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A S P O N S O R E D P U B L I C AT I O N F R O M Neutralizing Antibodies Making Their Mark in Next Wave of Biologics

Information-Rich Solutions to Transform Biologics Discovery Sartorius is transforming biologics discovery through innovation with our groundbreaking Octet®, iQue® and Incucyte® cell & protein analysis platforms that allow you to gain more insights and make better decisions. These unique, high capacity solutions provide information-rich data for tracking of complex biological processes to generate deeper, more biologically relevant insights, streamline workflows and accelerate biologics discovery and development. Learn more at: sartorius.com/biologics

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Information-Rich Solutions to Transform Biologics Discovery INTRODUCTION Sartorius is transforming biologics discovery through innovation with our groundbreaking Octet®, iQue® and Incucyte® cell & protein analysis platforms that allow you to gain more insights and make better decisions. I n the last couple of years, there has been heightened interest in the development of Monoclonal antibodies (mAbs) as therapeutics as they have shown great promise in the treatment of These unique, high capacity solutions provide information-rich data for tracking of complex biological processes to generate deeper, more biologically relevant serious or untreatable conditions such as cancer. This is partly due to insights, streamline workflows and accelerate biologics discovery and their highly specific targeting of antigens as well as their high efficacy development. and fewer side effects. Vaccines and effective antibody therapeutics Learn more at: sartorius.com/biologics will play an integral role in keeping the on-going COVID-19 pandemic and emerging variants under control. Neutralizing Antibodies are an example of molecules that will play a critical role in vaccine and therapeutics development as they can bind to a virus in a manner that blocks infection. Neutralizing Antibodies can be isolated from plasma of convalescent patients and have become a major focus for researchers around the globe in the race to a better understanding of the mechanisms of action (MOA) against the receptor-binding domains (RBD) of the SARS-CoV-2 Spike protein. In this collection we highlight the research efforts and related applications utilized to study binding affinity, potency and target specificities of neutralizing antibodies against SARS-CoV-2 and their potential for future use against diseases caused by viruses from the same coronavirus subgroup. The novel findings provide deeper insights into the immune responses to infection, and help further the understanding of the interactions between the spike protein on the SARS-CoV-2 virus and its receptor on host cells. The interruption of this interaction has become the key target of COVID-19 therapeutics. Sartorius continues to support these early stage investigations by providing innovative Incucyte® live-cell analysis and iQue® advanced flow cytometry and Octet® label-free biomolecular interactions analysis platforms to provide new critical information, with confidence, as quickly as possible. Learn more at — Monoclonal Antibody Discovery and Development | Sartorius GENengnews.com | 3

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS TABLE OF CONTENTS Neutralizing Antibodies Making Their Mark in Next Wave of Biologics 05 Coronavirus-Neutralizing Human Antibody Discovered Antibody Internalization Reagent Principles Outside cell: Inside cell: 08 Antibody InternalizatiopnH: ~ A7.4dvanced Flow pH ~7.0—7.2 Cytometry and Live-Cell Analysis Give Rich Insights During Antibody Profiling 20 AntiboAdntyib oRdye sponsMeix Atonti bFodluy ShapedA bddy la b eled test antibody Early Late Lysosome: Internalization Internalization to cells and read on iQue3 endosome: endosome: pH ~4.7 Pre-ExRiesatgienntg ImmuRneaigteynt with (iQue3 VBR, BR, BR HD) pH ~6.3 pH ~5.5 test antibody 24 SARS-CoV-2 Variants B.1.351 and B.1.1.7 Show Resistance to Neutralizing Antibodies 27 Synthetic Nanobodies, “Sybodies,” Found That Neutralize SARS-CoV-2 30 Octet® Bio-Layer Interferometry Systems: Advancing Development of Coronavirus Vaccine and Therapeutics Cover: KTSdesign / Science Photo Library / Getty Images © GEN Publishing • April 2021 4 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Coronavirus-Neutralizing Human Antibody Discovered R esearchers at Utrecht University, Erasmus antibody, which also neutralizes the related Medical Center and Harbour BioMed SARS-CoV coronavirus, represents an initial step (HBM) have identified a fully human towards developing a fully human antibody to monoclonal antibody that prevents SARS-CoV-2 treat or prevent COVID-19, and also potentially from infecting cultured cells. Discovery of the future diseases caused by viruses from the same coronavirus subgroup. ADDITIONAL CONTENT “This discovery provides a strong foundation App Note: Compendium— for additional research to characterize this Accelerated Antibody Discovery antibody and begin development as a potential COVID-19 treatment,” said Frank Grosveld, GENengnews.com | 5 7activestudio/Getty Images

