SARS-cov-2 camelid-derived nanobody or antigen-binding fragment thereof, and composition thereof and use thereof

Abstract

Provided are a SARS-CoV-2 camelid-derived nanobody or an antigen-binding fragment thereof, and a composition thereof and a use thereof. The nanobody has at least 95% homology to SEQ ID NO: 1. Mutation engineering of the multivalent nanobody obtained by screening and purifying provides a method for rapidly generating an efficient virus neutralizing agent, such that viral escape mutants can be effectively controlled.

Description

SARS-CoV-2 camelid nanobodies or antigen-binding fragments thereof, and compositions and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and particularly relates to SARS-CoV-2 camelid nanobodies or antigen-binding fragments thereof, and compositions and uses thereof. BACKGROUND

[0002] A betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the culprit of the unprecedented coronavirus disease 2019 (COVID-19) pandemic. As of October 21, 2023, there have been more than 771 million confirmed cases of COVID-19 worldwide, and a staggering 6.9 million people have died. In addition, this unprecedented pandemic has severely disrupted the global economy and overwhelmed the healthcare system. The scientific community has been working hard to develop effective treatments and vaccines against SARS-CoV-2. Therapeutic neutralizing antibodies are an important class of antiviral therapies that have been rapidly developed and applied for the prevention and treatment of SARS-CoV-2 in a short period of time. However, the constant adaptive evolution of SARS-CoV-2 has led to the emergence of several worrying variants of concern (VOCs), which pose a significant challenge to the effectiveness of antibody therapy. In particular, Omicron and its descendant subvariants not only weaken the efficacy of most neutralizing antibody therapies, but also weaken the efficacy of existing vaccines.

[0003] Omicron variant (B.1.1.529 / BA.1) was first discovered in South Africa at the end of 2021, and then rapidly spread to many countries and became the global dominant strain within a few weeks, triggering the fourth wave of global pandemic. Compared with other VOCs, the Omicron variant has the largest number of mutations, with more than 30 mutation sites in the S protein, and 15 of which are located on the RBD protein. These mutations impair the efficacy of many developed neutralizing antibodies, thus having higher transmissibility and stronger immune evasion ability. Over time, Omicron has expanded into multiple subvariants, such as BA.1, BA.2, BA.2.75, BA.4 and BA.5 (hereinafter referred to as BA.4 / 5, because the two viruses have the same spike sequence), BF.7, BQ.1.1 and XBB; these subvariants further exacerbate the public health problem. In particular, the BA.4 / 5 subvariant can further evade the acquired immunity generated by previous infection with BA.1 or BA.2 subvariant, leading to a surge of breakthrough infections in many regions around the world. Subsequently, new subvariants BQ.1.1 and XBB.1 emerged, which replaced BA.5 as the epidemic variant due to their stronger ability to evade antigen antibodies. Notably, all clinically authorized therapeutic antibodies are ineffective against BQ.1.1 and XBB.1 subvariants. In addition, the BA.5 bivalent booster also fails to elicit a strong immune response against BQ.1.1 and XBB.1, further highlighting the significant antibody evasion properties of these two subvariants. Therefore, it is urgent to develop potent and broad-spectrum neutralizing antibodies to effectively control the Omicron subvariants and actively prevent future emerging variants.

[0004] The S glycoprotein of SARS-CoV-2 is composed of S1 and S2 subunits and plays a crucial role in the process of infecting host cells. The S1 subunit can be divided into the N-terminal domain (NTD) and the receptor binding domain (RBD). There is a receptor binding motif (RBM) in the RBD, which is responsible for recognizing and binding to the specific receptor angiotensin-converting enzyme 2 (ACE2) of host cells. Once bound to ACE2, the S2 subunit facilitates the fusion of the virus with the host cell membrane. Based on this infection process, the S glycoprotein is considered an effective target for neutralizing antibody therapy. Currently, neutralizing antibodies against the S glycoprotein are divided into three categories: antibodies targeting the NTD, antibodies targeting the RBD, and antibodies targeting the S2 subunit. Most of the potent neutralizing antibodies developed so far mainly target the RBD. Their RBD epitopes are divided into seven core clusters, located on the cryptic inner surface, the solvent-exposed outer surface, and the RBM top surface of the RBD, respectively. Neutralizing antibodies that specifically bind to the RBM can directly inhibit the interaction between ACE2 and the S glycoprotein, effectively preventing viral entry into the body. Other neutralizing antibodies that bind to non-RBM regions prevent viral entry by steric inhibition of ACE2 binding, disrupting the stability of the spike trimer, or preventing spike-mediated syncytium formation. It is worth noting that most of the S glycoprotein mutations in Omicron are concentrated in the RBD region, which greatly reduces the potency of most RBD-targeting neutralizing antibodies. Recently, several broad-spectrum neutralizing antibodies have been re-proven to have strong potency in neutralizing BA.4 / 5, BQ, and XBB subvariants by binding to highly conserved RBD epitopes. These studies show that the RBD still contains protective conserved epitopes that can be used as a viable target for developing potent, broad-spectrum neutralizing antibodies.

[0005] Variable heavy domains of heavy chains (VHHs), also known as nanobodies, are derived from the pure heavy chain antibodies in Camelidae, including llamas, alpacas, and camels. Nanobodies are antigen-binding fragments derived from heavy chain antibodies in Camelidae, with a molecular weight of about 12-15 kDa. Compared with traditional antibodies, nanobodies have the advantages of high stability, good water solubility, low immunogenicity, easy and low-cost rapid production, and the ability to recognize hidden antigen epitopes. More importantly, nanobodies contain only one domain, so they are easier to engineer into multivalent molecules (including homo / hetero-multimers) than traditional antibodies, thereby enhancing binding affinity and neutralizing potency to viruses, and reducing or avoiding viral immune escape. Due to their small molecular weight and high stability, nanobodies can also be used to prevent and treat respiratory infectious diseases through inhalation. Based on the above advantages, nanobodies and their multivalent derivatives have great potential in the treatment of SARS-CoV-2 infection. Compared with traditional antibodies, nanobodies have many advantages, including high thermal stability and solubility, low immunogenicity, easy and rapid production, the ability to bind to hidden epitopes, and the ability to be engineered into multivalent forms with enhanced functions. In addition, nanobodies are small in size and stable, and can be effectively delivered by inhalation, so they are very suitable for the early prevention and treatment of respiratory diseases. Due to these advantages, nanobodies are considered an ideal choice for the next generation of anti-SARS-CoV-2 infection therapies. In recent years, significant progress has been made in the development of nanobodies for combating COVID-19. However, there are still very limited nanobodies that exhibit excellent potency and broad-spectrum activity.

