Anti-LASV virus nano antibody and application thereof

Abstract

The invention discloses a nano antibody for resisting an LASV virus and application of the nano antibody. The nano antibody disclosed by the invention can be specifically combined with GPC tripolymer proteins of various LASV viruses, has a broad-spectrum anti-LASV virus effect, and has a good application prospect in the aspect of treating and / or preventing LASV virus related diseases.

Description

Technical Field

[0001] This invention relates to the field of nanobody drug technology, specifically, to an anti-LASV nanobody and its application. Background Technology

[0002] Lassa fever (LF) is an acute viral hemorrhagic fever caused by Lassa virus (LASV), prevalent mainly in West Africa. Clinical manifestations primarily include fever, myalgia, and severe collapse, often accompanied by hemorrhage or neurological symptoms. This highly pathogenic virus affects 100,000 to 300,000 people annually and causes approximately 5,000 deaths. Currently, the commonly used treatment is the broad-spectrum antiviral drug ribavirin. There are currently no marketed neutralizing antibodies in clinical trials for the prevention and treatment of Lassa fever. The primary host of LASV is the multi-mammal mouse. Humans are infected through direct contact with infected rodents or their excrement; zoonotic transmission is the main factor in LASV outbreaks. The WHO's Epidemic Prevention Research and Development Action Plan has included LASV in its priority pathogen list for the urgent development of effective drugs.

[0003] Lassa virus is widespread in most parts of sub-Saharan Africa. Based on its geographical location of discovery and phylogenetic analysis of nucleotide variation sequences, Lassa virus can be divided into four lineages: lineages I–III are predominant in Nigeria, while lineage IV is predominant in Liberia, Guinea, Sierra Leone, and other countries; in the past 10 years, three new lineages (V–VII) have emerged that are not yet widely accepted. The S segment (encoding the GP protein) exhibits high variability among different lineages, affecting the virus's ability to bind to host cell receptors and potentially leading to differences in immune escape or transmissibility. The surface of the LASV virus is covered by a type I trimeric fusion glycoprotein complex (GPC), which is the sole target of neutralizing antibodies (NAbs). The functional GPC consists of three heterotrimers, each of which contains a receptor-binding GP1 subunit, a transmembrane GP2 subunit, and a stable associated signal peptide (SSP). The numerous glycans on GPC form a dense barrier that blocks the binding of antibodies to glycoprotein trimers, allowing LASV to evade neutralizing humoral immune responses and hindering the development of vaccines or infection-induced neutralizing antibodies.

[0004] Fully human nanobodies are antibody fragments consisting only of the heavy chain variable region. This single-domain structure lacks the light chain and Fc region of conventional antibodies, resulting in a much smaller size (approximately 15 kDa). They possess unique structural and functional properties, such as small size, high antigen-binding affinity, strong tissue penetration, easy identification of hidden sites, stability under extreme conditions, and ease of production, making them promising diagnostic and therapeutic tools.

[0005] Under natural conditions, after infection with LASV in humans and rodents, neutralizing antibodies play a crucial role in neutralizing the virus during the acute infection phase and blocking viral infection. An effective neutralizing antibody response can block LASV infection, thereby achieving the goal of infection prevention. Pinneo (Genbank: AAF86701.1) was the first LASV strain isolated from an infected individual, while the Josiah strain (Genbank: NP_694870.1) from lineage IV is also the immunogen for most LASV candidate vaccines and is the major LASV virus strain. In addition, there are other LASV virus strains such as 803213 (Genbank: AAF86703.1), CSF (Genbank: AAL13212.1), Togo (Genbank: AVN98153.1), AV (Genbank: AAG41802.1), and AYM (Genbank: AYM51697.1). Given that different predominant strains of LASV exist in different regions of the world, developing broad-spectrum neutralizing antibodies against LASV is a more promising treatment strategy for Lassa fever. Currently, there are no reported broad-spectrum antibodies against LASV; therefore, there is an urgent need to develop a fully human broad-spectrum neutralizing nanobody against LASV. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an anti-LASV nanobody and its application.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] The first aspect of this invention provides a nanobody against LASV virus, comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 1, 2, and 3, respectively; or, the amino acid sequences of which are shown in SEQ ID NO: 4, 5, and 6, respectively; or, the amino acid sequences of which are shown in SEQ ID NO: 7, 2, and 8, respectively; or, the amino acid sequences of which are shown in SEQ ID NO: 9, 10, and 11, respectively; or, the amino acid sequences of which are shown in SEQ ID NO: 1, 12, and 13, respectively; or, the amino acid sequences of which are shown in SEQ ID NO: 1, 12, and 13, respectively. As shown in SEQ ID NO: 14, 2, and 15; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 2, and 3, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 17, 18, and 13, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 2, and 13, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 20, 21, and 22, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23, 2, and 6, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 24, 25, and 8, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 2, and 3, respectively. As shown in SEQ ID NO: 26, 2 and 27; or, the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 28, 29 and 13, respectively.

[0009] In some implementations, the frame region of the heavy chain variable region is a human-source frame region.

[0010] In some preferred embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, and does not involve any alteration to the CDR sequence.

[0011] A second aspect of the present invention provides a binding molecule for LASV virus, said binding molecule comprising one or more nanobodies as described in the first aspect of the present invention.

[0012] The term "multiple" refers to ≥2 nanoantibodies.

[0013] In some preferred embodiments, the binding molecule comprises two, four, or six of the nanobodies; and / or, the binding molecule further comprises Fc.

