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Article

Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library

by
Isamu Tsuji
1,*,
Kumiko Okada
2,
Benjamin Kroppen
3,
Tetsufumi Katta
2,
Kaori Yamamura
2,
Takeshi Nishihama
2,
Ayako Miura
1,
Hansjörg Götzke
3,
Eric Crampon
1 and
Andrea Bertolotti-Ciarlet
1
1
Vaccine Bioanalytical Development, Vaccine Business Unit, Takeda Pharmaceutical Company Ltd., Cambridge, MA 02139, USA
2
Vaccine MS&T Japan, Global Vaccine Business Unit, Takeda Pharmaceutical Company Ltd., Hikari 743-8502, Japan
3
NanoTag Biotechnologies GmbH, 37079 Göttingen, Germany
*
Author to whom correspondence should be addressed.
Antibodies 2026, 15(2), 18; https://doi.org/10.3390/antib15020018
Submission received: 5 January 2026 / Revised: 9 February 2026 / Accepted: 11 February 2026 / Published: 24 February 2026
(This article belongs to the Section Antibody Discovery and Engineering)
Browse Figures
Review Reports Versions Notes

Abstract

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing.

Graphical Abstract

1. Introduction

The SARS-CoV-2 virus emerged in Wuhan, China, in December 2019. The virus rapidly spread worldwide; the first mortality case was reported in the United States in February 2020. The case fatality rate (CFR) from hospitalized patients was estimated at around 20% in the early stage of the pandemic [1]; however, the rate decreased by 96% over a period of 2.5 years after the pandemic’s onset [2]. There were two aspects to reducing the CFR. The first aspect was the development of various remedies, such as small molecules [3], monoclonal antibodies (mAbs) [4] and vaccines [5], which were developed in the early stages of the SARS-CoV-2 virus pandemic. The second aspect was the rapid mutation of the SARS-CoV-2 virus, allowing it to evade the human immune system. The Wuhan strain dominated in 2020, the strains Alpha (B.1.1.7 lineages) and Delta (B.1.6.17.2 lineages) in 2021, and the Omicron variants (BA.1, BA.2, BA.5) in 2022 [6]. In early 2023, XBB.1.5, an Omicron sub-lineage strain, dominated in the United States [7], and JN.1 strains prevailed in January 2024 [8]. The mutated virus gradually reduced its toxicity and reduced the CFR.
MAb therapy for SARS-CoV-2 was applied in the early stages of the pandemic [4] and was approved by the U.S. Food and Drug Administration (FDA) [9,10,11]. These mAbs were specific to Wuhan strain, and lost their neutralizing activity against the new, emerging variants, such as the Omicron variant [12]. Recent approaches have included the discovery of broad strain-neutralizing mAbs [13,14], but these have not been developed or approved as therapeutic. Therefore, vaccination remains one of the most effective approaches to eradicating the virus. Messenger RNA, virus vector, inactivated virus and protein vaccines have been developed against SARS-CoV-2 [5]. Due to the frequent mutation of SARS-CoV-2, vaccine manufacturers produce a new vaccine for each virus strain annually. Assay development is essential for every strain to confirm and distinguish the vaccine. The vaccine identification test is a qualitative assay that verifies the new SARS-CoV-2 vaccine strain, not previous strains, as stated in the FDA guidance [15]. Immunological assay techniques, such as enzyme-linked immunosorbent assay (ELISA) and Western blotting, are widely applied to prove the strains for protein-based vaccines [16]. And although mass spectrometry analysis has recently been reported to detect vaccine-strain-specific peptides [17], immunological assay approaches are still preferred for convenience in quality control laboratories at manufacturing sites. Subsequently, highly sensitive and specific mAbs must be created to ensure the vaccine’s strain specificity, even though neutralizing activity is not essential for its mAb character. There are two approaches to discovering mAbs [18]. Mice and rabbit immunization are widely applied, followed by hybridoma fusion with myeloma or B-cell sorting [19,20]. However, animal immunization takes several months, so applying this technology to critical reagents for the SARS-CoV-2 vaccine would be difficult. Another approach using synthetic antibody libraries can quickly discover mAbs, as the libraries do not require immunizing animals [21]. Human single-chain variable fragment (scFv) and fragment antigen-binding region (Fab) libraries are widely used as synthetic libraries. These libraries connect heavy and light chain variable regions with synthetic linkers. Aggregation, low solubility, and low expression in E. coli may occur in both libraries [22], and converting full antibodies is challenging for scFvs. Llama, alpaca, and camel immunoglobulins have unique features compared with other species. These antibodies lack light chains; thus, generating an antibody library as a single domain is straightforward [23,24,25]. Therefore, using a synthetic library, we have developed rapid methods to generate SARS-CoV-2 strain-specific single-domain antibodies (sdAbs) to overcome the frequent emergence of new strains.

2. Materials and Methods

2.1. Materials

The receptor-binding domain (RBD) of spike proteins of Wuhan (NCBI GenBank Accession No.: MN908947.3), XBB.1.5 (NCBI GenBank Accession No.: OR782922.1) and JN.1 (NCBI GenBank Accession No: PQ121600.1) strains and Angiotensin-converting enzyme 2 (ACE2) were produced by Trenzyme (Konstanz, Germany) with or without C-terminal biotinylation. BA.5 (NCBI GenBank Accession No: OR939741.1) RBD protein was obtained from The Native Antigen (Oxford, UK). High-precision streptavidin (SAX) biosensors were purchased from Sartorius (Fremont, CA, USA).