[0006] SUMMARY

[0007] The continuous evolution of SARS-CoV-2 antigens greatly hinders the efficacy of neutralizing antibody therapy. Due to a large number of spike mutations, the Omicron variant exhibits resistance to most existing neutralizing antibodies. Therefore, it is urgent to develop potent and broad-spectrum neutralizing antibodies that can effectively target the Omicron variant and future emerging variants.

[0008] In an embodiment, the present application provides a SARS-CoV-2 camelid nanobody or antigen-binding fragment thereof having at least 95% homology to the following SEQ ID NO: 1 : CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACCTCTGATATTTATATTACGGATTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTAAAATTGTCGCACGTTTTCACACTACTAGTGGAGACACATACGTCGACTCCTCCGTGAAGGGCCGATTCACCATATCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTGCTGCAAACGACAATGAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCTGCAGCGCACCACAGCGAAGACCCC.

[0009] The amino acid sequence of the antibody of SEQ ID NO: 1 is SEQ ID NO: 2: QLQLVESGGGLVQAGGSLRLSCAASGRTSDIYITDWVRQAPGKERKIVARFHTTSGDTYVDSSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAANDNEYWGQGTQVTVSAAHHSEDP.

[0010] In an embodiment, the nanobody is SEQ ID NO: 1.

[0011] In an embodiment, the nanobody is a tandem of at least two antibodies or fragments thereof of claim 1 or 2 connected by a flexible GlySer linker.

[0012] In an embodiment, the nanobody or antigen-binding fragment thereof is a tandem of two or three antibodies or fragments thereof of claim 1 or 2 connected by a flexible GlySer linker.

[0013] In one embodiment, the nanobody is a tandem of one of the above antibodies or fragments thereof and an antibody or fragment thereof having at least 95% homology to the sequence of SEQ ID NO: 3, CAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGGAGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCACTGGAAGCATCACCAGCATCAATGCCATGGGTTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAGTAGCAGTAATATTGGTACCACGTGGCATGCAGACGCCGTGAAGGGCCGATTCACCATCTCGCGAGGCAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTATTGTAATCTACGTCAAGTAGACCAACAAAATGACGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC, linked by a flexible GlySer linker.

[0014] The amino acid sequence of the antibody of SEQ ID NO: 3 is SEQ ID NO: 4: QVQLVESGGGLVQEGGSLRLSCAATGSITSINAMGWYRQAPGKQRELVASSSNIGTTWHADAVKGRFTISRGSAKNTVYLQMNSLKPEDTAVYYCNLRQVDQQNDVWGQGTQVTVSSAHHSEDP.

[0015] In one embodiment, the nanobody or antigen binding fragment thereof is a tandem of one antibody or fragment thereof of SEQ ID NO: 3 and one antibody or fragment thereof of SEQ ID NO: 3, linked by a flexible GlySer linker.

[0016] In one embodiment, there is provided an isolated nucleic acid molecule encoding any of the above camelid nanobodies or antigen binding fragments thereof.

[0017] In one embodiment, there is provided an antibody conjugate comprising the above antibody.

[0018] In one embodiment, there is provided a composition comprising the above camelid nanobody or antigen binding fragment thereof.

[0019] In one embodiment, there is provided a use of the camelid nanobody or antigen binding fragment thereof as described above in the manufacture of a medicament.

[0020] The present application constructs a highly diverse nanobody phage display library and identifies nine nanobodies against multiple different epitopes on RBD. Five of them can cross-neutralize SARS-CoV-2 WT and Omicron variants BA.1 and BA.4 / 5, and one specific nanobody (A14) can even effectively neutralize subvariants BQ.1.1 and XBB.1. In addition, we selected four neutralizing nanobodies against three non-overlapping epitopes to design various multivalent nanobodies, including two homodimers, five heterodimers, one homotrimer and one heterotrimer. Surprisingly, most of the multivalent nanobodies showed stronger neutralization ability and inhibited the escape of subvariants BQ.1.1 and XBB.1. The most effective B13-B13-B13 homotrimer can effectively neutralize all tested Omicron variants at low concentration. The mutation engineering of multivalent nanobodies provides a method for rapidly generating efficient virus neutralizers to effectively control virus escape mutants. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0022] FIG. 1 is a schematic diagram of the construction and screening strategy of the VHH phage display library;

[0023] FIG. 2 is a result graph showing the frequency of each sequence of 26 unique VHH sequences determined according to sequencing results;

[0024] FIG. 3 is a result graph of RBD-ACE2 blocking activity of nine nanobodies;

[0025] FIG. 4 is a result graph of neutralization activity of nine nanobodies at a concentration of 5 μg / mL on five SARS-CoV-2 pseudoviruses;

[0026] FIG. 5 is a schematic diagram of SARS-CoV-2 multivalent nanobodies of the present application;

[0027] FIG. 6 is a mechanism analysis diagram of neutralization breadth-enhanced B13-C2, B13-B13 and B13-B13-B13, wherein FIG. 6A and 6B are the binding activity results diagrams of B13, C2, B13-C2, B13-B13 and B13-B13-B13 with S trimer proteins of Omicron BA.1.1 and XBB.1, respectively;

[0028] FIG. 7 is a stability analysis diagram of B13-C2 and B13-B13-B13 multivalent nanobodies, wherein 7A and 7B are the thermal stability results diagrams of B13-C2 and B13-B13-B13, respectively. DETAILED DESCRIPTION

[0029] In order to make the technical field personnel better understand the technical solutions in the present application, the present application will be further described below in combination with examples. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by the ordinary skilled in the art without making creative efforts shall belong to the scope of protection of the present application.