[0014] In some preferred embodiments, the binding molecule satisfies one or more of the following conditions:

[0015] (1) Each nanobody in the binding molecule contains the same CDR1, CDR2 and CDR3 sequences;

[0016] (2) The nanobodies are connected by linker 1;

[0017] (3) The nanobody and the Fc are connected by linker 2;

[0018] (4) The Fc is derived from human IgG1; and,

[0019] (5) The nanobody is attached to the N-terminus of the Fc;

[0020] In some implementations, the heavy chain variable region of each nanobody has the same sequence.

[0021] In some embodiments, the amino acid sequence of linker 1 is shown in SEQ ID NO: 44.

[0022] In some embodiments, the amino acid sequence of the linker 2 is shown in SEQ ID NO: 46.

[0023] In some embodiments, the amino acid sequence of the Fc is shown in SEQ ID NO: 48.

[0024] In some further preferred embodiments, the amino acid sequence of the binding molecule is as shown in any of SEQ ID NO: 49-72.

[0025] A third aspect of the present invention provides an isolated nucleic acid that encodes a nanobody as described in the first aspect of the present invention, or a binding molecule as described in the second aspect of the present invention.

[0026] A fourth aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the third aspect of the present invention.

[0027] In some preferred embodiments, the backbone of the recombinant expression vector is pcomb3x.

[0028] A fifth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the third aspect of the present invention or recombinant expression vector as described in the fourth aspect of the present invention; the transformant is a non-plant or animal variety.

[0029] In some preferred embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0030] In some preferred embodiments, the prokaryotic cells are Escherichia coli TG1.

[0031] The sixth aspect of the present invention provides a method for preparing anti-LASV virus nanobodies or binding molecules comprising the same, the method comprising culturing a transformant as described in the fifth aspect of the present invention and obtaining the nanobodies or the binding molecules from the culture.

[0032] A seventh aspect of the present invention provides a pharmaceutical composition comprising a nanobody as described in the first aspect of the present invention or a binding molecule as described in the second aspect of the present invention, and optionally a pharmaceutically acceptable carrier and / or excipients.

[0033] An eighth aspect of the present invention provides a kit comprising one or more of the following: nanobody as described in the first aspect of the present invention, binding molecule as described in the second aspect of the present invention, and pharmaceutical composition as described in the seventh aspect of the present invention.

[0034] In some preferred embodiments, the kit further includes (i) means for administering the nanobody, the binding molecule, or the pharmaceutical composition; and / or (ii) instructions for use.

[0035] The ninth aspect of the present invention provides the use of nanobodies as described in the first aspect of the present invention, binding molecules as described in the second aspect of the present invention, isolated nucleic acids as described in the third aspect of the present invention, recombinant expression vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, or pharmaceutical compositions as described in the seventh aspect of the present invention in the preparation of LASV virus inhibitors, kits for detecting LASV virus, kits for diagnosing or predicting the prognosis of Lassa fever, or medicaments for treating and / or preventing Lassa fever.

[0036] The tenth aspect of the present invention provides a method for detecting LASV, the method comprising contacting a sample to be tested with a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention to detect the expression level of LASV, or using a kit as described in the eighth aspect of the present invention to detect the expression level of LASV in the sample to be tested.

[0037] In some preferred embodiments, the detection is for non-diagnostic purposes; and / or the method is based on Western blotting, ELISA, or flow cytometry.

[0038] The eleventh aspect of the present invention provides a method for treating and / or preventing LASV virus-related diseases, the method comprising administering to a patient a therapeutically effective amount of a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0039] The twelfth aspect of the present invention provides a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, a pharmaceutical composition as described in the seventh aspect of the present invention, or a kit as described in the eighth aspect of the present invention, for the treatment and / or prevention of disease.

[0040] In some preferred experimental protocols, the disease is LASV-related.

[0041] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0042] The reagents and raw materials used in this invention are all commercially available.

[0043] The positive and progressive effects of this invention are as follows:

[0044] This invention provides a nanobody against LASV virus with a unique CDR region, which can effectively neutralize multiple strains of currently prevalent LASV virus internationally, filling a market gap and having broad application value. Attached Figure Description

[0045] Figures 1-14 The binding activity (EC) of different nanobodies to the GPC trimer protein of four strains: Pinneo, Josiah, 803213, and CSF was measured. 50 );

[0046] Figure 15 The bivalent Fc antibody showed neutralizing activity against Josiah strain pseudovirus (IC50). 50 );

[0047] Figures 16-23 Neutralizing activity (IC50) of different tetravalent Fc antibodies against different strains of pseudoviruses 50 );

[0048] Figures 24-31Neutralizing activity (IC50) of different hexavalent Fc antibodies against different strains of pseudoviruses 50 ).

[0049] Figure 32 This is a schematic diagram of the structure of a multivalent nanobody Fc fusion antibody. Detailed Implementation

[0050] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0051] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0052] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all as defined by the Kabat numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTicsdatabase (IMGT, imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0053] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the Kabat numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0054] In this invention, "LASV virus binding molecules" are proteins that have the function of recognizing and binding to LASV virus, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, microbodies, affinity molecules, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0055] In this invention, "single-domain antibody," "VHH," and "nanobody" are used interchangeably, all referring to nanobodies that specifically recognize and bind to the LASV virus. A nanobody is the variable region of a heavy chain antibody. Typically, a nanobody contains three CDRs and four FRs. A nanobody is the smallest functional antigen-binding fragment. Typically, an antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region.

