Antibodies specifically binding to rabies virus and uses thereof

Abstract

The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to a rabies virus. The present disclosure also relates to the use of the antibodies or antigen-binding fragments thereof in the treatment, prevention and / or diagnosis of a rabies virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies or antigen-binding fragments thereof that specifically bind to rabies virus, and the application of such antibodies or antigen-binding fragments thereof. Background Technology

[0002] Rabies is an acute, zoonotic infectious disease of the central nervous system caused by the rabies virus (RABV). With a near 100% fatality rate, it is considered one of the deadliest infectious diseases. It is estimated that approximately 50,000 people die from rabies globally each year, with about 40% of these deaths occurring in children under the age of 15. To this day, rabies remains a serious public health challenge in more than 150 countries and regions worldwide, particularly severe in Asia and Africa.

[0003] While rabies vaccines have achieved some success in preventing the onset of rabies, passive immunization remains the most effective treatment for individuals already exposed to the rabies virus. Currently, the primary emergency prophylactic treatment for rabies is plasma-derived immunoglobulin (RIG). However, existing RIG treatments have several limitations, including high costs, supply shortages, potential pathogen contamination risks, and limited broad-spectrum coverage, failing to cover genetically distant strains within the Lyssavirus genus.

[0004] Therefore, there is an urgent need to develop novel humanized monoclonal antibodies with broad-spectrum neutralizing activity targeting the rabies virus G protein, and to establish an efficient antibody discovery, screening, and validation system to meet the global needs for rabies prevention and control. Summary of the Invention

[0005] This disclosure provides antibodies that specifically bind to rabies virus and their applications. The antibodies provided by this invention exhibit high affinity for the G protein of various classical rabies virus vaccine strains and demonstrate significant neutralizing ability against representative RABV strains and some non-classical Lyssavirus strains in pseudovirus systems, exhibiting broad coverage and broad-spectrum neutralizing activity.

[0006] According to one aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to rabies virus is provided, said antibody or antigen-binding fragment comprising: (1) The following three heavy chain variable region complementarity-determining regions (HCDRs): HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 8, 15, 22, 29 and 36, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region. HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 8, 15, 22, 29 and 36, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region. HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 1, 8, 15, 22, 29 and 36, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and (2) The following three light chain variable region complementarity-determining regions (LCDRs): LCDR1 having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in any one of SEQ ID NO: 2, 9, 16, 23, 30 and 37, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region. LCDR2 having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in any one of SEQ ID NO: 2, 9, 16, 23, 30 and 37, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region. LCDR3 having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in any one of SEQ ID NO: 2, 9, 16, 23, 30 and 37, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0007] In some implementations, the HCDR1-3 and / or the LCDR1-3 are defined by rules of Kabat, AbM, Chothia, Contact, IMGT or combinations thereof.

[0008] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 1, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 2, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0009] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 8, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 9, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0010] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 15, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 16, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 22, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 23, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0012] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 29, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 30, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises: three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 36, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three HCDRs contained in the heavy chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 37, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the three LCDRs contained in the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1 having an amino acid sequence as shown in any one of SEQ ID NO: 3, 10, 17, 24, 31 or 38, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the antibody, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the antibody; and / or, HCDR2 having an amino acid sequence as shown in any one of SEQ ID NO: 4, 11, 18, 25, 32 or 39, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the HCDR2, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the HCDR2; and / or, HCDR3 having an amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in any one of 5, 12, 19, 26, 33, or 40, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with it; and / or, LCDR1, which has as shown in SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 6, 13, 20, 27, 34 or 41, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with it; and / or, LCDR2, having an amino acid sequence as shown in WAS, ASS, GAS, EVT, EDN or AAS, or an amino acid sequence having one amino acid substitution, deletion or addition compared to it; and / or, LCDR3, having an amino acid sequence as shown in any one of SEQ ID NO: 7, 14, 21, 28, 35 or 42, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with it.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and / or, as in SEQ ID NO: 6, amino acid sequences of WAS and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in Figure 7, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and / or, as in SEQ ID NO: 13, amino acid sequences of ASS and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 14, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0017] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of said HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with said HCDR1, HCDR2, and HCDR3; and / or, as in SEQ ID NO: 20, with the amino acid sequence GAS and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 21, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0018] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of said HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with said HCDR1, HCDR2, and HCDR3; and / or, as in SEQ ID NO: 27, amino acid sequences of EVT and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 28, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of said HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with said HCDR1, HCDR2, and HCDR3; and / or, as in SEQ ID NO: 34, amino acid sequences of EDN and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 35, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0020] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40; or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of HCDR1, HCDR2, and HCDR3; or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them; and / or, as in SEQ ID NO: 41, amino acid sequences of AAS and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 42, or amino acid sequences having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences of LCDR1, LCDR2, and LCDR3, or amino acid sequences having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with them.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1, 8, 15, 22, 29 or 36, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and / or a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2, 9, 16, 23, 30 or 37, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0022] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 9, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0024] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 15, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 16, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 22, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 23, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 29, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the heavy chain variable region; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 30, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with the light chain variable region.

[0027] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 36, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 37, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with it.

[0028] In some embodiments, the antibody is of the IgA, IgD, IgE, IgG, or IgM type.

[0029] In some embodiments, the antibody or its antigen-binding fragment is scFv, Fab, Fab', (Fab')2, Fv fragment, dsFv, biantibody, bispecific antibody, and multispecific antibody.

[0030] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody, a human antibody, or a humanized antibody.

[0031] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0032] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4.

[0033] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of hIgG1, hIgG2, hIgG3, or hIgG4.

