Bovine dermatophilus antibody or antigen-binding fragment thereof and preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a bovine dermatophilus virus antibody or antigen-binding fragment thereof, a preparation method and application thereof. The antibody or antigen-binding fragment thereof can specifically recognize and combine with the bovine dermatophilus virus or A27L protein thereof and has good affinity therewith. Meanwhile, the antibody or antigen-binding fragment thereof has good neutralization activity and blocking activity on the bovine dermatophilus virus. The antibody or antigen-binding fragment thereof can be used for preparing a product for diagnosing, preventing and / or treating bovine dermatophilus virus infection or a disease caused by the bovine dermatophilus virus, detecting the presence or level of the A27L protein of the bovine dermatophilus virus in a sample or screening a drug for preventing and / or treating bovine dermatophilus virus infection or a disease caused by the bovine dermatophilus virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to bovine nodular dermatitis virus antibodies or their antigen-binding fragments, their preparation methods, and applications. Background Technology

[0002] Bovine lumpy skin disease (LSD) is a contagious bovine viral disease affecting cattle of all ages and breeds, with young and lactating calves being more susceptible. The causative agent of LSD, bovine lumpy skin disease virus (LSDV), is classified under the genus Capripoxvirus in the family Poxviridae, along with sheep poxvirus and goat poxvirus. This disease can cause severe reductions in milk production, decreased leather and meat quality, infertility, and abortion in dairy cows, resulting in significant economic losses to my country's livestock industry and making it a reportable disease of high economic importance. Currently, there is no effective treatment for this disease; vaccination and serological surveillance remain the most effective measures to control its spread. The vaccines currently available for prophylactic immunization of cattle are mainly live attenuated vaccines (LAVs) produced using the Neethling strain or its derivatives. Although LAVs have shown efficacy, they are affected by safety concerns, and adverse reactions are common after vaccination. Recent studies have explored inactivated LSD vaccines as an alternative treatment for the prevention and management of LSD infection. While these types of vaccines are considered safer than LSD, research reports indicate that inactivated LSD vaccine administration only provides short-term and partial protection. Therefore, safer LSD treatments still need to be developed.

[0003] LSDV ORF 117 and ORF 060 are orthologs of vaccinia virus (VV) proteins A27L and L1R. These proteins have been reported as potential antigens and targets for neutralizing antibodies. They can elicit immune responses in other poxviruses such as sheep pox and VV, and are known to play important roles in virus-cell binding and membrane fusion. Furthermore, A27L is involved in intracellular viral transport and the formation of intracellular enveloped virions (IEVs). Studies have shown that A27L can induce neutralizing antibodies as early as day 14 post-vaccination. Therefore, developing monoclonal antibodies with neutralizing effects can be used for both the treatment of LSD, compensating for the lack of long-term comprehensive protection provided by inactivated vaccines, and for the detection of LSD. This is of great significance for the prevention and control of LSD and the healthy development of my country's cattle industry. Therefore, there is an urgent need to develop antibodies against bovine nodular dermatitis virus or their antigen-binding fragments. Summary of the Invention

[0004] The first aspect of the present invention is to provide an antibody against bovine nodular dermatitis virus or an antigen-binding fragment thereof.

[0005] A second aspect of the present invention is to provide a chimeric antigen receptor.

[0006] A third aspect of the present invention aims to provide multispecific antibodies.

[0007] The fourth aspect of this invention aims to provide biomaterials.

[0008] The fifth aspect of this invention aims to provide a method for preparing the antibody or antigen-binding fragment thereof of the first aspect of this invention, the chimeric antigen receptor of the second aspect, or the multispecific antibody of the third aspect.

[0009] The sixth aspect of this invention aims to provide a coupling.

[0010] A seventh aspect of the present invention is to provide a pharmaceutical composition.

[0011] An eighth aspect of the present invention aims to provide a diagnostic or therapeutic reagent kit.

[0012] The object of the ninth aspect of the present invention is to provide the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the chimeric antigen receptor of the second aspect, the multispecific antibody of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0013] The object of the tenth aspect of this invention is to provide a method for preventing and / or treating bovine nodular dermatitis virus infection or diseases caused thereby.

[0014] The object of the eleventh aspect of this invention is to provide a method.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0016] In a first aspect, the present invention provides a bovine nodular dermatitis virus antibody or an antigen-binding fragment thereof, said bovine nodular dermatitis virus antibody or antigen-binding fragment thereof comprising:

[0017] a1) HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 5; and / or LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 6; (11C4) or

[0018] a2) HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 27; and / or LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 28; (28B5) or

[0019] a3) Having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in a1) or a2); and / or having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in a1) or a2).

[0020] In this invention, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0021] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0022] b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 8, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 9; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 10, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; (11C4) or

[0023] b2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 29, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 31; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 32, LCDR2 having the amino acid sequence DTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; (28B5) or

[0024] b3) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1) or b2); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1) or b2);

[0025] The CDR is defined according to the IMGT numbering system.

[0026] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0027] c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 12, HCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; (11C4) or

[0028] c2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 34, HCDR2 having the amino acid sequence shown in SEQ ID NO: 35, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; (28B5) or

[0029] c3) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1) or c2); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1) or c2);

[0030] The CDR is defined according to the Kabat numbering system.

[0031] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0032] d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; (11C4) or

[0033] d2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 39, HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; (28B5) or

[0034] d3) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1) or d2); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1) or d2);

[0035] The CDR is defined according to the Chothia numbering system.

[0036] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0037] e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; (11C4) or

[0038] e2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 41, HCDR2 having the amino acid sequence shown in SEQ ID NO: 42, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 43; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 44, LCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; (28B5) or

[0039] e3) The VH of the package includes the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1) or e2); and / or, the VL of the package includes the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1) or e2);

[0040] The CDR is defined according to the Contact numbering system.

[0041] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0042] In some embodiments, the heavy chain variable region of the bovine nodular dermatitis virus antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region.