Coronavirus-Neutralizing Human Antibody Discovered PhD, co-lead author on the study, Academy strain caused ~8000 infections, with a lethality of Professor of Cell Biology, Erasmus Medical 10%. There are currently no approved targeted Center, Rotterdam and Founding CSO at Harbour therapeutics are available for COVID-19, the BioMed. “The antibody used in this work is ‘fully disease caused by SARS-CoV-2. human,’ allowing development to proceed more Monoclonal antibodies targeting “vulnerable rapidly and reducing the potential for immune- sites” on viral surface proteins are increasingly related side effects.” recognized as a promising class of drugs against infectious diseases, and have shown “Using this collection of therapeutic efficacy for a number of viruses, SARS-CoV antibodies, the authors wrote. Coronavirus-neutralizing antibodies primarily target the trimeric spike (S) we identified an antibody glycoproteins on the coronavirus surface that that also neutralizes mediate entry into host cells. The S protein has infection of SARS-CoV-2 two functional subunits. The S1 subunit, which is composed of four core domains, S1A through in cultured cells.” to S1D, mediates attachment to the host cell. The — Frank Grosveld, PhD, S2 domain mediates fusion of the viral and cell Erasmus Medical Center, Rotterdam membranes. Grosveld and colleagues report on the The spike proteins of SARS-CoV-2 and antibody in Nature Communications, in a paper SARS-CoV share 77.5% identical amino acid titled, “A human monoclonal antibody blocking sequence, and are structurally very similar, the SARS-CoV-2 infection.” investigators continued. They commonly bind the Both SARS-CoV-2 and the SARS-CoV virus that human angiotensin converting enzyme 2 (ACE2) emerged in 2002, belong to the Sarbecovirus protein as the host receptor. “Potent neutralizing subgenus of the Betacoronavirus family of antibodies often target the receptor interaction coronaviruses. The two viruses crossed species site in S1, disabling receptor interactions,” the barriers from an animal reservoir, and can cause authors continued. life-threatening respiratory illness in humans. By For their reported antibody discovery effort, May 4th, 2020 there were more than 3.4 million Grosveld and colleagues built on work that the confirmed cases of SARS-CoV-2 worldwide, groups had carried out on antibodies targeting and in excess of 230,000 deaths. The SARS-CoV SARS-CoV, explained co-lead author Berend-Jan 6 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Bosch, Associate Professor, research leader at neutralization by RBD-targeting antibodies Utrecht University. “Using this collection of have been reported including spike inactivation SARS-CoV antibodies, we identified an antibody through antibody-induced destabilization of its that also neutralizes infection of SARS-CoV-2 in prefusion structure, which may also apply for cultured cells.” The human antibody identified, 47D11,” the team noted. 47D11, was generated using Harbour BioMed’s “In conclusion, this is the first report of a H2L2 transgenic mouse technology. (human) monoclonal antibody that neutralizes 47D11 was shown to bind to cells expressing SARS-CoV-2,” they concluded. “This antibody the full-length spike proteins of both SARS-CoV will be useful for development of antigen and SARS-CoV-2, and potently inhibited viral detection tests and serological assays targeting infection of cultured cells. Tests showed that SARS-CoV-2 … this antibody—either alone or the antibody targeted the S1B receptor-binding in combination—offers the potential to prevent domain (RBD) of the spike proteins of both and/or treat COVID-19, and possibly also other viruses. The fact that the antibody is cross-reactive future emerging diseases in humans caused by indicates that it likely targets the conserved viruses from the Sarbecovirus subgenus.” core structure of the S1B RBD, the investigators “This is groundbreaking research,” said suggested. “Such a neutralizing antibody has Jingsong Wang, PhD, founder, Chairman potential to alter the course of infection in the & Chief Executive Officer of HBM. “Much infected host, support virus clearance or protect more work is needed to assess whether this an uninfected individual that is exposed to the antibody can protect or reduce the severity virus,” Bosch stated. of disease in humans. We expect to advance Interestingly, the team’s results suggested that development of the antibody with partners. 47D11 neutralizes SARS-CoV and SARS-CoV-2 We believe our technology can contribute through “a yet unknown mechanism” that is to addressing this most urgent public health different from receptor-binding interference. need and we are pursuing several other “Alternative mechanisms of coronavirus research avenues.” n GENengnews.com | 7