[0034] In some embodiments, the antibody or its antigen-binding fragment is capable of binding to rabies virus genus. In some embodiments, the antibody or its antigen-binding fragment is capable of binding to RABV strains and / or non-classical Lyssavirus strains. In some embodiments, the antibody or its antigen-binding fragment is capable of binding to the G protein of rabies virus RABV strains.

[0035] In some embodiments, the antibody or its antigen-binding fragment exhibits strong neutralizing ability against G proteins of RABV virus strains (e.g., P08667.1, CAI43218.1, ACR39382.1, ADM32132.1, ACR39382.1, ADJ29911.1, ADD84785.1, CAI43218.1, P08667.1, etc.) and G proteins of non-classical Lyssavirus strains (ABLV: KU739052.1, DUVV: KU761302.1, EBLV1: EU352768.1, EBLV2: AAX62813.1, IRKV: EF614260.1, MOKV: NC006429.1, etc.).

[0036] According to another aspect of this disclosure, an antibody composition is provided, comprising a first antibody and a second antibody, wherein the first antibody and / or the second antibody are selected from the antibodies described in this disclosure or antigen-binding fragments thereof.

[0037] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 13 with the amino acid sequence ASS and SEQ ID NO: 14; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34 with the amino acid sequence EDN and SEQ ID NO: 35.

[0038] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6 with the amino acid sequence WAS and SEQ ID NO: 7.

[0039] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 41 with amino acid sequences AAS and SEQ ID NO: 42; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6 with amino acid sequences WAS and SEQ ID NO: 7.

[0040] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 27 with the amino acid sequence EVT and SEQ ID NO: 28.

[0041] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 41 with the amino acid sequence AAS and SEQ ID NO: 42; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 27 with the amino acid sequence EVT and SEQ ID NO: 28.

[0042] In some embodiments, the first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34 with the amino acid sequence EDN and SEQ ID NO: 35.

[0043] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 9, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 29 and a light chain variable region as shown in SEQ ID NO: 30.

[0044] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2.

[0045] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 37, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2.

[0046] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 23.

[0047] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 37, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 23.

[0048] In some embodiments, the first antibody includes a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody includes a heavy chain variable region as shown in SEQ ID NO: 29 and a light chain variable region as shown in SEQ ID NO: 30.

[0049] According to another aspect of this disclosure, a protein comprising an antigen-binding domain is provided, said antigen-binding domain comprising an antibody or an antigen-binding fragment thereof described in this disclosure or an antibody composition thereof.

[0050] In some embodiments, the protein is a chimeric antigen receptor, which includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding domain includes the antibody or its antigen-binding fragment as described in this disclosure, or the antibody composition described herein.

[0051] In some embodiments, the protein is a multispecific antibody or its antigen-binding fragment, the multispecific antibody or its antigen-binding fragment comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the antibody or its antigen-binding fragment described in this disclosure or the antibody composition described herein.

[0052] According to another aspect of this disclosure, biological materials are provided in relation to the antibodies or antigen-binding fragments thereof described herein, the antibody compositions thereof, the chimeric antigen receptors thereof, or the multispecific antibodies or antigen-binding fragments thereof, said biological materials comprising any one of a1) to a9): a1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, the antibody composition thereof, or the protein thereof as disclosed herein; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A carrier containing the nucleic acid molecule described in a1); a4) A carrier containing the expression box described in a2); a5) Cells containing the nucleic acid molecules described in a1); a6) Cells containing the expression cassette described in a2); a7) Cells containing the carrier described in a3); a8) Cells containing the carrier described in a4); a9) Cells containing the antibodies or antigen-binding fragments thereof described in this disclosure, the antibody compositions described herein, or the proteins described herein; None of the cells described in a5)-a9) contain reproductive material.

[0053] In some embodiments, the vector may be a eukaryotic cell vector and / or a prokaryotic cell vector, such as a retroviral vector, lentiviral vector, bacteriophage vector, adenovirus vector, adeno-associated vector, or herpes simplex vector.

[0054] In some embodiments, the carrier is present in nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0055] In some embodiments, the cells are conventional host cells in the art, as long as the expression vector stably expresses the carried nucleic acid molecules as the antibody or its antigen-binding fragment described in this invention. Preferably, the host cells are prokaryotic and / or eukaryotic cells. The prokaryotic cells are preferably E. coli cells such as TG1 or BL21 (expressing single-chain antibodies or Fab antibodies), and the eukaryotic cells are preferably HEK293 cells or CHO cells (expressing full-length IgG antibodies). Transforming the expression vector into the host cells yields the host cells of this invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation, heat shock, or electroporation.

[0056] According to another aspect of this disclosure, an antibody-drug conjugate is provided, comprising: an antibody or an antigen-binding fragment thereof as described in this disclosure or an antibody composition thereof; and a drug covalently linked to the antibody or the antigen-binding fragment thereof.

[0057] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising: the antibody or antigen-binding fragment thereof described in this disclosure, the antibody composition, the chimeric antigen receptor, the multispecific antibody or antigen-binding fragment thereof, the biological material, or the antibody-drug conjugate; and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a pharmaceutical excipient conventional in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or its antigen-binding fragment and / or other small molecule drugs or antibodies or peptides, and 0.01 to 99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0059] In some embodiments, the drug composition may be administered via parenteral, injection, or oral administration. The drug composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granule, injection, or infusion. It may be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasal, airway instillation, or intrapleural instillation. The drug composition may also be administered as an aerosol or spray, for example, via nasal administration; or via intrathecal, intramedullary, or intraventricular administration, or via transdermal, percutaneous, local, enteric, intravaginal, sublingual, or rectal administration. The drug composition may be formulated into various dosage forms as needed, and the physician may determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and route of administration.

