key: cord-0073531-5lknu5mo
authors: Zhou, Dongyan; Zhou, Runhong; Chen, Zhiwei
title: Human neutralizing antibodies for SARS-CoV-2 prevention and immunotherapy
date: 2021-12-30
journal: Immunother Adv
DOI: 10.1093/immadv/ltab027
sha: 7cd4aed6ad666e912968037b9a90f04a2726508b
doc_id: 73531
cord_uid: 5lknu5mo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 has been spreading worldwide since December 2019, resulting in the ongoing COVID-19 pandemic with 237 million infections and 4.8 million deaths by 11 October 2021. While there are great efforts of global vaccination, ending this pandemic has been challenged by issues of exceptionally high viral transmissibility, re-infection, vaccine-breakthrough infection, and immune escape variants of concerns. Besides the record-breaking speed of vaccine research and development, antiviral drugs including SARS-CoV-2-specific human neutralizing antibodies (HuNAbs) have been actively explored for passive immunization. In support of HuNAb-based immunotherapy, passive immunization using convalescent patients’ plasma have generated promising evidence on clinical benefits for both mild and severe COVID-19 patients. Since the source of convalescent plasma is limited, the discovery of broadly reactive HuNAbs may have significant impacts on the fight against the COVID-19 pandemic. In this review, therefore, we discuss the current technologies of gene cloning, modes of action, in vitro and in vivo potency and breadth, and clinical development for potent SARS-CoV-2-specific HuNAbs.

M a n u s c r i p t 

B cell receptor (BCR) repertoires exhibit high sequence diversity due to the somatic recombination and hypermutation during the B cell development. BCR is defined as a transmembrane receptor located on the B cell surface and interacts with a specific antigen epitope through its variable region to initiate antibody response. This variable region, therefore, shares the identical gene sequence with the antibody that is produced by this B cell. The somatic recombination of three gene segments of the heavy (H) chain locus (V, D, J) and two gene segments of the light (L) chain locus (V, J) to diversify the variable region gene. The variable region of an antibody immunoglobulin (Ig) determines the specificity for interaction with a corresponding viral antigenic epitope. The somatic hypermutation involves the B cell proliferation in the germinal center with random mutations in the genes encoding the variable region of individual monoclonal antibody (mAb), essential for high affinity binding to an antigenic epitope, so-called the antibody affinity maturation process. There are no identical BCRs between two different B cells. To ensure native pairing of antibody H and L chains, it is necessary to analyze one B cell at a time for cloning a mAb (5) . With the advancement of antibody gene cloning techniques, such as hybridoma technology, human B cell immortalization, antibody phage display, human immunoglobulin transgenic mice and single B cell antibody technology (6) , cloning of a functional HuNAb is no longer a search of a needle in the ocean. During the ongoing COVID-19 pandemic, most of the SARS-CoV-2 specific antibodies have been obtained through single B cell technology.

Single B cell-based antibody cloning is a technique to obtain the variable region of mAb H/L genes from individual memory B cell that has capacity to produce the high affinity antibody. It involves the amplification of auto-paired Ig H/L chain RNA sequences from the heterogeneous memory B cell population and in vitro construction into functional mAbs (7; 8) . This technique has been successfully M a n u s c r i p t 5 used for isolating neutralizing mAbs from convalescent patients against various viral infections such as the Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), Dengue and SARS-CoV-2 (9) (10) (11) (12) . Since 2009, the combination of single-cell RT-PCR and single B-cell sorting has improved the successful rate of antibody gene cloning greatly. The acquirement of single antigen-binding memory B cell by fluorescence-activated cell sorting (FACS) or opto-fluidics platform is the major technical improvement, allowing subsequent nested RT-PCR using primers to amplify naturally paired antibody H/L gene sequences from individual memory B cells (13) (14) (15) (16) . After COVID-19 outbreak, this method was quickly utilized to isolate HuNAb from SARS-CoV-2-infected patients (11; 17) . Moreover, there is the development of high-throughput single-cell RNA and VDJ deep sequencing of BCR repertoires accompanied by the bioinformatics analysis (18; 19) . This technique has outcompeted the single-cell RT-PCR in terms of high-throughput screening of a large pool of diverse memory B cells. Interestingly, immunization using RBD/S proteins or direct infection of mice with the genetically humanized immune system can also generate complete HuNAbs against SARS-CoV-2 (20) . This murine platform may simplify the source of antigen-specific human B cells although the antibody affinity maturation process remains to be improved in this model. Nevertheless, using this platform, some SARS-CoV-2-specific HuNAbs have been successfully cloned and screened (20) . The deep sequencing of variable regions and BCR repertoire has revealed H/L pairing of human antibody characteristics (20) . In addition, the combination of microfluidic-based technique and bioinformatics analysis can further improve the efficiency of identifying highly potent HuNAbs against specific viral antigens, which has implications to the fight against COVID-19 and other emerging infectious diseases.

