WO2023077287A1

WO2023077287A1 - High-affinity anti-egfp and anti-sars-cov-2 vnar single domain antibodies and use thereof

Info

Publication number: WO2023077287A1
Application number: PCT/CN2021/128297
Authority: WO
Inventors: Jiahai SHI; Likun Wei; Meiniang WANG; Naibo Yang
Original assignee: City university of hong kong shenzhen research institute; Bgi-Shenzhen
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2023-05-11

Abstract

The present invention provides vNAR single domain antibodies and the use thereof. The vNAR single domain antibodies according to the present invention has high affinity to EGFP and/or SARS-COV-2. Also provided are biparatopic vNAR single domain antibodies comprising the vNAR single domain antibodies according to the present invention.

Description

High-affinity anti-EGFP and anti-SARS-CoV-2 vNAR single domain antibodies and use thereof

Technical Field

The present invention relates to the field of single domain antibodies, more particularly, to high-affinity anti-EGFP and anti-SARS-COV-2 vNAR single domain antibodies and use thereof.

Background

As researchers strive to exploit novel targets, alternative or cryptic epitopes, readily engineering of antibodies, the innovative antibody fragments and novel scaffolds are being developed to fulfill diverse needs in life sciences. Single domain antibodies, also called nanobodies, include Camelid-derived VHHs and shark-derived vNARs (variable new antigen receptors) . They have unique features and advantages in potential diagnostic and therapeutic applications, such as nano-size, readily re-formatted, superior tissue penetration ability, great stability and solubility, potentials to reach into recessive epitopes, easy expression in prokaryotic cells. Besides, vNARs can be re-formatted upon different usages in biotechnology field, such as high-affinity capturing agents, super-resolution microscopy via nanobodies coupled to organic dyes, intrabodies interfering with protein function, nanobody-targeted enzymatic degradation, and crystallization chaperones.

Conventional antibodies (e.g., monoclonal IgGs) have emerged some limitations, such as high production costs, poor tissue penetration, and long serum half-life due to their large sizes and complex structures, which hinder their widespread uses as therapeutics and imaging reagents. In response, alternative antibody fragments (e.g., Fab, scFv, diabodies) are engineered to retain full capacity for antigen binding with smaller sizes. The smallest antibody fragments to date, single variable domain antibodies (sdAbs) including VHH and vNAR, have been developed from heavy-chain-only antibodies (HCAbs) found in Camelidae and cartilaginous fish, respectively. HcAbs are a heavy chain homodimeric isotype devoid of light chains. In cartilaginous fish they are called immunoglobulin new antigen receptor (IgNAR) .

Cartilaginous fish are the most ancient vertebrates possessing adaptive immunity with many close parallels to mammals. They have immunoglobulins (Igs) , alpha/beta or gamma/delta T-cell receptors, cytokines, and major histocompatibility complex molecules, etc. A recent study showed the immunological memory of nurse shark maintained for eight years implying the already-achieved sophisticated immune system in sharks. It is generally considered that the primary generation sites of antigen-specific immunity of cartilaginous fish are the epigonal organ, the Leydig organ, and the spleen with organized lymphoid follicles. Although without lymph nodes and germinal centers, sharks possess antigen-driven immune responses representing as the affinity maturation of antigen-specific antibodies by somatic hypermutation of Ig genes. Cartilaginous fish have three Ig isotypes, IgM, IgW, and IgNAR. All three Igs have both transmembrane forms and secretory forms. The studies on nurse shark showed that the adaptive humoral immune response is dominated by monomeric IgM (mIgM) and IgNAR, while pentameric IgM (pIgM) serves as an innate Ig rather than involving in the adaptive immune response. Sharks possess roughly equal amounts of mIgM and pIgM in serum at high concentration (>20mg/ml of both totally) and small amounts of IgNAR (0.1～1mg/ml) . Moreover, the two IgM isoforms mostly probably originate from different B cell lineages. Like pIgM containing J chain, adult nurse shark co-expresses IgW and J chain in epigonal organ indicating that IgW could also be a multimer. IgW is highly expressed in pancreas suggesting it may play a role in gut mucosal immunity.

IgNAR is first identified in nurse shark serum and present as multiple clusters in shark genome and only a few (<10) germline IgNAR clusters present in different sharks (Diaz, M., Stanfield, R., Greenberg, A. & Flajnik, M. Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR) : identification of a new locus preferentially expressed in early development. Immunogenetics 2002, 54, 501–512) . The last four C domains of IgNAR are homologous to IgW. Each IgNAR gene is organized with one variable (V) segment, three diversity (D) segments, one joining (J) segment, and one set of constant (C) segments. Therefore, at most four RAG-mediated rearrangements are involved in produce the primary repertoire of IgNAR, along with extensive N-region addition, P-nucleotide addition and exonuclease trimming. Therefore, the diversity of IgNAR repertoire mostly come from the CDR3 which vary greatly in length and sequence.

Previous studies have already shown that sharks can produce high affinity antigen-specific both mIgM and IgNAR responses upon immunization (Dooley, H. & Flajnik, M. Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur. J. Immunol. 2005, 35, 936–945) . vNAR elements of IgNAR can undergo somatic hypermutation in response to antigenic challenge. The most of the immunization protocols for sharks mainly take 5～6 times of immunization with a four-week interval and a subcutaneous immunization route. Unlike mammals, antigen-specific mIgM and IgNAR titers in shark serum increase slowly following immunization and take months to reach a significant level. Following cessation of immunization, the subsequent drop in titers is also very slow.

vNARs possess unique features, such as nano-level size (12～15 kDa) and elongated CDR3 loop (5～35 aa) . Due to lacking a CDR2 loop, vNAR has eight instead of ten β-strands. Compared to VHHs, vNARs are evolutionarily distant from mammals and possess longer CDR3 region and more non-canonical cysteines. The sequences of vNARs are highly homologous with shark TCR-NAR indicating as the possible origin of vNARs. Another study suggests that vNARs originated as cell-surface adhesion molecules due to some similar protein structure (Streltsov, V.A. et al. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12444–12449) . Four types of vNARs were identified in sharks based on the number and position of non-canonical cysteines. Type I exists only in nurse shark up to date. Type III is found primarily in infant nurse sharks with limited diversity. Type II is widely present in all sharks. Interestingly, more new types of vNARs outside the four known types have been recently reported in different sharks, such as nurse shark, horn shark, and whitespotted bamboo sharks. Further investigations are needed for their originations and functions. The affinity of vNARs to various antigens varies from sub-nanomolar to micromolar scales. The first vNAR generated from immunized library is specific to hen egg-white lysozyme with a 1.0 nM affinity (Dooley, H. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Molecular Immunology 2003, 40, 25–33) . The most of antigen-specific vNAR candidates isolated from different shark species are type II, followed by type IV, and other novel types.