[0060] In some embodiments, the antibody or its antigen-binding fragment in the pharmaceutical composition may be administered simultaneously or sequentially with other active ingredients.

[0061] According to another aspect of this disclosure, a diagnostic or therapeutic kit is provided, comprising: the antibody or antigen-binding fragment thereof described in this disclosure, the antibody composition, the chimeric antigen receptor, the multispecific antibody or antigen-binding fragment thereof, the biomaterial, the antibody-drug conjugate, or the pharmaceutical composition thereof.

[0062] In some embodiments, the kit includes one or more antibodies or antigen-binding fragments thereof described in this disclosure.

[0063] According to another aspect of this disclosure, the use of the antibody or antigen-binding fragment thereof described herein, the antibody composition thereof, the chimeric antigen receptor thereof, the multispecific antibody or antigen-binding fragment thereof, the biomaterial thereof, the antibody-drug conjugate thereof, or the pharmaceutical composition thereof in the preparation of a product for use in any one of b1)-b3): b1) Prevention, treatment and / or diagnosis of rabies virus infection, b2) Diagnose rabies virus infection or the disease it causes; b3) Detect the presence or level of rabies virus in the sample.

[0064] In some implementations, the rabies virus infection includes rabies.

[0065] In some embodiments, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

[0066] According to another aspect of this disclosure, a method for detecting rabies virus in a sample is provided, the method comprising the step of detecting rabies virus in the sample using the antibody or antigen-binding fragment thereof described in the first aspect.

[0067] In some embodiments, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

[0068] According to another aspect of this disclosure, a method for preventing, treating, and / or diagnosing rabies virus infection is provided, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof described in this disclosure, the antibody composition, the chimeric antigen receptor, the multispecific antibody or antigen-binding fragment thereof, the biomaterial, the antibody-drug conjugate, or the pharmaceutical composition thereof.

[0069] In some implementations, the rabies virus infection includes rabies. Attached Figure Description

[0070] Figure 1 The results of RABV pseudovirus neutralization in cell supernatant are shown.

[0071] Figure 2 The results of monoclonal antibody ELISA binding are shown.

[0072] Figure 3 The results of neutralization of RABV pseudoviruses by monoclonal antibody are shown.

[0073] Figure 4 The results of the BLI evaluation for the monoclonal antibody are shown.

[0074] Figure 5 The results of neutralization of street virus pseudoviruses by the monoclonal antibody RABV are shown.

[0075] Figure 6 The results of neutralization of other viruses and pseudoviruses by the monoclonal antibody Lyssavirus are shown. Detailed Implementation

[0076] To obtain specific antibodies against the rabies virus G protein, this disclosure utilizes flow cytometry to screen G protein-specific memory B cells from PBMCs of vaccine recipients with different immune backgrounds. Selected single B cells were sorted into 96-well PCR plates, subjected to single-cell lysis and RT-PCR, successfully constructing cDNA containing the variable regions of the naturally paired heavy and light chains. Subsequently, the full-length sequences of the antibody heavy and light chains were amplified using specific primers, and expression cassettes (TADs) were constructed through fragment splicing. The resulting products were transiently transfected into HEK293T cells for small-scale antibody expression in 96-well plates, and the cell supernatant was collected for pseudovirus neutralization experiments for initial functional screening. The neutralization experiment used the rabies virus VSV pseudovirus system to evaluate the neutralizing ability of the antibodies against representative rabies virus strains. Some expression products showed good neutralizing activity, thus initially screening a batch of positive candidate antibodies (…). Figure 1 ).

[0077] To further screen candidate antibodies with strong binding ability and neutralizing activity, ELISA binding assays and pseudovirus neutralization assays were performed on the initially screened positive antibodies. ELISA results showed that most antibodies exhibited good binding ability to the G proteins of classic vaccine strains (P08667.1, CAI43218.1, ACR39382.1), and some antibodies showed broad-spectrum binding ability between different strains. Figure 2 ).

[0078] The pseudovirus neutralization assay further evaluated the neutralizing activity of the candidate antibodies against the G proteins of multiple rabies virus strains (ADM32132.1, ACR39382.1, ADJ29911.1, ADD84785.1, CAI43218.1, P08667.1). The results showed that some antibodies had strong neutralizing capabilities against multiple strains (such as #230, #272, #249, #296, #213, #253), suggesting their potential as broad-spectrum neutralizing antibodies. Figure 3 ).

[0079] To determine the affinity of the purified antibodies for the rabies virus G protein, a biolayer interference (BLI) experiment was conducted. The results showed that most antibodies exhibited high affinity for the vaccine strain G protein (P08667.1). Figure 4 To verify the broad-spectrum neutralizing ability of candidate antibodies in real-world infection scenarios, a pseudovirus neutralization screening system containing 46 RABV street strains was constructed. Experimental results showed that most candidate antibodies retained neutralizing ability in the street virus background, and some antibody combinations (such as #230 / #272, #249 / #213, #296 / #213, #249 / #253, #296 / #253, #249 / #272) exhibited synergistic or complementary neutralizing effects among the strains. Figure 5These results indicate that antibody combination strategies may further expand the scope of protection and are a key direction for optimizing anti-rabies virus biologics.

[0080] Furthermore, to clarify whether these antibodies have neutralizing effects against other known human-infecting viruses of the rabies virus genus, the neutralizing activity of the antibodies against the G proteins of multiple rabies virus strains (ABLV: KU739052.1, ARAV: EF614259.1, BBLV: KU761304.1, EBLV1: EU352768.1, EBLV2: AAX62813.1, IRKV: EF614260.1, KHUV: EF614261.1) was further tested. The results showed that some antibodies maintained high neutralizing potency against multiple rabies virus strains, and suggested that some combinations may exhibit a potential broad-spectrum neutralizing advantage. Figure 6 ).