[0043] In this invention, the frame region of the heavy chain variable region includes the frame region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes the frame region of the heavy chain variable region or a mutant thereof derived from mouse immunoglobulins.

[0044] In some embodiments, the light chain variable region of the bovine nodular dermatitis virus antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0045] In this invention, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes the framework region of the light chain variable region or a mutant thereof derived from mouse immunoglobulins.

[0046] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0047] f1) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (11C4) or

[0048] f2) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it (28B5).

[0049] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0050] In this invention, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes at least a portion of the heavy chain constant region or a mutant thereof derived from human immunoglobulins.

[0051] In this invention, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes the light chain constant region or a mutant thereof derived from human immunoglobulins.

[0052] In this invention, the heavy chain constant region includes the heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin.

[0053] In this invention, the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:52, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0054] In this invention, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0055] In this invention, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0056] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0057] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment includes, but is not limited to, monoclonal antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

[0058] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment comprises:

[0059] g1) Heavy chain, comprising amino acid sequences 19-471 of SEQ ID NO: 55, the amino acid sequence shown in SEQ ID NO: 55, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising amino acid sequences 19-237 of SEQ ID NO: 56, the amino acid sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (11C4) or

[0060] g2) Heavy chain, comprising amino acid sequence 19-470 of SEQ ID NO: 57, amino acid sequence shown in SEQ ID NO: 57, or amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising amino acid sequence 19-229 of SEQ ID NO: 58, amino acid sequence shown in SEQ ID NO: 58, or amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it (28B5).

[0061] In some embodiments, the bovine nodular dermatitis virus antibody or its antigen-binding fragment specifically binds to the bovine nodular dermatitis virus A27L protein.

[0062] In some embodiments, the antigen-binding epitopes of the bovine nodular dermatitis virus antibody or its antigen-binding fragment include SEQ ID NO:1 (11C4) or SEQ ID NO:3 (28B5).

[0063] A second aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof from the first aspect of the invention.

[0064] A third aspect of the invention provides a multispecific antibody comprising two or more (e.g., three or four) protein functional regions targeting antigens, wherein one of the protein functional regions comprises the antibody or antigen-binding fragment of the first aspect of the invention.

[0065] In some embodiments, the multispecific antibody is a bispecific antibody, which includes: a first protein functional region and a second protein functional region;

[0066] The first protein functional region includes:

[0067] a1) having HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 5; and / or having LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 6; or

[0068] a11) having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1).

[0069] The second protein functional region includes:

[0070] a2) HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 27; and / or LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 28; or

[0071] a21) having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a2); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a2).

[0072] In some embodiments, the first protein functional region includes:

[0073] b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 8, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 9; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 10, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; or

[0074] b11) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1);

[0075] The second protein functional region includes:

[0076] b2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 29, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 31; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 32, LCDR2 having the amino acid sequence DTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; or

[0077] b21) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b2); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b2);

[0078] The CDR is defined according to the IMGT numbering system.

[0079] In some embodiments, the first protein functional region includes:

[0080] c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 12, HCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; or

[0081] c11) VH includes the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1); and / or VL includes the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1);

[0082] The second protein functional region includes:

[0083] c2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 34, HCDR2 having the amino acid sequence shown in SEQ ID NO: 35, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; or

[0084] c21) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c2); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c2);

[0085] The CDR is defined according to the Kabat numbering system.

[0086] In some embodiments, the first protein functional region includes:

[0087] d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; or

[0088] d11) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1);

[0089] The second protein functional region includes:

[0090] d2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 39, HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; or

[0091] d21) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d2); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d2);

[0092] The CDR is defined according to the Chothia numbering system.

[0093] In some embodiments, the first protein functional region includes:

[0094] e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; or

[0095] e11) includes VH with the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1); and / or includes VL with the following three CDRs: having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1);

[0096] The second protein functional region includes:

[0097] e2) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 41, HCDR2 having the amino acid sequence shown in SEQ ID NO: 42, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 43; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 44, LCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; or

[0098] e21) The VH of the package includes the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e2); and / or, the VL of the package includes the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e2);

[0099] The CDR is defined according to the Contact numbering system.

[0100] In some embodiments, the heavy chain variable region of the first protein functional region further includes the framework region of the heavy chain variable region.

[0101] In some embodiments, the heavy chain variable region of the second protein functional region further includes the framework region of the heavy chain variable region.

[0102] In some embodiments, the light chain variable region of the first protein functional region further includes the framework region of the light chain variable region.

[0103] In some embodiments, the light chain variable region of the second protein functional region further includes the framework region of the light chain variable region.

[0104] In some embodiments, the first protein functional region includes:

[0105] The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0106] In some embodiments, the second protein functional region includes:

[0107] The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0108] In some implementations, the first and second protein functional regions are directly connected or connected through a linker fragment.

[0109] In some implementations, the first protein functional region and the second protein functional region are independently one, two, or more.

[0110] In some embodiments, the first protein functional region and the second protein functional region are each independently an immunoglobulin or an antigen-binding fragment.

[0111] In some embodiments, the antigen-binding fragment includes a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, or a single-chain Fv (scFv); further, it is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv (scFv); even further, it is a single-chain Fv (scFv).

[0112] In some embodiments, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen-binding fragment; or, the first protein functional region is an antigen-binding fragment and the second protein functional region is an immunoglobulin.

[0113] In some embodiments, the heavy chain variable region (preferably the N-terminus of the heavy chain variable region) of the antigen-binding fragment is directly or via a linker to the CH1 region (preferably the C-terminus of the CH1 region of the immunoglobulin) and the light chain variable region (preferably the N-terminus of the light chain variable region) of the antigen-binding fragment is directly or via a linker to the light chain constant region CL region (preferably the C-terminus of the light chain constant region CL of the immunoglobulin); or the heavy chain variable region (preferably the N-terminus of the heavy chain variable region) of the antigen-binding fragment is directly or via a linker to the light chain constant region CL region (preferably the C-terminus of the light chain constant region CL of the immunoglobulin) and the light chain variable region (preferably the N-terminus of the light chain variable region) of the antigen-binding fragment is directly or via a linker to the heavy chain constant region CH1 region (preferably the C-terminus of the heavy chain constant region CH1 of the immunoglobulin).