[0056] In this invention, a binding molecule containing two or more nanobodies is a multivalent nanobody; a binding molecule containing two or more nanobodies with different specificities is a multispecific nanobody. Multiple nanobodies are linked together via linkers. The linkers typically consist of 1-15 amino acids selected from G and S, for example, (G4S)3.

[0057] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0058] The variants of the various antibodies described in this invention include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the various antibodies of this invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the various antibodies of this invention.

[0059] In some embodiments, the sequence of the variants described in this invention may have at least 95%, 96%, 97%, 98%, or 99% sequence identity with its source sequence. This sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. This invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) sequence identity with the CDRs identified herein.

[0060] The nanobodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques well known in the art. The nanobodies and binding molecules of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the present invention. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines suitable as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, and human hepatocellular carcinoma cells (e.g., HepG2). Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels and produce antibodies with essential LASV virus binding properties.

[0061] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0062] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0063] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0064] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as an animal or fungus; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0065] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0066] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0067] In this invention, "LASV virus-related disease" refers to a disease caused or related to infection with the LASV virus.

[0068] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0069] In this invention, the application scenarios for "non-diagnostic purposes" include, but are not limited to: for example, detecting the presence of antigens in vitro in the laboratory; or using it as a positive antibody to screen other antibodies with the same target; or competing with other antibodies with the same target to detect whether there is competition between antibodies, i.e., whether the antigen epitopes are the same or similar.

[0070] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0071] Example 1: Screening, identification, sequencing and purification of antibodies

[0072] I. Phage Library Design and Construction Methods:

[0073] Day 1:

[0074] (a) Preparation of electrocompetent states:

[0075] 1. Inoculate 1 L of 2×YT medium (16 g tryptone, 10 g yeast extract, 5 g sodium chloride, and deionized water to a final volume of 1 L, pH 7.4) with 10 mL of overnight TG1 bacterial culture (Solarbio, catalog number: C1170) and incubate at 37°C and 250 rpm for about 1 h.

[0076] 2. After culturing for 1 h, take 600 μL of bacterial culture and measure its OD using NanoDrop. 600 (Use cuvettes, select Cellculture, and check Use cuvette), use the culture medium as a blank control. When OD 600 Stop culturing when the value is between 0.4 and 0.5.

[0077] 3. OD 600 Once the bacterial culture reaches a pH of 0.4, immediately place it in prepared ice water and incubate for 30 minutes.

[0078] 4. Pre-cool the centrifuge: Place the 500 mL centrifuge bottle in a 4°C refrigerator for pre-cooling, and pre-cool it in ultrapure water and 10% glycerol ice.

[0079] 5. Remove the bacterial culture from the ice bath, pour it into a pre-cooled centrifuge bottle, balance it, and centrifuge at 5500×g for 10 min at 4℃.

[0080] 6. After centrifugation, discard the supernatant, add about 50 mL of pre-cooled ultrapure water, tighten the cap, immerse the centrifuge bottle in ice water and shake in the same direction, being careful not to use too much force, so that the bacteria can be resuspended.

[0081] 7. Add pre-cooled ultrapure water to 500 mL, balance the mixture, centrifuge at 5500×g for 10 min at 4℃.

[0082] 8. Carefully discard the supernatant, and wash the bacteria once with 500 mL of ultrapure water using the same method.

[0083] 9. After washing the bacterial cells twice, carefully discard the supernatant, add about 50 mL of pre-cooled 10% glycerol, tighten the cap, immerse the centrifuge bottle in ice water and shake in the same direction, being careful not to shake too vigorously, so that the bacterial cells are resuspended.

[0084] 10. Transfer the bacterial culture to a pre-cooled 50 mL centrifuge tube, tighten the cap, and centrifuge at 5000×g for 5 min at 4℃.

[0085] 11. Discard the supernatant and use a pipette to remove any remaining solution.

[0086] 12. Add 0.8 mL of pre-cooled 10% glycerol, and use a pipette tip to stir and suspend the bacterial cells. The electroporation competent cells are now ready.

[0087] (ii) Purification and concentration of ligation products

[0088] 1. Add 5 volumes of Buffer PB to 1 volume of the ligation product of the fully human VH fragment and pcomb3x vector (Newp Biotech, catalog number: V008299), mix well, load onto the column, and centrifuge at 17900×g for 1 min.

[0089] 2. Discard the liquid in the collection tube. Add 750 μL of Buffer PE to the column and centrifuge at 17900×g for 1 min;

[0090] 3. After repeating step 2, centrifuge the empty column at 17900×g for 1 min;

[0091] 4. Replace with a 1.5 mL EP tube, add 30-50 μL H2O, let stand for 4 min, and centrifuge at 17900×g for 1 min.

[0092] 5. The ligation products were purified using the QIAquick PCR Purification Kit (catalog number: 28104), and finally eluted with 100 μL H2O. The detection concentration was 56.4 ng / μL, 1.92 for 260 / 280, and 1.86 for 260 / 230. The ligation products were identified by electrophoresis before and after purification.

[0093] (III) Conversion of Linkage Products

[0094] 1. Prepare 100 mL of antibiotic-free 2×YT medium + 10×SOC glucose solution (25 mM KCl, 100 mM MgCl2·6H2O, 200 mM Glucose) and incubate at 37℃ in a shaker.

[0095] 2. Mix the purified and concentrated ligation product with the electrotransfer competent cells at a volume ratio of 1:10, incubate on ice for 5 min, and then take 200 μL into a pre-cooled electrotransfer cup and incubate on ice for another 10 min.

[0096] 3. Set the electroporator to the Eco 2 option. After electroporating the electroporator cuvette, quickly add 1 mL of 2×YT medium kept at 37℃. After blowing and aspiration, add 75 mL of 2×YT medium kept at 37℃.