[0081] The humanized antibodies obtained in this public screening showed high affinity for the G proteins of various classical vaccine strains in ELISA binding experiments, and also demonstrated significant neutralizing ability against representative RABV strains and some non-classical Lyssavirus strains in pseudovirus systems, covering a wide range and showing potential as broad-spectrum neutralizing antibodies.

[0082] In a screening system of 46 naturally derived RABV street strains for neutralization, the protective efficacy of candidate antibodies and their combinations under a background closer to natural infection was further validated. Some antibody combinations exhibited synergistic or complementary neutralizing effects among different strains, significantly expanding the neutralization coverage. Furthermore, the antibodies obtained in this invention are all fully humanized IgG1 frameworks, suitable for large-scale mammalian cell expression, with low safety and immunogenicity risks, and mature production processes, making them suitable candidates for clinical prophylaxis or treatment in drug development.

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0084] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0085] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0086] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0087] The term "rabies virus" as used in this article refers to the genus Lyssavirus. The genus Lyssavirus is a genus of viruses within the family Rhabdoviridae. Its members are viruses that can infect mammals, and their core characteristic is high pathogenicity.

[0088] Rabies virus (RABV) is the most classic, highly pathogenic, and widely distributed virus species in the genus Lyssavirus, and the main pathogen causing rabies in humans worldwide. Non-classical Lyssaviruses are the other virus species in the genus Lyssavirus besides RABV.

[0089] Rabies virus (RABV) belongs to the genus *Rabiesvirus* in the family Rhabdoviridae. It is the pathogen that causes rabies, a highly contagious zoonotic disease, primarily transmitted through the saliva of infected animals (such as dogs, cats, and bats). The mortality rate after infection is almost 100%. The viral particle structure consists of a nucleocapsid and an envelope. The nucleocapsid contains a single-stranded, negative-sense RNA genome encoding five proteins: N, P, M, G, and L. The envelope surface is embedded with the crucial glycoprotein G, which is the "key" for the virus to invade host cells and is also the core antigen that induces the body to produce neutralizing antibodies.

[0090] Street viruses refer to wild-type RABVs isolated from naturally infected hosts (such as brain tissue or saliva of diseased animals), as opposed to "fixed viruses" (adapted to cell culture, with reduced pathogenicity, and used for vaccine production) that have been passaged and domesticated in the laboratory over a long period. Street viruses possess natural pathogenicity, high virulence, and a wide host range (able to infect various mammals such as dogs, cats, humans, and bats). Their genomes may exhibit natural variations, making them closer to the characteristics of truly circulating viral strains.

[0091] Pseudoviruses, also known as "pseudoviruses," are artificially constructed, non-replicating viral mimics. Their core characteristic is that they are infectious but not pathogenic. Structurally, they retain only the virus's infection-related structures (such as the envelope glycoprotein G in RABV), while replacing or deleting essential genomic segments for replication (e.g., the RABV RNA genome is replaced with a reporter gene, or the coding sequences for key replication proteins such as L and P are missing). Functionally, they can bind to host cell receptors and invade cells like natural viruses through surface antigens (such as the G protein), but because they lack a complete genome, they cannot replicate within cells, form new infectious particles, or cause disease.

[0092] As used herein, the term "antibody" encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0093] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0094] As used herein, the terms “antibody or antigen-binding fragment thereof” and “antibody” are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant regions). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: k-light chains and λ-light chains.

[0095] Within the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0096] As used in this article, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of antigen-binding molecules to antigens. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0097] As used in this paper, the term "variable" refers to the fact that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the variable domains of the light and heavy chains. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are held together tightly by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). Constant domains do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0098] As used herein, the term "hypervariant region" or "HVR" refers to a region in the variable domain region of an antibody that exhibits high sequence variability and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the variable domain (H1, H2, H3) and three in the variable chain (L1, L2, L3). The term "complementarity-determining region" or "CDR" as used herein refers to the region in the variable domain where amino acid variation is most concentrated; each VH and VL contains three CDRs (HCDR1-HCDR3 and LCDR1-LCDR3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the complementarity-determining region (CDR), which has the highest sequence variability and / or participates in antigen recognition.

[0099] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The variable domain FR is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0100] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0101] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0102] "Humanized antibodies" have an amino acid sequence that corresponds to that of antibodies produced by humans or human cells, or derived from non-human antibodies using sequences encoded by human antibody libraries or other human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0103] This invention also relates to amino acid sequence variants that can be prepared by introducing appropriate modifications into the nucleotide sequence of a coding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain a final construct with desired properties, such as antigen-binding activity. Sites used for substitution typically include HVRs and frames (FRs). See the table below for possible amino acid substitutions.

[0104] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0105] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0106] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0107] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0108] As used herein, the term "specific binding" refers to binding selectivity for antigens, distinguishable from unwanted or nonspecific binding. The term "affinity" or "binding affinity" as used herein refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed using the dissociation constant (Kd), which is the sum of the dissociation rate constant and the association rate constant (K0, K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K2 ... off and K onThe ratio of the rate constants to the antigen-binding molecule. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In some embodiments, the dissociation constant (Kd) of the antigen-binding molecule is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10). -7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). As used herein, the term "affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed using the dissociation constant (Kd), which is the sum of the dissociation rate constant and the association rate constant. A smaller Kd indicates less dissociation and a stronger affinity between the antibody and the antigen.

[0109] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding a polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0110] As used herein, the term "vector" or "expression vector" refers to a DNA molecule to which a specific gene operatively linked is introduced into a target cell and directed to its expression. The vector includes vectors that function as self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. As used herein, the term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in the target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter.