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Antibody Internalization: Advanced Flow Cytometry and Live-Cell Analysis Give Rich Insights During Antibody Profiling CLARE SZYBUT1*, CAROLINE WELDON2*, NICOLA BEVAN1*, LORI KING 1. 1. Essen BioScience, Ltd., Part of the Sartorius Group, Units 2 & 3 The Quadrant, Newark Close, Royston Hertfordshire SG8 5HL, UK 2. 2. Essen BioScience, Inc., Part of the Sartorius Group, 5700 Pasadena Ave. NE, Albuquerque, NM 87113 USA Introduction candidates. For instance, binding and rapid T he natural characteristics of antibodies, internalization are desirable properties for such as high binding affinity, specificity antibody-drug conjugates (ADCs), as cells must to a wide variety of targets, and be selectively targeted and killed via delivery good stability, make them ideal therapeutic of a cytotoxic payload. In contrast, if the goal is candidates for many diseases. Monoclonal to induce antibody-dependent cell-mediated antibodies (mAbs), in particular, deliver promising cytotoxicity (ADCC), the antibody must remain therapeutic results in several different disease bound to the cell surface, rather than being areas, such as autoimmunity, oncology, and internalized, in order to activate an immune chronic inflammation. Researchers’ abilities to response. improve the breadth of antibodies have been When designing ADCs, one key attribute to aided by innovative technologies for antibody predicting efficacy is the antibody internalization discovery, for instance, through humanization of (ABI) rate and the associated kinetics. These mouse antibodies and phage display. However, internalization kinetics are influenced by factors advanced antibody design techniques create such as the epitope on the target antigen, affinity the need for new screening methods so that of the ADC-antigen interaction, and intracellular lead candidates can be quickly and effectively trafficking. Evaluating for these factors is critical identified as early in the development process as throughout the antibody screening pathway possible. (Figure 1); however, here, we concentrate on Part of an effective screening strategy is to evaluating antibodies’ ABI during functional identify the desired therapeutic goal for your profiling via rate comparisons during the 8 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Scale up of key Ab Early cellular Ab raised as Check for binding (~50) testing for Humanization multiple hybridomas to target (500–1000 functionality, Further functional of Ab of interest (1000s Ab) Ab) affinity ranking evaluation (<10) Advanced High- Single concentration Throughput screening of 100s Full concentration Ab, non-purified, range profiling Flow Cytometry direct comparison Full concentration Kinetic Live- Full concentration range profiling, range profiling, rate mechanistic studies Cell Analysis comparison multiplexed with additional readouts Figure 1: Inclusion of ABI in the screening pathway for antibodies. Selecting for ABI throughout the antibody screening pathway, whether during functional profiling and rate comparisons or mechanistic studies. development process, as well as multiplexed insight by using a novel, pH-sensitive reagent mechanistic studies later in the screening process. for characterizing ABI via both advanced flow Traditionally, antibody screening cytometry and live-cell imaging. workflows have required labor intensive, time consuming, end-point-only methods, such Challenges with Traditional as FACS, ELISA, or microscopy. One solution Antibody Screening Workflows to address these screening challenges is to Monoclonal antibodies (mAbs) are large use advanced screening methods that offer (about 150 kDa), complex biologic molecules maximum insight through high-content data. that require post-translational modifications Herein, we demonstrate an advanced antibody for their activity (Chames et al., 2009). screening method that focuses on speed and Therefore, researchers face several challenges GENengnews.com | 9