Phage display has been one of the widely used methods for cloning human antibodies. The two key steps of the phage display technique include the construction of antibody gene library and the screening of antigen-specific antibodies. The human Fab library can be generated from peripheral blood mononuclear cell (PBMC) derived from COVID-19 patients. A set of primers targeting the variable H/L chain regions are used to amplify the total antibody gene pool for construction of the phage library (21) . In solution panning, SARS-CoV-2 RBD can be used as the bait to fish out the RBDspecific human Fabs, followed by construction of the Fabs into full-length IgG1 for subsequent biochemical and functional testing. Alternatively, the human Fab antibody library can also be synthesized using the selected human germline immunoglobulin variable segments. The diversity in the complementarity-determining regions (CDR) 3 of H/L chains (CDR-L3 and CDR-H3) can be introduced by well-designed mutagenic oligonucleotides. A competitive phage screening strategy has been successfully used to obtain RBD-specific HuNAbs against SARS-CoV-2 (22) . Interestingly, to obtain a cross-reactive HuNAb against both SARS-CoV and SARS-CoV-2. Mice immunized with SARS-CoV RBD were used for the establishment of the phage display library. Then, this library was screened using the SARS-CoV-2 RBD as the bait. Since these two RBDs share 75% similarity in their amino acid sequence, the cross-reactive murine mAbs were successfully obtained for subsequent humanization against both SARS-CoV and SARS-CoV-2 (23) . The drawbacks for the phage displaybased antibody cloning technique include unnatural pairing of VH/VL and time-consuming panning procedure. Nevertheless, this technique remains very useful for obtaining antigen-specific antibodies including potent SARS-CoV-2-specific HuNAbs. M a n u s c r i p t 6 

The S glycoprotein of SARS-CoV-2 exists in a prefusion trimer conformation that may rearrange during the fusion of virus-cell membrane. This viral entry is a dynamic process beginning with the binding of the viral S1 portion to the host cell receptor ACE2. The interaction between S1 and ACE2 makes the prefusion trimer unstable, leading to the cleavage of S into S1 and S2 by cellular proteases (e.g., Furin) with the transition into a stable post-fusion conformation. The RBD in S1 transiently hides or exposes the determinants of receptor binding, due to its hinge-like conformational movement, displaying two structural states with "up" and "down" conformations. The "up" conformation is an active state for ACE2 binding (24) . Once in the "down" conformation, RBD is usually stable but is inaccessible to ACE2. Because of this unique property, the "up" conformation serves as the major target for SARS-CoV-2 NAbs. Interestingly, two major conformational structural regions within the S1 have been identified as SARS-CoV-2 neutralizing domains. Besides RBD, the Nterminal domain (NTD) also possesses binding sites for HuNAbs.