2.2. Discovery of Anti-SARS-CoV-2 sdAb

A synthetic sdAb library was developed based on alpaca sdAb frameworks with degrees of freedom in the three complementarity-determining regions (CDRs). Antigen-specific sdAb clones were selected from the library through three rounds of panning, consisting of one initial ribosome display followed by two rounds of phage display. In each panning, biotinylated XBB.1.5 or JN.1 SARS-CoV-2 RBD proteins were used as bait at decreasing concentrations. In addition, Wuhan RBD protein for XBB.1.5 selection and Wuhan, BA.5, and XBB.1.5 RBD proteins for JN.1 selection were used as counter-selectors, which were previously prepared vaccine strain RBDs, during the panning to remove universal RBD protein binders, thereby achieving strain specificity. Following the panning process, single-positive clones were selected based on their lack of cross-reactivity with counter-selected proteins or streptavidin, as indicated by an absorbance below 0.1 at 450–620 nm in the ELISA. As a result, 384 out of 450 single clones of XBB.1.5 and three out of 96 single clones of JN.1 were selected from the criteria, and the CDR sequences were subsequently used to further narrow down the search. The final clones were selected based on the specificity, affinity, and uniqueness of their amino acid sequences among the positive clones. Finally, five unique XBB.1.5-specific sdAbs and two unique JN.1-specific sdAbs were selected from these selections. Two sdAb-rabbit fragment crystallizable region (Fc) fusion proteins were selected from five XBB.1.5 sdAbs due to the homology of CDR3 regions and longer CDR3 amino acid sequences. SdAbs and sdAb–rabbit Fc fusion proteins were expressed by E. coli, and mammalian cells. Protein purifications were conducted using affinity purification resins.

2.3. CDR Amino Acid Sequence Analysis

CDR amino acid sequence analysis of anti-XBB.1.5 sdAb and anti-JN.1 sdAb was conducted by IMGT-V-Quest, The International ImMunoGeneTics Information System, Montpellier, France, https://www.imgt.org/IMGT_vquest/input (accessed on 10 February 2026) [26].

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

An ELISA was conducted by capturing the RBD protein using Neutravidin (Thermo Fisher, Waltham, MA, USA), which was coated on a 96-well ELISA plate (MaxiSorp, Thermo Fisher, Waltham, MA, USA). Briefly, 100 µL of 5 µg/mL Neutravidin in phosphate-buffered saline (PBS) buffer was coated on the ELISA plate overnight, followed by blocking with 100 µL Superblock T20 (PBS) blocking buffer (Thermo Fisher, Waltham, MA, USA) at 37 °C for 60 min. After washing three times with PBS-0.05% Tween 20 (PBS-T), 100 µL of 1 µg/mL RBD protein in 20% Superblock T20 (PBS) blocking buffer in PBS-T was added and the plate was incubated at 37 °C for 60 min. After washing three times with PBS-T, 100 µL of anti-XBB.1.5 and JN.1 sdAb or sdAb-Fc fusion proteins at various concentrations were incubated at 37 °C for 60 min. After washing three times with PBS-T, horseradish peroxidase-conjugated DYKDDDDK tag mAb (1 µg/mL 20% Superblock T20 (PBS) blocking buffer in PBS-T: Thermo Fisher, Waltham, MA, USA) for sdAb and anti-rabbit IgG (5000 times dilution to PBS-T: Jackson Immuno Research Laboratories, West Grove, PA, USA) for sdAb-Fc fusion protein were added to 100 µL/well and incubated at 37 °C for 60 min. After washing three times with PBS-T, the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) substrate kit (Sera-care, Milford, MA, USA) was used for detection, and absorbance was measured at 405 nm using a plate reader (SpectraMax: Molecular Devices, Silicon Valley, CA, USA). The assay was performed as a single run for each concentration and its data was analyzed; reproducibility was confirmed in independent runs.

2.5. Equilibrium Dissociation Constant, KD, Measurement

Antibody kinetic analyses were conducted using Octet HTX systems (Sartorius, Fremont, CA, USA). Briefly, 5 µg/mL biotinylated XBB.1.5 or JN.1 RBD proteins were captured on a SAX biosensor (Sartorius, Fremont, CA, USA). Then, the excess streptavidin in the biosensor was blocked by 50 µg/mL biocytin (Thermo Fisher, Waltham, MA, USA) and 0.18–12 µg/mL anti-XBB.1.5 or JN.1 sdAb or sdAb-Fc fusion protein in PBS-T containing 0.1% bovine serum albumin (BSA Millipore Sigma Burlington, MA, USA) was associated with RBD protein for 600 s and dissociated for 900 s. The assays were performed in three independent runs, and the average values were calculated.