[0030] I. Materials and methods

[0031] 1. Cell lines, plasmids and recombinant proteins

[0032] HEK293T and HEK293-hACE2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, TransGen Biotech). Additional 200 pg / mL (Geneticin) was required when culturing HEK293-hACE2 cells. Plasmids encoding SARS-CoV-2 spike glycoprotein (WT, BA.1, BA.4 / 5, BQ.1.1 and XBB.1), lentivirus packaging plasmid psPAX2 and reporter plasmid pLenti-GFP expressing GFP and luciferase were generously provided by Qian Zhaohui (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing). Plasmid encoding Omicron BA.4 / 5 spike glycoprotein was subjected to site-directed mutagenesis, introducing single-point mutations as previously described. All SARS-CoV-2 proteins used were provided by Zhongsheng Biotech, including WT RBD with Fc tag, Omicron BA.1 RBD with mFc tag, Omicron BQ.1.1 spike trimer with His tag, Omicron XBB.1 spike trimer with His tag and biotinylated hACE2 with His tag.

[0033] HEK293T and HEK293-hACE2 cells were both cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, TransGen Biotech). Additional 200 pg / mL G418 (Geneticin) was required for culturing HEK293-hACE2 cells. Plasmids encoding SARS-CoV-2 spike glycoprotein (WT, BA.1, BA.4 / 5, BQ.1.1, and XBB.1), lentivirus packaging plasmid psPAX2, and reporter plasmid pLenti-GFP expressing GFP and luciferase were provided by Zhan Zhao-hui (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing). The plasmid encoding the spike glycoprotein of Omicron BA.4 / 5 was subjected to site-directed mutagenesis to introduce single-point mutations as previously described. All SARS-CoV-2 proteins used, including WTRBD with Fc tag, Omicron BA.1 RBD with mFc tag, Omicron BQ.1.1 S trimer with His tag, Omicron XBB.1 S trimer with His tag, and biotinylated hACE2 with His tag, were provided by Microbix.

[0034] 2. Construction of Camelid VHH phage display library

[0035] Four llamas were immunized with recombinant S1 and RBD proteins by Andes Antibody Biotechnologies. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of these llamas, and then reverse-transcribed into cDNA using gene-specific primers, oligo(dT), or random hexamers (see Table 1). VHH genes were amplified by nested PCR using specific oligonucleotides, and then purified VHH fragments were inserted into the phage vector pHEN1 (Hedgehog Bio) through Sfil / Notl restriction sites (see Table 1).

[0036] Table 1 Primer sequences for amplifying VHH genes

[0037] These recombinant plasmids were introduced into E. coli TG1 cells (Chengdu Renyu Biotechnology Co., Ltd.) by electroporation at 2.5 kilovolt voltage and 5 millisecond duration. After recovery in a shaker, the transformed strains were evenly spread on six 15 centimeter diameter 2xYT agar plates containing 100 pg / mL ampicillin (LABLEAD). After incubation at 37°C overnight, the colonies were harvested and transferred to culture medium. An aliquot of the sequence dilution was cultured to calculate the size of the VHH library. Twenty-four colonies were randomly picked from the serially diluted plates for colony PCR detection to verify the VHH insert. When preparing the phage library, the E. coli library was added to 250 mL 2xYT medium with 2% glucose and 100 pg / mL ampicillin (2xYT-GA) and cultured to an OD600 of 0.5. At this time, M13KO7 helper phage (New England Biolabs) was added to the bacterial culture for rescue. After overnight culture of the bacterial culture, the VHH phage display library was obtained using the PEG / NaCl precipitation method.

[0038] 3. Phage bio-panning

[0039] We performed three rounds of bio-panning to obtain high-affinity RBD-targeting VHHs. After each round of panning, the amount of coated WTRBD antigens in the microwells gradually decreased. After three PBST rinses, the RBD wells were blocked with 300 pL blocking solution (2% BSA in PBST) and incubated at 37°C for one hour. At the same time, the VHH phage display library (about 1011 phage particles, about 10000x library size) was added to the control wells without RBD antigen for pre-incubation to eliminate non-specific phages. After three PBST rinses, the pre-incubated VHH phage display library was added to the RBD wells and incubated at 37°C for two hours. Then, the wells were rinsed with PBST for 20 times, and the phages were placed in a 10 pg / mL trypsin solution (Solarbio) for incubation to release the bound phages. After infecting exponentially growing E. coli TG1, the released phage particles were amplified and used for the subsequent round of bio-panning.

[0040] 4. Phage ELISA

[0041] The 106 VHH colonies obtained from the third round of bio-panning were assayed using phage ELISA. Each VHH clone was inoculated into 1 mL 2xYT-GA solution and cultured to an OD600 of 0.5. After infection with M13KO7 helper phage, the bacterial culture was incubated at 30°C overnight. After centrifugation, 500 pL of phage supernatant was collected for subsequent phage ELISA detection.

[0042] WTRBD was coated in the wells of a 96-well microplate (Costar) at 50 ng per well, and the negative control was 5% BSA. After incubation at 4°C overnight, the microplate was blocked with 300 μL per well of blocking solution. Subsequently, 100 μL of phage supernatant was added to each well of the RBD and BSA containing wells. After incubation at 37°C for one hour, the microplate was washed three times with PBST to remove unbound phage. Bound phage was detected with HRP-conjugated anti-M13 antibody (1:10,000, SinoBiological) for one hour at 37°C. TMB substrate and stop solution (Solarbio) were then added sequentially. The absorbance at 450 nm wavelength was measured using a SpectraMax i3x plate reader (Molecular Devices, USA). The RBD and BSA well readings were recorded and the binding rate was calculated.

[0043] 5. Reformattmg, expression and purification of nanobodies

[0044] For monomeric nanobodies, the VHH sequences were amplified by PCR and cloned into pET-22b(+) vector (Hedgehog Bio) with a C-terminal His-tag in tandem. For multivalent nanobodies, two or three VHH sequences were connected head-to-tail by a (Gly4Ser)4linker and cloned into pET-22b(+) vector. All recombinant plasmids were transformed into competent BL21(DE3) cells (Solarbio). Single colonies were inoculated into 5 mL LB medium containing 100 μg / mL ampicillin (LB-A). After overnight incubation at 37°C, the pre-culture was diluted 1:100 with 50 mL LB-A and incubated to an OD600 of 0.5 to 1.0. Subsequently, the bacterial culture was treated with 0.5 mM IPTG (Solarbio) and incubated at 30°C for 4 hours to induce the production of recombinant nanobodies. After centrifugation, the bacterial culture was resuspended in lysis buffer and sonicated on ice. The clarified bacterial lysate was collected by centrifugation at 12,000 x g for 20 minutes. To obtain purified nanobodies, the bacterial lysate was loaded onto Ni Sepharose 6 Fast Flow (GE Healthcare) and eluted with a gradient of pre-cooled elution buffer (pH 7.4) containing 100-300 mM imidazole (Solarbio). The eluted nanobodies were concentrated using a centrifugal filter device (Pall) with a 3 kDa molecular weight cut-off and their buffer was exchanged to 1x PBS. The final concentration of all nanobodies was determined by the BCA method and their purity was determined on a 12% SDS-PAGE gel.