[0114] In some embodiments, the heavy chain variable region (preferably the C-terminus of the heavy chain variable region) of the antigen-binding fragment is directly or via a linker to the heavy chain (preferably the N-terminus of the heavy chain of the immunoglobulin) and the light chain variable region (preferably the C-terminus of the light chain variable region) of the antigen-binding fragment is directly or via a linker to the light chain (preferably the N-terminus of the light chain of the immunoglobulin); or the heavy chain variable region (preferably the C-terminus of the heavy chain variable region) of the antigen-binding fragment is directly or via a linker to the light chain (preferably the N-terminus of the light chain of the immunoglobulin) and the C-terminus of the light chain variable region of the antigen-binding fragment is directly or via a linker to the heavy chain (preferably the N-terminus of the heavy chain of the immunoglobulin).

[0115] In some embodiments, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain Fv; or, the first protein functional region is a single-chain Fv and the second protein functional region is an immunoglobulin.

[0116] In some embodiments, when the first protein functional region is an immunoglobulin, the first protein functional region includes a heavy chain constant region and / or a light chain constant region.

[0117] In some embodiments, when the second protein functional region is an immunoglobulin, the second protein functional region includes a heavy chain constant region and / or a light chain constant region.

[0118] In some embodiments, the heavy chain constant region of the first protein functional region may be the same as or different from the heavy chain constant region of the second protein functional region.

[0119] In some embodiments, the light chain constant region of the first protein functional region may be the same as or different from the light chain constant region of the second protein functional region.

[0120] In some embodiments, the single chain Fv includes a heavy chain variable region, a light chain variable region, and a connecting segment for connecting the heavy chain variable region and the light chain variable region.

[0121] In some embodiments, the single-chain Fv has the following structure: NH2-VL-linker fragment-VH-COOH or NH2-VH-linker fragment-VL-COOH.

[0122] In some embodiments, the single-chain Fv is attached to the C-terminus of the heavy chain of the immunoglobulin, the N-terminus of the heavy chain of the immunoglobulin, or the C-terminus of CH1 of the immunoglobulin via a linker fragment; more preferably, the single-chain Fv is attached to the C-terminus of the heavy chain of the immunoglobulin via a linker fragment.

[0123] In some embodiments, the number of immunoglobulins is 1, and the number of single-chain Fv molecules is 2, that is, one immunoglobulin molecule is linked to two single-chain Fv molecules.

[0124] In some implementations, the two single-chain Fv molecules are identical.

[0125] In this invention, the connection segment is (GGGGS)-(GGGGT)n-(GGGGS)m-1; m is a positive integer, for example: 1, 2, 3, 4, 5, 6; n is 0 or a positive integer, for example: 1, 2, 3, 4, 5.

[0126] In some implementations, the linker segment used to connect the first and second protein functional regions may be the same as or different from the linker segment in the single-chain Fv used to connect the heavy chain variable region and the light chain variable region.

[0127] In some embodiments, the amino acid sequence of the linker fragment used to connect the first and second protein functional regions is shown in SEQ ID NO:60.

[0128] In some embodiments, the amino acid sequence of the linker segment in the single-chain Fv used to connect the heavy chain variable region and the light chain variable region is shown in SEQ ID NO:61.

[0129] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain:

[0130] The first polypeptide chain comprises (preferably from the N-terminus to the C-terminus): the heavy chain of the immunoglobulin, the linker fragment, and the single-chain Fv; the second polypeptide chain comprises: the light chain of the immunoglobulin.

[0131] In some embodiments, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0132] In some embodiments, the first polypeptide chain comprises amino acids 19-733 of SEQ ID NO: 59, the amino acid sequence shown in SEQ ID NO: 59, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0133] The second polypeptide chain includes the amino acid sequence of positions 19-237 of SEQ ID NO: 56, the amino acid sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0134] A fourth aspect of the invention provides a biological material comprising any one of n1)-n9):

[0135] n1) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, or a multispecific antibody of the third aspect;

[0136] n2) An expression cassette containing the nucleic acid molecule described in n1);

[0137] n3) A carrier containing the nucleic acid molecule described in n1);

[0138] n4) A carrier containing the expression box described in n2);

[0139] n5) A cell containing the nucleic acid molecules described in n1);

[0140] n6) Cells containing the expression cassette described in n2);

[0141] n7) Cells containing the carrier described in n3);

[0142] n8) Cells containing the carrier described in n4);

[0143] n9) Cells containing an antibody or antigen-binding fragment thereof from the first aspect of the present invention, a chimeric antigen receptor from the second aspect, or a multispecific antibody from the third aspect;

[0144] None of the cells described in n5)-n9) contain reproductive material.

[0145] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0146] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0147] In some embodiments, the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 25, the nucleotide sequence shown in SEQ ID NO: 47, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0148] In some embodiments, the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 26, the nucleotide sequence shown in SEQ ID NO: 48, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0149] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment of the first aspect of the present invention further comprises a nucleic acid molecule encoding the heavy chain constant region of the antibody or antigen-binding fragment of the first aspect of the present invention and a nucleic acid molecule encoding the light chain constant region of the antibody or antigen-binding fragment of the first aspect of the present invention.

[0150] In some embodiments, the nucleic acid molecule encoding the heavy chain constant region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 51, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0151] In some embodiments, the nucleic acid molecule encoding the light chain constant region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 53, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0152] In some embodiments, any of the vectors n3)-n4) can be expression vectors. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0153] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0154] In some embodiments, any of the cells (n5)-n9) can be host cells conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody of the present invention. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0155] In some embodiments, any of the cells (n5)-n9) can be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, DC cells, and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptor (i.e., modified immune cells) described above in this invention.