2.6. Inhibitory Assay with ACE2/RBD Protein

Inhibitory assay of anti-XBB.1.5 and JN.1 sdAb-Fc fusion proteins to ACE2 were conducted by Octet HTX systems (Sartorius, Fremont, CA, USA). Briefly, 5 µg/mL biotinylated XBB.1.5 or JN.1 RBD proteins were captured using a SAX biosensor (Sartorius, Fremont, CA, USA). Then, excess streptavidin in the biosensor was blocked by 50 µg/mL biocytin (Thermo Fisher, Waltham, MA, USA). A total of 0.06–6 µg/mL anti-XBB.1.5 or JN.1 sdAb-Fc fusion proteins in 0.1% BSA PBS-T were bound with RBD protein for 600 s, then 1 µg/mL ACE2 protein was associated for 600 s. ACE2 binding responses were calculated by Octet Data Analysis Software HT (ver. 11.1.2.48 Sartorius, Fremont, CA, USA). The following equations were calculated for the inhibitory activity:
I n n i b i t o r y   a c t i v i t y % = ( R e s p o n s e   a t   0   µ g m L s d A b   F c   f u s i o n   p r o t e i n − R e s p o n s e   a t   X   µ g m L s d A b   F c   f u s i o n   p r o t e i n ) ÷ R e s p o n s e   a t   0   µ g m L s d A b   F c   f u s i o n   p r o t e i n × 100
The assay was performed in three independent experiments, and the results are presented as means with standard deviations in the graphs.

2.7. Western Blotting Analysis

SARS-CoV-2 vaccine drug substances were denatured under reduced or non-reduced conditions with β-mercaptoethanol. These samples were loaded at 3.5 µg per lane and run on NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher, Waltham, MA, USA). After running, the gel was transferred to the PVDF membrane. The blotting membrane was blocked with 5% skimmed milk and then reacted with 8 µg/mL anti-JN.1 1B9 sdAb, 1:500, for 60 min. After washing the membrane with PBS-T, the membrane was incubated with 1:5000 anti-FLAG M2–alkaline phosphatase conjugate (Millipore Sigma, Burlington, MA, USA) for 60 min. After washing with PBS-T, colorimetric development was conducted using a BCIP/NBT phosphatase substrate kit (Thermo Fisher, Waltham, MA, USA).

2.8. Protein and Antibody Preparation for Binding Epitope Analysis

Two solutions of JN.1 RBD protein were prepared. JN.1 RBD protein alone (12 µM, 150 µL) and JN1 RBD protein mixed with anti-JN.1 1B9 sdAb (ratio 1:2) with a respective final concentration 24 µM (150 µL). These solutions were introduced into the hydrogen–deuterium exchange (HDX) robotic system, and 4 µL of each solution was injected for HDX liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

2.9. Hydrogen–Deuterium Exchange, On-Line Digestion, and LC-MS Analysis

HDX reactions were initiated by diluting JN.1 RBD protein alone or the JN.1 RBD protein:anti-JN.1 sdAb complex into D2O buffer (5 µM K2HPO4, 5 mM KHPO4, D2O, pH 6.6) at 0 °C. The exchange was quenched at 0.5, 1, 5, 10, and 60 min by adding 30 µL of quench buffer (1.5 M NaCl, 0.8% formic acid, 100 mM tris(2-carboxyethyl) phosphine, pH 2.3, 0 °C). The HDX sample-handling robotics (Waters, Milford, MA, USA) performed all necessary pipetting, quenching and proteolysis steps before direct injection into the Q-ToF Xevo G2-XS mass spectrometer (Waters, Milford, MA, USA) in positive ion mode. Digestion was conducted with an immobilized pepsin column (Enzymate BEH Pepsin 2.1 × 30; Waters, Milford, MA, USA). Peptic peptides were trapped and separated on a reversed-phase column (BEH C18, 1.0 mm × 100 mm, 1.7 µm particles; Waters, Milford, MA, USA) after a VanGuard trap column (BEH C18, 1.7 µm; Waters, Milford, MA, USA). Peptide identification was performed using ProteinLynx Global SERVER (Waters, Milford, MA, USA) and deuterium uptake was calculated using DynamX software (Version 3.0 Waters, Milford, MA, USA). Epitope mapping was determined by comparing uptake between the free JN.1 RBD protein and JN.1 RBD protein–anti-JN.1sdAb complex. Differential uptake greater than 10% between JN.1 RBD protein and JN.1 RBD protein–anti-JN.1 sdAb complex was determined to be significant [27]. Epitope mapping peptides were mapped to the Wuhan RBD protein (Protein Data Base (PDB):6M0J) for visualization.

2.10. Statics Analysis

The half-maximal inhibitory concentration (IC50) and half-maximal effective concentration (EC50) were calculated using GraphPad Prism (Ver 9.3.1, GraphPad Software, San Diego, CA, USA). Kinetic parameters, association constant (ka), and dissociation constant (kdis) were analyzed by Octet Data Analysis Software HT (ver. 11.1.2.48 Sartorius, Fremont, CA, USA) with the 1:1 monovalent binding model for sdAb and 1:2 bivalent binding model for sdAb-Fc fusion protein.

3. Results

3.1. Discovery of Anti-SARS-CoV-2 sdAbs

Five unique anti-XBB.1.5 sdAbs and two unique anti-JN.1 sdAbs were discovered from a synthetic sdAb library developed using alpaca sdAb frameworks (Figure 1). These sdAb discovery processes took 4 weeks to select the candidate clones and 2–3 weeks for the expression and purification of sdAb and sdAb-Fc fusion proteins. Table 1 shows the CDR amino acid sequences of anti-SARS-CoV-2 sdAbs. The lengths of CDR1 and CDR2 were eight amino acids, and CDR3′s length varied, ranging from 12 (anti-XBB.1.5: O_P3B7) to 17 amino acids (anti-XBB.1.5: MixP2B3, MixP2B7 and CK_P3C10). Anti-XBB.1.5 sdAb clones MixP2B6 and CK_P3C10 were selected for sdAb-Fc fusion protein due to the uniqueness of CDR3 amino acid sequence and longer CDR3 length. These two clones of anti-XBB.1.5 sdAb-Fc fusion proteins were named as clone B6 and C10. Clones of 1B9 and 1A10 of anti-JN.1 sdAb also fused with rabbit Fc for future analysis.