Compared to conventional antibodies, vNARs have better thermal and chemical stability (stable in 350mM urea) , good solubility, better tissue penetration, and can be easily expressed by E. coli and readily re-formatted to catering to different applications. These advantages make vNARs promising candidates in the applications of diagnostics, biotechnology and even clinical therapeutics. vNARs against various antigens have been developed in the recent two decades for different purposes, such as drug candidates (targeting cancer, auto-immune disease, infectious viral) , blood-brain barrier transporters, clinical diagnosis and crystallization chaperones. Most antigen-specific vNARs are selected via phage display, and some via yeast display.

vNAR as intrabodies have previously been reported to target hepatitis B virus precore antigen, transferrin receptor 1 for transfer across the blood-brain barrier, delta-like ligand 4 (DLL4) for treating DLL4-overexpressing tumours, and induced costimulatory ligand for treating auto-inflammatory non-infectious uveitis (Walsh, R. et al. Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 2011, 411, 132–141; Stocki, P. et al. Blood-brain barrier transport using a high-affinity, brain-selective VNAR (Variable Domain of New Antigen Receptor) antibody targeting transferrin receptor 1. bioRxiv 816900, 2020, doi: 10.1101/816900; Leach, A. et al. Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature. Nanoscale 2020, 12, 14751–14763; Kovaleva, M., Johnson, K., Steven, J., Barelle, C.J. & Porter, A. Therapeutic Potential of Shark Anti-ICOSL VNAR Domains is Exemplified in a Murine Model of Autoimmune Non-Infectious Uveitis. Front. Immunol. 2017, 8, 1121) . These studies demonstrate the efficacy of vNAR nobodies for clinical biologics development. The drug or Fc-domain conjugated vNARs as novel biotherapeutics could offer many potential benefits over conventional antibodies including increased tumor penetration, readily production and conjugation, and improved pharmacokinetics. Recently, an anti-human RA biparatopic vNARs (named as Quad-X ^TM) showed a complete disease control with 10x the in vivo potency of a marketed drug in a mouse model and notably, is of low inherent immunogenicity (Ubah, O.C., Porter, A.J. & Barelle, C.J. In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development. Journal of Immunology Research vol. 2020 e7283239 https: //www. hindawi. com/journals/jir/2020/7283239) . Undoubtedly, more studies on vNARs as therapeutics will continue to surface in future years.

However, the potential immunogenicity of vNARs could limit their application in clinical therapeutics because of their limited sequence homology shared with human VH/VL domains (30%to 40%overall sequence identity) . Some studies on the humanization of vNARs are addressing this issue. The principle is to graft the antigen binding regions of vNAR to a human framework. Kovalenko described the humanized type I and type IV vNARs grafted on human germline VL scaffold, DPK9 (Kovalenko, O.V. et al. Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis. J. Biol. Chem. 2013, 288, 17408–17419) . They found the changes in the HV2 and HV4 resulted in partial loss of vNAR activity and the improper human framework can result in the dimerization or affinity reduction of vNARs. Thus, the design of human framework for vNARs is still needed to be optimized. However, a mostly-recent publication reported that the biparatopic/bispecific vNAR constructs are of low immunogenicity in a transgenic mouse model as validated by in vitro ELISA and cell-based assays (Ubah, 2020) . It is speculated that the small, simple and stable architecture of vNAR contribute to this low immunogenicity property. When the immunogenicity issue is addressed, the application of vNARs in clinical therapeutics can be further expanded.

The second issue for vNARs to be solved is the short serum half-life due to the inherent small size. This will limit vNARs in targeting cancer cells because of its rapid clearance in vivo by glomerular filtration. One solution is the fusion of vNAR candidates with an anti-human serum albumin (HSA) vNAR developed by Müller (Müller, M.R. et al. Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain. MAbs 2012, 4, 673–685) . This relies on the unique advantages of HSA in human, such as the long half-life (19d) , relative abundance in blood and accumulation in disease-related regions. Other strategies may be worth trying, including increasing the size of vNARs by multimerization or conjugation to other biologics, and alterations to site-specific glycosylation.

The simple architecture of vNARs allow various re-formatting to the desired constructs. Some multivalent formats have been demonstrated, including dimeric and trimeric vNARs, and Fc-fusion vNARs derived from nurse shark and spotted wobbegong shark respectively. Generally, the affinity of bivalent/biparatopic vNARs is 10 to 80 folds higher than that of their monomeric formats. Moreover, vNARs can be re-formatted upon different usages in the biotechnology field, such as high-affinity capturing agents, super-resolution microscopy via nanobodies coupled to organic dyes, intrabodies interfering with protein function, nanobody-targeted enzymatic degradation, and crystallization chaperones.

There are three types of vNAR libraries, including

libraries, synthetic libraries (CR3-randomization) , and immunized libraries. The library size ranges from 10 ⁸ to 10 ¹¹ for a synthetic library, and 10 ⁶ to 10 ⁹ for an immunized library. The size, type and diversity of a vNAR library are important to isolate high affinity vNARs. The higher the library size and diversity, the more likelihood the high affinity candidates isolated. Most vNAR libraries are semi-synthetic libraries, followed by immunized libraries and

libraries. Previous studies have shown that CDR1, HV2 and HV4 were also involved in antigen-driven affinity maturation though mostly occurred in CDR3 in immunized sharks (Zielonka, S. et al. The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope. Mar. Biotechnol. 2015, 17, 386–392) . However, the sequence variability of CDR1, HV2 and HV4 in

vNAR libraries are pretty low. Therefore, the four hypervariable regions of vNARs especially CDR3 could be randomly mutated to create high diversity semi-synthetic libraries.

vNARs have been generated from seven shark species so far, including nurse shark, spotted wobbegong, horn shark, spiny dogfish, banded houndshark, dusky smooth-hound, and white-spotted bamboo shark. Not every shark species can produce antigen-specific IgNAR response by passive immunization, such as small-spotted catshark. Immunized libraries have been reported so far only in nurse shark, ornate wobbegong and horn shark. However, some shark species, such as nurse shark, wobbegong and spiny dogfish, are of hard operability and large raising cost due to their large body size or aggressive temper or fast movement. The most researched shark species, nurse shark, is listed as vulnerable or critically endangered by the IUCN Red List of Threatened Species. Therefore, there is an urgent need for finding an ideal shark species without the above issues.

White-spotted bamboo shark (Chiloscyllium plagiosum) is a small inshore bottom-dwelling shark species (up to 1m of adult length) living in the coast areas of Southeast China and Southeast Asia. They are small, sedentary, harmless, robust, easily captured and readily adaptive to small water body and tagging. They can be bred artificially and domesticated to eat artificial feed. They can be kept captive in self-designed aquarium in a long-term steady state which ensure the completion of shark immunization. Lower farming cost and easy-to-operate in immunization and maintenance make bamboo shark an ideal animal model for sdAbs production in high market competitiveness.

The vNARs generated from white-spotted bamboo shark are rarely reported to date and the library type is all semi-synthetic (CDR3-randomization) . However, the vNAR candidates isolated from this library is type IV or other types, but not type II which represents the majority and the most diverse vNAR type in bamboo shark. Therefore, a higher diverse vNAR library is needed to be constructed on the basis of the comprehensive knowledge of IgNAR genes in bamboo shark genome. The vNARs from an immunized library are generally higher in affinity than that from a

library and synthetic library because of the vNARs having experienced iterative affinity-maturation in shark body during immunization.

Summary of Invention

In order to address the above issues in the prior art, an objective of the present invention is to provide an isolated high affinity anti-EGFP vNAR single domain antibody.

Another objective of the present invention is to provide an isolated high affinity anti-SARS-COV-2 vNAR single domain antibody.

Still another objective of the present invention is to provide a biparatopic vNAR single domain antibody comprising the isolated high affinity anti-EGFP vNAR single domain antibody according to the present invention.

Yet another objective of the present invention is to provide a biparatopic vNAR single domain antibody comprising the isolated high affinity anti-SARS-COV-2 vNAR single domain antibody according to the present invention.

In one aspect, the present invention provides an isolated vNAR single domain antibody derived from a bamboo shark library by phage display. In certain embodiments, the bamboo shark library is an immunized bamboo shark library. In certain embodiments, the bamboo shark is white-spotted bamboo shark.

In certain embodiments, the isolated vNAR single domain antibody according to the invention comprises an amino acid sequence which is at least 90%homologous to an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7. In preferred embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to EGFP. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to EGFP with a nanomolar or sub-nanomolar binding affinity.

In certain embodiments, the isolated vNAR single domain antibody according to the invention comprises an amino acid sequence which is at least 90%homologous to an amino acid sequence selected from SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11, SEQ. ID No. 12, SEQ. ID No. 13, and SEQ. ID No. 14. In preferred embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence selected from SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11, SEQ. ID No. 12, SEQ. ID No. 13, and SEQ. ID No. 14. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to at least one of SARS-CoV-2 spike S1 and spike RBD. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a micromolar or nanomolar binding affinity.