[0045] 6. Enzyme-linked immunosorbent assay

[0046] WTRBD, BA.1 RBD, BQ.1.1 S-trimer or XBB.1 S-trimer proteins were added to 96-well microplates at 50 ng / well and incubated overnight at 4°C. After three PBST washes, 300 μL / well of blocking solution was added to the microplates for blocking. Nanobodies and ACE2 were added to the microplates after 0.5% BSA / PBST 5-fold dilution and incubated at 37°C for one hour. The subsequent experiments were performed using HRP-conjugated anti-His antibody (1:5000, Proteintech), TMB substrate, stop solution and plate reader. The experiments were performed in duplicate and the average of the OD450 readings was used to calculate the EC50 values.

[0047] 7. SPR affinity measurements

[0048] SPR measurements were performed using a Biacore T200 instrument (Cytiva Life Sciences) at 25°C. SARS-CoV-2 WTRBD and BA.1 RBD were immobilized on a CM5 Series S sensor chip (Cytiva Life Sciences) at approximately 300 response units (RU) after dilution in sodium acetate buffer (pH 5.5). Five serial dilutions of purified nanobodies were used as flow phase at a flow rate of 30 μL / min for 180 seconds over the sensor chip, followed by a dissociation flow for 300 seconds. After each cycle, 10 mM glycine (pH 1.5) was injected for 90 seconds to regenerate the sensor chip. Binding data were determined by subtracting the background signal of the blank cycle and the reference flow cell. Data were fitted according to a 1:1 binding model and binding and dissociation curves were plotted for different concentrations of nanobodies to RBD.

[0049] 8. Epitope grouping competitive phage ELISA

[0050] WTRBD protein (50 ng / well) was coated on 96-well microplates and incubated overnight at 4°C. Subsequently, 300 μL / well of blocking solution was added to the microplates for blocking. After three PBST washes, phage-displayed nanobody supernatant (1:10 dilution) and purified nanobodies (10 μg / mL) 1:1 mixed samples were added to the wells at 100 μL per well. In addition, non-competitive binding was set up in wells without purified nanobodies. After one hour of incubation at 37°C, the subsequent experiments were performed using HRP-conjugated anti-M13 antibody, TMB substrate, stop solution and plate reader as described above.

[0051] 9. Detection of RBD-ACE2 binding inhibition by competitive ELISA

[0052] WTRBD proteins (50 ng / well) were coated on 96-well microplates at 4 °C overnight. After blocking, a 1:1 mixture of purified nanobodies (1 pg / mL or 10 pg / mL) and biotinylated ACE2 (0.4 pg / mL) was added to the wells with a volume of 100 pL per well. In addition, 50 pL biotinylated ACE2 and 50 pL 0.5% BSA / PBST were added to the remaining wells as non-competitive binding controls. After incubation at 37 °C for one hour, the subsequent experiments were performed as described above using HRP-conjugated streptavidin antibody (1:10000, Proteintech), TMB substrate, stop solution, and plate reader.

[0053] 10. Pseudovirus neutralization assay

[0054] To generate SARS-CoV-2 pseudoviruses, HEK293T cells were seeded into 6-well plates one day before transfection. The next day, cells were co-transfected with three plasmids including the one encoding SARS-CoV-2 spike glycoprotein (WT, BA.1, BA.4 / 5, BQ.1.1, XBB.1, or single mutants), psPAX2, and pLenti-GFP. Six hours after transfection, the culture medium was replaced with fresh DMEM containing 10% FBS. Forty-eight hours after transfection, the supernatant containing pseudoviruses was collected and filtered to remove cell debris using a 0.45 pm syringe filter (Merck).

[0055] In the pseudovirus neutralization assay, nanobodies were diluted three-fold and added to 96-well white opaque plates (Beyotime) with a volume of 50 pL per well. Then, 50 pL of pseudovirus solution was added to each well and mixed with the nanobodies, followed by incubation at 37 °C for one hour. Virus control wells contained only the pseudovirus solution. Subsequently, 2 x 104HEK293-hACE2 cells were seeded into the white opaque plates and incubated at 37 °C and 5% CO2 for 48 hours. After the white opaque plates were equilibrated at ambient temperature for 10 minutes, 100 pL of luciferase detection reagent was added to each well. After incubation for 5 minutes, chemiluminescence detection was performed using a SpectraMax i3x plate reader to quantify the intracellular fluorescent signal in each well. The percentage of neutralization was calculated by comparison with the virus control. Then, IC50 values were determined using a 4-parameter logistic regression. Experiments were performed in triplicate and repeated twice or more.

[0056] 11. Stability analysis

[0057] To evaluate the stability of multivalent nanobodies in vitro, they were stored at 4 °C, room temperature (RT), and 37 °C for 2 weeks or 4 weeks, respectively. Subsequently, their binding affinity to Omicron XBB.1 S-trimer protein was determined using an enzyme-linked immunosorbent assay.

[0058] To determine the thermal stability, the multivalent nanobodies were incubated in a water bath at 25, 37, 50, 60, 70, 80, and 90 °C for half an hour, and then equilibrated to room temperature. All the treated multivalent nanobodies were diluted to a concentration of 8 ng / μL, and then their binding affinity to the Omicron XBB.1 S trimeric protein was determined by enzyme-linked immunosorbent assay. The ratio of the OD450 value after heat treatment to the OD450 value before treatment was calculated, and then the result was multiplied by 100% to obtain the relative binding activity.

[0059] 12. Statistical analysis

[0060] All statistical analyses were performed using GraphPad Prism software (version 8.0). Data were presented as mean ± SD of at least two replicates. A 4-parameter logistic regression analysis was used to determine EC50 and IC50 values.

