Antibody Affinity Maturation Research Articles

Immunoglobulin VDJ repertoires reveal hallmarks of germinal centers in unique cell clusters isolated from zebrafish (Danio rerio) lymphoid tissues.

DNA mutagenesis during antibody affinity maturation has potentially oncogenic or autoimmune outcomes if not tightly controlled as it is in mammalian germinal centers. Cold blooded vertebrates lack germinal centers, yet have a functional Ig gene mutator enzyme, Aicda. In fish there are clusters of Aicda+ cells encircled by pigmented 'melano-macrophages' and we test the hypothesis that these clusters are functionally analogous to germinal centers. Sequenced IgH VDJ repertoire libraries from individual isolated clusters showed evidence of B-cell clonal expansion and VDJ somatic hypermutation. Construction of Ig clonal lineage trees revealed that unlike surrounding lymphoid tissue, each cluster is dominated by a few B-cell VDJ clonotypes having hundreds of mutated variants. Recruitment of B-cells to the clusters appears to be ongoing, as there are additional Ig clones having smaller lineages. Finally, we show evidence for positive selection for replacement mutations in regions encoding the antigen contact loops, but not in the framework regions, consistent with functional antibody modification. Melano-macrophages appear to trap the Ag used for post-mutation B-cell selection, performing a role analogous to the follicular dendritic cells of mammalian germinal centers. These findings provide insights into the evolution of the affinity maturation process, the improvement of fish vaccines and possibly also the workings of atypical ectopic germinal centers generated in several human diseases.

A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure-guided engineering

The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependentanti-EGFR antibodies can overcome multiple limitationswith antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigenbinding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.

Structure-Based Affinity Maturation of Antibody Based on Double-Point Mutations.

Structure-based site-directed affinity maturation of antibodies can be expanded by multiple-point mutations to obtain various mutants. However, selecting the appropriate number of promising mutants for experimental evaluation from the vast number of combinations of multiple-point mutations is challenging. In this report, we describe how to narrow candidate mutants using the so-called weak interaction analysis such as CH-π and CH-O in addition to widely recognized interactions such as hydrogen bonds.

Antibody Affinity Maturation Using Computational Methods: From an Initial Hit to Small-Scale Expression of Optimized Binders.

Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes.

Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses.

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of "first responders" (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and Tcell responses invivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.

Hallucinating structure-conditioned antibody libraries for target-specific binders.

Antibodies are widely developed and used as therapeutics to treat cancer, infectious disease, and inflammation. During development, initial leads routinely undergo additional engineering to increase their target affinity. Experimental methods for affinity maturation are expensive, laborious, and time-consuming and rarely allow the efficient exploration of the relevant design space. Deep learning (DL) models are transforming the field of protein engineering and design. While several DL-based protein design methods have shown promise, the antibody design problem is distinct, and specialized models for antibody design are desirable. Inspired by hallucination frameworks that leverage accurate structure prediction DL models, we propose the FvHallucinator for designing antibody sequences, especially the CDR loops, conditioned on an antibody structure. Such a strategy generates targeted CDR libraries that retain the conformation of the binder and thereby the mode of binding to the epitope on the antigen. On a benchmark set of 60 antibodies, FvHallucinator generates sequences resembling natural CDRs and recapitulates perplexity of canonical CDR clusters. Furthermore, the FvHallucinator designs amino acid substitutions at the VH-VL interface that are enriched in human antibody repertoires and therapeutic antibodies. We propose a pipeline that screens FvHallucinator designs to obtain a library enriched in binders for an antigen of interest. We apply this pipeline to the CDR H3 of the Trastuzumab-HER2 complex to generate in silico designs predicted to improve upon the binding affinity and interfacial properties of the original antibody. Thus, the FvHallucinator pipeline enables generation of inexpensive, diverse, and targeted antibody libraries enriched in binders for antibody affinity maturation.

Structural basis of interleukin-17B receptor in complex with a neutralizing antibody for guiding humanization and affinity maturation

Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.

Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVCEnv) covalently complexed to two-domain CD4S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits

Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.

Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry.

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO22+ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity.

Potential of conserved antigenic sites in development of universal SARS-like coronavirus vaccines.

Given pandemic risks of zoonotic SARS-CoV-2 variants and other SARS-like coronaviruses in the future, it is valuable to perform studies on conserved antigenic sites to design universal SARS-like coronavirus vaccines. By using antibodies obtained from convalescent COVID-19 patients, we succeeded in functional comparison of conserved antigenic sites at multiple aspects with each other, and even with SARS-CoV-2 unique antigenic sites, which promotes the cognition of process of humoral immune response to the conserved antigenic sites. The conserved antigenic sites between SARS-CoV-2 and SARS-CoV can effectively induce affinity maturation of cross-binding antibodies, finally resulting in broadly neutralizing antibodies against multiple variants of concern, which provides an important basis for universal vaccine design, however they are subdominant, putatively due to their lower accessibility relative to SARS-CoV-2 unique antigenic sites. Furthermore, we preliminarily design RBDs to improve the immunogenicity of these conserved antigenic sites. Our study focusing on conserved antigenic sites provides insights for promoting the development of universal SARS-like coronavirus vaccines, thereby enhancing our pandemic preparedness.

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape pathways

SummaryThe rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

Antibody affinity and cross-variant neutralization of SARS-CoV-2 Omicron BA.1, BA.2 and BA.3 following third mRNA vaccination

There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in naïve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with naïve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with naïve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants.

On the Rapid Calculation of Binding Affinities for Antigen and Antibody Design and Affinity Maturation Simulations.

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process.

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display.

Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.

Targeting TFH cells in human diseases and vaccination: rationale and practice.

The identification of CD4+ T cells localizing to B cell follicles has revolutionized the knowledge of how humoral immunity is generated. Follicular helper T (TFH) cells support germinal center (GC) formation and regulate clonal selection and differentiation of memory and antibody-secreting B cells, thus controlling antibody affinity maturation and memory. TFH cells are essential in sustaining protective antibody responses necessary for pathogen clearance in infection and vaccine-mediated protection. Conversely, aberrant and excessive TFH cell responses mediate and sustain pathogenic antibodies to autoantigens, alloantigens, and allergens, facilitate lymphomagenesis, and even harbor viral reservoirs. TFH cell generation and function are determined by T cell antigen receptor (TCR), costimulation, and cytokine signals, together with specific metabolic and survival mechanisms. Such regulation is crucial to understanding disease pathogenesis and informing the development of emerging therapies for disease or novel approaches to boost vaccine efficacy.

Boosting corrects a memory B cell defect in SARS-CoV-2 mRNA-vaccinated patients with inflammatory bowel disease.

Immunosuppressed patients with inflammatory bowel disease (IBD) generate lower amounts of SARS-CoV-2 spike antibodies after mRNA vaccination than healthy controls. We assessed SARS-CoV-2 spike S1 receptor binding domain–specific (S1-RBD–specific) B lymphocytes to identify the underlying cellular defects. Patients with IBD produced fewer anti–S1-RBD antibody–secreting B cells than controls after the first mRNA vaccination and lower amounts of total and neutralizing antibodies after the second. S1-RBD–specific memory B cells were generated to the same degree in IBD and control groups and were numerically stable for 5 months. However, the memory B cells in patients with IBD had a lower S1-RBD–binding capacity than those in controls, which is indicative of a defect in antibody affinity maturation. Administration of a third shot to patients with IBD elevated serum antibodies and generated memory B cells with a normal antigen-binding capacity. These results show that patients with IBD have defects in the formation of antibody-secreting B cells and affinity-matured memory B cells that are corrected by a third vaccination.

Isotype-specific plasma cells express divergent transcriptional programs

Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell-indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation-modulating programs dictated by type 1 IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1+ and type 1 inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic type 3 IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.