In another aspect, the present invention provides a biparatopic vNAR single domain antibody comprising at least one segment consisted of the isolated vNAR single domain antibody according to the invention. In certain embodiments, the biparatopic vNAR single domain antibody comprises a first segment consisted of the isolated vNAR single domain antibody according to the invention and a second segment consisted of the isolated vNAR single domain antibody according to the invention. In certain embodiments, the isolated vNAR single domain antibodies consisting both segments of the biparatopic vNAR single domain antibody are the same or different. In certain embodiments, the isolated vNAR single domain antibodies consisting both segments of the biparatopic vNAR single domain antibody are different.

In yet another aspect, the present invention provides a construct encoding an amino acid sequence comprising the isolated vNAR single domain antibody according to the invention.

In still another aspect, the present invention provides a nucleic acid or nucleotide sequence encoding the isolated vNAR single domain antibody or the biparatopic vNAR single domain antibody according to the invention.

Brief Description of Drawings

The invention will become more fully understood from the following description taken in conjunction with the accompanying drawings.

Figure 1 shows the procedures of phage library construction and biopanning for antigen-specific vNAR isolation.

Figure 2 shows four rounds of biopanning to select EGFP-specific vNAR binders. (A) Polyclonal phage ELISA against EGFP indicates the successful enrichment of EGFP-specific vNARs during panning. (B) Phylogenetic analysis of 105 EGFP-specific vNAR candidates. The finally selected seven unique anti-EGFP vNARs are indicated.

Figure 3 shows the production and specificity validation of EGFP-specific vNAR candidates with the comparison with anti-EGFP VHHs. (A) SDS-PAGE analysis of seven unique anti-EGFP vNARs purified by nickle affinity chromatography. (B) SDS-PAGE analysis of five anti-EGFP VHHs purified by nickle affinity chromatography. (C) Validation of EGFP specificity of seven vNARs and five VHHs by western blotting. Five VHHs, including GFP_enhancer, GFP_miminizer, LaG2, LaG16, and LaG41 are five anti-GFP VHHs as positive controls.

Figure 4 shows the binding curves of seven vNARs and five VHHs to EGFP for the determination of ELISA EC50. (A) represents the results of vNARs; (B) represents the results of VHHs. Three repeats were performed for each value.

Figure 5 shows the SPR sensorgrams of seven vNARs and five VHHs binding with EGFP. The KD measurements were obtained using Biacore T200.

Figure 6 shows the validation of EGFP-specific vNARs as intrabodies in mammalian cells. (A) Procedures of the functional analysis of EGFP-specific vNARs as intrabodies in HEK293T cells. WB, western blotting; IP, immunoprecipitation. (B) Expression of EGFP-specific vNARs in HEK293T cells validated by WB. All vNARs or VHH contain a C-terminal 3xFLAG tag. NC (negative control) were transfected with the same expression plasmid but without vNAR or VHH insertion. GFP_minimizer is an anti-EGFP VHH as a positive control. (C) Affinity pull-down of EGFP-specific vNARs functioning as intrabodies in HEK293T cells. 293T cells co-express EGFP and nanobodies with a C-terminal 3xFLAG tag. vNAR or VHH binding with EGFP in cell lysate was captured by anti-FLAG magnetic beads and the eluate was then subjected to WB analysis. ‘-’ represents pre-pulldown by anti-Flag magnetic beads. ‘+’ represents post-pulldown by anti-Flag magnetic beads.

Figure 7 shows the overlapping/non-overlapping epitopes prediction of twelve EGFP-specific nanobodies by competitive epitope binding assay. Three repeats were performed for each value. Significant level is marked as asterisk.

Figure 8 shows the design and production of anti-EGFP bivalent vNARs. (A) Schematic diagram of the design of anti-EGFP bivalent vNARs. The bivalent vNAR BsG3-BsG98 is taken as an example to represent their non-overlapping epitopes on EGFP. (B) SDS-PAGE analysis of four anti-EGFP bivalent vNARs purified by nickle affinity chromatography.

Figure 9 shows the SPR sensorgrams of four bivalent vNARs binding with EGFP. The KD measurements were obtained using Biacore T200.

Figure 10 shows the two targets for SARS-Cov-2, Spike S1 and Spike RBD.

Figure 11 shows four rounds of biopanning to select S1/RBD-specific vNAR binders. Polyclonal phage ELISA against Spike RBD (A) and Spike S1 (B) indicate the successful enrichment of target-specific vNARs during panning. (C) SDS-PAGE analysis of seven anti-S1/RBD vNARs purified by nickle affinity chromatography.

Figure 12 shows the SPR sensorgrams of seven vNARs binding with S1 and RBD. The KD measurements were obtained using Biacore T200.

Detailed Description of Invention

Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the related field. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

As used herein, a “vNAR” refers to the variable domain of an immunoglobulin new antigen receptor (IgNAR) .

As used herein, a “biparatopic (vNAR) ” refers to a vNAR comprising two antigen binding domains (both are variable domain of immunoglobulin new antigen receptor) in which each one recognizes unique, non-overlapping epitopes on the same target antigen.

As used herein, an “immune library” or “immunized library” refers to an antibody library constructed from the animal immunized with antigens of interest.

As used herein, “diversity” means the sequence variety of antibody which results in different target binding specificity and affinity.

As used herein, a “construct” encompasses antibodies of many different formats that are obtained by protein engineering techniques.

In the present invention, an isolated vNAR single domain antibody derived from a bamboo shark library by phage display is provided.

In certain embodiments, the bamboo shark library is an immunized bamboo shark library. In certain embodiments, the bamboo shark is white-spotted bamboo shark.

In certain embodiments, the isolated vNAR single domain antibody according to the invention comprises an amino acid sequence which is at least 90%homologous to an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7. In preferred embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 1. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 1, and has a CDR3 of the amino acid sequence YEAWDESDSWNCGDYY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG3 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 1, in which CDR3 is the amino acid sequence YEAWDESDSWNCGDYY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 1.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 2. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 2, and has a CDR3 of the amino acid sequence YRGPSCGGNWAY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG73 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 2, in which CDR3 is the amino acid sequence YRGPSCGGNWAY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 2.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 3. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 3, and has a CDR3 of the amino acid sequence YKCSWERNPWDDYY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG80 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 3, in which CDR3 is the amino acid sequence YKCSWERNPWDDYY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 3.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 4. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 4, and has a CDR3 of the amino acid sequence YPPLDGGCYTANIA. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG89 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 4, in which CDR3 is the amino acid sequence YPPLDGGCYTANIA. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 4.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 5. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 5, and has a CDR3 of the amino acid sequence YPQMGCRSAGIG. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG93 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 5, in which CDR3 is the amino acid sequence YPQMGCRSAGIG. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 5.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 6. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 6, and has a CDR3 of the amino acid sequence SRDQYCGGRWVY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG98 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 6, in which CDR3 is the amino acid sequence SRDQYCGGRWVY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 6.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 7. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 7, and has a CDR3 of the amino acid sequence FRDEYCGGEFPY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsG105 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 7, in which CDR3 is the amino acid sequence FRDEYCGGEFPY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 7.

In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to EGFP. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to EGFP with a nanomolar or sub-nanomolar binding affinity. Preferably, the isolated vNAR single domain antibody according to the invention binds to EGFP with a binding affinity (KD value) of 10 ^-7 or more, 10 ^-8 or more, or 10 ^-9 or more.

In certain embodiments, the isolated vNAR single domain antibody according to the invention can be expressed in mammalian cells. In certain embodiments, the isolated vNAR single domain antibody according to the invention can be used as a targeted intrabody binding with native intracellular EGFP in mammalian cells. Preferably, the mammalian cells are cells derived from human.