Antibody Internalization when engineering and producing mAbs their fluorescence properties, it cannot be used as therapeutics. Engineering antibodies to to perform high-throughput, time-dependent optimize their biological potencies during studies on individual cells because the cells the discovery phase can address many of must be sorted into single colonies before any these challenges. However, these attempts to further analysis can be performed (Doerner optimize one attribute can have profound and et al., 2014). unintended consequences on other antibody ELISA, on the other hand, is adaptable to attributes. For instance, optimizing an antibody’s high-throughput screening (Saeed et al., 2017), specificity may negatively affect activity and thus has historically been used to screen (Tiller and Tessier, 2015). hybridomas and other libraries for antibody One way of simultaneously optimizing binding to each of its targets. ELISAs are multiple antibody properties is by using performed by coating a single target antigen mutagenesis to produce large screening onto the wells of assay plates, followed by the libraries. However, large screening libraries addition of individual samples from an antibody necessitate a thorough in vitro, high- library (for instance, from hybridoma or phage throughput screening method to quickly display). Antibodies that bind to the immobilized identify the most suitable drug candidates antigen are detected by a color change due to an for further development early in their indirect enzyme/substrate reaction. discovery process. In addition to its adaptability to high- Workflows for antibody screening commonly throughput screening, ELISA is rapid, consistent, include fluorescence-activated cell sorting and relatively easy to analyze (Saeed et al., 2017). (FACS), enzyme-linked immunosorbent assays However, ELISA has several disadvantages (ELISA), and microscopy (such as confocal) that can limit its successful use in a modern techniques. Yet, these in vitro antibody screening antibody screening lab. First, primary screens methods have several drawbacks: (1) they can that test binding to a single antigen often require be labor intensive with limited throughput, subsequent secondary and sometimes even (2) they do not allow direct, head-to-head tertiary screens with control antigens to confirm comparisons of antibodies, and (3) they need their specificity and cross-reactivity. Second, ELISA large amounts of reagents. is not the best method for screening antibodies For instance, even though FACS can be used that bind to cell surface antigens because these to sort hundreds of thousands of cells based on antigens are extracted from the cell membrane 10 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS and purified before adsorbing to the plastic ELISA thorough functional analysis of a smaller set plate. Extracting antigens from the membrane of antibody candidates using microscopy often leads to disruption of conformational (Doerner et al., 2014). epitopes that can be important targets for Like FACS, microscopy techniques also require therapeutic antibodies. Finally, to minimize labeling each antibody with a fluorescent tag, background signal, ELISA requires multiple which must be separated from the free label via wash steps to remove unbound antibodies and a column or wash step because analysis requires detection reagents, resulting in long, labor- robust isolation of internalized antibodies intensive screening workflows. from those outside the cells. To aid isolation Also, although ELISA can provide data on the of the positive signal, researchers often resort immunoglobulin G (IgG) titer, it offers no reflec- to perturbing techniques, such as washing tion on how the therapeutic antibody candi- cells, using blocking dyes, and reducing the dates affect the health of the test cells. i.e. how temperature to slow cellular activity. However, quickly or efficiently they induce death. Thus, cells can be lost during washing steps, and the candidates that appear to be productive based associated reductions in temperature perturb the on ELISA screening may be carried forward into cellular environment. the next step of the production process even A further drawback to almost all the though they are unideal candidates in terms of techniques used for antibody screening is function or vigour. that they only enable end-point analysis, Microscopy techniques, in contrast to FACS, which means that multiple experiments are are useful for single-cell analysis, as well as required to follow an antibody attribute, such as for such localization and temporal studies as internalization, over time. antibody internalization and live-cell imaging The best approach to address all the for monitoring individual cell behavior. limitations of traditional antibody screening Microscopy techniques, however, have limited is to combine data from advanced screening throughput due to data acquisition time, and methods; using advanced methods, they have limited multiplexing capabilities. researchers can choose their candidates Thus, for antibody discovery, some other based on a thorough evaluation of all relevant method, preferably high-throughput, is characteristics, such as IgG titer, cell health, generally used for the initial selection and internalization, and their associated kinetics screening of large libraries, followed by a more early in their discovery process. GENengnews.com | 11