According to the CoV-AbDab, the coronavirus antibody database, there are amazingly a total number of 3152 SARS-CoV-2-specific antibodies by September 23, 2021, including 2693 mAbs and 459 nanobodies. 1048 of the 2639 mAbs have neutralizing activities. In a separate report, among a total of 1584 RBD-specific antibodies, 902 have the neutralization ability (25) . By mapping the binding sites of a collection of mAbs against the S protein, NAbs bind to multiple S epitopes including ones in RBD, NTD and quaternary regions (26; 27) . While RBD is the primary target for NAb, these antibodies can be classified into three main groups according to the distinct binding sites at the molecular level (28) . The first group of NAbs have epitopes within the RBD overlapping largely with the binding site of ACE2. These NAbs are described as receptor binding site (RBS) antibodies. Furthermore, due to different interacting angles with viral RBD, RBS NAbs are divided into three indepth subclasses including RBS-A, RBS-B and RBS-C (28) . As demonstrated in the crystal structures (Figure 1) , the binding sites and interaction direction of RBS-A antibodies with viral RBD shared the highest similarity with those of ACE2 binding. RBS-A antibodies bind to RBD like ACE2 on the "left side" of the ridge. Typically, both Fab-RBD and ACE2-RBD interactions show on one side of the ridge in the crystal complex. Comparing to RBS-A antibodies, the epitope of RBS-B antibodies shows less overlapping with the ACE2 binding site. RBS-B antibodies are positioned more upright and straddle the central of the ridge (28) . The RBS-C antibodies are the subclass of antibodies that have the least overlapping epitopes with ACE2 binding site as they bind to viral RBD on the opposite side of the RBS-A antibodies. It should be emphasized that all three subclasses of RBS antibodies can compete with human ACE2 for SARS-CoV-2 neutralization. While several RBS-C antibodies can bind to the "up" or "down" RBD, the RBS-A antibodies can only interact with the "up" RBD. The binding mode of RBS-B antibodies is in between as some can only bind to RBD in the "up" state while others show in the "down" state (28; 29) . The second group of NAb is represented by the cross-reactive CR3022 antibody that recognizes a cryptic site in RBD. CR3022 is an antibody isolated from a phage library of a convalescent SARS patient in 2003. CR3022 binds to a cross-reactive epitope on SARS-CoV-2 RBD but it does not neutralize SARS-CoV-2. The position of the CR3022 cryptic site is highly conserved near the "tail" part of an "up" RBD. This binding site is common for cross-reactive antibodies against both SARS-CoV and SARS-CoV-2. Different from CR3022, however, several CR3022-like antibodies present various neutralizing activities against both SARS-CoV and SARS-CoV-2 (28) . The third group M a n u s c r i p t 7 of NAb is represented by the S309 antibody that neutralizes both SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the RBD of the S glycoprotein through binding to an epitope containing the N343 glycan, without competing with ACE2 interaction. The S309 antibody was identified from a SARS convalescent patient in 2003 as well. It binds to either the "up" or the "down" RBD. The binding site of S309 is not as conserved as the one for the CR3022 antibody (28) . Besides three groups of RBD-specific NAbs, the mode of action for NTD-directed NAbs remains incompletely revealed (26; 27) . It is possible that the binding of these NAbs to NTD may affect indirectly the conformational interaction between RBD and ACE2. Since S2 subunit is more conserved among coronaviruses than S1, some cross-reactive neutralizing antibodies specific to S2 of the S protein have been reported (30; 31) . Interestingly, SARS survivors have developed potent cross-clade pan sarbecovirus neutralizing antibodies after immunization with the BioNTech mRNA vaccine (32) . The findings of multiple NAb targets of vulnerability on SARS-CoV-2 S protein are useful not only for designing vaccine and antiviral but also for building the basis for HuNAb-cocktailed treatment.