In certain embodiments, the isolated vNAR single domain antibody according to the invention comprises an amino acid sequence which is at least 90%homologous to an amino acid sequence selected from SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11, SEQ. ID No. 12, SEQ. ID No. 13, and SEQ. ID No. 14. In preferred embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence selected from SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11, SEQ. ID No. 12, SEQ. ID No. 13, and SEQ. ID No. 14.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 8. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 8, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS3 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 8, in which CDR3 is the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 8.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 9. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 9, and has a CDR3 of the amino acid sequence YRCLTAGRDRWDTIDGGSDYY. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS11 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 9, in which CDR3 is the amino acid sequence YRCLTAGRDRWDTIDGGSDYY. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 9.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 10. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 10, and has a CDR3 of the amino acid sequence RYSWYCYSPDSSNYI. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS12 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 10, in which CDR3 is the amino acid sequence RYSWYCYSPDSSNYI. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 10.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 11. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 11, and has a CDR3 of the amino acid sequence YSDCWVEDGGAPYI. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS18 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 11, in which CDR3 is the amino acid sequence YSDCWVEDGGAPYI. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 11.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 12. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 12, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS19 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 12, in which CDR3 is the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 12.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 13. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 13, and has a CDR3 of the amino acid sequence RYSWYCYSPDSSNYI. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS29 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 13, in which CDR3 is the amino acid sequence RYSWYCYSPDSSNYI. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 13.

In certain embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 14. In other embodiments, the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to the amino acid sequence of SEQ. ID No. 14, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody is an isolated vNAR single domain antibody BsS45 comprising an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 14, in which CDR3 is the amino acid sequence YTCWDSGHTGGYK. In a preferred embodiment, the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 14.

In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to at least one of SARS-CoV-2 spike S1 and spike RBD. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a micromolar binding affinity. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a nanomolar binding affinity. Preferably, the isolated vNAR single domain antibody binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a binding affinity (KD value) of 10 ^-6 or more, 10 ^-7 or more, 10 ^-8 or more, or 10 ^-9 or more.

In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to both of SARS-CoV-2 spike S1 and spike RBD. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to both of SARS-CoV-2 spike S1 and spike RBD with a micromolar binding affinity. In certain embodiments, the isolated vNAR single domain antibody according to the invention binds to both of SARS-CoV-2 spike S1 and spike RBD with a nanomolar binding affinity. In certain embodiments, the isolated vNAR single domain antibody binds to both of SARS-CoV-2 spike S1 and spike RBD with a binding affinity (KD value) of 10 ^-6 or more, 10 ^-7 or more, 10 ^-8 or more, or 10 ^-9 or more.

In another aspect according to the present invention, provided is a biparatopic vNAR single domain antibody comprising at least one segment consisted of the isolated vNAR single domain antibody according to the invention.

In certain embodiments, the biparatopic vNAR single domain antibody comprises a first segment consisted of the isolated vNAR single domain antibody according to the invention and a second segment consisted of the isolated vNAR single domain antibody according to the invention. In certain embodiments, the isolated vNAR single domain antibodies consisting both segments of the biparatopic vNAR single domain antibody are the same or different. In certain embodiments, the isolated vNAR single domain antibodies consisting both segments of the biparatopic vNAR single domain antibody are different.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7. In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7, and a second segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, SEQ. ID No. 2, SEQ. ID No. 3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, and SEQ. ID No. 7. In some embodiments, in the biparatopic vNAR single domain antibody, the isolated vNAR single domain antibody consisting the first segment is the same as the isolated vNAR single domain antibody consisting the second segment. In other embodiments, in the biparatopic vNAR single domain antibody, the isolated vNAR single domain antibody consisting the first segment is different from the isolated vNAR single domain antibody consisting the second segment.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, and a second segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 2. In a preferred embodiment, the biparatopic vNAR single domain antibody is a biparatopic vNAR single domain antibody BsG3-BsG73 comprising a first segment having the amino acid sequence of SEQ. ID No. 1 and a second segment having the amino acid sequence of SEQ ID. No. 2.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, and a second segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 4. In a preferred embodiment, the biparatopic vNAR single domain antibody is a biparatopic vNAR single domain antibody BsG3-BsG89 comprising a first segment having the amino acid sequence of SEQ. ID No. 1 and a second segment having the amino acid sequence of SEQ ID. No. 4.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, and a second segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 6. In a preferred embodiment, the biparatopic vNAR single domain antibody is a biparatopic vNAR single domain antibody BsG3-BsG98 comprising a first segment having the amino acid sequence of SEQ. ID No. 1 and a second segment having the amino acid sequence of SEQ ID. No. 6.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention comprises a first segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 1, and a second segment consisted of an isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to an amino acid sequence selected from SEQ. ID No. 7. In a preferred embodiment, the biparatopic vNAR single domain antibody is a biparatopic vNAR single domain antibody BsG3-BsG105 comprising a first segment having the amino acid sequence of SEQ. ID No. 1 and a second segment having the amino acid sequence of SEQ ID. No. 7.

In certain embodiments, the biparatopic vNAR single domain antibody according to the invention binds to EGFP. In preferred embodiments, the biparatopic vNAR single domain antibody according to the invention binds to EGFP in different epitopes.

In certain embodiments, the first and the second segments of the biparatopic vNAR single domain antibody according to the invention are linked by a four GGGGS repeat.

Further provided in the present invention is a construct encoding an amino acid sequence comprising the isolated vNAR single domain antibody according to the invention.

In another aspect, the present invention provides a nucleic acid or nucleotide sequence encoding the isolated vNAR single domain antibody or the biparatopic vNAR single domain antibody according to the invention.

According to the present invention, high affinity EGFP-specific vNAR binders are generated and isolated from immunized bamboo shark library by phage display. The bamboo shark immune library according to the invention has a diversity of 10 ⁸ to10 ⁹ (amoderate level for a

library) and therefore it could be used as a

library to select vNAR binders specific to many other antigens. Seven vNAR binders against the spike proteins of pandemic SARS-CoV-2 are successfully isolated. Few studies reported the dimerization and intrabody usage of vNARs. In the present invention, it is demonstrated that the functional monovalent vNARs can be expressed well in mammalian cells and the dimeric/biparatopic vNARs have a fundamental increasing of binding affinity compared to their parental monovalent vNARs.

The library type and size are critical for the success of the isolation of high affinity vNARs. A highly diverse phage-displayed vNAR library is constructed from immunized bamboo sharks and is useful for isolating high affinity antigen-specific vNARs. Our success in the isolation of EGFP-specific vNARs with affinity up to sub-nanomolar level is owing to the immune library with a diversity of 10 ⁸ to 10 ⁹ (for example, a diversity of 4.67x10 ⁸) . The isolated EGFP-specific vNARs are all type II and they are the first vNARs targeting EGFP so far. The binding kinetics and affinity performance of these vNARs are comparable to VHHs.

To assess the intracellular expression and functionality of vNAR in mammalian cells, the inventor demonstrates the vNAR utility in target antigen recognition as an anti-GFP intrabody. vNAR as intrabodies have previously been reported in targeting different disease-related antigens. These studies demonstrate the efficacy of vNAR nobodies for clinical biologics development.

The simple architecture of vNARs allow various re-formatting to the desired constructs. Some formats have been demonstrated, including dimeric and trimeric vNARs, and Fc-fusion vNARs derived from nurse shark and spotted wobbegong shark respectively. The EGFP-specific biparatopic vNARs according to the invention reach a picomolar affinity, which is much higher than the biparatopic VHH equivalents, and recognize unique conformational epitopes. Generally, the affinity of bivalent/biparatopic vNARs is 10 to 80 folds higher than that of their parental monomeric formats.