[0156] A fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment thereof from the first aspect of the present invention, a chimeric antigen receptor from the second aspect of the present invention, or a multispecific antibody from the third aspect of the present invention, obtained by culturing cells from the fourth aspect of the present invention.

[0157] A sixth aspect of the invention provides a conjugate comprising an antibody or antigen-binding fragment thereof from the first aspect of the invention or a multispecific antibody from the third aspect of the invention; and a conjugation portion.

[0158] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0159] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention or multispecific antibodies of different lengths via linkers of varying lengths to reduce potential steric hindrance.

[0160] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0161] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of bovine nodular dermatitis virus infection or the disease it causes.

[0162] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0163] A seventh aspect of the invention provides a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof of the first aspect of the invention, a chimeric antigen receptor of the second aspect, a multispecific antibody of the third aspect, a biomaterial of the fourth aspect, or a conjugate of the sixth aspect; and a pharmaceutically acceptable carrier.

[0164] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0165] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating bovine nodular dermatitis virus infection or the disease it causes.

[0166] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0167] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0168] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0169] An eighth aspect of the present invention provides a diagnostic or therapeutic kit comprising: an antibody or antigen-binding fragment thereof of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a multispecific antibody of the third aspect, a biomaterial of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0170] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0171] In some embodiments, the kit can be used to diagnose bovine nodular dermatitis virus infection or the disease it causes, to detect the presence or level of bovine nodular dermatitis virus or its A27L protein in a sample, or to screen for drugs to prevent and / or treat bovine nodular dermatitis virus infection or the disease it causes.

[0172] In some embodiments, the kit can be used to prevent and / or treat bovine nodular dermatitis virus infection or the disease it causes.

[0173] A ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the chimeric antigen receptor of the second aspect, the multispecific antibody of the third aspect, the biomaterial of the fourth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in any one of c1)-c6):

[0174] c1) Prepare products for the diagnosis of bovine nodular dermatitis virus infection or diseases caused by it;

[0175] c2) Prepare products for the prevention and / or treatment of bovine nodular dermatitis virus infection or diseases caused by it;

[0176] c3) Prepare products for detecting the presence or level of bovine nodular dermatovirus or its A27L protein in samples;

[0177] c4) Detect the presence or level of bovine nodular dermatosis virus or its A27L protein in the sample;

[0178] c5) Screening for drugs to prevent and / or treat bovine nodular dermatitis virus infection or diseases caused by it;

[0179] c6) Products for the preparation of screening drugs for the prevention and / or treatment of bovine nodular dermatitis virus infection or diseases caused by it.

[0180] In some implementations, the applications described in c4) and c5) do not involve the diagnosis or treatment of diseases.

[0181] In this invention, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the object to be tested.

[0182] In this invention, the body fluid includes at least one of blood and lymph.

[0183] In this invention, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0184] In this invention, the excrement includes at least one of urine, feces, and tears.

[0185] In this invention, the test subject includes mammals, such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0186] In this invention, the object to be tested includes a cow.

[0187] A tenth aspect of the present invention provides a method for preventing and / or treating bovine nodular dermatitis virus infection or the disease caused therefrom, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment of the first aspect of the present invention, a chimeric antigen receptor of the second aspect, a multispecific antibody of the third aspect, a biomaterial of the fourth aspect, a conjugate of the sixth aspect, or a pharmaceutical composition of the seventh aspect.

[0188] The eleventh aspect of the present invention is to provide a method comprising, under conditions that allow the antibody of the first aspect of the present invention or its antigen-binding fragment or the multispecific antibody of the third aspect of the present invention to form a complex with bovine nodular dermatosis virus A27L protein, contacting a sample with the antibody of the first aspect of the present invention or its antigen-binding fragment or the multispecific antibody of the third aspect of the present invention, and detecting the formation of the complex;

[0189] The method is used for any one of f1)-f3):

[0190] f1) Diagnose bovine nodular dermatitis virus infection or the disease it causes;

[0191] f2) Detect the presence or level of bovine nodular dermatosis virus or its A27L protein in the sample;

[0192] f3) Screening for drugs for the prevention and / or treatment of bovine nodular dermatitis virus infection or the disease it causes.

[0193] In some embodiments, the sample is the sample of the ninth aspect of the present invention.

[0194] In this invention, the A27L protein comprises the amino acid sequence encoded by nucleotides 110,419-110,865 of the full sequence of accession number OP508345.1.

[0195] In this invention, the disease caused by bovine nodular dermatitis virus infection includes bovine nodular dermatitis.

[0196] The beneficial effects of this invention are:

[0197] This invention provides an antibody against bovine nodular dermatitis virus or its antigen-binding fragment, which can specifically recognize and bind to bovine nodular dermatitis virus or its A27L protein and has good affinity for it; at the same time, it has good neutralizing and blocking activity against bovine nodular dermatitis virus; it can be used to prepare products for diagnosing, preventing and / or treating bovine nodular dermatitis virus infection or diseases caused by it, detecting the presence or level of bovine nodular dermatitis virus A27L protein in samples, or screening for drugs to prevent and / or treat bovine nodular dermatitis virus infection or diseases caused by it.