3.2. Binding Activities of Anti-XBB.1.5 and JN.1 sdAbs and sdAb-Fc Fusion Proteins

ELISA confirmed the cross-reactivity of sdAb and sdAb-Fc fusion proteins to other serotypes. All five anti-XBB.1.5 sdAbs were specifically bound to the XBB.1.5 RBD protein, and two anti-JN.1 sdAbs can be bound to the JN.1 RBD protein but do not cross-react to absorbed serotype RBD proteins (Table 2). Anti-XBB.1.5 sdAb-Fc fusion protein clone C10 bound to the XBB.1.5 RBD protein, but B6 cross-reacted to Wuhan RBD protein (Table 3, Figure 2A). Anti-JN.1 sdAb-Fc fusion protein clones 1A10 and 1B9 were specifically bound to the JN.1 RBD protein (Figure 2B). EC50 values for these sdAb-Fc fusion proteins were smaller: C10: 3.6, B6: 42.6, 1A10: 3.3, and 1B9: 3.6 pM (Table 3).

3.3. KD, of Anti-XBB.1.5 and JN.1 sdAbs and sdAb-Fc Fusion Proteins

The equilibrium dissociation constant of anti-XBB.1.5 and JN.1 sdAbs were measured by Octet HTX. KD ranged from 495 nM for anti-XBB.1.5 sdAb clone P2B3 to 12.6 nM for anti-JN.1 sdAb clone 1A10 (Table S1). Anti-JN.1 sdAb-Fc fusion proteins improved affinity, but anti-XBB.1.5 sdAb-Fc fusion proteins did not. Anti-Anti-JN.1 sdAb clone 1A10 reduced KD value from 12.6 to 0.76 nM, and clone 1B9 reduced KD value from 14.4 to 0.19 nM when fused with Fc proteins (Table S1, Figure 2C–E). The change may be caused by the avidity effect [28]. Conversely, the fusion with Fc protein did not improve the affinity of anti-XBB.1.5 sdAb (KD: sdAb clone MixP2B6: 4.13 nM; sdAb-Fc fusion protein clone B6:14.4 nM; sdAb clone CK_P3C10: 12.9 nM; sdAb-Fc fusion protein C10: 265 nM: Table S1 and Figure 2E).

3.4. ACE2 Binding Inhibition Assay by Anti-XBB.1.5 and JN.1 sdAb-Fc Fusion Protein

The ACE2/RBD protein binding assay was developed using Octet HTX. First, biotinylated RBD proteins were captured to SAX biosensors, and then ACE2 binding with no rapid dissociation was confirmed. The inhibition assay was conducted for the binding of the anti-SARS-CoV-2 sdAb-Fc fusion protein to biotinylated RBD protein on the SAX biosensor, followed by the ACE2 binding inhibition (Figure 3A). Anti-XBB.1.5 and JN.1 sdAb-Fc fusion proteins inhibited the binding of ACE2 to RBD proteins using Octet HTX systems (Figure 3A–C). IC50 values of anti-XBB.1.5 sdAbs-Fc fusion proteins were 1.68 to 1.88 nM and anti-JN.1 sdAb-Fc fusion proteins were 2.59 to 2.95 nM (Figure 3D).

3.5. Western Blotting of SARS-CoV-2 Vaccine Candidate

We confirmed that these anti-SARS-CoV-2 sdAbs were used for vaccine identification testing. The assay was conducted by Western blotting. Anti-XBB.1.5 sdAb clone C10 detected XBB.1.5 vaccine substances but not Wuhan strains. Anti-JN.1 sdAb clone 1A9 detected JN. 1 vaccine drug substance with a theoretical molecular weight of 170 kDa, but did not react to Wuhan, BA.5, and XBB.1.5 drug substances under non-reduced conditions, and none were not detected in reduced conditions (Figure 4).

3.6. Binding Epitope Analysis by Hydrogen–Deuterium Exchange Mass Spectrometry

Four peptides of JN.1 RBD protein were identified for binding epitopes of anti-JN.1 sdAb clone 1B9: amino acids 400–422, 430–433, 453–470, and 491–495 (amino acid number: Wuhan spike protein: Figure 5A,B). These peptides contained four ACE2 contact amino acids (N417, Y453, S455 and Q493) [29]. One unique amino acid mutation—L455S of JN.1 from Wuhan, BA.5, and XBB.1.5—enhanced JN.1’s ability to evade neutralizing antibodies [30,31]. Various anti-SARS-CoV-2 mAbs binding epitopes were compared with anti-JN.1 sdAb clone 1B9 (Figure 5C): LY-CoV016 [32], LY-CoV556 [33], COV2-2196 [34], CT-P59 [35], REGN10933 [36], REGN10987 [36], SARS2-38 [37], COV2-2130 [34], S2H97 [13], and S309 [14]. Anti-JN.1 sdAb clone 1B9 binding epitopes were similar to Wuhan-specific mAbs LY-CoV016, LY-CoV556, and COV2-2196, but were different from cross-reactive mAbs’ binding epitopes, such as 2H97 and S309. LY-CoV016 shares the most binding epitopes with anti-JN.1 sdAb clone 1B9.