The inventor finds that the immune vNAR phage library according to the invention can also serve as a

library for many antigens. The two selected anti-S1/RBD nanobodies can bind to both S1 and RBD and therefore can be used to detect SARS-CoV-2 or possibly prevent the virus entering human cells. Easy production in prokaryotic and eukaryotic cells with high efficiency makes nanobody a powerful tool for treating infectious virus pandemics. For example, an inhaled nanobody neutralizing SARS-CoV-2 can reach a 20g/L yield in yeast.

The present invention will be described hereinafter in more details by means of specific examples. However, these examples are merely illustrative, and the invention is not intended to be limited by these examples.

Examples

Experimental

Animals and Husbandry

Adult white-spotted bamboo sharks were wildly captured from the coastal area of Xiamen city, China and raised at City University of Hong Kong, Hong Kong Special Administrative Region of the People's Republic of China. The body length of sharks was about 50～85cm. All animal-related experiments were performed in accordance with protocols approved by the Department of Health of Hong Kong.

Antigen Preparation

EGFP with an N-terminal 6*His tag was used for bamboo shark immunization. The EGFP gene was cloned into pET32 (a+) plasmid and then transformed into Shuffle T7 cells for protein expression under the 0.1mM IPTG induction. The protein expression was checked by Coomassie blue stained protein gel, western blot and activity assay. The E. coli cell pellet was collected after o/n induction by centrifugation and then lysed for 4-6 rounds using high pressure homogenizer (1000psi) . The cell lysate was then centrifuged (8000rpm, 60min, 4℃) to collect the supernatant. The protein was firstly purified by Ni affinity chromatography (Bio-rad, 5mL Ni-Charged cartridge, #780-0812) and then further by size exclusion chromatography (GE, Superdex 200 16/60, #28-9893-35) using FPLC (Biorad FPLC NGC QUEST 10 High Pressure Chromatography System) . The purified protein was concentrated by centrifugal filter device (Merck Millipore, #UFC900324 or #UFC800324) . The protein concentration was measured by Thermo Scientific NanoDrop 2000 spectrophotometers or BCA method (Pierce ^TM BCA Protein Assay Kit, #23225) . The proteins were subjected to endotoxin removing by Detoxi-gel endotoxin removing columns (Thermo, #20344) . The endotoxin level was then tested by Bioendo KC Endotoxin Test Kit (Xiamen Bioendo, China) . The final endotoxin level in 100ug antigen was limited to ≤ 0.5EU/kg shark in each injection.

Immunization Strategies

The sharks were anesthetized with MS-222 (0.1g/L seawater) prior to performing administration and bleeding. Freund’s adjuvant is used as an immunopotentiator. The sharks were immunized biweekly subcutaneously with antigen emulsions in Complete Freund’s Adjuvant (CFA) or Incomplete Freund’s Adjuvant (IFA) (v: v=1: 1) as scheduled. 200ug EGFP were injected with CFA in the first immunization and then 100ug for the subsequent eleven times of boosters with IFA.

Shark PBMCs Collection during Immunization

Each bleeding is operated two weeks after the latest injection. Blood are drawn from the caudal vein and centrifuged at 300g for 5min to separate the peripheral blood mononuclear cells (PBMC) and plasma for subsequence analysis. All samples collected are stored at -80℃.

Construction of vNAR-Phage Library

Total RNAs are extracted from PBMC of bamboo sharks and then reverse transcribed to cDNA following the protocol of SuperScript III First-Strand Synthesis System (Invitrogen, 18080051) . The vNAR DNAs are amplified by PCR following the previous report () . The PCR products are performed with clean-up using QIAEXII gel extraction kit (QIAGEN, 20051) . The purified vNAR DNAs and the phagemid pMECS are double-digested by restriction endonucleases PstI-HF (NEB, R3140M) and NotI-HF (NEB, R3189M) respectively at 37℃ for overnight. Next, the digestion products are performed with clean-up using QIAquick gel extraction kit (QIAGEN, 28706) . The purified vNAR DNAs are ligated into the pMECS vector by T4 DNA ligation (1ug vNAR DNA, 3ug pMECS, 10U T4 DNA ligase in 200ul ligation buffer) at 16c for overnight. The ligation reaction is used for transformation after heat inactivation at 70℃for 15 mins. Electroporation is carried out in a 0.1cm gap cuvette using 1ul ligation reaction in 25ul of E. coli TG1 electrocompetent cells (Lucigen, ER2738) . The TG1 cells are then plated on Amp-selective medium to generate a vNAR library of more than 10 ⁷ individual transformants. Following this, the TG1 cells were collected for the subsequent phage display and panning.

Phage Display and Panning

The TG1 cells bearing the phagemid library (～10 ¹⁰ cells) are cultured in 2xTY/Amp-Glu medium (16g tryptone, 10g yeast extract, 5g NaCl, 100ug/ml Amp, 2%D-glucose in 1 L MilliQ water) at 37C for 3 h. Then the cells are infected with M13K07 helper phages (NEB, N0315S) at multiplicity of infection of about 20 to produce a phage-displayed vNAR library. After an over-night culture, the amplified phage particles are precipitated using PEG/NaCl solution (20%polyethylene glycol 6000, 2.5M NaCl in MilliQ water) at 4℃ for 1 h. About 1x10 ¹¹ phage particles are incubated in each EGFP-coated well of MaxiSorp plate (BioLegend, 423501) for vNAR binding with EGFP at room temperature for 2 h. The unbound phage particles are washed away by PBS/0.05%Tween and the EGFP-bound phages are eluted for the consecutive rounds of panning using the same protocols mentioned above. A total of three to four rounds of panning is sufficient to enrich EGFP-specific phage particles.

Phage ELISA

Phage ELISA is used for assessing the enrichment of antigen-specific phage particles. 100ng EGFP was coated per well at 4℃ for overnight and then blocked with 5%skimmed milk-PBS at room temperature for 3 h. The phage particles amplified after each round of panning are diluted into 2x10 ¹⁰ phages in 100ul 3%skimmed milk-PBS and then incubated in wells at room temperature for 2 h. Anti-M13 mAb HRP conjugate (Abcam, ab50370) diluted 1: 3000 in 5%skimmed milk-PBS was then added for incubating at room temperature for 1 h. The ELISA was developed with TMB substrate (Abcam, ab171522) and then read at 450 nm.

Identification of Antigen-Specific vNARs

96 to 192 TG1 colonies randomly picked from LB-Amp agar plates are individually cultured in 1 ml TB-Amp medium (1.15g KH2PO4, 8.2g K2HPO4.3H20, 6g tryptone, 12g yeast extract, 2ml glycerol, 100ug/ml Amp in 0.5L MilliQ water) in each well of a 96 deep well plate. Steak the same cells on a reference master LB-Amp-Glu plate for temporary cell conservation. Incubate the 96 deep well plate at 37℃ with shaking at 250rpm for 3 to 5h until OD600 growing to 0.6. Then add 1ul 1M IPTG to induce the vNAR expression overnight at 37℃ with shaking at 200rpm. Next morning, centrifuge the plate to pellet bacteria and then use TES-TES/4 buffers (TES: 0.2M Tris-HCl pH 8.0, 0.5mM EDTA, 0.5M sucrose; TES/4: 1 volume TES buffer, 3 volumes MilliQ water) to lysis the cells following the protocol from Pardon et al. (2014) ¹⁴. Then the supernatant of cell lysate is used to perform ELISA to identify EGFP specific clones. Reference can be made to the previous section for ELISA procedures.