[0198] Furthermore, this invention provides a bispecific antibody comprising two antibodies against bovine nodular dermatitis virus (BND) or their antigen-binding fragments having different antigen-binding epitopes, retaining the biological functions of both antibodies. This achieves a single bispecific antibody molecule possessing the biological functions of two monoclonal antibodies simultaneously, recognizing different sites on the BND A27L protein. Its neutralizing activity against BND is significantly higher than that of the maternal monoclonal antibody, and its blocking activity against BND is also higher than that of the maternal monoclonal antibody. Moreover, it exhibits low toxicity and good stability; it also improves the selectivity and neutralizing activity of the maternal monoclonal antibody. This improved biocompatibility enhances the safety and efficacy of monoclonal antibody drugs, enabling the preparation of products for the diagnosis, prevention, and / or treatment of bovine nodular dermatitis virus infection or its associated diseases, the detection of the presence or level of bovine nodular dermatitis virus A27L protein in samples, or the screening of drugs for the prevention and / or treatment of bovine nodular dermatitis virus infection or its associated diseases. It possesses promising market application prospects. Furthermore, the well-developed in vitro recombinant expression technology provides a key production technology for the production of bispecific antibodies, enabling batch control, high yield, and high efficiency, thus solving many challenges related to uncontrollable batches and individual variability in mice. This bispecific antibody retains the complete monoclonal antibody structure and possesses a highly stable symmetrical structure. During host expression, it does not produce protein isoforms with other structures, significantly reducing the difficulty of extraction and purification processes, and offering advantages such as simple preparation and high yield. Attached Figure Description

[0199] Figure 1 The image shows the SDS-PAGE results of the A27L recombinant protein.

[0200] Figure 2 This is a schematic diagram showing the positional distribution of the four polypeptides in the full-length A27L protein.

[0201] Figure 3 The images show the ELISA results of monoclonal antibodies 11C4 and 28B5 reacting with peptides.

[0202] Figure 4A This is a construction map of the 11C4 heavy chain recombinant expression plasmid.

[0203] Figure 4B This is a construction map of the 11C4 light chain recombinant expression plasmid.

[0204] Figure 4C This is a construction map of the 28B5 heavy chain recombinant expression plasmid.

[0205] Figure 4D This is a construction map of the 28B5 light chain recombinant expression plasmid.

[0206] Figure 4E This is a construction map of the recombinant expression plasmid for the bispecific antibody 11C4-28B5 heavy chain.

[0207] Figure 5 This is a schematic diagram of the structure of the bispecific antibody (11C4-28B5).

[0208] Figure 6 The images show the SDS-PAGE and size exclusion chromatography results of monoclonal and bispecific antibodies: A represents the SDS-PAGE results of monoclonal and bispecific antibodies; B represents the size exclusion chromatography results of monoclonal and bispecific antibodies.

[0209] Figure 7 The graphs show the results of the binding activity assays for monoclonal antibodies and bispecific antibodies: A represents the binding activity assay for bispecific antibody 11C4-28B5; B represents the binding activity assay for monoclonal antibody 11C4; and C represents the binding activity assay for monoclonal antibody 28B5.

[0210] Figure 8 The figure shows the specificity identification results of monoclonal antibodies and bispecific antibodies.

[0211] Figure 9 The graph shows the results of the neutralizing activity evaluation of monoclonal antibodies and bispecific antibodies.

[0212] Figure 10 IC50 for monoclonal antibodies and bispecific antibodies 50 Fitted curve. Detailed Implementation

[0213] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] As used in this article, "antibody" refers to a globulin produced by plasma cells, which are formed from the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0218] 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.

[0219] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0220] The terms “full-length antibody” and “intact antibody” as used herein 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, CH). 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 region). 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: the κ (kappa) light chain and the λ (lambda) light chain.

[0221] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing 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.

[0222] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0223] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0224] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0225] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0226] The CDR of the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0227] As used in this article, the term "variable" refers to the fact that certain segments of the variable region 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 region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native 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 tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0228] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0229] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0230] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0231] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0232] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0233] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0234] The instruments involved in the following embodiments are as follows:

[0235] AKTA protein purification system (Cytiva, UK); inverted microscope (Olympus, Japan); pure water treatment system (PURLABclassic, UK); high-speed refrigerated centrifuge (Beckman, Germany); ultrafiltration concentrator (SARTORIUS, Germany); biosafety cabinet (Thermo Fisher Scientific, USA); electrophoresis apparatus (Beijing Liuyi Biotechnology Co., Ltd.); CO2 shaking incubator / shaker (Shanghai Zhichu Instrument Co., Ltd.); BioTeck multi-label microplate analyzer (BioTeck, USA); flow cytometer (GuavaeasyCyte, USA); automated cell counter (Invitrogen, USA); micropipette (Eppendorf, Germany); molecular interaction analyzer (Fortebio, USA); multi-mod microplate reader (BMGLABTECH, Germany); NanoDrop1000 ultra-micro UV spectrophotometer (Thermo Fisher Scientific, USA); -80℃ ultra-low temperature freezer (Thermo Fisher Scientific, USA); electronic balance (Shimadzu, Japan).

[0236] The reagents involved in the following examples are as follows:

[0237] Fetal bovine serum (Gibco, USA); DMEM medium (Hyclone, USA); PRMI 1640 medium (Hyclone, USA); BSA (Solarbio, China); TMB (Solarbio, China); PBS (Solarbio, China); HRP-labeled goat anti-human IgG (Sigma, Germany); FITC-labeled goat anti-human IgG (Sigma, Germany); PE anti-human IgG Fc (BioLegend, USA); Anti-Human IgG Fc Capture (Fortebio, USA); pCAGGS vector, pcDNA3.4 vector (Newp Biotech).

[0238] The reagents involved in the following examples are as follows:

[0239] 293F cells (Shangen Biotechnology Co., Ltd.), Expi293™ cells (Gibco, Inc., USA).

[0240] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0241] Example 1. Screening of monoclonal antibodies against bovine nodular dermatitis virus

[0242] 1. Expression, purification, and identification of recombinant A27L protein

[0243] The full-length gene sequence (447 bp) of A27L (nucleotides 110, 419-110, 865 of the full sequence of bovine nodular dermatitis virus (GenBank accession number: OP508345.1) published in GenBank was selected. After codon optimization, it was inserted between EcoR I and Xho I of the pCAGGS vector (10 His were introduced at the N-terminus of A27L) to obtain the recombinant plasmid pCAGGS-A27L. The plasmid was transformed into E. coli DH5α competent cells, plated and cultured overnight. The next day, positive clones were picked and sequenced. The correctly sequenced positive bacterial cultures were extracted to obtain the correctly sequenced recombinant plasmid pCAGGS-A27L for later use.