To study the neutralizing potency of HuNAbs targeting the S glycoprotein of SARS-CoV-2, both pseudovirus and authentic virus neutralization assays have been developed. For the pseudovirus assay, two systems have been established through co-transfection with the functional S glycoprotein of SARS-CoV-2. One system employs the luciferase reporter in the backbone of lentivirus (e.g., HIV-1) (33; 34). The other system employs the luciferase reporter in the backbone of vesicular stomatitis virus (VSV) (26; 35) . The authentic neutralization assay uses primary SARS-CoV-2 strains isolated from COVID-19 patients directly. It should be noted that different research groups may use various types of target cell for in vitro neutralization assays, which may generate incomparable data to compare the neutralization potency. For example, various Vero cell lines and 293T cells stably expressing human ACE2 have been extensively used for viral neutralization assays. Bearing this in mind, one may refer to some representative HuNAbs in (Table 1) . All these HuNAbs are generated in the native form of IgG1. Usually, HuNAbs with the half-maximal inhibitory concentrations (IC 50 ) less than 0.1 µg/mL is categorized as potent neutralizers whereas those with IC 50 values of 0.1-1 µg/mL and 1-10 µg/mL is considered as moderate and weak neutralizers, respectively (36) . The most potent SARS-CoV-2 HuNAb may display IC 50 values at the single digit ng/mL. For example, one of the most potent HuNAbs, namely 2-15, displays the IC 50 value of as low as 0.7 ng/mL (26) .

Several animal models have been established to mimic the natural course of SARS-CoV-2 infection in humans. Since ACE2 contains natural variation in different animal species, their susceptibility to live SARS-CoV-2 infection may vary significantly. Based on the structural analysis, there are 29 amino acid residues at the interface of ACE2 and SARS-CoV-2 RBD, which determines the binding affinity of these two proteins. Sequence alignment of these 29 amino acid residues reveals high similarity between human ACE2 with homologues of Syrian golden hamster, rhesus macaque and common marmoset (37) . Since hamster ACE2 exhibits the high binding affinity with the S glycoprotein of SARS-CoV-2 by in silico prediction, this animal model exhibits acute clinical and histopathological manifestations that models the upper and lower respiratory tract infection in humans (37) . Non-M a n u s c r i p t 8 human primates share 100% identity of these 29 amino acid residues with human ACE2 but only displays a transient and mild clinical manifestation after live SARS-CoV-2 challenge (38) (39) (40) . The amount of ACE2 and cellular protease expression in upper and lower respiratory tracts of rhesus monkeys likely affects their susceptibility to live SARS-CoV-2 infection, which explains why the intratracheal viral inoculation has been used for viral challenge in the experiments. Mouse is generally resistant to wildtype SARS-CoV-2 challenge because mouse ACE2 does not effectively bind the viral S glycoprotein (41) . However, after transduction with adenovirus-or adeno-associated virus-vectored human ACE2 (Ad5-hACE2 or AAV-hACE2), mouse becomes susceptible to SARS-CoV-2 infection (42) . Meantime, transgenic mice with human ACE2 expression leads to mild or lethal SARS-CoV-2 infection (43) . Subsequently, SARS-CoV-2 with the N501Y mutation can interact with murine ACE2 and infect mouse species directly (44; 45) . These animal models have been widely used to evaluate the in vivo efficacy of various HuNAbs and vaccines against SARS-CoV-2 ( Table 2) . We and others reported that most of these HuNAbs can suppress viral loads in lungs and alleviate lung injury in animal models. We, however, found that systemic HuNAb injection or DNA vaccination has limited efficacy in preventing SARS-CoV-2 nasal infection in Syrian hamsters likely due to insufficient biodistribution of antibody at the site of viral transmission (21) . Using the same model, we recently found that potent neutralizing dimeric IgA may enhance SARS-CoV-2 nasal infection probably by engaging alternative cellular entry and cell-to-cell transmission mechanisms (46) . In fact, few animal studies have showed significant reduction of nasal viral loads in various animal models post HuNAb injection and systemic vaccination (26; 47; 48) , implicating that exceptionally high dose is likely required for improved protection. Some published studies claim sterile protection without actual evaluation of SARS-CoV-2 nasal infection. These results explain the rising number of re-infections and over thousands of vaccine-breakthrough infections in humans (49) (50) (51) . The data generated in animal models, therefore, may have significant implications to human vaccine and passive immunization studies.