vNAR Expression and Purification

TG1 cells bearing the vNAR expression plasmid are cultured in 0.5L TB-Amp medium (1.15g KH ₂PO ₄, 8.2g K ₂HPO ₄ ^.3H ₂O, 6g tryptone, 12g yeast extract, 2ml glycerol, 100ug/ml Amp in 0.5L MilliQ water) at 16℃ for overnight under the 1mM IPTG induction. For cell lysis, the cell pellet collected by centrifuge are firstly resuspended in 8 ml TES buffer (0.2M Tris-HCl pH 8.0, 0.5mM EDTA, 0.5M sucrose) at 4℃ for 6 h with rotation at 200 rpm and then mixed in 16ml TES/4 buffer (1 volume TES buffer, 3 volumes MilliQ water) at 4℃ for 2 h with rotation at 200 rpm. After centrifuge, the supernatant is collected and the pellet can be performed with a second cell lysis in TES-TES/4 buffer. Next, the periplasmic extracts are filter by 0.22um syringe filters (Merck, SLGS033SB) and then added with 1ml IMAC nickel resin (Bio-Rad, 1560135) for affinity capture of His-tagged nanobodies. After an overnight gentle shaking at 4℃, the nickel resin is collected by gravity and washed with 30ml PBS by draining at gravity. The protein is eluted in 5ml PBS-Imidazole buffer (150mM imidazole in PBS) . The imidazole can be removed by Amicon Ultra 3kDa Centrifugal Filters (Merck, UFC900308) and the protein can be further purified using size exclusion chromatography. The protein purity is checked by CBB-stained SDS-PGAGE gel.

The bivalent vNARs are cloned into pMECS vector and then transformed into TG1 cells. The other procedures for bivalent vNARs expression and purification are the same to that of monovalent vNARs.

EC50 Determination of vNARs

For EC50 determination of vNARs, 100ng EGFP was coated per well and blocked with 5%skimmed milk-PBS. Tenfold serial dilutions of purified His-tagged vNARs (10 ^-3 to 10 ⁴ nM) were prepared in 5%skimmed milk-PBS and then incubated at room temperature for 2 h. Mouse anti-His tag mAb HRP conjugate (Sino Biological, 105327-MM02T-H) diluted 1: 3000 in 5%skimmed milk-PBS was then added for incubating at room temperature for 1 h. The ELISA was developed with TMB substrate (Abcam, ab171522) and then read at 450 nm.

SPR for KD Determinations

The kinetics binding and dissociation process between nanobody and antigen is monitored with surface plasmon resonance (SPR) by Biacore T200 (Cytiva, USA) . Briefly, One-minute pulse of the Ni solution (0.5 mM NiCl ₂ in water) is injected to saturate the NTA chip with nickel. Then, his-tagged nanobodies (20ug/ml in HBS-P buffer: 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005%v/v Surfactant P20) are captured on the flow cell of Sensor Chip NTA (Cytiva, 28994951) . Next, a serial of double-folds dilution of GFP are injected and the sensorgrams were globally fitted with a floating R _max using the built-in evaluation software. The binding and dissociation time are set at 120s and 300s, respectively. Lastly, the regeneration solution (350 mM EDTA) is used to remove nickel and any chelated molecules on the chip surface. The binding affinity (KD) is calculated as KD (nM) = Kd (1/s) /Ka (1/Ms) . Kd is the dissociation constant; Ka is the association constant.

Overlapping/Non-Overlapping Epitope Prediction

For overlapping/non-overlapping epitope prediction of vNARs and VHHs, one His-tagged nanobody (named as the 1 ^st Nb) was coated with 40ng per well and then blocked with 5%skimmed milk-PBS. 400ng EGFP were then incubated per well for nanobody binding. The other His-tagged nanobodies (named as the 2 ^nd Nb) were then individually incubated with 400ng per well for competitive epitope binding. The 1 ^st Nb was added either as a control. Mouse anti-His tag mAb HRP conjugate (Sino Biological, 105327-MM02T-H) was then added for binding with His-tagged nanobodies. The ELISA was developed with TMB substrate (Abcam, ab171522) and then read at 450 nm. The non-overlapping epitopes existing between the 1 ^st Nb and the 2 ^nd Nb will be identified if the well added with the 2 ^nd nanobody has higher absorbance values than the control.

Validation of Intrabody Expression

For the validation of intrabody expression, 12 well plate of 293T cells were transfected with plasmids bearing vNAR and VHH insertion by Lipofectamine 3000 (Invitrogen, L3000015) according to the product protocol. Transfected 293T cells were cultured at 37℃, 5%CO ₂ for 48 h. Cell lysates were prepared by incubation with RIPA buffer containing protease inhibitor cocktail (Bimake, B14001) . After incubation on ice for 30 min, cell lysates were centrifuged for 10 min at 10,000 rpm, and supernatants was boiled with SDS loading buffer for 5 min. Then total protein samples were separated by SDS-PAGE gel, and transferred to PVDF membrane (Millipore) . Membranes were blocked in TBST containing 5%skimmed milk and incubated in primary antibodies diluted in blocking buffer at 4℃ for overnight. Primary antibodies for immunodetection were sourced as follows: Flag antibody (CST, 2368S) , β-actin (ABBKINE, A01010) .

Immunoprecipitation

293T cells expressing EGFP in 10cm plate were transfected with plasmids bearing vNAR and VHH insertion by Lipofectamine 3000 (Invitrogen, L3000015) according to the product protocol. Transfected 293T cells were cultured at 37℃, 5%CO ₂ for 48 h. Cells were lysed in 500ul lysis buffer (1%NP40, 25 mM Tris pH 7.5, 150 mM NaCl, and protease inhibitor cocktail (Bimake, B14001) ) . After incubation on ice for 30 min, cell lysates were centrifuged for 10 min at 10,000 rpm, and supernatants were diluted to 500ul binding buffer (25 mM Tris pH7.5, 150 mM NaCl) . 4μg Flag antibody (Sigma, F3165) was incubated with 50ul magnetic protein G beads (Bio-Rad, 1614023) for 30 mins at room temperature, followed by the addition of the diluted cell lysates, and further incubated at 4℃ overnight. The beads were washed four times with washing buffer (0.5%NP40, 25 mM Tris pH 7.5, and 300 mM NaCl) before analysis. Beads was boiled with SDS loading buffer for 5 min, then loading supernatants into SDS- PAGE gel. Primary antibodies for immunodetection were sourced as follows: Flag antibody (CST, 2368S) , GFP antibody (CST, 2956S) .

Example 1. Construction of Phage-Displayed vNAR Library

The peripheral blood mononuclear cell (PBMC) from the last three bleeding time-points of eight immunized bamboo sharks were used to clone the antigen-specific vNARs from IgNAR in a phage display vector (Fig. 1) . The vNAR-phage library totally contained 4.67x10 ⁸ individual transformants. Hundreds of full-length vNAR sequences obtained by colony sequencing revealed that about 85%of vNARs were unique and the most of the sequence diversity were from CDR3 as presented by the sequence logos (data not shown) .

Example 2. Biopanning of EGFP-specific vNARs

Antigen-specific vNARs can be retrieved by successive rounds of selection and their functions were then validated by ELISA/WB/SPR and intracellular experiment. Four rounds of panning against GFP was conducted. An obvious enrichment of GFP-bound clones was observed during panning, as shown by the increasing values of polyclonal phage ELISA against GFP (Fig. 2 A) .

Based on the periplasmic extract ELISA data, total 105 GFP-positive vNAR candidates were identified from 196 randomly-selected clones from the 2 ^nd to 4 ^th panning rounds. Finally, seven CDR3-unique (and also CDR1-unique) GFP-specific vNARs were selected. By the phylogenetic analysis, the 85%of 105 clones share the same CDR3 with BsG3 and very few residual differences with each other, indicating they were likely expanded from the same ancestral B cell, and the other six vNARs are more homologous though their CDR3 sequences are quite different (Fig. 2 B) .