[0111] As used herein, the term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, and also includes the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the antibodies or antigen-binding fragments of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, and also include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0112] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antibody- or antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC comprises a binding protein, a drug, and a connector linking the binding protein to the drug.

[0113] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0114] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0115] The terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human.

[0116] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antibodies or antigen-binding fragments of the present disclosure, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0117] The term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0118] The term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a patient having one or more diseases or symptoms, and the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce the regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0119] The embodiments of the present invention will now be described in detail with reference to examples.

[0120] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0121] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0122] Materials and methods: 1. Cell Culture HEK293F cells (Thermo Fisher Scientific) were cultured in suspension in shake flasks in OPM-293 serum-free medium (OPMBiosciences) at 37 °C, 8% CO2, and 135 rpm. The cell density reached 4 × 10⁻⁶ cells / year. 6At a cell / mL concentration, HEK293T cells were passaged at a 1:4 ratio. HEK293T cells (Thermo Fisher Scientific) were cultured adherently in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. Once cells reached approximately 80% confluence, they were digested with 0.25% trypsin. After cell detachment was observed, 4 mL of complete culture medium was immediately added to terminate the reaction. The cells were gently pipetted to mix, centrifuged at 1,000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended in the culture medium and passaged at a 1:5 ratio.

[0123] 2. Expression and purification of rabies virus G protein Molecular construction: G protein sequences (containing signal peptides) from six rabies virus strains (P08667.1, CAI43218.1, ADM32132.1, ACR39382.1, ADJ29911.1, and ADD84785.1) were selected, codon-optimized, and then artificially synthesized. Twin-Strep-tag II and an 8-His-tag were added to the C-terminus of each sequence to facilitate purification and detection. The synthesized fragments were ligated into the pCAGGS vector after enzyme digestion and, after sequencing verification, were used for expression.

[0124] Cell transfection and protein expression: HEK 293F cells were counted and viability was assessed to ensure cell viability ≥95%. Cells were diluted to 1.7-2 × 10⁻⁶ cells / mL using OPM-293 medium. 6 Prepare two 10 mL OPM-293 culture media in sterile 50 mL centrifuge tubes. Add 1.5 mg PEI and 0.5 mg of the plasmid DNA dropwise to each tube, and mix gently. Then, add the PEI-containing medium dropwise to the plasmid-containing solution, mix thoroughly, and incubate at room temperature for 20 minutes to form a complex. Add the DNA / PEI complex dropwise to 500 mL of diluted 293F cell suspension, mix well, and incubate at 37 °C, 8% CO2, and 135 rpm. After transfection, culture for 3–4 days and collect cells for protein purification.

[0125] Protein purification: Transfected 293F cells were collected by centrifugation at 3,800 rpm for 20 minutes at 4 °C. The cell pellet was resuspended in 20 mL of lysis buffer (20 mM HEPES, 150 mM NaCl), thoroughly lysed, and then centrifuged at 36,000 rpm for 30–45 minutes at 4 °C. The supernatant was discarded. The resulting pellet was resuspended in dissolution buffer (20 mM HEPES, 150 mM NaCl, 1% n-octyl-β-D-glucoside, containing protease inhibitors), thoroughly dispersed, and then magnetically stirred at 4 °C for 3 hours to dissolve the membrane. Subsequently, the cells were centrifuged again at 36,000 rpm for 30–45 minutes at 4 °C, and the supernatant was collected for subsequent purification steps. After incubating the supernatant with a Strep-Tactin affinity chromatography column, non-specifically bound contaminating proteins were washed away with protein buffer (20 mM HEPES, 150 mM NaCl, 1% n-octyl-β-D-glucoside). Finally, the target protein was eluted with protein elution buffer (50 mM Biotin, 20 mM HEPES, 150 mM NaCl, 1% n-octyl-β-D-glucoside), and the protein was identified by SDS-PAGE and peptide mapping to obtain the target membrane protein suitable for downstream experiments.

[0126] Example 1: Obtaining Antibodies 1. Human peripheral blood mononuclear cell , PBMC separation Blood was collected from vaccine recipients with different immune backgrounds (volunteers had no underlying chronic diseases, were aged 23-41 years, and received either a 4-dose or 5-dose rabies vaccination schedule for PV / PM / CTN vaccines). The blood was collected in anticoagulated blood bags, stored at 4°C, and transported. 15 mL of Ficoll-Paque PLUS density gradient centrifugation medium (Cytiva) was added to a lymphocyte separation tube (Sigma-Aldrich), and centrifuged at 400 g for 5 minutes at room temperature. Then, approximately 25 mL of anticoagulated whole blood was slowly added, and the tube was centrifuged at 900 g for 15 minutes at room temperature with the centrifuge speed set to the lowest setting. After centrifugation, a flat-tipped Pasteur pipette was gently inserted into the mononuclear cell layer, and the cells were carefully aspirated by rotating along the tube wall. The cells were transferred to a 50 mL centrifuge tube, and D-Hanks buffer (Solarbio) without calcium and magnesium was added to a total volume of 45 mL. The tube was centrifuged at 400 g for 10 minutes to remove residual plasma. If red blood cell contamination is present, use ACK cleavage buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM C). 10 H16 The cells were treated with N2O8 and then brought to a final volume of 45 mL with D-Hanks. The mixture was centrifuged at 400 g for 10 minutes. The cells were resuspended in D-Hanks, filtered to remove aggregated cells, and counted for later use. The cells were resuspended in cryopreservation medium (fetal bovine serum to DMSO, volume ratio 9:1), aliquoted into labeled cryovials, and placed in a programmed temperature-controlled cryopreservation box at -80 °C overnight before being transferred to liquid nitrogen for long-term storage.