[0097] 4. After culturing the electrocompetent cells at 37℃ and 250 rpm for 20 min, perform 10 [processes / steps] on an Amp plate. -3 10 -4 10 -5 and 10 -6 Dilution flow line (add 10 μL at each point), and take 100 μL of the solution. -4 The bacterial culture was plated on Amp plates and incubated overnight at 37°C. The volume of the fully human VH antibody library was calculated and sequenced.

[0098] (iv) Bacteriophage production

[0099] 1. After culturing electrocompetent cells at 37℃ and 250 rpm for 20 min, add 2×YT medium to 600 mL, add 600 μL Amp (100 mg / mL), and incubate at 37℃ and 250 rpm for 2 h before measuring the OD value.

[0100] 2. When OD 600 When the concentration is 0.8, take 300 mL and add 20 times the amount of helper phage (M13K07), incubate at 37°C for 45 min, inverting and mixing once every 15 min. Incubate the remaining 300 mL of bacterial culture overnight at 30°C and 220 rpm.

[0101] 3. Let stand for 45 minutes. After that, centrifuge at 4000 rpm for 10 minutes. Discard the supernatant and replace with 500 mL of 2×YT medium. Add Amp and Kana, and incubate overnight at 30℃ and 220 rpm.

[0102] the next day:

[0103] 1. Centrifuge 300 mL of the overnight culture at 8000 rpm for 5 min, discard the supernatant, and resuspend the cells in 9.38 mL of 40% glycerol. Finally, store the cells in 15 mL of 25% glycerol at -80℃.

[0104] 2. Replace with 500 mL of 2×YT culture medium, centrifuge at 8000 rpm for 15 min, and collect the supernatant.

[0105] 3. Add 1 / 4 volume of PEG / NaCl solution (containing 20% ​​(mass percentage) PEG8000 in 2.5 M NaCl solution) to the supernatant, mix well and let stand at 4°C for 1 h.

[0106] 4. Centrifuge at 8000×g, 4℃ for 15 min, discard the supernatant and centrifuge empty for 5 min to allow the precipitate to aggregate together.

[0107] 5. Dissolve the precipitate in 30 mL of PBST containing 10% glycerol, and pipette it using a 1 mL pipette tip.

[0108] 6. Centrifuge at 10000×g, 4℃ for 10 min to remove residual cell debris. Aliquot the supernatant into 1.5 mL EP tubes and measure the absorbance at 268 nm using the UV-Vis option of Nanodrop to determine the phage OD value. Label the library volume and OD value, and store at -80℃.

[0109] 7. Phage lib was inoculated into 50 mL of 2×YT medium (containing 2% glucose and 100 μg / mL ampicillin amprolium). Initial OD... 600 Incubate at 0.05-0.1 μL, 30℃, 220 rpm for 3 h until OD reaches zero. 600 Approximately 0.5.

[0110] 8. Add the amount according to [(OD 600 ×8×10 8 mL -1 The helper phage was calculated as [×50 mL (bacterial volume) × 20) ÷ helper phage titer] mL. The cells were incubated at 37℃ for 1 h, centrifuged at 3800 rpm for 10 min, and resuspended on 250 mL of 2×YT medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin). The cells were then incubated overnight at 30℃ and 220 rpm (more than 16 h).

[0111] 9. Centrifuge the overnight cultured phage library at 12,000 rpm for 30 min. Mix the supernatant with the precipitant at a volume ratio of 4:1. The precipitant is a 2.5 M NaCl solution containing 20% ​​(w / w) PEG8000. Incubate at 4℃ for 2-3 h, centrifuge at 8,000 rpm for 30 min, discard the supernatant, invert the tube onto sterile paper and pat dry. Resuspend the phage pellet in 1 mL of 1×PBS, and centrifuge several times at 12,000 rpm until no cell debris precipitates.

[0112] 10. Titer Detection: Dilute the phage 10-fold in 1×PBS. Take 10 μL of phage diluted 1:10000 and mix it with TG1 competent cells (OD10000). 600 Mix approximately 0.5 g of ampicillin with water, let stand at 37°C for 30 min, then spread onto a plate (2×YT agar medium containing 100 μg / mL ampicillin amp) and incubate overnight at 37°C. Observe the plaques on the plates.

[0113] II. Phage Library Screening:

[0114] (a) First round of screening

[0115] 1. GPC trimer protein expression: GPC trimer protein encoding plasmids of four Lassa virus strains—Pinneo (Genbank: AAF86701.1), Josiah (Genbank: NP_694870.1), CSF (Genbank: AAL13212.1), and 803213 (Genbank: AAF86703.1)—were synthesized and transfected into HEK 293F cells. After culturing for 4–6 days, the cell culture supernatant was collected, and the protein was purified by Streptactin affinity chromatography (Cytiva, catalog number: 29401320). The protein was analyzed by SDS-PAGE, stained with Coomassie brilliant blue, and used for subsequent antibody screening.

[0116] 2. One day before screening, TG1 competent cells were seeded into 20 mL of 2×YT medium and cultured at 37°C and 250 rpm until OD500. 600 Store at approximately 0.6, 4°C. Take 50 μg of GPC trimer protein from Pinneo, Josiah, 803213 and CSF strains respectively, and biotinylate the protein using a biotinylation kit (Thermo, catalog number: 21445).

[0117] 3. Add 500 μL of phage library to a 1.5 mL EP tube, add 500 μL of blocking buffer (1×PBS buffer containing 6% skim milk) to the phage solution, add 5 μg of biotinylated Pinneo strain GPC trimer protein to the phage-blocking buffer and incubate at room temperature for 2 h by rotation.