4. Discussion

Monoclonal antibodies are indispensable in the clinic and laboratory. They are used at the bedside as a therapeutic mAbsto treat a variety of diseases. At the lab bench, mAbs serve as essential reagents for experimental procedures. Research progress is reliant on the high quality of mAbs. Similar to the progress of research, tool mAbs play a crucial role in the manufacture of biologics, including therapeutic monoclonal antibodies, recombinant proteins, and vaccines. They are widely utilized for quality control purposes, ensuring that biologic products meet necessary standards and specifications before they are released for clinical use. Highly selective and sensitive mAbs are also critical success factors for the production of biologics.
The SARS-CoV-2 virus mutates rapidly and escapes human immune surveillance. Vaccine manufacturers produce a new vaccine for each strain annually, and assay development is crucial for confirming and distinguishing from other vaccine strains. Identification assays are essential for vaccine quality control, as stated by the FDA. Alpaca synthetic antibody libraries, which do not require animal immunization, were selected to expedite the vaccine development timeline. Five anti-XBB.1.5 and two JN.1 sdAbs were discovered in 8 to 9 weeks, which aligns with the short vaccine development timeline, and these sdAbs bound specifically to the strains.
A vaccine identification test was conducted by Western blotting using anti-JN.1 sdAb clone 1B9. The clone detected JN.1 drug substances but not Wuhan, BA.5, and XBB.1.5 vaccines that were produced in previous seasons. We found that using an alpaca synthetic antibody library is the most efficient strategy to generate tool antibodies for vaccine identification tests, providing highly specific and strong sdAbs in around two months.
Focusing on the monoclonal character, one of the features of alpaca sdAb is its longer CDR3 regions; the average CDR3 length is 18 amino acids, which is longer than those of humans (14 amino acids) and mice (13 amino acids), respectively [38,39]. Longer CDR3 lengths were critical to acquiring high and broad neutralizing activity of virus mAbs to recognize complex virus surfaces [40,41,42]. Therefore, we speculated that a highly specific sdAb had been discovered from the library at the beginning of the research. In fact, our anti-SARS-CoV-2 sdAbs were highly specific to the strains and had longer CDR3 amino acid sequences, ranging from 12 to 17 amino acids. Barnet et al. reported that anti-SARS-CoV-2 mAbs can be categorized into four classes [43]. Anti-JN.1 sdAb clone 1B9 was bound to the “up” RBD protein conformation classified as Class 1, the same as LY-CoV016 [44]. They defined Class 1 mAbs as having shorter CDR-H3 amino acids with the human VH3-53 allele; LY-CoV016 was the VH3-53 allele with 13 amino acids having the short CDR-H3 amino acids. However, other clones, 1-57 and BG10-19, were also classified as Class 1 with different alleles and longer CDR-H3: 1-57, VH3-72/21 CDR-H3 amino acids and BG10-19, VH5-59/18 CDR-H3 amino acids [45,46,47]. Interestingly, several researchers reported that VH3-53 was the dominant human allele for neutralizing anti-SARS-CoV-2 antibodies; the allele structure may fit the surface to neutralize the virus [48,49,50]. If antibodies selected the different alleles, not VH3-53, the CDR-H3 length may be important in covering the virus surface. Therefore, we concluded that longer CDR3 amino acids are essential for highly specific anti-SARS-CoV-2 mAbs.
Antibody affinity maturations are crucial processes that enhance the binding and neutralizing activities of antibodies, such as those against human immunodeficiency virus type 1 [51], influenza [52], Zika [53], dengue [54,55,56], and the SARS-CoV-2 virus [57]. Our alpaca synthetic sdAb library did not induce affinity maturation. Surprisingly, anti-JN.1 sdAb-Fc fusion protein clone 1B9 bound strongly to RBD protein, KD: 0.19 nM, without an affinity maturation process. In contrast, the anti-XBB.1.5 sdAb-Fc fusion protein clone C10 (KD: 265 nM) exhibited weaker binding compared to the anti-JN.1 sdAb-Fc fusion clone 1B9 (KD: 0.19 nM), even though their CDR3 amino acid lengths were similar. Since neither sdAb underwent affinity maturation, studying the differences in their binding mechanisms is important for developing high specificity and high affinity sdAbs in the future. In summary, the alpaca sdAb library is one of the most effective resources for discovering potent anti-SARS-CoV-2 monoclonal antibodies.
The binding epitopes of anti-JN.1 sdAb clone 1B9 to JN.1 RBD protein were identified as four peptides by HDX-MS. We speculated that anti-JN.1sdAb clone 1B9 could bind to frequently mutated regions of JN.1 that differ from those of Wuhan, BA.5, and XBB.1.5 strains, including V445H, N450D, and L452W. Interestingly, these amino acids were not selected, but a peptide, including L455S, was identified. The L455S mutation is a key mutation that enhances immune evasion capability against neutralizing antibodies [58,59], reduces hydrophobicity, and induces conformational changes in the RBD protein [58]. We concluded that anti-JN.1 sdAb clone 1B9 recognized the most critical conformational changes induced by L455S and acquired high specificity to JN.1 strains.
Historically, many anti-SARS-CoV-2 mAbs were discovered against the Wuhan strain to cease the pandemic [4]. Unfortunately, most of these mAbs lost neutralizing activity with the Omicron variants [12], including the XBB.1.5 and JN.1 strains. Walker et al. and Guselnikov et al. reported mAbs bound to JN.1 and XBB.1, and XBB.1.5, respectively [60,61]. However, they focused on broad neutralizing mAbs to eradicate multiple strains. Therefore, this report is the first to discover XBB.1.5 and JN.1 strain-specific mAbs.
In summary, we discovered strain-specific anti-SARS-CoV-2 single-domain antibodies (sdAbs) from alpaca synthetic libraries within 8–9 weeks, without immunization. These sdAbs can be used in vaccine identification tests and align with vaccine development timelines. Utilizing an alpaca synthetic library is among the most effective methods for discovering tool antibodies that ensure quality control in biologics, supporting the release of high-quality therapeutic products.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antib15020018/s1, Table S1: Summary of kinetic studies of anti-SARS-CoV-2 sdAbs.