[0061] II. Experimental results

[0062] 1. Construction and screening of VHH phage display library

[0063] To rapidly identify RBD-specific nanobodies with high affinity and appropriate diversity, we tried to construct a highly diverse VHH phage display library, see Fig. 1. Initially, we immunized four llamas with SARS-CoV-2 S1 and RBD proteins for six rounds. A total of 5 μg intact RNA was extracted from 2 x 107 PBMCs of the immunized llamas as a template for cDNA synthesis). Then, the VHH genes were amplified using nested PCR. In the first round of PCR, 1000 bp (VH-CH1-CH2) and 750 bp (VHH-CH2) gene fragments were amplified from the cDNA template, respectively. The 750 bp gene fragment was subsequently used as a template for the next round of PCR amplification, resulting in a VHH gene fragment of approximately 400 bp in length. The VHH genes were then cloned into the pHEN1 vector and transformed into TG1 cells, resulting in a VHH library of approximately 1.3 x 107 cfu.

[0064] Twenty-four clones were randomly picked for verification by colony PCR, and the results showed that the insertion rate of the VHH library was 100%. Subsequently, all 24 clones were sequenced. A total of 23 clones contained correct VHH gene framework regions and had different complementary determining region (CDR) compositions, indicating that the library we established had a 96% in-frame insertion rate and 100% VHH gene diversity. In summary, the above results showed that the VHH library we constructed had high quality and satisfactory diversity. Then, this VHH library was revived with M13KO7 helper phage, resulting in a VHH phage display library with a size of 4.4 x 1014 pfu / mL.

[0065] To isolate potential RBD-specific binders from the VHH phage display library, we performed three rounds of biopanning using RBD protein as bait. We calculated the enrichment factor for each round of biopanning, and the results showed that RBD-binding phage was effectively enriched, see Table 2 below.

[0066] Table 2. Panning of anti-SARS-CoV-2 nanobody phage display library

[0067] Subsequently, 106 VHH clones were randomly picked from the third round of biopanning and evaluated for their binding to WTRBD using phage enzyme-linked immunosorbent assay. Notably, 101 out of 106 VHH clones were identified as positive with a binding ratio higher than 3 and were sent for sequencing. Sequence analysis showed that 26 different VHHs were obtained according to the classification of CDRs’ amino acid sequences, see Figure 2. Phylogenetic tree analysis showed the sequence diversity of these VHHs. According to the results of sequencing and phage enzyme-linked immunosorbent assay, nine nanobodies (named A14, A31, B13, B18, B20, B27, C2, C8 and D11, respectively) were selected as candidates for further expression and purification. SDS-PAGE analysis showed that the nine nanobodies were highly isolated and pure, with a molecular weight of about 15 kDa, consistent with the theoretical value.

[0068] 2. Characterization of nanobody candidates and epitope grouping

[0069] We first investigated the binding capacity of the nine candidate Nanobodies to WTRBD and BA.1 RBD using enzyme-linked immunosorbent assay. As shown in Table 3, the results indicated that all Nanobodies were able to bind strongly to WTRBD with EC50 values ranging from 0.26 to 23.8 ng / mL. Compared to ACE2, most Nanobodies (except B27) showed stronger positive signals with EC50 values lower than 0.9 ng / mL. Strikingly, we found that most Nanobodies (except B27) still bound strongly to BA.1 RBD with EC50 values lower than 107 ng / mL. However, all Nanobodies except C8 showed lower binding activity to BA.1 RBD than to WTRBD. SPR experiments were also performed to calculate the kinetic rate constants and affinity constants, which showed that all Nanobodies (except B27) bound to WTRBD with high affinity. The binding affinity (KD) ranged from 0.013 to 0.775 nM. Consistent with the results of enzyme-linked immunosorbent assay, most Nanobodies (7 out of 8) showed relatively weak binding affinity to Omicron BA.1 RBD, but their KD values (except B27) were still in the single-digit nanomolar range or lower. Notably, A14 and B20 showed strong binding affinity to Omicron BA.1 RBD with KD values of 0.065 nM and 0.026 nM, respectively (Table 3). Unfortunately, the binding affinity of C8 could not be successfully measured by SPR, possibly due to the inappropriateness of CM5 chip.

[0070] Table 3. Affinity constants of eight Nanobodies binding to WT-RBD and BA.1 RBD proteins

[0071] To explore whether the nine Nanobodies bind to different epitopes, we evaluated their ability to compete for RBD binding using phage competitive enzyme-linked immunosorbent assay. High concentrations (up to 5 pg / mL) of purified Nanobodies were used to show Nanobody competition for binding to WTRBD. Table 4 summarizes the competition, with a residual binding rate of less than 20% for a pair of competing Nanobodies, indicating that the two Nanobodies can target the same or similar epitopes. Conversely, if the residual binding rate of a competing pair is greater than 90%, it indicates that the two Nanobodies recognize non-overlapping RBD epitopes. We found that the nine Nanobodies were highly diverse and could be divided into five competition groups, indicating that they recognized five non-overlapping epitopes on RBD (Table 4).

[0072] [Corrected according to Rule 26 23.07.2024] Table 4. Antigenic epitope grouping of nine Nanobodies (Nanobodies in the left column, phage Nanobodies in the top row)

[0073] Subsequently, the blocking activity of these nanobodies against RBD-ACE2 was evaluated by competitive enzyme-linked immunosorbent assay. As shown in Figure 3, A14 and D11 could effectively block the binding of ACE2 to RBD with a blocking rate of more than 50% when the excess concentration was 5 μg / mL. Other nanobodies had lower blocking activity at a concentration of 5 μg / mL and had no blocking effect at a concentration of 0.5 μg / mL. These results suggest that the epitopes of A14 and D11 may partially overlap with RBM (also known as ACE2 binding site), while the epitopes bound by the remaining nanobodies (A31, B13, B18, B20, B27, C2 and C8) may be far away from RBM. These experiments show that the nine nanobodies target five non-overlapping epitopes on RBD and exhibit satisfactory cross-binding activity against WT RBD and BA.1 RBD, thereby laying a solid foundation for the development of cross-reactive nanocluster antibodies.