Antibody Internalization Antibody Internalization Reagent Principles Outside cell: Inside cell: pH ~7.4 pH ~7.0—7.2 Antibody Mix Antibody Add labeled test antibody Early Late Lysosome: Internalization Internalization to cells and read on iQue3 endosome: endosome: pH ~4.7 Reagent Reagent with (iQue3 VBR, BR, BR HD) pH ~6.3 pH ~5.5 test antibody Figure 2: The pH-sensitive fluorescent probe principle. A novel pH-sensitive fluorescent probe enables one-step, no-wash labeling of isotype-matched antibodies. A fluorescent signal is generated as internalized antibody is processed into the acidic endosome and lysosome pathway. A Simple Way of Labeling Antibodies Fab fragments conjugated to a pH-sensitive for Addressing Challenges with fluorescent probe (Nath et al., 2016). This type FACS, ELISA, and Microscopy of novel reagent enables a generic, one-step, One of the challenges presented above with no-wash labeling protocol for all isotype- FACS, ELISA, and microscopy techniques is matched, Fc-containing test antibodies when the requirement for wash steps to minimize optimized for use on specific instruments. background signal. Among other undesirable Figure 2 shows how this reagent works: labeled effects, such as increased time to results, this antibodies are added to cells, and a fluorogenic need for wash steps also increases reagent signal is produced as the Fab-Ab complex is requirements (and cost) and makes cell loss internalized and processed via acidic (pH 4.5-5.5) inevitable. However, an advanced method for lysosomes and endosomes. labeling cells could reduce reagent requirements, Antibodies of interest are quickly and as well as simplify protocols when analyzing effectively labeled, with low reagent antibody internalization. requirements, by incubating in growth media ABI Assays Made Simple, With No-Wash Labeling with this novel, pH-sensitive dye (Figure 3). and Low Reagent Requirements via a Novel, Cells are then added to 384-well plates, along Ph-Sensitive Reagent with the dye-conjugated antibodies, and The key to this simple, no-wash protocol is a incubated again. This reagent, when used with novel reagent composed of Fc-region targeting an advanced flow cytometry platform, provides 12 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Multiplexed Viability/Encoding Antibody Internalization Reagent Workflow Violet Encoding Dye (V/Blue) Cell Preparation Rinse Plate cells cells Label test antibodies with Encode cell populations with Add labeled antibody Acquire data Antibody Internalization Violet Encoding Dye and plate and Membrane Integrity on iQue3 VBR. Reagent and prepare dilutions. encoded cells. Dye (B/Green). Incubate two hours. Figure 3: The assay consists of 3 components, each 10 µL: labeled test antibody, cells (encoded or not), and Cell Membrane Integrity Dye (B/Green). Each component is prepared at 3X before addition for a final concentration of 1X and an assay volume of 30 µL. a comprehensive, integrated solution for rapid characteristics within the same workflow. For profiling of antibody internalization and instance, you can measure cell viability using a other critical antibody attributes using small membrane integrity dye to assess general cell sample volumes in 96- or even 384-well plate health, as well as cell death due to cargo delivery, formats (Riedl et al., 2016). Much of the data when optimizing ADCs. Or you can characterize acquisition and analysis, including generation cell specificity using encoding dyes (for cell lines) of serial dilution curves and EC50 calculations, is or directly conjugated fluorescent antibodies automated with the help of advanced software (for complex cell models with a variety of cell packages, for example, as included in the iQue® types). Other reagents, such as those for assessing advanced flow cytometry platform. cytokine release, are also available for more detailed antibody assessments on the sample. Functional Profiling for Therefore, analysis with an advanced flow Comprehensive Cell and Antibody cytometry platform can be optimized to deliver Characterization Early in the rich content very quickly while only using a low Process with Multiplex Analysis sample volume. Combining the above-described pH-sensitive dye Sartorius produces such a pH-sensitive with other reagents in one assay on an advanced reagent for use on our iQue® advanced flow cytometry platform enables simultaneous flow cytometry platform. The combination analysis of a variety of cell and antibody of non-perturbing and validated reagents GENengnews.com | 13