[0118] 4. Take 200 μL of magnetic beads (nanomicroorganisms, catalog number: MPHCAS-300) into a 1.5 mL EP tube, place it on a magnetic rack, and wait for the magnetic beads to be completely adsorbed onto the tube wall. Then, remove the supernatant, wash twice with 1×PBS, resuspend the magnetic beads in 500 μL of 1×PBS buffer, add blocking solution at a 1:1 ratio, and incubate at room temperature for 1 h by rotation.

[0119] 5. Place the EP tube containing the magnetic beads on a magnetic rack, discard the solution, add the blocked phage-protein solution, resuspend and mix well, and incubate at room temperature for 30 min by rotation.

[0120] 6. Place the EP tube on a magnetic rack, aspirate the solution, wash 5 times with 0.05% PBST solution, and finally wash 2 times with 1×PBS buffer.

[0121] 7. Add 1 mL of TG1 competent cells (OD) to the cleaned magnetic beads. 600 After resuspending the sample at approximately 0.5 g, add 20 mL of TG1 competent cells for incubation at 37°C for 45 min, inverting and mixing once every 15 min.

[0122] 8. Titer Detection: Dilute 1 μL of phage solution infecting TG1 competent cells 1:100 and plate it on 2×YT agar medium (containing 100 μg / mL ampicillin amprolium). Incubate overnight at 37°C. Observe the phage plaques on the plates. Add ampicillin amprolium and glucose to the remaining bacterial solution to make the final solution contain 2% glucose and 100 μg / mL ampicillin amprolium by weight. Incubate at 37°C and 250 rpm for 1 h.

[0123] 9. Add the amount according to [(OD 600 ×8×10 8 mL -1 The helper phage titer was calculated as [×50 mL (bacterial volume) × 20) ÷ helper phage titer] mL. The mixture was incubated at 37°C for 45 min, inverted and mixed every 15 min. After centrifugation at 3800 rpm for 10 min, the bacterial culture was resuspended on 20 mL of 2×YT medium (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin), and incubated overnight at 30°C and 220 rpm (at least 16 h).

[0124] (II) Second round of screening

[0125] 1. The next day, centrifuge the overnight bacterial culture at 8000 rpm for 15 min and collect the supernatant.

[0126] 2. Add 1 / 4 volume of PEG / NaCl solution to the supernatant, mix well, and let stand at 4°C for 20 min.

[0127] 3. Centrifuge at 8000×g, 4℃ for 15 min, discard the supernatant and centrifuge for 5 min to allow the precipitate to aggregate.

[0128] 4. Dissolve the precipitate with 1 mL of 1×PBS and pipette it with a 1 mL pipette tip.

[0129] 5. Take 300 μL of resuspension and add it to the blocking buffer (6% skim milk dissolved in 1×PBS) at a 1:1 ratio to continue the first round of operation steps (i.e., the aforementioned step (II) phage library screening).

[0130] In the second, third and fourth rounds, 5 μg of GPC trimer protein antigen from Josiah, 803213 and CSF strains were added, respectively. After the phages were adsorbed by magnetic beads, the number of elutions with 0.05% PBST by volume gradually increased from 5 times, 10 times and 15 times.

[0131] III. ELISA detection of bacteriophages

[0132] I. Phage amplification

[0133] 1. Dilute the phage library obtained in the last round and spread it on a large plate containing amp, and incubate overnight at 37°C.

[0134] 2. Add 100 μL of 2×YT medium to each well of the round-bottom plate, pick up the single clones from the last round of plating and transfer them to the round-bottom plate, and incubate at 37℃ and 250 rpm for 4 h until the logarithmic growth phase.

[0135] 3. Add the corresponding amount of helper phage to each well, incubate at 37℃ for 45 min, and then incubate at 250 rpm for 1 h.

[0136] 4. Add 100 μL of 2×YT medium (containing 200 μg / mL ampicillin and 100 μg / mL kanamycin) to each well and incubate overnight at 30°C and 220 rpm.

[0137] II. Antigen coating and ELISA detection

[0138] 1. Coat 50 ng / 100 μL of GPC trimer protein of the antigen Pinneo, Josiah, 803213 and CSF strains and incubate overnight at 4°C.

[0139] 2. The next day, remove the coating solution, wash three times with 0.05% PBST solution, add 200 μL of blocking solution (3% skim milk) to each well, and block at 37°C for 1 h.

[0140] 3. Centrifuge the overnight cultured bacterial solution from step I (4) at 3800 rpm for 10 min and retain the supernatant as the phage solution.

[0141] 4. Remove the blocking solution, wash three times with 0.05% PBST solution, add 100 μL of phage solution to each well, and incubate at 37°C for 2 h.

[0142] 5. Remove the phage solution, wash 5 times with 0.05% PBST solution, add 50 μL of M13 phage mouse monoclonal antibody anti-M13 (Sinochem, catalog number: 11973-MM05T-H) (1:15000 diluted in blocking buffer) to each well, and incubate at 37°C for 1 h.

[0143] 6. Remove the anti-M13 phage mouse monoclonal antibody solution, wash 5 times with 0.05% PBST solution, add 50 μL of TMB chromogenic solution to each well (protect from light), wait for about 5 min (until the color change is moderate), and then add 50 μL of chromogenic stop solution.