[0074] 3. Cross-neutralization ability of nanobodies against multiple SARS-CoV-2 Omicron subvariants

[0075] During the identification of these nanobodies, the Omicron subvariants BA.4 / 5, BQ.1.1 and XBB.1 have successively become dominant variants in many regions around the world and have been found to be able to evade most existing neutralizing antibody responses. To evaluate the cross-neutralization ability of nanobodies against these Omicron subvariants, we performed lentivirus-based pseudovirus infection assays. Initially, we successfully packaged five pseudoviruses carrying SARS-CoV-2 WT, BA.1, BA.4 / 5, BQ.1.1 or XBB.1 spike glycoprotein. At a fixed concentration (5 μg / mL), the neutralization potency of the nine nanobodies against the SARS-CoV-2 pseudoviruses was quantified, as shown in Figure 4. Nanobodies with a neutralization efficiency of less than 50% were defined as non-neutralizing nanobodies. Then, nanobodies with a neutralization efficiency of more than 50% were diluted three-fold, and the IC50 values of each pseudovirus are shown in Table 5.

[0076] [Corrected according to Rule 26 23.07.2024] Table 5 Summary of neutralization activity (IC50) of nine nanobodies against five SARS-CoV-2 pseudoviruses

[0077] Encouragingly, A14 in Group C exhibited cross-protection activity against all pseudoviruses with IC50 values ranging from 83.93 to 316.6 ng / mL, suggesting its potential to target a conserved RBD epitope, see Table 5. Three nanobodies from Group A (A31, B13 and B18) exhibited potent neutralization activity against SARS-CoV-2 WT, Omicron BA.1 and BA.4 / 5 with IC50 values between 117.6 and 1026 ng / mL, while C2 from Group B showed limited neutralization capacity (IC50 > 2 pg / mL). In addition, D11 was able to neutralize SARS-CoV-2 WT and Omicron BA.1, while B27 could only neutralize the WT pseudovirus. Notably, B20 and C8 had strong binding to both WT RBD and BA.1 RBD; however, they failed to neutralize any of the tested pseudoviruses even at a high concentration of 5 pg / mL. This suggests that they are specifically directed against non-neutralizing epitopes on RBD.

[0078] We obtained a total of five nanobodies (A14, A31, B13, B18 and C2) that specifically target three non-overlapping epitopes (A, B and C) on RBD, effectively neutralizing the Omicron BA.1 and BA.4 / 5 pseudoviruses. Among them, A14 retains neutralization activity against Omicron BQ.1.1 and XBB.1. However, they all lack the potency required for therapeutic applications and need to be further optimized through multimerization strategies to enhance their potency and broad-spectrum neutralization. Compared to A31 and B13, B18 from the same competition group exhibits relatively weak neutralization potency and thus is not considered for further study. Finally, A14, A31, B13 and C2 are selected as modular units for further multivalent engineering.

[0079] 4. Generating diversified multivalent nanobodies with enhanced neutralization potency and breadth

[0080] To evaluate the potential of multivalency in enhancing the neutralization potency and breadth of A14, A31, B13 and C2 monomers, we designed nine multivalent nanobodies, including homo- or hetero-dimeric nanobodies and homo- or hetero-trimeric nanobodies, see Figure 5. These multivalent nanobodies were prepared by linking two or three nanobody monomers with an optimized flexible GlySer linker of 20 amino acids [(G4S)4]. We found that they can be easily expressed in BL21 cells with a purified yield of about 20-50 pg per mL in shake flask culture. SDS-PAGE analysis showed that the dimeric (~30 kDa) and trimeric (~45 kDa) nanobodies have high purity and the expected molecular weight.

[0081] Next, we evaluated the neutralization potency of the generated multivalent nanobodies against SARS-CoV-2 WT, Omicron BA.1, BA.4 / 5, BQ.1.1, and XBB.1 pseudoviruses, and the corresponding IC50 values are given in Table 6, Summary of neutralization activities (IC50) of nine multivalent nanobodies against five SARS-CoV-2 pseudoviruses, and the fold improvement relative to monovalent nanobodies.

[0082] [Amended according to Rule 26 23.07.2024] Table 6 Summary of neutralization activities (IC50) of nine multivalent nanobodies against five SARS-CoV-2 pseudoviruses

[0083] Surprisingly, except for A31-A31, these multivalent nanobodies were able to neutralize all the tested pseudoviruses. The B13-B13-B13 homotrimer had the strongest neutralization activity with a GMIC50 value of 20.83 ng / mL (Table 6). Compared to the B13 monomer, its neutralization capacity was significantly enhanced, with an 86.1- to 268-fold increase in neutralization capacity against all the tested pseudoviruses. The homodimers of A31-A31 and B13-B13 and the heterodimers of A31-C2 and B13-C2 had potent neutralization activities against SARS-CoV-2 WT, Omicron BA.1, and BA.4 / 5, with IC50 values lower than 24 ng / mL. Again, compared to the corresponding monomers A31 and B13, their neutralization capacity was significantly enhanced by about 15.4-113-fold. Notably, the heterodimers A31-C2 and B13-C2 exhibited higher potency against Omicron BQ.1.1 and XBB.1 compared to the homodimers A31-A31 and B13-B13. Although the four multivalent nanobodies containing the A14 module unit (A14-A31, A14-B13, A14-C2, and A14-A31-C2) exhibited broad neutralization against all five tested pseudoviruses, their enhanced neutralization potency was weak or insignificant compared to the monomer A14. In summary, these data suggest that the multimerization strategy can enhance the neutralization capacity and breadth of monomeric nanobodies.

[0084] 5. Mechanistic analysis of multivalent nanobodies' improved neutralization breadth

[0085] To further explore the mechanism of multivalent nanobodies to improve the neutralization breadth, we selected B13-B13, B13-C2 and B13-B13-B13 as representative antibodies for further study. See FIG. 6, first, we investigated whether the binding affinity of these three multivalent nanobodies to Omicron BQ.1.1 (FIG. 6A) and XBB.1S trimers (FIG. 6B) was higher than that of their monomers. As expected, compared with B13, the binding affinity of B13-C2, B13-B13 and B13-B13-B13 to Omicron BQ.1.1S trimer protein increased by 643-fold, 482-fold and 965-fold, respectively. Similarly, their binding to Omicron XBB.1S trimer protein was also 157-fold, 94-fold and 471-fold tighter than B13. Compared with C2, the binding activity of B13-C2 to Omicron BQ.1.1 and XBB.1S trimer proteins also increased by 12.2-fold and 15.7-fold, respectively. These results indicate that the combined structure of our multivalent nanobodies can significantly enhance the binding affinity of monomeric nanobodies to Omicron S-trimer, thereby improving their neutralization ability.