[0244] 293F cells in normal suspension culture were selected, and a mixture containing 2 mg pCAGGS-A27L plasmid and 12 mg PEI was prepared in 1 L of cell culture medium. The cell density was 2 × 10⁶ cells / year. 6 The mixture was transfected into cells at a concentration of 1000×g for 20 min at 48 hours post-transfection. The supernatant of transfected cells was collected, and impurities were removed using a 0.22 μm filter. The supernatant was added to pretreated Ni-NTA beads and purified according to the nickel column purification procedure. The effluent and protein eluent of the target protein were collected and concentrated using a 10 kDa ultrafiltration tube. 10 μL of the sample was prepared for SDS-PAGE analysis, and the protein concentration was determined using the BCA method. The sample was aliquoted and stored at -20℃ for later use.

[0245] SDS-PAGE test results are as follows: Figure 1 As shown, there is a clear target band around 17KD, which matches the expected size, meaning that the A27L recombinant protein with the expected target size was obtained.

[0246] 2. Mouse immunization and preparation of monoclonal antibodies

[0247] Ten 8-week-old BALB / c mice were immunized with the A27L recombinant protein obtained in step 1. After three booster immunizations, blood titers were measured. Once the serum titer stopped rising, the mice were immunized with twice the dose of A27L recombinant protein without adjuvant. The mice were then euthanized by cervical dislocation three days later. Spleen cells were prepared from the spleen under sterile conditions and mixed with rapidly growing mouse myeloma cells at a ratio of 8:1 in a 50 mL centrifuge tube. 30 mL of serum-free IPMI 1640 medium was added, and the mixture was centrifuged at 1100 rpm for 5 min. The supernatant was discarded, the cell clumps were gently loosened, and the cells were placed in a 37°C water bath. Slowly add 1 mL of 50% PEG-4000 to the cells over 1 minute, while gently stirring the precipitate at the bottom. After standing for 1 minute, slowly and evenly add 1 mL of serum-free culture medium along the tube wall for the first 30 seconds, then add 2 mL for the next 30 seconds, and finally quickly add 27 mL to terminate the fusion process. Centrifuge at 1100 rpm for 5 minutes, discard the supernatant, resuspend the cells in HAT selective medium, and then add the solution to a 96-well cell culture plate containing feeder cells. Incubate at 37°C with 5% CO2.

[0248] Seven days later, the medium was changed to HT. When the number of hybridoma cells in the wells reached more than 300, indirect ELISA was used for screening. Wells with strong positive results, good inhibition effect, and vigorous cell growth were selected for limiting dilution cloning. After more than three cloning cultures and tests, the cells in the wells that were positive were identified as hybridoma cells secreting monoclonal antibodies. The hybridoma cells were expanded for monoclonal antibody preparation. A total of 6 positive hybridoma cell lines were obtained, and antibody subtypes were identified (see Table 1). The purified monoclonal antibody was obtained using the in vivo ascites induction method and the octanoic acid-ammonium sulfate precipitation method. The concentration of the purified monoclonal antibody was determined by the BCA method.

[0249] Table 1. Identification of Monoclonal Antibody Subtypes

[0250]

[0251] 3. Screening of neutralizing antibodies

[0252] A neutralization assay was performed on the six purified antibodies to detect their neutralizing activity using an IC50 assay. 50 As an evaluation indicator, IC 50 The value is expressed in µg / mL, representing the minimum antibody concentration required to inhibit 50% cell infection. A lower value indicates a better effect. IC50 (Inhibitory Concentration) 50 Antibodies with a concentration >50 μg / mL are considered to lack neutralizing activity. The specific experimental method is as follows:

[0253] (1) LT cells (cells disclosed in patent document CN117165530A with accession number CGMCC NO.45500) were pre-coated in 96-well plates and the experiment was conducted when the cell confluence was about 80%.

[0254] (2) The virus (the virus is LSDV used in the IFA experiment in the literature: Yu Haoyang, Li Haoxuan, Sun Yu, Geng Yujing, Yang Zhen, Wang Caixia, Zhao Xuejin, Zhang Zhou, Qiu Songyin, Wang Huiyu, Lin Xiangmei, Wu Shaoqiang, Feng Chunyan. Preparation and immunological identification of polyclonal antibody against bovine nodular dermatitis virus A33R [J / OL]. Chinese Journal of Animal Infectious Diseases. https: / / doi.org / 10.19958 / j.cnki.cn31-2031 / s.20250902.001) was diluted with DMEM to four concentrations: 200 TCID50 / 50 μL, 20 TCID50 / 50 μL, 2 TCID50 / 50 μL and 0.2 TCID50 / 50 μL.

[0255] (3) The antibody was serially diluted 2-fold in a 96-well plate (1:4-1:512). After dilution, 50 μL of the diluted antibody was mixed with an equal volume of 200 TCID50 / 50 μL of virus. Virus control groups were set up with 200 TCID50 / 50 μL, 20 TCID50 / 50 μL, 2 TCID50 / 50 μL, and 0.2 TCID50 / 50 μL. 50 μL of virus was mixed with 50 μL of DMEM. After mixing, the plate was incubated in a 37℃ 5% CO2 incubator for 1 h.

[0256] (4) After washing the 96-well cell culture plate three times with PBS, add 100 μL of virus-antibody mixture or virus-DMEM mixture.

[0257] (5) Place in an incubator at 37℃ and 5% CO2 and incubate for 48 h to observe cell lesions.

[0258] The neutralization assay results (Table 2) showed that antibodies 11C4, 17E2, and 28B5 had neutralizing activity, with 11C4 and 28B5 exhibiting higher neutralizing titers and 17E2 showing lower neutralizing titers. Antibodies 16H5, 23C3, and 25D7 showed no neutralizing activity; therefore, monoclonal antibodies 11C4 and 28B5 were selected for further research.