Bach2 regulates B cell survival to maintain germinal centers and promote B cell memory

The transcription factor Bach2 serves as a crucial regulator of the germinal center (GC) reaction, which is required for production of high-affinity antibodies and establishment of long-lived B cell memory. However, the stage at which Bach2 controls the GC programs and the precise mechanism underlying these processes remain poorly understood. In this study, we show that genetic ablation of Bach2 in GC B cells of mice impairs their survival and maintenance, and memory B cell formation. These defects can be rescued by enforced expression of anti-apoptotic gene Bcl2. As expected, Bach2-deficient GC B cells are defective in antibody affinity maturation, but have normal somatic hyper mutation and class switch recombination of immunoglobulin genes. Mechanistically, Bach2 controls the GC programs by directly repressing pro-apoptotic gene Bim and a set of genes involved in cell stress response and metabolic processes. Thus, our work reveals the precise roles of Bach2 in the GC biology, and demonstrates that Bach2 acts as a crucial survival regulator of GC B cells, providing a key mechanism underlying GC B maintenance and B cell memory formation.

Germinal Center-Related G Protein-Coupled Receptors in Antibody-Mediated Autoimmune Skin Diseases: from Basic Research to Clinical Trials.

Germinal center (GC) reaction greatly contributes to the humoral immune response, which begins in lymph nodes or other secondary lymphoid organs after follicular B cells are activated by T-dependent antigens. The GCs then serve as a platform for follicular B cells to complete clonal expansion and somatic hypermutation and then interact with follicular dendritic cells (FDC) and follicular helper T cells (Tfh). Through the interaction between the immune cells, significant processes of the humoral immune response are accomplished, such as antibody affinity maturation, class switching, and production of memory B cells and plasma cells. Cell positioning during the GC reaction is mainly mediated by the chemokine receptors and lipid receptors, which both belong to G protein-coupled receptors (GPCRs) family. There are some orphan GPCRs whose endogenous ligands are unclear yet contribute to the regulation of GC reaction as well. This review will give an introduction on the ligands and functions of two types of GC-relating GPCRs-chemokine receptors like CXCR4 and CXCR5, as well as emerging de-orphanized GPCRs like GPR183, GPR174, and P2RY8. The roles these GPCRs play in several antibody-mediated autoimmune skin diseases will be also discussed, including systemic lupus erythematosus (SLE), pemphigus, scleroderma, and dermatomyositis. Besides, GPCRs are excellent drug targets due to the unique structure and vital functions. Therefore, this review is aimed at providing readers with a focused knowledge about the role that GPCRs play in GC reaction, as well as in provoking the development of GPCR-targeting agents for immune-mediated diseases besides autoimmune diseases.

Structure-guided affinity maturation of a novel human antibody targeting the SARS-CoV-2 nucleocapsid protein

The continuous mutation of SARS-CoV-2 has presented enormous challenges to global pandemic prevention and control. Recent studies have shown evidence that the genome sequence of SARS-CoV-2 nucleocapsid proteins is relatively conserved, and their biological functions are being confirmed. There is increasing evidence that the N protein will not only provide a specific diagnostic marker but also become an effective treatment target. In this study, 2G4, which specifically recognizes the N protein, was identified by screening a human phage display library. Based on the computer-guided homology modelling and molecular docking method used, the 3-D structures for the 2G4 scFv fragment (VH-linker-VL structure, with (G4S)3 as the linker peptide in the model), SARS-CoV-2 N protein and its complex were modelled and optimized with a suitable force field. The binding mode and key residues of the 2G4 and N protein interaction were predicted, and three mutant antibodies (named 2G4-M1, 2G4-M2 and 2G4-M3) with higher affinity were designed theoretically. Using directed point mutant technology, the three mutant antibodies were prepared, and their affinity was tested. Their affinity constants of approximately 0.19 nM (2G4-M1), 0.019 nM (2G4-M2) and 0.075 nM (2G4-M3) were at least one order of magnitude lower than that of the parent antibody (3 nM; 2G4, parent antibody), as determined using a biolayer interferometry (BLI) assay. It is expected that high-affinity candidates will be used for diagnosis and even as potential therapeutic drugs for the SARS-CoV-2 pandemic.