[0144] 7. OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 450 nm absorbance values ​​were read from the plate. Positive clones were sent to the company for sequencing to obtain the sequences encoding nanobodies. The sequences were analyzed and processed to prepare the following nanobodies (LS23-2E9, LS23-2A2, LS23-1G7, LS23-1H11, LS23-2A4, LS23-1C9, LS23-2D11, LS23-1A11, LS23-1D3, LS23-1H3, LS23-2C1, LS23-1H12, LS23-2H6, LS23-2H1). The CDR sequences were defined according to the Kabat numbering system (see Table 1).

[0145] Table 1. CDR sequences (Kabat numbers) of nanobodies

[0146] Nanobody CDR1 SEQ ID NO: CDR2 SEQ ID NO: CDR3 SEQ ID NO: LS23-2E9 DYAMG 1 EINHSGSTNYNPSLKS 2 GHYQLEV 3 LS23-2A2 SYAMS 4 EISHSGSTYYNPSLKS 5 ERVTMDV 6 LS23-1G7 NYDMS 7 EINHSGSTNYNPSLKS 2 GHFQLEV 8 LS23-1H11 NFGMN 9 AISGSGGSTYYADSVKG 10 DRDGMEV 11 LS23-2A4 DYAMG 1 EINRVGATHYNPSLKS 12 ERITMDV 13 LS23-1C9 SYDMG 14 EINHSGSTNYNPSLKS 2 GAYRLDV 15 LS23-2D11 DSSVG 16 EINHSGSTNYNPSLKS 2 GHYQLEV 3 LS23-1A11 DYGMH 17 DIDPSGGTNYNPSLKS 18 ERITMDV 13 LS23-1D3 DYAMH 19 EINHSGSTNYNPSLKS 2 ERITMDV 13 LS23-1H3 SYAMN 20 SIYHSGNTYYNPSLKS 21 VPWSSGMDV 22 LS23-2C1 GYTMS 23 EINHSGSTNYNPSLKS 2 ERVTMDV 6 LS23-1H12 NSDMS 24 EIGHSGSTNYNPSLKS 25 GHFQLEV 8 LS23-2H6 SYGMH 26 EINHSGSTNYNPSLKS 2 DRVANSNAIDV 27 LS23-2H1 DYTMH 28 EINHSGGTNYKPSLKS 29 ERITMDV 13

[0147] >LS23-2E9

[0148] Amino acid sequence (SEQ ID NO: 30)

[0149] EVQLVESGGGLVQPGGSLRLSCTVSGFTFSDYAMGWVRQAPGKGLEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCARGHYQLEVWGQGTTVTVSS

[0150] >LS23-2A2

[0151] Amino acid sequence (SEQ ID NO: 31)

[0152] EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYAMSWVRQAPGKALEWIGEISHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERVTMDVWGPGTTVTVSS

[0153] >LS23-1G7

[0154] Amino acid sequence (SEQ ID NO: 32)

[0155] EVQLVESGGGLVQPGGSLRLSCVASGFPFSNYDMSWVRQAPGKALEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCARGHFQLEVWGQGTTVTVSS

[0156] >LS23-1H11

[0157] Amino acid sequence (SEQ ID NO: 33)

[0158] EVQLVESGGGLVQPGGSLRLSCVASGFTFSNFGMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAKDRDGMEVWGQGTTVTVSS

[0159] >LS23-2A4

[0160] Amino acid sequence (SEQ ID NO: 34)

[0161] EVQLVESGGGLVQPGGSLRLSCVASGFSFGDYAMGWVRQAPGKALEWIGEINRVGATHYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERITMDVWGPGTTVTVSS

[0162] >LS23-1C9

[0163] Amino acid sequence (SEQ ID NO: 35)

[0164] EVQLVESGGGLVQPGGSLRLSCVASGFSFSSYDMGWVRQAPGKALEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCARGAYRLDVWGQGTTVTVSS

[0165] >LS23-2D11

[0166] Amino acid sequence (SEQ ID NO: 36)

[0167] EVQLVESGGGLVQPGGSLRLSCVASGFTFSDSSVGWVRQAPGKALEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHYQLEVWGQGTTVTVSS

[0168] >LS23-1A11

[0169] Amino acid sequence (SEQ ID NO: 37)

[0170] EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYGMHWVRQAPGKALEWVGDIDPSGGTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTALYYCAKERITMDVWGPGTTVTVSS

[0171] >LS23-1D3

[0172] Amino acid sequence (SEQ ID NO: 38)

[0173] EVQLVESGGGLVQPGGSLRLSCVASGFDFNDYAMHWVRQAPGKALEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCAKERITMDVWGPGTTVTVSS

[0174] >LS23-1H3

[0175] Amino acid sequence (SEQ ID NO: 39)

[0176] EVQLVESGGGLVQPGGSLRLSCKGSGFTFSSYAMNWVRQAPGKGLEWIGSIYHSGNTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARVPWSSGMDVWGQGTTVTVSS

[0177] >LS23-2C1

[0178] Amino acid sequence (SEQ ID NO: 40)

[0179] EVQLVESGGGLVQPGGSLRLSCVASGFTFSGYTMSWVRQAPGKALEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERVTMDVWGPGTTVTVSS

[0180] >LS23-1H12

[0181] Amino acid sequence (SEQ ID NO: 41)

[0182] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNSDMSWVRQAPGKALEWIGEIGHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCARGHFQLEVWGQGTTVTVSS

[0183] >LS23-2H6

[0184] Amino acid sequence (SEQ ID NO: 42)

[0185] EVQLVESGGGLVQPGGSLRLSCKGSGFRFDSYGMHWVRQAPGKGLEWIGEINHSGSTNYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCAKDRVANSNAIDVWGQGTQVTVSS