[0127] 2. Sorting of single-specific memory B cells by RABV G antigen binding To obtain rabies virus G protein (RABV-G)-specific human memory B cells, PBMCs were sorted using multicolor flow cytometry. PBMCs were incubated with the following fluorescently labeled antibodies: PE / Cyanine7-labeled anti-human CD19 antibody (BioLegend) for B lymphocyte recognition; PE-labeled anti-human IgG antibody and PerCP / Cyanine5.5-labeled human IgM / D antibody for screening IgG-positive, IgM / D-negative B cells; and APC-labeled anti-human CD27 antibody for memory B cell recognition. Rabies virus G protein was labeled with Alexa Fluor 488 (BioLegend) for antigen-specific B cell recognition. After staining, cell sorting was performed using a BD FACSAria III flow cytometer (BD Biosciences). The final result was a single CD19 cell bound to the RABV G protein. + IgM / IgD - IgG + CD27 + Memory B cells were sorted into 96-well PCR plates for subsequent single-cell RT-PCR amplification of antibody variable region sequences.

[0128] 3. Antibody gene cloning and transient expression Single-cell antibody gene amplification was performed on isolated memory B cells. First-strand cDNA was synthesized, followed by amplification of full-length cDNA and variable region fragments of the IgG heavy and light chains. Based on this, transcriptional-active DNA (TAD) cassettes containing the CMV promoter and polyA tail of the IgG heavy and light chains were constructed via fragment splicing reactions. The obtained TADs containing the IgG heavy and light chains were co-transfected into HEK293T cells. The culture supernatant was collected 72 hours after transfection; this was the antibody expression product, used for subsequent binding and neutralizing function assays.

[0129] Example 2: Initial screening of positive candidate antibodies The cell supernatant (antibody expression product) obtained in Example 1 was used for initial functional screening in a pseudovirus neutralization experiment. The neutralization experiment used the rabies virus VSV pseudovirus system to evaluate the antibody's neutralizing ability against representative rabies virus strains.

[0130] 1. Preparation of rabies virus pseudovirus The rabies virus G protein sequences (ADM32132.1, ACR39382.1, ADJ29911.1, ADD84785.1, CAI43218.1, P08667.1) were codon-optimized and synthesized into the pcDNA3.1 plasmid, which was then amplified. The plasmid was transfected into HEK293T cells at 70-80% confluence. TCID2 was then... 50 7×10 4 Vesicular stomatitis virus pseudovirus (G) ΔG luciferase (VSV pseudovirus) was diluted with 2% FBS in DMEM and added to cells, then incubated at 37 ℃ in a 5% CO2 incubator for 3-5 hours. After washing with PBS, the cell culture medium was replaced with fresh 2% FBS in DMEM, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for another 24 hours. The culture supernatant was centrifuged at 1,000 rpm at room temperature for 5 minutes, aliquoted, and stored at -80 ℃.

[0131] 2. Antibody neutralization experiment based on rabies virus pseudovirus The antibody to be tested (supernatant from transiently expressed cells obtained in Example 1) was initially diluted 30-fold using DMEM, and then serially diluted 5-fold, resulting in a total of 8 dilution gradients. Dilutions were performed in 96-well plates, with cell controls (100 μL DMEM) and virus controls (50 μL pseudovirus and 50 μL DMEM) included. Each dilution and the diluted pseudovirus were incubated at 37°C and 5% CO2 for 1 hour. Subsequently, well-grown HEK293T cells were digested and the concentration adjusted to 4 × 10⁻⁶. 5 Cells / mL, add 100 μL of cell suspension to each well. Incubate at 37°C, 5% CO2 for 24 hours. After 24 hours, discard the supernatant, add 100 μL of ONE-Glo™ EX Reagent (Promega), and incubate in the dark for 2 minutes. Use a PerkinElmer EnSight multi-functional imaging microplate reader to read the fluorescence signal (RLU) to assess the neutralization effect. Plot a neutralization curve based on RLU changes and calculate IC50. 50 value.

[0132] Figure 1The results showed that some of the expression products exhibited good neutralizing activity, thus initially identifying a batch of positive candidate antibodies.

[0133] Example 3: Construction and expression of antibody expression vector Based on the candidate supernatants selected in Example 2 and showing significant effects, heavy and light chain variable region sequencing was performed (antibody sequences are shown in Tables 1-6 below), and naturally paired variable region sequences were synthesized. The heavy and light chains were cloned into the expression vector pcDNA3.1 containing the human IgG1 constant region, and the plasmids were amplified. Transient transfection expression was performed using HEK293F cells. Five days after transfection, cells were collected by centrifugation at 3,800 rpm for 40 minutes at 4 °C. The cell pellet was discarded, and the supernatant was collected and filtered (using a 0.45 μm filter). The filtered supernatant was incubated with a protein A affinity chromatography column at 4 °C for 1 hour. After washing away non-specifically bound proteins with PBS, the target antibody was eluted with 0.1 M glycine solution at pH 2.5 and immediately neutralized with 1 M Tris solution at pH 8.0. The purified antibody was validated by SDS-PAGE and used for subsequent functional validation.