[0086] Compared with BA.4 / 5, the RBD proteins of BQ.1.1 and XBB.1 contain an additional 7 mutations. To verify the impact of these additional mutations, we constructed seven mutant pseudoviruses based on BA.4 / 5 to determine the key residues that confer resistance to B13 and C2. Compared with BA.4 / 5, the neutralization ability of B13 against R346T mutant pseudovirus decreased significantly, with an IC50 value decrease of more than 10.8-fold. In addition, the neutralization activity of monomeric C2 against N460K mutant pseudovirus also decreased, with an IC50 > 5 μg / mL (change in fold cannot be calculated). The simultaneous presence of R346T and N460K mutations in BQ.1.1 and XBB.1 is the reason why these two subvariants are resistant to the neutralization of B13 and C2. In sharp contrast, however, the B13-C2, B13-B13 and B13-B13-B13 multivalent nanobodies exhibited significantly improved neutralization breadth and stronger potency (Table 7). They could effectively neutralize all seven single mutants, with IC50 values below 100 ng / mL. Therefore, the R346T and N460K mutations enable the virus to evade monomeric nanobodies but not multivalent nanobodies. Notably, the neutralization activity of these three multivalent nanobodies against R346T and / or N460K mutant pseudoviruses decreased slightly (3.1-4.4-fold) (Table 7), which is the IC50 values of B13, C2, B13-C2, B13-B13 and B13-B13-B13 against the seven single mutants and the respective fold reduction in IC50 relative to BA.4 / 5.

[0087] [Corrected according to Rule 26 23.07.2024] Table 7 IC50 values of B13, C2, B13-C2, B13-B13 and B13-B13-B13 against seven single mutants

[0088] These findings elucidate the reason why these multivalent nanobodies have lower neutralization activity against BQ.1.1 and XBB.1 than BA.4 / 5. In short, our multivalent nanobodies (B13-C2, B13-B13 and B13-B13-B13) have stronger neutralization potency that can adapt to the R346T and N460K escape mutations present on Omicron BQ.1.1 and XBB.1 subvariants, thus resulting in good neutralization breadth.

[0089] 6. Superior stability of multivalent nanobodies

[0090] Among the bivalent and trivalent nanobodies, B13-C2 and B13-B13-B13 exhibited the highest neutralization activity, respectively. Therefore, we selected them as representative antibodies for further stability analysis. We evaluated the in vitro stability of B13-C2 and B13-B13-B13 by storing them at three different temperatures (4°C, RT and 37°C) for 2 or 4 weeks. Subsequently, their binding capacity to S-trimer was determined by enzyme-linked immunosorbent assay. As shown in Table 8, the binding capacity of B13-C2 and B13-B13-B13 remained almost unchanged after 2 weeks of storage at three different temperatures. After 4 weeks at RT or 37°C, their binding activity decreased slightly but still remained in the range of single-digit ng / mL. In addition, as shown in Figure 7, B13-C2 and B13-B13-B13 exhibited excellent thermal stability (Figures 7A and 7B, respectively). After incubation at 70°C for 30 minutes, the relative binding activity of B13-C2 and B13-B13-B13 remained above 90%. However, after incubation at 80°C for 30 minutes, their relative binding activity decreased significantly but still remained above 50%. The above results indicate that our designed multivalent nanobodies have significant drug stability.

[0091] Table 8 Binding activity of multivalent nanobodies to Omicron XBB.1 S-trimer protein after storage under different conditions

[0092] In this study, our goal was to develop potent nanobodies against different epitopes on RBD and effective against various Omicron subvariants. The highly diverse nanobody phage display library can increase the possible diversity of epitopes. Therefore, we used multiple primers to amplify the VHH genes to introduce CDR diversity when generating the phage library. After three rounds of biopanning, we identified nine nanobodies that tightly bind to WT RBD and BA.1 RBD. According to epitope grouping analysis, these nanobodies can be divided into five competitive groups (A-E). Pseudovirus neutralization assay showed that only nanobodies in groups A-C can effectively neutralize SARS-CoV-2 WT strain. Four nanobodies in groups A and B (A31, B13, B18 and C2) showed effective neutralization activity against BA.1 and BA.4 / 5 subvariants, but completely lost the neutralization ability against BQ.1.1 and XBB.1 subvariants, which indicated that they were directed against non-conserved epitopes on RBD. Encouragingly, A14 in group C showed broad-spectrum neutralization against all tested Omicron subvariants, which indicated that it might target a conserved epitope on RBD. Notably, the discovery of broad-spectrum neutralizer A14 suggested that SARS-CoV-2 antigen prototype immunization can also induce broad-spectrum neutralizing antibodies against conserved epitopes.

[0093] Currently, most available RBD-targeting neutralizing antibodies can directly or sterically inhibit the binding of ACE2 to S protein, thus preventing viral entry. Similarly, A14 showed moderate ACE2-RBD blocking activity in competitive enzyme-linked immunosorbent assay, indicating that its main neutralization mechanism is blocking ACE2. However, all nanobodies in groups A and B, except D11, failed to inhibit ACE2-RBD binding. This suggests that their binding epitopes are located outside the RBM. The neutralization mechanism of these non-RBM-targeting nanobodies remains to be further studied, possibly by disrupting the stability of SARS-CoV-2 spike trimer or blocking the formation of potential syncytia. In summary, we obtained three groups of RBD-targeting neutralizing nanobodies with different epitopes and different neutralization mechanisms, which laid a solid foundation for developing potent and broad-spectrum multivalent nanobodies.