Example 3. Comparison of Binding Affinities of VHH and vNAR

All candidates are type II vNARs (MW: 11.7～12.5 kDa) and were successfully purified after prokaryotic expression indicating their good solubilities (Fig. 3 A) . Five GFP-specific VHHs derived from llama were incorporated here as positive controls (Fig. 3 B) . These nanobodies, including vNARs and VHHs, all recognize denatured GFP with different binding levels as validated by western blotting (Fig. 3 C) .

The binding affinity (KD) and ELISA EC50 values of 12 nanobodies were measured by SPR and respectively and the values from the two methods are consistent in general for each nanobody (Fig. 4; Fig. 5) . BsG3 shows a sub-nanomolar lever of binding affinity to GFP and other vNARs all reach a nanomolar level (Table 1) . Their binding affinity, association constants, and dissociation constants are comparable with the VHHs.

Table 1. Binding affinities and EC50 values of seven vNARs and three VHHs to EGFP.

Notes: ka, association rate; kd, dissociation rate; KD, a ratio of kd/ka

Example 4. vNAR Dimerization and Functions as Intrabody

Except for BsG80, all other six vNARs can be well expressed in 293T cells as validated by western blotting (Fig. 6 A-B) . Each vNAR and GFP were co-expressed in 293T cells and then the native vNAR-GFP complexes in all examined vNARs and VHH control were successfully isolated in the anti-FLAG pull-down assay (Fig. 6 C) . This data confirmed that vNARs can be used as functional intrabodies in mammalian cells.

For the seven unique anti-EGFP vNARs selected from the immunized bamboo shark library, we did an overlapping epitope prediction firstly and then chosen the vNARs with non-overlapping epitopes to construct the biparatopic vNARs. We want to see if their binding affinities have a fundamental improvement. Some vNARs have non-overlapping epitopes with VHHs (Table 2) , predicted by competitive epitope binding assay (Fig. 7) . Especially BsG3 recognizes a unique epitope which doe not fit for the three major epitope types on EGFP recognized by five anti-EGFP VHHs. By virtue of the unique structural features of vNARs, vNAR can therefore enrich the epitope diversity to other types of antibodies.

BsG3 was chosen as the first vNAR because it has a sub-nanomolar level of affinity and then a second vNAR with a non-overlapping epitope was selected. The biparatopic vNARs were linked by a four GGGGS repeats (Fig. 8 A) . They were purified well after the expression in E. coli (Fig. 8 B) . By the SPR analysis, there are significant changes in the association rate and dissociation rate. The two rates became slower than the monovalent BsG3 vNAR (Fig. 9; Fig. 5 A) . The four biparatopic vNARs all reach a picomolar level of binding affinity with GFP and the increasing times is 17 to 63 compared to the monovalent BsG3 vNAR (Table 3) .

Table 2. Nanobodies targeting overlapping or non-overlapping epitopes on EGFP predicted by epitope competition ELISA.

Table 3. Binding affinities of four anti-EGFP bivalent vNARs to EGFP.

Example 5. Anti-SARS-Cov-2 vNAR isolation

Human coronaviruses are the major cause of upper respiratory tract illness. Spike S is a multifunctional glycoprotein that mediates SARS-CoV-2 (2019-nCoV) entry into target host cells through the binding of the host receptor ACE2 and the action of host proteases (e.g. TMPRSS2) . Spike protein contains two subunits, S1 and S2. S1 contains a receptor binding domain (RBD) , which is responsible for recognizing and binding with human cell surface receptor (Fig. 10) . Therefore, the S protein is considered a key target for vaccine development.

Four rounds of biopanning were conducted and the obvious enrichment of S1-specific and RBD-specific VHH clones were observed (Fig. 11 A-B) . Finally, seven candidates with the effective KD values (Fig. 11 C; Fig. 12) were obtained. The BsS3 and BsS11 have a nanomolar level of binding affinity with RBD. And some candidates bind only with RBD and some only with S1 (Table 4) . The higher affinity to RBD than to S1 may suggest that the steric hindrance of RBD-excluded parts of S1 results a lower affinity to S1. Thus, our bamboo shark vNAR library exhibits its potential to serve as a

library to isolate vNARs specific to various antigens.

Table 4. Binding affinities of seven vNARs to S1 and RBD.

The invention has been described in conjunction with the above specific examples, but many alternatives, modifications and variations will be apparent to those skilled in the art or are otherwise intended to be embraced. Accordingly, the embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention as defined in the appended claims. All patents, patent application, scientific articles and other published documents cited herein are hereby incorporated in their entirety for the substance of their disclosures.

References

1. Ubah, O.C. et al. Novel, Anti-hTNF-α Variable New Antigen Receptor Formats with Enhanced Neutralizing Potency and Multifunctionality, Generated for Therapeutic Development. Front. Immunol. 8, (2017) .

2. Ubah, O.C., Porter, A.J. & Barelle, C.J. In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development. Journal of Immunology Research vol. 2020 e7283239 https: //www. hindawi. com/journals/jir/2020/7283239/ (2020) .

3. Camacho-Villegas, T.A. et al. Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization. Mar Drugs 16, (2018) .

4. Arbabi-Ghahroudi, M. Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front Immunol 8, (2017) .

5. Zare, H. et al. Nanobodies, the potent agents to detect and treat the Coronavirus infections: A systematic review. Molecular and Cellular Probes 55, 101692 (2021) .

6. English, H., Hong, J. & Ho, M. Ancient species offers contemporary therapeutics: an update on shark VNAR single domain antibody sequences, phage libraries and potential clinical applications.

Antibody Ther

3, 1–9 (2020) .

7. Uth, C. et al. A chemoenzymatic approach to protein immobilization onto crystalline cellulose nanoscaffolds. Angew Chem Int Ed Engl 53, 12618–12623 (2014) .

8. Ries, J. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. nature methods 6 (2012) .

9. Fang, T. et al. Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure. Nat Methods 15, 1029–1032 (2018) .

10. Aguilar, G., Matsuda, S., Vigano, M.A. & Affolter, M. Using Nanobodies to Study Protein Function in Developing Organisms. Antibodies 8, 16 (2019) .

11. Lim, S. et al. bioPROTACs as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA) . Proc Natl Acad Sci USA 117, 5791–5800 (2020) .

12. McClelland, L.J. et al. Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1. Nature Communications 11, 1077 (2020) .

13. Shi, J., Chan, L.L., Wei, L. & Feng, L. Method of Producing Antibody Fragment. (2017) .

14. Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9, 674–693 (2014) .

15. Kubala, M.H., Kovtun, O., Alexandrov, K. & Collins, B.M. Structural and thermodynamic analysis of the GFP: GFP-nanobody complex. Protein Science 19, 2389–2401 (2010) .

16. Fridy, P.C. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nature Methods 11, 1253–1260 (2014) .

17. Walls, A.C. et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281-292. e6 (2020) .

18. Walsh, R. et al. Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 411, 132–141 (2011) .

19. Stocki, P. et al. Blood-brain barrier transport using a high-affinity, brain-selective VNAR (Variable Domain of New Antigen Receptor) antibody targeting transferrin receptor 1. bioRxiv 816900 (2020) doi: 10.1101/816900.

20. Leach, A. et al. Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature. Nanoscale 12, 14751–14763 (2020) .

21. Kovaleva, M., Johnson, K., Steven, J., Barelle, C.J. & Porter, A. Therapeutic Potential of Shark Anti-ICOSL VNAR Domains is Exemplified in a Murine Model of Autoimmune Non-Infectious Uveitis. Front. Immunol. 8, 1121 (2017) .

22. Steven, J. et al. In Vitro Maturation of a humanized shark Vnar Domain to improve its Biophysical Properties to Facilitate clinical Development. Front. Immunol. 8, 1361 (2017) .