[0259] Table 2 Neutralization Experiment Results

[0260]

[0261] 4. Determination of antibody recognition epitopes

[0262] The secondary structure of the A27L protein was preliminarily analyzed and predicted using online sequence analysis software: https: / / services.healthtech.dtu.dk / services / NetSurfP-2.0 / and http: / / bioinf.cs.ucl.ac.uk / psipred / . Based on the predictions from NetSurfP-2.0, the hydrophilic exposed region of the A27L protein was obtained. Based on the predictions from PSIPRED, the predicted secondary structure of the A27L protein was obtained. Combined with the antigen recognition epitope prediction results from http: / / sysbio.unl.edu / SVMTriP / , after preliminary screening, four peptides were synthesized. The positional distribution of the four peptides in the A27L protein is shown in [Figure missing]. Figure 2 The peptide sequences are shown in Table 3. The peptide synthesis was commissioned to Nanjing Peptide Valley Biotechnology Co., Ltd. The synthesized peptide powder was dissolved in 1 ml of DMSO, vortexed to a final concentration of 1 mg / ml, aliquoted into 100 μL portions, and stored at -80℃ for later use. The peptide was coated onto ELISA plates with carbonate coating buffer at a concentration of 1 μg / mL. Indirect ELISA was performed using monoclonal antibody cell supernatant as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody. Mouse negative serum was used as a control. OD values ​​were read after the assay. 450nm Value, calculate P / N value, detection results as follows Figure 3 As shown: 11C4 monoclonal antibody showed a strong reaction with peptide 1, but no reaction with peptides 2, 3, and 4, indicating that the antigen recognition site of monoclonal antibody 11C4 is the amino acid sequence of peptide 1: "IFPGDDDETNERNINHREKT"; 28B5 monoclonal antibody showed a strong reaction with peptide 3, but no reaction with peptides 1, 2, and 4, indicating that the antigen recognition site of monoclonal antibody 28B5 is the amino acid sequence of peptide 3: "KKIINERYSNYISIDDDEISDILKDSFISNEEMQI".

[0263] Table 3. Peptide Sequences

[0264]

[0265] Example 2. Sequencing of monoclonal antibodies and construction, recombinant expression, and functional verification of monoclonal and bispecific antibodies.

[0266] 1. Sequencing of monoclonal antibodies

[0267] Once the hybridoma cells have grown into a monolayer, discard the culture medium, pipette and count the cells using 3 mL of sterile PBS, and collect 1×10⁶ cells. 6Each cell was transferred to a 1.5 mL centrifuge tube and centrifuged at 800 r / min for 5 min. After discarding the supernatant, RNA was extracted from the hybridoma cells using the Trizol method. The VH and VL regions of the hybridoma cells were amplified using the Frdbio® mouse hybridoma cell monoclonal antibody variable region fragment amplification (sequencing) kit. The obtained target bands were recovered from the gel and cloned into the T vector. Positive clones were selected for sequencing, and the sequences of VH and VL were obtained. The amino acid / nucleotide sequences of the variable regions of 11C4 and 28B5 and CDR are shown in Table 4.

[0268] Table 4. Amino acid / nucleotide sequences of the variable regions and CDRs of 11C4 and 28B5.

[0269]

[0270]

[0271]

[0272]

[0273] 2. Construction of monoclonal antibodies and bispecific antibody expression vectors

[0274] 2.1 Construction of recombinant plasmids for monoclonal antibodies

[0275] The heavy chain (SEQ ID NO: 55) and light chain (SEQ ID NO: 56) of monoclonal antibody 11C4, and the heavy chain (SEQ ID NO: 57) and light chain (SEQ ID NO: 58) of monoclonal antibody 28B5 were synthesized and inserted into the pcDNA3.4 eukaryotic expression vector, respectively, to obtain recombinant expression plasmids of the heavy chain of monoclonal antibody 11C4, the light chain of monoclonal antibody 11C4, the heavy chain of monoclonal antibody 28B5, and the light chain of monoclonal antibody 28B5 (see the construction diagram of the recombinant expression plasmids). Figures 4A-4D The recombinant expression plasmids were transformed into DH10Bac competent cells and plated. Single colonies were picked and sequenced to identify positive colonies. The plasmids were then extracted for later use.

[0276] 2.2 Construction of recombinant plasmids for bispecific antibodies

[0277] The heavy chain of the bispecific antibody (SEQ ID NO: 59) was synthesized and inserted into the pcDNA3.4 eukaryotic expression vector to obtain a recombinant expression plasmid expressing the heavy chain of the bispecific antibody. After transforming DH10Bac competent cells, the plasmid was plated, and single colonies were picked, identified by sequencing, and the plasmid was extracted for later use. The light chain of the bispecific antibody was constructed using a recombinant expression plasmid with the same sequence as SEQ ID NO: 56 (i.e., the recombinant expression plasmid of the light chain of the aforementioned monoclonal antibody 11C4). The construction diagram of the 11C-28B5 bispecific antibody recombinant expression plasmid is shown below. Figure 4B , 4E The structural diagram of the bispecific antibody (11C4-28B5) is shown below. Figure 5 As shown, a scFv antibody of 28B5 is linked to the C-terminus of the 11C4 monoclonal antibody via a hinge structure, forming a bivalent bispecific antibody. This antibody can bind to both antigenic sites of the LSDV A27L protein.

[0278] 3. Expression and purification of monoclonal antibodies (11C4, 28B5) and bispecific antibodies (11C4-28B5).