Antibody Internalization A. CD19 CD20 CD79b CD71 CD19 CD20 CD79b CD71 CD19 CD20 CD79b CD71 mlgG1 CD22 CD3 mlgG1 CD22 CD3 mlgG1 CD22 CD3 100000.0 60000.0 100000.0 80000.0 50000.0 80000.0 60000.0 40000.0 60000.0 30000.0 40000.0 40000.0 20000.0 20000.0 20000.0 10000.0 0 0 0 2 3 2 3 2 3 log(Concentration) μg/mL log(Concentration) μg/mL log(Concentration) μg/mL B. Jurkat Raji Ramos mlgG1 CD71 CD3 CD20 CD19 CD22 CD79b Figure 4: Serial dilution curves for internalization-labeled antibodies with a top concentration of 1 mg/mL in different cell types after a three-hour incubation. Multiplexed positive and negative cell lines may be used in an ABI assay to generate high-content data in one assay. Median fluorescent intensity (MFI) for Internalization Reagent-labeled antibodies after three hours. A serial dilution of each antibody with a top concentration of 1 mg/mL was prepared and incubated with encoded Jurkat, Raji, and Ramos cells in the same well. Jurkat cells (a T lymphocyte cell line) show a concentration-dependent increase in internalization of anti-CD3, but not the two B cell markers. Conversely, Raji cells show a concentration-dependent increase in internalization of anti-CD19 and anti-CD22, but not anti-CD3. Ramos cells show a concentration-dependent increase in internalization of anti-CD79b, an ADC drug target for non-Hodgkin’s lymphoma. for multiplexing, no-wash protocols, high- visualization expedite the process of screening throughput capabilities, flexibility for a drug candidates for potential efficacy and robotic interface, and integrated Forecyt® toxicity to accelerate antibody discovery and software for multiparametric data analysis and development. 14 | GENengnews.com Median internalization (RL1-H) of Jurkat Median internalization (RL1-H) of Raji Median internalization (RL1-H) of Ramos

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS To demonstrate the multiplexing alone or when we mixed them. This result capabilities of this novel pH-sensitive dye on shows that multiplexing positive and negative the iQue® platform, we used Ramos and Raji cell lines does not interfere with the antibody cells stained with two intensities of violet internalization assay. encoding dye, combined with unstained Compared to performing a series of singleplex Jurkat cells. We incubated this for three hours assays, a multiplexed assay approach enables with a serial dilution of dye-conjugated you to analyze multiple readouts (internalization, specificity antibodies (isotype-matched viability, cell type) from a single well, decreasing anti-CD3 as a T cell marker, anti-CD19, anti- the number of tests needed to perform a CD20, anti-CD22, or anti-CD79b as B cell comprehensive functional characterization of the markers, anti-CD71 as a positive control, and Ab candidate. IgG as a negative control), then added a cell membrane integrity dye before acquiring data Full Concentration Profiling with from the plate. Using this strategy, we were Live-Cell Imaging and Analysis able to identify viable cells, then spectrally A pH-sensitive dye-coupled antibody fragment separate Ramos, Raji, and Jurkat cells. designed according to the same principle as We then assessed antibody internalization that used above (Figure 2) can also be optimized for each cell line. We generated series dilution and used in other instruments. For instance, curves for the specificity markers of each cell using this pH-sensitive reagent in a real-time, type (Figure 4A). As expected, Jurkat cells showed live-cell imaging system such as the Incucyte® internalization of anti-CD3, but not anti-CD19 Live-Cell Analysis System, allows visualization and or anti-CD22, whereas the Raji cells internalized automatic quantification of the full time-course of anti-CD19 and anti-CD22, but not anti-CD3. Only ABI. This combination of reagent and platform thus Ramos cells showed a concentration-dependent provides a simple method for directly profiling and increase in internalization of anti-CD79b, an ADC comparing ABI for a large number of antibodies drug target for non-Hodgkin’s lymphoma. In the (10–100s at a time in a miniaturized format). three-hour assay time frame, we did not observe To demonstrate the power of the pH-sensitive anti-CD20 internalization, but we did see an dye approach for high-throughput antibody increase in the two B cell lines by 24 hours (data internalization assays in a real-time, live-cell not shown). Importantly, we saw little difference analysis system, we performed a head-to-head when we assessed the cells for internalization comparison of the internalization properties of GENengnews.com | 15