[0186] >LS23-2H1

[0187] Amino acid sequence (SEQ ID NO: 43)

[0188] EVQLVESGGGLVQPGGSLRLSCAASGFFFEDYTMHWVRQAPGKALEWIGEINHSGGTNYKPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERITMDVWGPGTTVTVSS

[0189] Example 2: ELISA detection of the binding activity of nanobodies to the GPC trimer protein of four strains: Pinneo, Josiah, 803213, and CSF (ECG). 50 )

[0190] GPC trimer proteins from four strains (Pinneo, Josiah, 803213, and CSF) were diluted to 500 ng / mL with 1×PBS and coated onto ELISA plates at 100 μL / well overnight at 4°C. The plates were then blocked with 3% skim milk blocking buffer at 37°C for 60 min, and washed three times with PBST. Serial dilutions of LS23-1D3, LS23-1H3, LS23-1G7, LS23-2C1, LS23-1H12, LS23-2D11, LS23-2A4, LS23-2E9, LS23-2H6, LS23-2H1, LS23-1C9, LS23-1A11, LS23-1H11, and LS23-2A2 were performed, starting with 1 μM and followed by eight 5-fold dilutions. The plates were incubated at 37°C for 2 hours. h, wash the plate 3 times with PBST; add diluted HRP anti-Flag secondary antibody (Beyotime, catalog number: AF2855) and react for 1 h, wash the plate 3 times with PBST; finally add 50 μL of TMB substrate for color development, incubate at room temperature in the dark for 10 min, stop the reaction with 50 μL of stop solution, and read and record the absorbance of the plate at a wavelength of 450 nm using a microplate reader. In the assay, the irrelevant antibody SA protein (Sino Biological, catalog number: 40948-ANAE) was used as the NC (blank control) (currently there are no antibodies against LASV under development or in clinical trials).

[0191] The results are as follows Figures 1-14 As shown, the binding activity (EC) of different nanobodies to the GPC trimer protein of four strains (Pinneo, Josiah, 803213, and CSF) was calculated. 50 (as shown in Table 2).

[0192] Table 2. Binding activities (EC50) of different nanobodies to GPC trimer proteins of four strains: Pinneo, Josiah, 803213, and CSF. 50 )

[0193]

[0194] Example 3: Neutralization experiment of nanobodies with seven pseudovirus strains: Pinneo, Josiah, 803213, CSF, Togo, AYM, and AV.

[0195] GPC-encoding plasmids of synthesized Lassa virus strains Pinneo (Genbank: AAF86701.1), Josiah (Genbank: NP_694870.1), 803213 (Genbank: AAF86703.1), CSF (Genbank: AAL13212.1), Togo (Genbank: AVN98153.1), AYM (Genbank: AYM51697.1), and AV (Genbank: AAG41802.1) were transfected into HEK-293T cells and infected with G*ΔG-luciferase (MOI=5). After 24 hours, the supernatant was collected and treated with 20% I1 hybridoma cell supernatant for 1 h to neutralize residual VSV-G. The pseudoviruses were titrated, aliquoted, and stored at -80°C.

[0196] Different nanobodies were linked using linker 1 to construct bivalent, tetravalent, and hexavalent nanobodies; the bivalent, tetravalent, and hexavalent nanobodies were then fused with Fc (SEQ ID NO: 62) to prepare Fc fusion antibodies containing multivalent nanobodies (see [link to Fc fusion antibody formula]). Figure 32 The full-length amino acid sequences of the multivalent nanobodies are shown in Tables 3-5. In the Fc fusion antibody, the multivalent nanobodies and Fc are linked by linker 2. The cells are transfected into 293F cells, the Fc antibody is purified, and PBS is replaced.

[0197] Linker 1 amino acid sequence (SEQ ID NO: 44):

[0198] GGGGSGGGGSGGGGS

[0199] Linker 1 nucleotide sequence (SEQ ID NO: 45):

[0200] GGCGGCGGCGGTTCCGGTGGTGGTGGTAGCGGCGGCGGCGGTAGC

[0201] Linker 2 amino acid sequence (SEQ ID NO: 46):

[0202] GGGGSG

[0203] Linker 2 nucleotide sequence (SEQ ID NO: 47):

[0204] GGTGGTGGCGGTTCCGGA

[0205] Fc fragment amino acid sequence (SEQ ID NO: 48):

[0206] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0207] Table 3 Full-length amino acid sequence of the bivalent Fc fusion protein

[0208]

[0209]

[0210] Table 4 Full-length amino acid sequence of the tetravalent Fc fusion protein

[0211]

[0212]

[0213]

[0214] Table 5. Full-length amino acid sequence of the hexavalent Fc fusion protein

[0215]

[0216]

[0217]

[0218] In the neutralization experiment, pseudoviruses were mixed with serially diluted antibodies, incubated, and then added to Vero-E6 cells (Pronos, catalog number: CL-0491). Luciferase signal was detected after 24 hours, and IC50 was measured. 50 The value was defined as the concentration of antibody that reduced the relative luminescent unit (RLU) by 50% (relative to the virus control well, minus the cell blank value), and was calculated using nonlinear regression analysis using GraphPad Prism software.

[0219] The results are as follows Figures 15-31As shown, the IC50 neutralizing activity of different antibodies against seven strains—Pinneo (Genbank: AAF86701.1), Josiah (Genbank: NP_694870.1), 803213 (Genbank: AAF86703.1), CSF (Genbank: AAL13212.1), Togo (Genbank: AVN98153.1), AYM (Genbank: AYM51697.1), and AV (Genbank: AAG41802.1)—was calculated. 50 (As shown in Tables 3, 4 and 5).