23. Simmons, D.P. et al. Dimerisation strategies for shark IgNAR single domain antibody fragments. Journal of Immunological Methods 315, 171–184 (2006) .

24. Zhang, Z., Wang, Y., Ding, Y. & Hattori, M. Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification.

Scientific Reports

10, 1–10 (2020) .

25. Gai, J. et al. A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential. bioRxiv 2020.08.09.242867 (2020) doi: 10.1101/2020.08.09.242867.

26. Diaz, M., Stanfield, R., Greenberg, A. & Flajnik, M. Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR) : identification of a new locus preferentially expressed in early development. Immunogenetics 2002, 54, 501–512.

27. Dooley, H. & Flajnik, M. Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur. J. Immunol. 2005, 35, 936–945.

28. Streltsov, V.A. et al. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc. Natl. Acad. Sci. U.S. A. 2004, 101, 12444–12449.

29. Dooley, H. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

Molecular Immunology

2003, 40, 25–33.

30. Kovaleva, M., Johnson, K., Steven, J., Barelle, C.J. & Porter, A. Therapeutic Potential of Shark Anti-ICOSL VNAR Domains is Exemplified in a Murine Model of Autoimmune Non-Infectious Uveitis. Front. Immunol. 2017, 8, 1121.

31. Kovalenko, O.V. et al. Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis. J. Biol. Chem. 2013, 288, 17408–17419.

32. Müller, M.R. et al. Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain. MAbs 2012, 4, 673–685.

33. Zielonka, S. et al. The Shark Strikes Twice: Hypervariable Loop 2 of Shark IgNAR Antibody Variable Domains and Its Potential to Function as an Autonomous Paratope. Mar. Biotechnol. 2015, 17, 386–392.

Sequence list

Claims

An isolated vNAR single domain antibody derived from a bamboo shark library by phage display.
The isolated vNAR single domain antibody according to claim 1, wherein the bamboo shark library is an immunized library with a diversity of 10 ⁸ to 10 ⁹.
The isolated vNAR single domain antibody according to claim 1 or 2, wherein the bamboo shark is a white-spotted bamboo shark.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 1, and has a CDR3 of the amino acid sequence YEAWDESDSWNCGDYY.
The isolated vNAR single domain antibody according to claim 4, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 1.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 2, and has a CDR3 of the amino acid sequence YRGPSCGGNWAY.
The isolated vNAR single domain antibody according to claim 6, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 2.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 3, and has a CDR3 of the amino acid sequence YKCSWERNPWDDYY.
The isolated vNAR single domain antibody according to claim 8, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 3.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 4, and has a CDR3 of the amino acid sequence YPPLDGGCYTANIA.
The isolated vNAR single domain antibody according to claim 10, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 4.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 5, and has a CDR3 of the amino acid sequence YPQMGCRSAGIG.
The isolated vNAR single domain antibody according to claim 12, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 5.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 6, and has a CDR3 of the amino acid sequence SRDQYCGGRWVY.
The isolated vNAR single domain antibody according to claim 14, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 6.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 7, and has a CDR3 of the amino acid sequence FRDEYCGGEFPY.
The isolated vNAR single domain antibody according to claim 16, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 7.
The isolated vNAR single domain antibody according to any of claim 1 to 17, wherein the isolated vNAR single domain antibody binds to EGFP with a nanomolar or sub-nanomolar binding affinity.
The isolated vNAR single domain antibody according to any of claim 4 to 7 and 10 to 17, wherein the isolated vNAR single domain antibody can be expressed in mammalian cells.
The isolated vNAR single domain antibody according to any of claim 4 to 7 and 10 to 17, wherein the isolated vNAR single domain antibody can be used as a targeted intrabody binding with native intracellular EGFP in mammalian cells.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90% homologous to the amino acid sequence of SEQ. ID No. 8, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK.
The isolated vNAR single domain antibody according to claim 21, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 8.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 9, and has a CDR3 of the amino acid sequence YRCLTAGRDRWDTIDGGSDYY.
The isolated vNAR single domain antibody according to claim 23, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 9.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 10, and has a CDR3 of the amino acid sequence RYSWYCYSPDSSNYI.
The isolated vNAR single domain antibody according to claim 25, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 10.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 11, and has a CDR3 of the amino acid sequence YSDCWVEDGGAPYI.
The isolated vNAR single domain antibody according to claim 27, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 11.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 12, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK.
The isolated vNAR single domain antibody according to claim 29, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 12.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90% homologous to the amino acid sequence of SEQ. ID No. 13, and has a CDR3 of the amino acid sequence RYSWYCYSPDSSNYI.
The isolated vNAR single domain antibody according to claim 31, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 13.
The isolated vNAR single domain antibody according to any of claims 1 to 3, wherein the isolated vNAR single domain antibody comprises an amino acid sequence which is at least 90%homologous to the amino acid sequence of SEQ. ID No. 14, and has a CDR3 of the amino acid sequence YTCWDSGHTGGYK.
The isolated vNAR single domain antibody according to claim 33, wherein the isolated vNAR single domain antibody comprises the amino acid sequence of SEQ. ID No. 14.
The isolated vNAR single domain antibody according to any of claims 21 to 34, wherein the isolated vNAR single domain antibody binds to at least one of SARS-CoV-2 spike S1 and spike RBD.
The isolated vNAR single domain antibody according to any of claims 21 to 34, wherein the isolated vNAR single domain antibody binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a micromolar binding affinity.
The isolated vNAR single domain antibody according to any of claims 21 to 34, wherein the isolated vNAR single domain antibody binds to at least one of SARS-CoV-2 spike S1 and spike RBD with a nanomolar binding affinity.
The isolated vNAR single domain antibody according to any of claims 35 to 37, wherein the isolated vNAR single domain antibody binds to both of SARS-CoV-2 spike S1 and spike RBD.
A biparatopic vNAR single domain antibody comprising at least one segment consisted of the isolated vNAR single domain antibody according to any of claims 1 to 17.
The biparatopic vNAR single domain antibody according to claim 39, wherein the biparatopic vNAR single domain antibody comprises a first segment having the amino acid sequence of SEQ. ID No. 1.
The biparatopic vNAR single domain antibody according to claim 39 or 40, wherein the biparatopic vNAR single domain antibody comprises a first segment having the amino acid sequence of SEQ. ID No. 1, and a second segment having the amino acid sequence of SEQ. ID No. 2.
The biparatopic vNAR single domain antibody according to claim 39 or 40, wherein the biparatopic vNAR single domain antibody comprises a first segment having the amino acid sequence of SEQ. ID No. 1, and a second segment having the amino acid sequence of SEQ. ID No. 4.
The biparatopic vNAR single domain antibody according to claim 39 or 40, wherein the biparatopic vNAR single domain antibody comprises a first segment having the amino acid sequence of SEQ. ID No. 1, and a second segment having the amino acid sequence of SEQ. ID No. 6.
The biparatopic vNAR single domain antibody according to claim 39 or 40, wherein the biparatopic vNAR single domain antibody comprises a first segment having the amino acid sequence of SEQ. ID No. 1, and a second segment having the amino acid sequence of SEQ. ID No. 7.
The biparatopic vNAR single domain antibody according to any of claims 39 to 44, wherein the two entities bind to EGFP in different epitopes.
The biparatopic vNAR single domain antibody according to any of claims 39 to 45, wherein the two entities are linked by a four GGGGS repeat.
The biparatopic vNAR single domain antibody according to any of claims 39 to 46, wherein the biparatopic vNAR single domain antibody binds to EGFP with a picomolar binding affinity.
A construct encoding an amino acid sequence comprising the isolated vNAR single domain antibody according to any of claims 1 to 38.
A nucleic acid or nucleotide sequence encoding the isolated vNAR single domain antibody according to any of claims 1 to 38 or the biparatopic vNAR single domain antibody according to any of claims 39 to 46.