Antibody Internalization Ab1a Ab2 Ab3 Controls Ab1b Ab4 Ab5 Controls Figure 5: Screening test of Abs for internalization. The pH-sensitive reagent is suitable for high-throughput antibody internalization assays in a real-time, live-cell analysis system. six different commercially available anti-CD71 same clone from two different sources). antibodies into HT1080 fibrosarcoma cells. We We found that three antibodies (Ab1a, Ab2, labeled the anti-CD71 with our pH-sensitive and Ab1b) gave internalization signals that we reagent before adding to cells in 96-well plates. detected at low concentrations (< 0.05 μg/ml) We then captured the internalization signal in a (Figure 5). Reassuringly, Ab1a and Ab1b gave live-cell analysis system every 30 minutes over similar internalization responses. Antibodies 12 hours using a 10X magnification. 3, 4, and 5 were internalized more weakly and The plate view in Figure 5A shows clear only at higher concentrations. From the control positive and negative control responses responses, we calculated a mean Z’ value of in column 11 and 12, with concentration- 0.82 (two plates: 0.75, 0.87), indicating high dependent responses for each antibody across robustness for this microplate assay. the two plates (antibodies 1a and 1b are the These data show our method is suitable 16 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS for comparing the internalization of multiple monoclonal antibody, Herceptin (Trastuzumab). antibodies at a single target, or one antibody We constructed a concentration-response curve in various cell types. The assay precision and by labeling Herceptin with the pH-sensitive workflow is such that it would be possible to reagent, then serially diluting (1:2) before adding compare 100s of different antibodies at once, and to BT-474 cells. further throughput could be achieved through In BT-474 Her2-positive breast carcinoma miniaturization to a 384-well format. cells, we saw definite time and concentration- Showing a Simple Pharmacological and Kinetic dependent internalization of Herceptin over 48 Quantification of Antibody Internalization Using hours. From an area under the curve (AUC) time- Herceptin course analysis, we calculated the EC50 value for In addition, we experimented to determine EC50 internalization at 323 ng/mL = 2.1 nM (Figure 6). values for the internalization of a clinically used Our calculated EC50 value is similar to the known Figure 6: Quantitative pharmacological analysis of pH-sensitive dye-labeled Herceptin shows time and concentration-dependent internalization and an EC50 value of 2.1 nM. Quantitative pharmacological analysis of pH-sensitive dye-labeled Herceptin. BT-474 Her2- positive cells were treated with increasing concentrations of pH-sensitive dye-labeled Herceptin. The time course graph displays an increasing normalized red area over time with increasing Herceptin concentrations (A). Area under the curve analysis of this response displays a clear concentration dependent response with an EC50 of 323 ng/mL (B). All data shown as a mean of 3 wells ± SEM, time course data shown as normalized red area. A. B. 80 80 3 3 80008 n0g0/m0 Lng/mL 40004 n0g0/m0 Lng/mL 20002 n0g0/m0 Lng/mL 60 60 1000 1n0g0/m0 Lng/mL 2 2 500 n5g0/m0 Lng/mL EC50 3E2C35 n0 g3/2m3 Lng/mL 40 40 250 n2g5/m0 Lng/mL (21 nM(2)1 nM) 1 1 20 20 125 ng12/m5 Lng/mL 62.5 n6g2/.m5 Lng/mL 0 0 0 0 0 0 12 12 24 24 36 36 48 48 -8 -8 -7 -7 -6 -6 -5 -5 TimeT (ihm)e (h) Log [LHoegr [cHeeprtcine]p (tgin/m] (Lg)/mL) GENengnews.com | 17 Normalized red area (%) Normalized red area (%) AUC (0-48 h) x 103 AUC (0-48 h) x 103

Antibody Internalization KD value for Herceptin for its target receptor process is a simple, fast, and insightful way to (approximately 5 nM). identify candidates that meet your therapeutic goals early in the drug discovery process. Speed and Insight through Advanced Antibody Screening Combining Advanced Flow Cytometry Quick and accurate identification of suitable and Live-Cell Imaging and Analysis drug candidates is key to the development of for Complete Antibody Profiling: therapeutic antibodies. ABI is an essential part A Real-World Screening Strategy of the selection criteria for ADC candidates. It Employing Our pH-Sensitive Dye can be used for functional profiling and rate and Instrument Platforms comparisons, as well as mechanistic studies Researchers at LifeArc used the iQue® platform when coupled with additional multiplexed and