Author Contributions

Conceptualization, I.T., E.C., A.M. and A.B.-C.; Methodology, I.T., B.K., K.O. and T.K.; Software, I.T. and B.K.; Validation, I.T., B.K., K.O. and T.K.; Formal Analysis, I.T., K.O. and B.K.; Investigation, I.T., B.K., K.O. and T.K.; Resources, I.T., K.O. and B.K.; Data Curation, I.T., K.O. and B.K.; Writing—Original Draft Preparation, I.T., B.K. and K.O.; Writing—Review and Editing, all authors; Visualization, I.T., B.K., K.O. and T.K.; Supervision, I.T., A.M., K.Y., H.G. and A.B.-C.; Project Administration, A.M., K.Y. and T.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Takeda Pharmaceutical Company Limited, Tokyo, Japan. The authors declare that this study received funding from the Ministry of Health, Labour and Welfare, Tokyo, Japan. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We appreciate Yoshio Okubo, the global program lead overseeing the projects; Matthew Downey, responsible for medical writing support; and Sethu Alexander, for suggesting the project. We also thank the Vaccine Bioanalytical Development team, the MS&T Japan team, the Global Vaccine Business Unit, the Vaccine Quality Japan Vaccine Testing Group, and Global Quality at Takeda. We thank NanoTag Biotechnologies for discovering and supplying Anti-SARS-CoV-2 sdAbs and Trenzyme for antigen preparation. We thank Ryan Wenzel, Kelsey Sharp and Alexis Nazabal from CovalX for analyzing sdAb binding epitope analysis by HDX-MS.

Conflicts of Interest

I.T., A.M. and A.B.-C. are employees of Takeda Vaccines and may hold stock/stock options in Takeda. K.O., T.K., K.Y. and T.N. are employees of Takeda Pharmaceutical and may hold stock/stock options in Takeda. E.C. is a former employee of Takeda Vaccines. H.G. and B.K. are NanoTag Biotechnologies GmbH employees and may hold stock/stock options in NanoTag.