[0134] Example 4: Evaluation using enzyme-linked immunosorbent assay (ELISA) The purified rabies virus G proteins P08667.1, CAI43218.1, and ACR39382.1 were diluted to 2 μg / mL with PBS buffer and coated onto 96-well ELISA plates, incubated overnight at 4 °C. The plates were washed three times with PBST (PBS containing 0.05% Tween-20), and each well was blocked with 5% skim milk blocking buffer at room temperature for 2 hours. The test antibody (positive candidate antibody obtained in Example 2) was then added, diluted, and incubated at room temperature for 1 hour. After washing, 5000-fold diluted HRP-labeled anti-human IgG antibody (Sigma-Aldrich) was added, and the plates were incubated at room temperature for 1 hour. After washing again, TMB chromogenic buffer (Beyotime) was added, and the reaction was allowed to proceed for approximately 10 minutes. The reaction was terminated with stop solution, and the absorbance was read at 450 nm.

[0135] Figure 2 ELISA results showed that most antibodies (such as #230, #272, #249, #296, #213 and #253) had good binding ability to the G protein of classic vaccine strains (P08667.1, CAI43218.1, ACR39382.1), and some antibodies showed broad-spectrum binding ability between different strains.

[0136] Example 5: Antibody Neutralization Experiment Based on Rabies Virus Pseudovirus 1. Preparation of rabies virus pseudovirus The rabies virus G protein sequences (GenBank: ADM32132.1, ACR39382.1, ADJ29911.1, ADD84785.1, CAI43218.1, P08667.1) were codon-optimized and synthesized into the pcDNA3.1 plasmid, which was then amplified. The plasmid was transfected into HEK293T cells at 70-80% confluence. TCID2 was then used. 50 7×10 4 Vesicular stomatitis virus pseudovirus (G) ΔG luciferase (VSV pseudovirus) was diluted with 2% FBS in DMEM and added to cells, then incubated at 37 ℃ in a 5% CO2 incubator for 3-5 hours. After washing with PBS, the cell culture medium was replaced with fresh 2% FBS in DMEM, and the cells were incubated at 37 ℃ in a 5% CO2 incubator for another 24 hours. The culture supernatant was centrifuged at 1,000 rpm at room temperature for 5 minutes, aliquoted, and stored at -80 ℃.

[0137] 2. Antibody neutralization experiment based on rabies virus pseudovirus The antibody to be tested (the positive candidate antibody obtained in Example 2) was initially diluted 30-fold using DMEM, and then serially diluted 3-fold or 5-fold, for a total of 8 dilution gradients. The dilutions were performed in 96-well plates, with cell controls (100 μL DMEM) and virus controls (50 μL pseudovirus and 50 μL DMEM). Each dilution and the diluted pseudovirus were incubated at 37°C and 5% CO2 for 1 hour. Subsequently, HEK293T cells in good growth condition were digested and the concentration was adjusted to 4 × 10⁻⁶. 5 Cells / mL, add 100 μL of cell suspension to each well. Incubate at 37°C, 5% CO2 for 24 hours. After 24 hours, discard the supernatant, add 100 μL of ONE-Glo™ EX Reagent (Promega), and incubate in the dark for 2 minutes. Use a PerkinElmer EnSight multi-functional imaging microplate reader to read the fluorescence signal (RLU) to assess the neutralization effect. Plot a neutralization curve based on RLU changes and calculate IC50. 50 value.

[0138] Figure 3 The results showed that some antibodies (such as #230, #272, #249, #296, #213, and #253) had strong neutralizing capabilities against multiple strains, suggesting their potential as broad-spectrum neutralizing antibodies. The heavy chain variable region, light chain variable region, and CDR sequences of antibodies #230, #272, #249, #296, #213, and #253 are shown in Tables 1-6 below.

[0139] Table 1

[0140] Table 2

[0141] Table 3

[0142] Table 4

[0143] Table 5

[0144] Table 6

[0145] Example 6 Determination of antigen-antibody affinity dissociation constant The affinity between purified antibodies and rabies virus G protein was assessed using biolayer interferometry (BLI).

[0146] The purified G protein (P08667.1) was diluted to 20 μg / mL, and the initial concentration of the candidate antibody was 100 nM, followed by 2-fold serial dilutions. Experiments were performed using an Octet Red96 sensor. The G protein was immobilized using a Ni-NTA sensor, and a program was set to sequentially detect affinity dissociation of each candidate antibody screened in Example 5. Between each detection round, the sensor was regenerated using a glycine solution at pH 1.5, followed by neutralization with 10 mM NiSO4 solution. The affinity dissociation curves were fitted using the Octet system's Data Analysis software, and the affinity dissociation constant was calculated to evaluate the antibody binding activity.

[0147] The results are as follows Figure 4 As shown, antibodies #230, #272, #249, #296, #213, and #253 exhibit good affinity and binding activity to the rabies virus G protein.

[0148] Example 7: Neutralization Experiment of Pseudovirus Antibody Based on Street Rabies Virus To verify the broad-spectrum neutralizing ability of candidate antibodies in real infection scenarios, this embodiment constructed a pseudovirus neutralization screening system containing 46 RABV street viruses.

[0149] The test antibody (the monoclonal antibody identified in Example 5) and the antibody combination (antibody mass concentration 1:1 combination) were initially diluted 30-fold using DMEM, and then serially diluted 3-fold or 5-fold, for a total of 8 dilution gradients. Dilutions were performed in 96-well plates, with cell controls (100 μL DMEM) and virus controls (50 μL pseudovirus and 50 μL DMEM). Each dilution and the diluted pseudovirus were incubated at 37 °C and 5% CO2 for 1 hour. Subsequently, HEK293T cells in good growth condition were digested and the concentration adjusted to 4 × 10⁻⁶. 5 Cells / mL, add 100 μL of cell suspension to each well. Incubate at 37°C, 5% CO2 for 24 hours. After 24 hours, discard the supernatant, add 100 μL of ONE-Glo™ EX Reagent (Promega), and incubate in the dark for 2 minutes. Use a PerkinElmer EnSight multi-functional imaging microplate reader to read the fluorescence signal (RLU) to assess the neutralization effect. Plot a neutralization curve based on RLU changes and calculate IC50. 50 value.