Incucyte Live-Cell Analysis System as part readouts, thus reducing the time required for of their strategy to develop a new ADC targeting lead generation. To illustrate the capabilities of neuroblastoma. Neuroblastoma is a rare cancer advanced antibody screening, we have described that nevertheless is the most common extra- our solution for efficiently interrogating libraries of cranial solid tumor in children, with a 5-year candidates early in the screening process. survival rate of 50% for patients with high-risk For comprehensive cell and antibody disease. The researchers found they were able characterization, we used our novel, pH-sensitive to use these systems in combination, not only reagent and the iQue® advanced flow cytometry for screening, but also for assay development, platform. This combination is best for screening lead candidate profiling, and characterization. and early full concentration profiling because it They found that the iQue® platform offered fast, enables simultaneous analysis of a variety of cell high-content analysis, while the Incucyte live- and antibody characteristics within the same cell analysis system offered kinetic, image-based workflow. For quantitative, pharmacological analysis. Combining data from the two systems analysis and direct, head-to-head comparisons of gave them a complete antibody profile. ABI, we used a version of our novel, pH-sensitive In brief, the researchers identified anaplastic reagent and the Incucyte live-cell analysis system. lymphoma kinase (ALK) as a target for their This combination is useful for further functional therapeutic approach because level of ALK profiling that requires spatial and temporal expression correlates with disease stage and resolution. This advanced antibody screening ALK antibodies show surface expression 18 | GENengnews.com

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS in patient samples. In collaboration with advanced screening methods that incorporate a colleagues at Mt. Sinai, researchers at LifeArc novel, innovative reagent for characterizing ABI produced 1,152 candidate hybridoma clones via both advanced flow cytometry and live-cell that they were able to narrow to 53 candidates imaging, they gained maximum insight through that ELISA and flow cytometry showed bound high-content data and were able to make critical to ALK. Using the advanced flow cytometry decisions early in their process, thus saving time, capabilities of the iQue® platform, the material, and money. n researchers were able to further narrow this to 20 candidates that bound ALK at the surface References of cells, since this is a critical attribute of the Tiller KE and Tessier PM. Advances in antibody design. Annu Rev Biomed Eng, Feb 5; 17: 191-216 (2015) PMID: 26274600 mechanism of action of ADCs. Doerner A, et al. Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett, Jan 21; 588(2); 278-87 (2014) PMID: 24291259 Of particular interest, these researchers used Nath N, et al. Homogeneous plate based antibody internalization internalization assays on both the iQue® and assay using pH sensor fluorescent dye. J Immun Meth, Feb 3; 431: 11-21 (2016) PMID: 26851520 Incucyte cell analysis platforms to further narrow Reidl T, et al. High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe. J their lead candidates to two. Critically, they Biomol Screen, Jan; 21(1): 12-23 (2016) PMID: 26518032 gained kinetic imaging data and high- content Chames P, et al. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmaco, May; 157(2): 220-33 (2009) PMID: analysis using multiplexed cell viability assays 19459844 Saeed A, et al. Antibody Engineering for Pursuing a Healthy Future. early in their ADC discovery process. Using Front Microbiol, Mar; 8(495) (2017) PMID: 28400756 GENengnews.com | 19

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS Antibody Response to Flu Shaped by Pre-Existing Immunity Receiving the seasonal flu vaccine each year, or vaccination. The authors found that a in addition to seasonal infections, exposes person’s antibody response to influenza viruses people to a lifetime of building up immune is dramatically shaped by their pre-existing responses to influenza antigens. Yet, it remains immunity, and that the quality of this response unclear whether infection and vaccination differs in individuals who are vaccinated or induce distinct influenza-specific immunological naturally infected. Their results highlight the memory. A team led by researchers at the importance of receiving the annual flu vaccine to University of Chicago compared antibodies induce the most protective immune response. produced by individuals after influenza infection The study is published in Science 20 | GENengnews.com