Several anti-SARS-CoV-2 HuNAbs have been developed for clinical studies. Paired HuNAbs have been formulated to improve the breadth of neutralization due to emerged SARS-CoV-2 variants and to minimize HuNAb escape variants. Most neutralizing antibodies under Emergency Use Authorization (EUA) showed live virus neutralization IC 50 below 0.1 µg/mL. The paired antibodies often include one RBS-A HuNAb plus either one RBS-B or one RBS-C HuNAb. For example, the pair from the Eli Lily company included the RBS-B LY-CoV555 (bamlanivimab, BAM) and the RBS-A LY-CoV016 (etesevimab, ETE) with live virus neutralizing IC 50 around 0.02 µg/mL and 0.036 µg/mL, respectively (52; 53). LY-CoV555 and LY-CoV016 were probably the first paired HuNAbs administered into COVID-19 patients in early June 2020 (54) . The randomized phase 2/3 trial evaluated the efficacy of combined LY-CoV555 (2800 mg) and LY-CoV016 (2800 mg) as compared with the LY-CoV555 monotherapy. A statistically significant reduction in viral load was found at day 11 among non-hospitalized patients with mild to moderate illness by the combination therapy but not by the monotherapy (54) . Subsequently, another clinical trial reported the REGN-CoV2 HuNAbs for prophylaxis and immunotherapy of COVID-19 patients. This phase 1/2/3 trial showed that both 2400 and 8000 mg of REGN-COV2 antibody cocktail showed approximately 2-log reduction of viral loads in patients with baseline viral load higher than 10 7 copies/mL compared with the placebo group (55) . It should be noted that these clinical M a n u s c r i p t 9 dosages are indeed much higher than 10-20 mg/kg tested in small animal models. Then, both companies have acquired the permission from the U.S. Food and Drug Administration (FDA) for EUA of their HuNAbs. Now, these HuNAbs are used for emergency treatment of mild-tomoderate adult and paediatric patients (age 12 and older with body weight at least 40 kg) who have positive viral loads and are at high risk for progressing to severe COVID-19. 700 mg LY-CoV555 is recommended as monotherapy, or it can be combined with 1400 mg LY-CoV016. As for the REGN-CoV2, it consisted of paired RBS-A REGN10933 (casirivimab, CAS) and RBS-C REGN10987 (imdevimab, IMD) at 1:1 ratio. REGN10933 and REGN10987 displayed live virus neutralizing IC 50 around 0.0056 µg/mL and 0.0063 µg/mL, respectively (20) . The recommendation dosage of REGN-CoV2 is 2400 mg (1200 mg each) (56) . Recently, the combination of BRII-196 and BRII-198 was developed by the BRII Biosciences including one RBS-A and one RBS-C antibody, both with live virus neutralizing IC 50 around 0.03 µg/mL. This antibody combination showed 78% efficacy in trials and has been approved for clinical use on 9 December 2021 by China FDA. Since antibody combination therapy may face reduced neutralizing abilities against SARS-CoV-2 variants of concerns (VOC), more broadly reactive HuNAbs and next generation antibodies, such as bispecific antibodies and engineered antibodies are on the list for clinical trials in different regions and countries. 6 Impact of SARS-CoV-2 VOCs on HuNAbs SARS-CoV-2 is an RNA virus that is usually prone to mutate during the natural course of infection in humans. SARS-CoV-2, however, has a self-proof-correcting machinery system that maintains the relative genomic fidelity during viral replication (57) . Analysis on global phylogenies indicates a slow mutation rate of approximately two mutations per month in viral genome (58) . Unlike SARS in 2003, since COVID-19 has not been eliminated till now, there is growing concern on viral escapes due to immune pressure conferred by natural infection or vaccination. Indeed, the increasing and longlasting COVID-19 pandemic has already resulted in several VOCs with various resistance profiles to HuNAbs. (59) . Since late November, 2021, a new VOC, B.1.1.529 (Omicron, South Africa) has been predicted to become another dominated strain. The Omicron variant was first discovered in South Africa with heavily mutated S glycoprotein containing around 26 to 32 mutations mainly in the RBD region, in addition to 3 deletions and one insertion (60) . The mutations or deletions of amino acids in the S glycoprotein of VOCs have led to not only HuNAb resistance but also increased the affinity binding to human ACE2 for higher viral transmissibility (61; 62) . Critically, all these five VOCs contain the D614G mutation, which enhances viral transmissibility and infectivity (62) . Moreover, the E484 position in RBD is an important