (ATCC, CCL-134, IPF134) were cultured in fibroblast basal medium supplemented with FGM TM -2 SingleQuots TM pack (Lonza). In the fibrosis assay screen, SAEpC and IPF134 cells were cocultured in a 1:1 ratio in renal epithelial cell basal medium (ATCC) supplemented with renal epithelial cell growth kit (ATCC). Cells were grown at 37°C, 5% CO 2 .

Fibrosis screen assay
To generate Fab-K D -Fabs, Fab-X and Fab-Y molecules were mixed in renal epithelial cell growth medium at an equimolar ratio and incubated for 1 h at 37°C. BYbe TM proteins were diluted in renal epithelial cell growth medium. Fab-K D -Fab or BYbe TM proteins were added to 384-well black, clear bottom plates (Greiner Bio-One). Both SAEpC and IPF134 cell cultures were then added, seeded at a density of 2000 cells/well. SAEpC monocultures and untreated cocultures were included as controls. Plates were incubated at 37°C, 5% CO 2 for 7 d. Cell viability was assessed using PrestoBlue® cell viability reagent according to the manufacturer's instructions.

High-content analysis of ECM
Image analysis of fibronectin and collagens I and III was conducted using Pipeline Pilot (version 9.2, Biovia). Size, shape, intensity, and texture statistics were collected from each raw image and averaged across the four fields collected for each well. A Pareto scale was then applied to each of the 64 collected statistics and positive (epithelial cell monoculture) and negative control wells (epithelial-fibroblast coculture) were then used to build a Partial Least Square (PLS) model using R software 54 for analysis. Test well statistics were then analyzed by the PLS model to generate a score which was multiplied by 100 to generate a percentage inhibition; 100 representing 100% inhibition of matrix accumulation, and 0 representing no inhibition of matrix accumulation as compared to controls on each plate. In this model it was possible to obtain negative values and values greater than 100 if there was increased or decreased fibronectin and/or collagen I and III in the treatment wells compared to the control wells, respectively. Data from Pipeline Pilot were exported into Spotfire® (version 7.11.1, TIBCO®) for visualization and analysis, or to GraphPad Prism (version 8.1.1, GraphPad Software, Inc.). In concentration-response assays, fibronectin, and collagen I and III fluorescence intensity measurements were taken from the "Cellomics Cell Health Profiling V4" bio-application. Non-linear curve fitting was performed in GraphPad Prism (version 8.1.1, GraphPad Software, Inc.) where possible.

T cell analysis by flow cytometry
To generate Fab-K D -Fabs, Fab-X and Fab-Y molecules were mixed in TexMACs cell culture medium at an equimolar ratio and incubated for 1 h at 37°C. PBMC (1.00 × 10 5 ) were added to 384-well tissue culture plates, followed by addition of the Fab-K D -Fab proteins. The cells were then activated via T cell receptor stimulation either by addition of 250 ng/mL (final assay concentration) mouse anti-human CD3 (eBioscience, UCHT1) or 1 µg/mL (final assay concentration) SEB (Sigma-Aldrich). Medium alone was added to unstimulated cells. Cells were incubated at 37°C, 5% CO 2 for 48 h and then centrifuged at 500 × g for 5 min and the conditioned medium transferred to a storage plate and placed at −80°C. Cell plates were then washed twice in flow buffer using an ELX automated plate washer (BioTek). Cells were resuspended by vortexing prior to antibody staining. Antibody details are shown in Table S3. Cells were stained at 4°C in the dark for 45 min and washed twice with flow buffer before fixing in BD CellFix overnight. The plates were then centrifuged at 500 × g for 5 min, the supernatant was removed, and the pellet resuspended in FACS buffer before acquiring on the iQue® Screener Plus flow cytometer (IntelliCyt®). ForeCyt® software (version 6.3, IntelliCyt®) was used to gate on CD14+ monocytes, CD56 + NK cells, CD19+ B-cells, CD4+ and CD8+ memory and naïve T cells. For each cell population, the cellular expression of CD69, CD25, CD71 and CD137 was measured as reported median fluorescence intensity values. The data were then used to calculate the log2 fold changes in expression relative to control well values and exported into Spotfire® (version 7.11.1, TIBCO®) for analysis and visualization.

PBMC soluble mediator analysis
Conditioned medium was quantified for granzyme B, IFNγ and IL-2 using the IntelliCyt® QBead PlexScreen kit. The conditioned medium was removed from −80°C storage and allowed to thaw. Plates stimulated with either anti-CD3 or SEB were diluted 1 in 20 in TexMACs media, while unstimulated plates were diluted 1 in 5. The samples were then added to plates containing analyte-specific capture beads. The plates were vortexed and incubated at room temperature in the dark for 1 h. The detection antibodies were added to the wells and the plates vortexed and incubated for a further 2 h. Wash buffer was then added before centrifugation at 1100 × g for 5 min. The supernatant was removed using an ELX automated plate washer (BioTek) and the beads resuspended by vortexing before acquisition on the iQue® Screener. Bead populations were identified using ForeCyt® software (version 6.3, IntelliCyt®) and median fluorescence intensity staining values for each population were generated. The data were then used to calculate the log2 fold changes in expression relative to control well values and exported into Spotfire® (version 7.11.1, TIBCO®) for analysis and visualization.

Statistics and reproducibility
Experiments analyzing BCR activity in healthy versus SLE patient samples were performed with n = 12 donors and three technical replicates per donor. Geometric mean fluorescence values of intracellular phosphorylated protein expression were calculated for each activation readout. p-Values were determined by a two-tailed unpaired t-test.