[0150] Figure 5 The experimental results showed that most candidate antibodies (such as #230, #272, #249, #296, #213 and #253) retained their neutralizing ability in the context of street viruses, and some antibody combinations (such as #230 / #272, #249 / #213, #296 / #213, #249 / #253, #296 / #253, #249 / #272) showed synergistic or complementary neutralizing effects among virus strains.

[0151] Example 8: Antibody Neutralization Experiment Based on Rabies Virus Pseudovirus To clarify whether the candidate antibody combination has a neutralizing effect on some viruses of the Rabies virus genus, the neutralizing activity of the antibody against a variety of Rabies virus strains (ABLV: KU739052.1, ARAV: EF614259.1, BBLV: KU761304.1, EBLV1: EU352768.1, EBLV2: AAX62813.1, IRKV: EF614260.1, KHUV: EF614261.1) was further tested according to the method in Example 7.

[0152] Figure 6 The results showed that some antibody combinations (such as #230 / #272, #249 / #213, #296 / #213, #249 / #253, #296 / #253, and #249 / #272) maintained high neutralizing efficacy in multiple rabies virus strains, suggesting that some combinations may exhibit a potential broad-spectrum neutralizing advantage.

[0153] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to rabies virus, said antibody or antigen-binding fragment comprising: The three HCDRs contained in the heavy chain variable region as shown in SEQ ID NO: 1, and the three LCDRs contained in the light chain variable region as shown in SEQ ID NO: 2; or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 8, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 9; or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 15, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 16; or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 22, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 23; or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 29, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 30; or For example, the heavy chain variable region shown in SEQ ID NO: 36 contains 3 HCDRs, and the light chain variable region shown in SEQ ID NO: 37 contains 3 LCDRs. in, The HCDR1-3 and LCDR1-3 are defined by the rules of Kabat, AbM, Chothia, Contact, or IMGT.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6 with the amino acid sequences WAS and SEQ ID NO: 7; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20, with the amino acid sequence GAS, and SEQ ID NO: 21; or As shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, HCDR1, HCDR2 and HCDR3; and as shown in SEQ ID NO: 27, the amino acid sequences EVT and SEQ ID NO: 28, LCDR1, LCDR2 and LCDR3; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34, SEQ ID NO: 35; or HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 41, with amino acid sequences of AAS and SEQ ID NO: 42; The HCDR1-3 and LCDR1-3 are defined by the rules of IMGT.

3. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 8, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 9, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 22, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 23, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 29, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 30, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; or The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 37, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it.

4. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody is of the IgG type.

5. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment is scFv, Fab, Fab', (Fab')2, Fv fragment or dsFv.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment is a chimeric antibody, a human antibody, or a humanized antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4.

9. An antibody composition comprising a first antibody and a second antibody, wherein the first antibody and the second antibody are selected from the antibodies or antigen-binding fragments thereof according to any one of claims 1-8. in, The first antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 13 with the amino acid sequence ASS and SEQ ID NO: 14; and the second antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34 with the amino acid sequence EDN and SEQ ID NO: 35; or The first antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6 with the amino acid sequence WAS and SEQ ID NO: 7; or The first antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 41 with amino acid sequences AAS and SEQ ID NO: 42; and the second antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6 with amino acid sequences WAS and SEQ ID NO: 7; or The first antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 27 with the amino acid sequence EVT and SEQ ID NO: 28; or The first antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 41 with the amino acid sequence AAS and SEQ ID NO: 42; and the second antibody comprises: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 27 with the amino acid sequence EVT and SEQ ID NO: 28; or The first antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20 with the amino acid sequence GAS and SEQ ID NO: 21; and the second antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34 with the amino acid sequence EDN and SEQ ID NO:

35.

10. The antibody composition according to claim 9, wherein, The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 9, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 29 and a light chain variable region as shown in SEQ ID NO: 30; or The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2; or The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 37, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2; or The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 23; or The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 37, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 23; or The first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 29 and a light chain variable region as shown in SEQ ID NO:

30.

11. A biomaterial relating to the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the antibody composition of claim 9 or 10, wherein the biomaterial comprises any one of a1)-a9): a1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, or the antibody composition of claim 9 or 10; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A carrier containing the nucleic acid molecule described in a1); a4) A carrier containing the expression box described in a2); a5) Cells containing the nucleic acid molecules described in a1); a6) Cells containing the expression cassette described in a2); a7) Cells containing the carrier described in a3); a8) Cells containing the carrier described in a4); a9) Cells comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, or the antibody composition of claim 9 or 10; None of the cells described in a5)-a9) contain reproductive material.

12. A pharmaceutical composition comprising: an antibody or an antigen-binding fragment thereof according to any one of claims 1-8, or an antibody composition according to claim 9 or 10; and a pharmaceutically acceptable carrier.

13. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof of any one of claims 1-8, the antibody composition of claim 9 or 10, or the pharmaceutical composition of claim 12.

14. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, the antibody composition according to claim 9 or 10, or the pharmaceutical composition according to claim 12 in the preparation of a product, wherein the product is used in any one of b1)-b3): b1) Prevention, treatment and / or diagnosis of rabies virus infection or related diseases, b2) Diagnose rabies virus infection or related diseases; b3) Detect rabies virus in the sample.

15. The application according to claim 14, wherein, The rabies virus in the test sample refers to the presence or level of the rabies virus G protein in the test sample.

16. The application according to claim 14, wherein, The rabies virus infection-related diseases include rabies.

17. The application according to claim 14, wherein, The samples are whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

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