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Antibodies 15 00018 g001
Figure 1. Alpaca synthetic single-domain antibody (sdAb) selection. (A1–A4) Selection of binders by ribosomal display. (B) Construction of the sdAb-library phages. (C1–C4) Selection of binders by phage display. (D) Construction of the sdAb-library expression vectors. (E) Expression of sdAb by prokaryote. (F) Screening sdAb binding clones by ELISA.
Figure 1. Alpaca synthetic single-domain antibody (sdAb) selection. (A1–A4) Selection of binders by ribosomal display. (B) Construction of the sdAb-library phages. (C1–C4) Selection of binders by phage display. (D) Construction of the sdAb-library expression vectors. (E) Expression of sdAb by prokaryote. (F) Screening sdAb binding clones by ELISA.
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Figure 2. Binding activity of anti-SARS-CoV-2 sdAbs and sdAb-Fc fusion proteins. (A,B) ELISA of anti-XBB.1.5 sdAb-Fc fusion protein clone C10 (A) and anti-JN.1 sdAb-Fc fusion protein clone 1B9: EC50 values (pM) were calculated by GraphPad Prism (B). (C,D) KD measurements of anti-XBB.1.5 sdAb-Fc fusion protein clone C10 (C) and anti-JN.1 sdAb-Fc fusion protein clone 1B9 (D). The assay used Octet-HTX (Sartorius). Kinetic parameters, association constant (ka), and dissociation constant (kdis) were analyzed with the 1:2 bivalent binding model. KD values were calculated by kdis/ka. The biosensor grams are generated in each sdAb-Fc concentration (nM), and red line is the fitting model. (E) kdis/ka plot of anti-SARS-CoV-2 sdAbs and sdAb-Fc fusion proteins. The dotted line indicates the KD values (KD = kdis/ka). For the ELISA, the assay was performed in a single run, and its reproducibility was confirmed with independent runs. For the kinetic analysis, the assay was conducted in triplicate, and the average values were calculated (detailed data is in Table S1).
Figure 2. Binding activity of anti-SARS-CoV-2 sdAbs and sdAb-Fc fusion proteins. (A,B) ELISA of anti-XBB.1.5 sdAb-Fc fusion protein clone C10 (A) and anti-JN.1 sdAb-Fc fusion protein clone 1B9: EC50 values (pM) were calculated by GraphPad Prism (B). (C,D) KD measurements of anti-XBB.1.5 sdAb-Fc fusion protein clone C10 (C) and anti-JN.1 sdAb-Fc fusion protein clone 1B9 (D). The assay used Octet-HTX (Sartorius). Kinetic parameters, association constant (ka), and dissociation constant (kdis) were analyzed with the 1:2 bivalent binding model. KD values were calculated by kdis/ka. The biosensor grams are generated in each sdAb-Fc concentration (nM), and red line is the fitting model. (E) kdis/ka plot of anti-SARS-CoV-2 sdAbs and sdAb-Fc fusion proteins. The dotted line indicates the KD values (KD = kdis/ka). For the ELISA, the assay was performed in a single run, and its reproducibility was confirmed with independent runs. For the kinetic analysis, the assay was conducted in triplicate, and the average values were calculated (detailed data is in Table S1).
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Figure 3. Inhibitory activity of anti-SARS-CoV-2 sdAbs-Fc fusion proteins with ACE2/RBD protein binding. (A) Biosensor grams of ACE2/RBD protein binding inhibition of anti-JN.1 sdAb-Fc fusion protein clone 1B9. Red line: JN.1 RBD/anti-JN.1 clone 1B9 (40 nM)/ACE2; blue line: JN.1 RBD/no anti-JN.1 clone 1B9 (0 nM)/ACE2. (B,C) ACE2/RBD protein binding inhibitory activity of anti-XBB.1.5 sdAb-Fc fusion proteins (B) and anti-JN.1 sdAb-Fc fusion proteins (C); the average values ± standard deviation are shown in the graphs. (D) IC50 values of anti-SARS-CoV-2 sdAb-Fc fusion proteins. The assay was conducted as triplicate independent runs and the average values are shown.
Figure 3. Inhibitory activity of anti-SARS-CoV-2 sdAbs-Fc fusion proteins with ACE2/RBD protein binding. (A) Biosensor grams of ACE2/RBD protein binding inhibition of anti-JN.1 sdAb-Fc fusion protein clone 1B9. Red line: JN.1 RBD/anti-JN.1 clone 1B9 (40 nM)/ACE2; blue line: JN.1 RBD/no anti-JN.1 clone 1B9 (0 nM)/ACE2. (B,C) ACE2/RBD protein binding inhibitory activity of anti-XBB.1.5 sdAb-Fc fusion proteins (B) and anti-JN.1 sdAb-Fc fusion proteins (C); the average values ± standard deviation are shown in the graphs. (D) IC50 values of anti-SARS-CoV-2 sdAb-Fc fusion proteins. The assay was conducted as triplicate independent runs and the average values are shown.
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Figure 4. Vaccine identification test of SARS-CoV-2 JN.1 drug substances. Western blotting using anti-JN.1 sdAb clone 1B9. Lanes 1 and 5: Wuhan drug substance (DS); lanes 2 and 6: BA.5 DS; lanes 3 and 7: XBB.1.5 DS; and lanes 4 and 8: JN.1 DS. Lanes 1–4: reduced condition. Lanes 5–8: non-reduced condition (theoretical molecular weight: 170 kDa). Apply: 3 µg/lane. Anti-JN.1 sdAb clone 1B9 was used as primary antibody; anti-FLAG M2–alkaline phosphatase conjugate was applied as secondary antibody. Detected by BCIP/NBT phosphatase substrate kit.