binding site for many RBD-specific HuNAbs (61) . Unfortunately, VOCs of B.1.1.7, B.1.351 and P1 all have the E484K mutation. Moreover, some amino acid deletions in the S protein, such as the 242-244del in B.1.351 variants and M a n u s c r i p t 10 the 144del in B.1.1.7 variants reduced more than 1000-fold neutralizing activity of the NAbs that target the supersite of NTD domain (35) . To date, the impact of S mutations in Omicron variants on the ongoing pandemic remains unknown. Various combinations of these mutations and deletions in emerging VOCs, however, have substantially reduced potency of HuNAbs elicited by vaccines and passive immunization, and therefore, becomes new challenges to public health and vaccine responses. Hopefully, the boost vaccine and immunotherapy using cocktailed HuNAbs can overcome these VOCs. 7 Future perspectives With continuously emerged VOCs, vaccine-induced correlates of immune protection remain to be comprehensively investigated. Due to urgency of the COVID-19 pandemic, huge global efforts have been placed for vaccine development. Till now, six vaccines have been approved by regulatory agencies for emergency use within just one year of the pandemic including (1) two mRNA-based vaccines, namely BNT162b2 (by Pfizer Inc. and BioNTech SE) and mRNA-1273 (by Moderna), expressing full S glycoprotein with an efficacy rate of 95% (63; 64), (2) the chimpanzee adenovirus-vectored vaccine, named ChAdOx1 nCoV-19 (by the Oxford University and AstraZeneca Inc.), encoding the full S glycoprotein with an efficacy rate of 70.4% (65), (3) the human adenovirus-vectored vaccine, namely Ad26.COV2.S (by Johnson & Johnson Inc.), encoding the full S glycoprotein with an efficacy rate of 73.1%, (4) two inactivated vaccines CoronaVac and BIBP (by Sinovac Biotech and SinoPharm) with an efficacy rate of 83.5% and 78.1%, respectively. Since these efficacy rates were mainly determined for prevention of severe diseases, their potency in preventing SARS-CoV-2 nasal infection and eliciting broadly reactive HuNAbs against VOCs remains to be carefully investigated. In recent studies, the B.1.351 variant was significantly resistant (10.3-12.4-fold) to neutralization by sera derived from vaccinated individuals (Moderna or BioNTech) compared to the D614G strain (35) . Although vaccinations reduced death rates, vaccine-induced attenuation of peak viral burden has decreased for the B.1.617.2 variant (absolute difference of 10-13% for BNT162b2 and 16% for ChAdOx1) compared to the B.1.1.7 variant in UK (66) . These results are in line with the increasing number of breakthrough infections among fully vaccinated population (49) . Nevertheless, extensive vaccination programs among general populations have contributed greatly to reduced numbers of hospitalization, mortality, and infections in countries like Israel, UK, and USA even when some VOCs have displayed reduced neutralization sensitivity (35) . Future boost vaccination and HuNAb-based immunotherapy should focus on broadly reactive immune protection. Since systemic HuNAb did not completely prevent SARS-CoV-2 nasal infection, the role of mucosal immunity especially tissue resident memory T cells in upper respiratory tract should be studied for long-term protection. Lastly, antibody-mediated enhancement of SARS-CoV-2 infection and immunopathogenesis should still be carefully monitored in the context of human infections and clinical care. M a n u s c r i p t 11 M a n u s c r i p t 19 Figure 1 

Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

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Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

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The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates

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SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody

REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters

Potently neutralizing and protective human antibodies against SARS-CoV-2

This review was submitted on behalf of the research group from AIDS Institute and Department of Microbiology, Li Ka Shing Faculty of Medicine, the University of Hong Kong and Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park. The Editor-in-Chief, Tim Elliott, and handling editor, Tao Dong, would like to thank the following reviewer, Ricardo Fernandes, and an anonymous reviewer, for their contribution to the publication of this article.

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