[0094] Given the natural trimeric presentation of the spike protein on the SARS-CoV-2 surface, multimeric nanobodies have the potential to target multiple RBD antigens simultaneously, thus greatly enhancing potency. The multimeric designs mainly include homo-dimers, hetero-dimers, homo-trimers and hetero-trimers. Therefore, we focused on the above four designs and selected four neutralizing nanobodies, A31 and B13 from group A, C2 from group B and A14 from group C, to construct twelve different multivalent nanobodies. However, due to the low protein expression of three multivalent nanobodies (A14-A14, A14-A14-A14 and A31-A31-A31), we could only obtain nine purified multivalent nanobodies. As expected, most of the multivalent nanobodies showed significantly improved resistance to Omicron variants compared to monomers. Notably, two homo-dimers (A31-A31, B13-B13), two hetero-dimers (A31-C2, B13-C2) and one homo-trimer (B13-B13-B13) showed very high neutralization potency against SARS-CoV-2 WT and Omicron BA.1 and BA.4 / 5 with IC50 values lower than 24 ng / μL. In addition to A31-A31, they also effectively neutralized Omicron BQ.1.1 and XBB.1, while the corresponding monomers (A31, B13 and C2) could not do so. Furthermore, we found that heterodimers A31-C2 and B13-C2 were more effective against BQ.1.1 and XBB.1 than homodimers A31-A31 and B13-B13. These results suggest that fusing nanobodies against different epitopes into heterodimeric form is an effective strategy to overcome escape mutants, which is consistent with previous research results. We also observed that the in vitro neutralization potency of nanobodies against pseudoviruses gradually increased with increasing valency. Taking B13 as an example, from B13 to B13-B13 and then to B13-B13-B13, its neutralization against SARS-CoV-2 WT, Omicron BA.1, BA.4 / 5, BQ.1.1 and XBB.1 pseudoviruses increased by 109-fold, 120-fold, 268-fold, >146-fold and >86.1-fold, respectively. Compared with the corresponding A14 monomer, the neutralization potency of the other four multivalent nanobodies (A14-A31, A14-B13, A14-C2 and A14-A31-C2) increased less, which may be due to the spatial limitation of the linker. Notably, in this study, the most effective multivalent nanobody B13-B13-B13 outperformed previously reported broad-spectrum neutralizing antibodies such as S728-1157 and VacBB-551. Our study shows that rationally designed multivalent nanobodies are a quick way to produce potent virus neutralizers, which can enhance the efficacy of controlling virus escape mutants.

[0095] Determination of the binding affinity of our multivalent nanobodies to the S-trimer proteins of Omicron BQ.1.1 and XBB.1 is undoubtedly crucial to elucidate their enhanced breadth of neutralization. ELISA assays showed that the binding affinity of B13-C2, B13-B13, and B13-B13-B13 multivalent nanobodies to the S-trimer of BQ.1.1 and XBB.1 was significantly higher than that of B13 and C2, which was basically consistent with the neutralization results. Compared with BA.4 / 5 subvariants, the RBD proteins of BQ.1.1 and XBB.1 subvariants have seven more mutations (R346T, L368I, K444T, V445P, G446S, N460K, and F490S). These additional mutations enhance the immune escape ability of BQ.1.1 and XBB.1 to therapeutic antibodies. The BQ.1.1 subvariant showed stronger anti-neutralization ability, mainly due to the key N460K mutation, and the R346T and K444T mutations also had some influence. Another study showed that the R346X mutation (mainly R346T) in the RBD provided additional fitness for Omicron subvariants such as XBB and BQ, which might help antibody escape. In our study, the R346T mutation significantly weakened the neutralization ability of B13, while the reduction of C2 neutralization activity was attributed to the N460K mutation. However, the B13-C2, B13-B13, and B13-B13-B13 multivalent nanobodies showed significant efficacy in neutralizing R346T and N460K mutants due to their rational multimerization design. Notably, the R346T mutation did not produce drug resistance to B13-B13-B13. The neutralization potency of B13-C2 heterodimer against BQ.1.1 and XBB.1 was affected by one mutation (R346T), while that of B13-B13 homodimer was affected by two mutations (R346T and N460K). This explains why the potency of B13-B13 is weaker than that of B13-C2. These findings again confirm that the connection of two different paratopes is a prerequisite for improving the ability to resist viral escape. In summary, the multimerization of nanobodies can expand the interface area, enhance the affinity between a single nanobody and the spike protein, and better tolerate escape mutations, which may be the reason for the broad neutralization mechanism mediated by our multivalent nanobodies.

[0096] Those skilled in the art will further appreciate that the invention described herein is susceptible to variations and / or modifications as will be evident to those of ordinary skill in the art. It is to be understood that the invention disclosed in this patent specification includes all such variations and modifications and is limited only by the claims.

Claims

1. A SARS-CoV-2 camelid nanobody or antigen-binding fragment thereof, characterized in that, The nanobody has at least 95% homology with SEQ ID NO: 1: CAGTTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGGACCTCTGATATTTATATTACGGATTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTAAAATTGTCGCACGTTTTCACACTACTAGTGGAGACACATACGTCGACTCCTCCGTGAAGGGCCGATTCACCATATCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTACTGTGCTGCAAACGACAATGAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCTGCAGCGCACCACAGCGAAGACCCC.

2. The camelid nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The nanobody is SEQ ID NO:

1.

3. The camelid nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The nanobody is formed by connecting at least two antibodies or fragments thereof of claim 1 or 2 in series through a flexible GlySer connector.

4. The camelid nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The nanobody is formed by connecting two or three antibodies or fragments thereof of claim 1 or 2 in series through a flexible GlySer connector.

5. The camelid nanobody or antigen-binding fragment thereof according to claim 2, characterized in that, The nanobody is formed by connecting one antibody or fragment thereof of claim 1 or 2 and one antibody or fragment thereof having at least 95% homology with SEQ ID NO: 3 in series through a flexible GlySer connector, the SEQ ID NO: 3 sequence being CAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGGAGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCACTGGAAGCATCACCAGCATCAATGCCATGGGTTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAGTAGCAGTAATATTGGTACCACGTGGCATGCAGACGCCGTGAAGGGCCGATTCACCATCTCGCGAGGCAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTATTGTAATCTACGTCAAGTAGACCAACAAAATGACGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGCACCACAGCGAAGACCCC.

6. The camelid nanobody or antigen-binding fragment thereof according to claim 5, characterized in that, The nanobody is formed by concatenating one antibody or fragment thereof according to claim 2 with one antibody or fragment thereof of SEQ ID NO: 3 via a flexible GlySer linker.

7. An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a polypeptide of claim 1. which codes for a camelid nanobody or antigen binding fragment thereof according to any one of claims 1 to 6.

8. An antibody conjugate comprising an antibody according to any one of claims 1 to 6.

9. A composition comprising a camelid nanobody or antigen binding fragment thereof according to any one of claims 1 to 6.

10. Use of a camelid nanobody or antigen binding fragment thereof according to any one of claims 1 to 6 for the manufacture of a medicament.

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