Figure 4. Vaccine identification test of SARS-CoV-2 JN.1 drug substances. Western blotting using anti-JN.1 sdAb clone 1B9. Lanes 1 and 5: Wuhan drug substance (DS); lanes 2 and 6: BA.5 DS; lanes 3 and 7: XBB.1.5 DS; and lanes 4 and 8: JN.1 DS. Lanes 1–4: reduced condition. Lanes 5–8: non-reduced condition (theoretical molecular weight: 170 kDa). Apply: 3 µg/lane. Anti-JN.1 sdAb clone 1B9 was used as primary antibody; anti-FLAG M2–alkaline phosphatase conjugate was applied as secondary antibody. Detected by BCIP/NBT phosphatase substrate kit.
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Figure 5. Binding epitope analysis of anti-JN.1 sdAb-Fc clone 1B9. (A) Binding epitope peptides of anti-JN.1 sdAb clone 1B9 determined by hydrogen–deuterium exchange mass spectrometry (HDX-MS); red amino acid: unique amino acids of JN.1 from Wuhan, BA.5 and XBB.1.5; red arrow: ACE2 contact amino acids. (B) Epitope peptide mapped to Wuhan RBD (PDB: 6M0J). (C) Comparison of binding epitopes of anti-SARS-CoV-2 mAbs. Red: unique amino acids of JN.1 from Wuhan, BA.5 and XBB.1.5; turquoise: binding epitope of anti-SARS-CoV-2 mAbs. LY-CoV016, PDB: 7C01; LY-CoV555, PDB: 7KMG; COV2-2196, PDB: 7L7D; CT-P59, PDB: 7CM4; REGN10933, PDB: 6XDG; REGN10987, PDB: 6XDG; SARS2-38, PDB: 7MKM; COV2-2130, PDB: 7L7E; S2H97, PDB: 9ATM; S309, PDB: 6WPS. * ACE2 contact amino acid. Amino acid numbers were from Wuhan spike protein: NCBI: MN908947.3. JN.1 spike protein: NCBI: PQ121600.1.
Figure 5. Binding epitope analysis of anti-JN.1 sdAb-Fc clone 1B9. (A) Binding epitope peptides of anti-JN.1 sdAb clone 1B9 determined by hydrogen–deuterium exchange mass spectrometry (HDX-MS); red amino acid: unique amino acids of JN.1 from Wuhan, BA.5 and XBB.1.5; red arrow: ACE2 contact amino acids. (B) Epitope peptide mapped to Wuhan RBD (PDB: 6M0J). (C) Comparison of binding epitopes of anti-SARS-CoV-2 mAbs. Red: unique amino acids of JN.1 from Wuhan, BA.5 and XBB.1.5; turquoise: binding epitope of anti-SARS-CoV-2 mAbs. LY-CoV016, PDB: 7C01; LY-CoV555, PDB: 7KMG; COV2-2196, PDB: 7L7D; CT-P59, PDB: 7CM4; REGN10933, PDB: 6XDG; REGN10987, PDB: 6XDG; SARS2-38, PDB: 7MKM; COV2-2130, PDB: 7L7E; S2H97, PDB: 9ATM; S309, PDB: 6WPS. * ACE2 contact amino acid. Amino acid numbers were from Wuhan spike protein: NCBI: MN908947.3. JN.1 spike protein: NCBI: PQ121600.1.
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Table 1. Amino acid sequence of anti-XBB.1.5 and anti-JN.1 sdAbs.
Table 1. Amino acid sequence of anti-XBB.1.5 and anti-JN.1 sdAbs.
CloneCDR1CDR2CDR3Amino acid residue of CDR3
anti-XBB.1.5 (O_P3B7)GFPVFWQQIESWGSSTNVKDGGAIWYDY12
anti-XBB.1.5 (MixP2B6)GRINTIEYLQTDSGGTAAAVWGRQFPLWYMYSY17
anti-XBB.1.5 (MixP2B3) and (MixP2B7) *GEIRAIEYLSTYRGFTAAAYGGHHYPLYSNYYY17
anti-XBB.1.5 (CK_P3C10)GQIEHIEYLATYFGETAAAYGGHHYPLADTYSY17
anti-JN.1 1B9 and 1A10 *GEIASIHYLFTIEGSTAAAQAGIYNPLTAYYY16
* Mutations in the framework region were confirmed.
Table 2. Summary of anti-XBB.1.5 and JN.1 sdAb ELISA.
Table 2. Summary of anti-XBB.1.5 and JN.1 sdAb ELISA.
sdAb/SARS-CoV-2 StrainEC50 (nM)
WuhanXBB.1.5BA.5JN.1
anti-XBB.1.5 (MixP2B3) sdAbND672.1NANA
anti-XBB.1.5 (MixP2B6) sdAb *ND15,824NANA
anti-XBB.1.5 (MixP2B7) sdAbND2909NANA
anti-XBB.1.5 (O_P3B7) sdAbND721.7NANA
anti-XBB.1.5 (CK_P3C10) sdAb *ND1325NANA
anti-JN.1 1B9 sdAbNDNDND5.96
anti-JN.1 1A10 sdAbNDNDND2.48
NA: not assigned for the assay; ND: not detected. * sdAb fused with Fc-protein: MixP2B6: B6 for sdAb-Fc; and CL_P3C10: C10 for sdAb-Fc. EC50 values were demonstrated in a single analysis, and reproducibility was verified through independent assay runs.
Table 3. Summary of anti-XBB.1.5 and JN.1 sdAb-Fc fusion protein ELISAs.
Table 3. Summary of anti-XBB.1.5 and JN.1 sdAb-Fc fusion protein ELISAs.
sdAb-Fc/SARS-CoV StrainEC50 (nM)
WuhanXBB.1.5BA.5JN.1
anti-XBB.1.5 B6 sdAb-Fc698.70.0426NANA
anti-XBB.1.5 C10 sdAb-FcND0.0036NANA
anti-JN.1 1B9 sdAb-FcNDNDND0.0036
anti-JN.1 1A10 sdAb-FcNDNDND0.0033
NA: not assigned the assay; ND: not detected. EC50 values were demonstrated in a single analysis and reproducibility was verified through independent assay runs.
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Tsuji, I.; Okada, K.; Kroppen, B.; Katta, T.; Yamamura, K.; Nishihama, T.; Miura, A.; Götzke, H.; Crampon, E.; Bertolotti-Ciarlet, A. Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library. Antibodies 2026, 15, 18. https://doi.org/10.3390/antib15020018

AMA Style

Tsuji I, Okada K, Kroppen B, Katta T, Yamamura K, Nishihama T, Miura A, Götzke H, Crampon E, Bertolotti-Ciarlet A. Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library. Antibodies. 2026; 15(2):18. https://doi.org/10.3390/antib15020018

Chicago/Turabian Style

Tsuji, Isamu, Kumiko Okada, Benjamin Kroppen, Tetsufumi Katta, Kaori Yamamura, Takeshi Nishihama, Ayako Miura, Hansjörg Götzke, Eric Crampon, and Andrea Bertolotti-Ciarlet. 2026. "Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library" Antibodies 15, no. 2: 18. https://doi.org/10.3390/antib15020018

APA Style

Tsuji, I., Okada, K., Kroppen, B., Katta, T., Yamamura, K., Nishihama, T., Miura, A., Götzke, H., Crampon, E., & Bertolotti-Ciarlet, A. (2026). Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library. Antibodies, 15(2), 18. https://doi.org/10.3390/antib15020018

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