[0220] Table 3. Neutralizing activity (IC50) of different bivalent Fc fusion antibodies against Josiah strain pseudoviruses 50 )

[0221]

[0222] Table 4. Neutralizing activities (IC50) of different tetravalent Fc fusion antibodies against different strains of pseudoviruses. 50 )

[0223]

[0224] Table 5. Neutralizing activities (IC50) of different hexavalent Fc fusion antibodies against different pseudovirus strains. 50 )

[0225]

[0226] As shown in Tables 3-5, within a certain concentration range, after fusion of bivalent nanobodies with Fc, 1H11, 2E9, 2A2, 1A11, and 2D11 all exhibited a certain neutralizing effect, reaching the 50% threshold. Since multivalent nanobodies can enhance neutralizing efficacy, tetravalent and hexavalent nanobodies were subsequently constructed and fused with Fc. All antibodies showed improved neutralizing activity against different LASV strains. Among them, the tetravalent Fc fusion antibody 2A2 showed the highest IC50 value. 50 Values ​​ranged from 35.18 to 78.39 μg / ml, and the IC50 value for the hexavalent Fc fusion antibody 2A2 was [value missing]. 50 The IC50 of the tetravalent Fc fusion antibody 2E9 was 53.96-133.49 μg / ml. 50 The IC50 of the hexavalent Fc fusion antibody 2E9 was 32.32-63.73 μg / ml. 50 The concentrations ranged from 8.01 to 21.21 μg / ml. The results showed that the IC50 values ​​of the tetravalent Fc fusion antibodies 2A2 and 2E9 were... 50 Similarly, the hexavalent Fc fusion antibody 2E9 has a more pronounced neutralizing effect and can be further studied as a means of preventing or treating LASV virus infection.

[0227] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A nanobody against LASV virus, comprising a heavy chain variable region, said heavy chain variable region comprising CDR1, CDR2 and CDR3, characterized in that, The amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 1, 2, and 3, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 4, 5, and 6, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 7, 2, and 8, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 9, 10, and 11, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 1, 12, and 13, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 14, 2, and 15, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 16, 2, and 3, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO: 16, 2, and 3, respectively. As shown in SEQ ID NO: 17, 18, and 13; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 2, and 13, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 20, 21, and 22, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23, 2, and 6, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 24, 25, and 8, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 26, 2, and 27, respectively; or, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, 29, and 13, respectively.

2. The nanobody as described in claim 1, characterized in that, The framework region of the heavy chain variable region is a human-derived framework region; Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 and does not involve any alteration to the CDR sequence.

3. A binding molecule for LASV virus, characterized in that, The binding molecule comprises one or more nanobodies as described in claim 1 or 2; Preferably, the binding molecule comprises two, four, or six of the nanobodies; and / or, the binding molecule further comprises Fc; More preferably, the binding molecule satisfies one or more of the following conditions: (1) The CDR1, CDR2 and CDR3 sequences contained in each nanobody in the binding molecule are the same; preferably, the heavy chain variable region sequences of each nanobody are the same; (2) The nanobodies are connected by linker 1; preferably, the amino acid sequence of linker 1 is as shown in SEQ ID NO: 44; (3) The nanobody and the Fc are connected by linker 2; preferably, the amino acid sequence of linker 2 is as shown in SEQ ID NO: 46; (4) The Fc is derived from human IgG1; preferably, the amino acid sequence of the Fc is as shown in SEQ ID NO: 48; and, (5) The nanobody is attached to the N-terminus of the Fc; More preferably, the amino acid sequence of the binding molecule is as shown in any of SEQ ID NO: 49-72.

4. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the nanobody as described in claim 1 or 2, or the binding molecule as described in claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 4; Preferably, the backbone of the recombinant expression vector is pcomb3x.

6. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 4 or the recombinant expression vector as described in claim 5; the transformant is a non-plant or animal variety; Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the prokaryotic cell is Escherichia coli TG1.

7. A method for preparing anti-LASV virus nanobodies or binding molecules comprising them, characterized in that, The method comprises culturing the transformant as described in claim 6, and obtaining the nanobody or the binding molecule from the culture.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody as described in claim 1 or 2 or a binding molecule as described in claim 3, and optionally a pharmaceutically acceptable carrier and / or excipients.

9. A reagent kit, characterized in that, The kit comprises one or more of the nanobody as described in claim 1 or 2, the binding molecule as described in claim 3, and the pharmaceutical composition as described in claim 8; Preferably, the kit further includes (i) means for administering the nanobody, the binding molecule, or the pharmaceutical composition; and / or (ii) instructions for use.

10. The use of the nanobody as described in claim 1 or 2, the binding molecule as described in claim 3, the nucleic acid as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, or the pharmaceutical composition as described in claim 8 in the preparation of LASV virus inhibitors, kits for detecting LASV virus, kits for diagnosing or predicting the prognosis of Lassa fever, or medicaments for treating and / or preventing Lassa fever.

11. A method for detecting LASV, characterized in that, The method includes contacting a sample to be tested with a nanobody as described in claim 1 or 2, a binding molecule as described in claim 3, or a pharmaceutical composition as described in claim 8 to detect the expression level of LASV, or using a kit as described in claim 9 to detect the expression level of LASV in the sample to be tested. Preferably, the detection is for non-diagnostic purposes; and / or, the method is based on Western Blot, ELISA, or flow cytometry techniques.

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