[0279] 90 mL of 293F cells were adjusted to a density of 1.1 × 10⁻⁶. 6 100 μg of plasmid (50 μg heavy chain and 50 μg light chain plasmid, i.e., transient transfection with heavy chain and light chain plasmids at a 1:1 ratio) and 300 μL of PEI were added to 5 mL of Freestyle 293 Expression medium. After mixing thoroughly, the mixture was incubated at room temperature for 5 min. The solution containing plasmid and PEI was then mixed and incubated at room temperature for 20 min. After incubation, the mixture was added to 293F cells, and the cell supernatant was collected after 5 days. The cell supernatant was filtered and purified using a protein A gel, eluted with 0.1 mol / L Glycine-HCl (pH 3.0), neutralized with 1 mol / L Tris-HCl (pH 9.0), concentrated using a 30,000 μL ultrafiltration tube, and then replaced with phosphate buffer. The purified protein underwent reductive denaturation, and its size and purity were determined by SDS-PAGE. The light chain was approximately 26,000, the heavy chain of the monoclonal antibody was approximately 50,000, and the heavy chain of the bispecific antibody was approximately 78,000, all consistent with the expected relative molecular mass. Figure 6 (A). Size exclusion chromatography was used to analyze the aggregation state and purity of monoclonal and bispecific antibodies. Neither the monoclonal nor the bispecific antibodies showed any heavy-chain or light-chain byproducts, and no severe aggregation was observed. Figure 6 (B)

[0280] 4. Verification of the antigen-binding properties of monoclonal antibodies and bispecific antibodies.

[0281] The A27L recombinant plasmid from Example 1 was transiently transfected into HEK293T cells using a Lipo3000 to construct HEK293T-A27L cells. Wild-type HEK293T cells and HEK293T-A27L cells were digested and then incubated with different concentrations of the bispecific antibody 11C4-28B5 and monoclonal antibodies 11C4 and 28B5 (prepared in step 3), respectively. After washing twice with PBS, the cells were stained with APC-anti-human hIgG1Fc antibody (Biolegend, catalog number 410711), washed twice with PBS, and then the cell fluorescence intensity was detected by flow cytometry.

[0282] The results are as follows Figure 7 As shown, the bispecific antibody 11C4-28B5 and the monoclonal antibodies 11C4 and 28B5 can bind to HEK293T-A27L cells, but their binding to wild-type HEK293T cells is very weak. Furthermore, the bispecific antibody has a stronger binding ability to HEK293T-A27L cells than the monoclonal antibodies 11C4 and 28B5.

[0283] 5. Specificity identification of monoclonal antibodies and bispecific antibodies

[0284] The specificity of the bispecific antibody 11C4-28B5 was identified using an ELISA method, specifically as follows: The target antigen A27L-His (nucleotides 110,419-110,865 of the full-length sequence of accession number OP508345.1) and irrelevant antigens p32-His (nucleotides 65011-65979 of the full-length sequence of accession number OP508345.1), and L1R-His (accession number...) were analyzed. Nucleotides 53187-53924 of the full sequence of OP508345.1 and 122-His (nucleotides 113468-114058 of the full sequence of OP508345.1) were coated onto 96-well ELISA plates (NC indicates coated BSA), 200 ng / 50 μL per well, and incubated overnight at 4°C; the solution was discarded, the plates were washed twice with PBS, and blocked with PBS containing 5% skim milk powder at 200 μL / well. h; Discard the solution, add 4 nmol / L of the antibody to be tested (diluted with PBS containing 5% skim milk powder and 0.1% Tween-20, the same below) at 50 μL / well, and react at 37℃ for 1 h; Discard the solution, wash 3 times with PBS containing 0.1% Tween-20, add HRP-labeled goat anti-human IgG (1:2000 dilution) at 50 μL / well, and react at 37℃ for 45 min; Discard the solution; wash 3 times with PBS containing 0.1% Tween-20; add freshly prepared TMB chromogenic solution at 50 μL / well, and react at room temperature for 3-10 min; stop the chromogenic reaction by adding 2 mol / L concentrated sulfuric acid at 50 μL / well, and measure the absorbance value at 450 nm as the detection wavelength; perform differential analysis of the results using one-way ANOVA. The results are as follows. Figure 8 As shown, monoclonal antibodies 11C4, 28B5, and bispecific antibody 11C4-28B5 all exhibit strong binding activity to A27L-His, but show almost no binding with irrelevant antigens p32-His, L1R-His, and 122-His. This indicates that 11C2-28B5 prepared in this invention still possesses good specificity, and the realization of the dual epitope recognition function has not altered its specificity.

[0285] 6. Evaluation of neutralizing activity of monoclonal antibodies and bispecific antibodies

[0286] The test antibodies, including bispecific antibody 11C4-28B5, control antibody 11C4, control antibody 28B5, and a mixture of two control antibodies in equal proportions (11C4+28B5), were sequentially added to a 96-well cell culture plate in serially diluted form. A negative control well with an antibody concentration of 0 was also included. Each reaction was performed in duplicate. Then, 50 μL of the solution diluted to 10⁻⁶ was added to each well. -3LSDV red fluorescent virulence (the same LSDV red fluorescent virulence used in the polyclonal antibody neutralization activity assay in the literature: Yu Haoyang, Li Haoxuan, Sun Yu, Geng Yujing, Yang Zhen, Wang Caixia, Zhao Xuejin, Zhang Zhou, Qiu Songyin, Wang Huiyu, Lin Xiangmei, Wu Shaoqiang, Feng Chunyan. Preparation and immunological identification of polyclonal antibodies against bovine nodular dermatitis virus A33R [J / OL]. Chinese Journal of Animal Infectious Diseases. https: / / doi.org / 10.19958 / j.cnki.cn31-2031 / s.20250902.001) was used. Different concentrations of antibodies were mixed with the fluorescent virulence and reacted in a 37℃, 5% CO2 incubator for 1 h. A virus regression control was set up, with 3 replicates for each gradient. After 1 h, the logarithmic growth phase LT cells were diluted to 2×10⁶ cells with culture medium. 5 / mL, seeded at 100μL / well into 96-well cell culture plates pre-incubated with the above antibody and fluorescent virus for 1h and with virus return control, and cultured for 2 days. After 2 days, observe the fluorescence count in each well. The above neutralization experiment was repeated three times, and the results were recorded and statistically analyzed.

[0287] The results are as follows Figure 9 As shown, 11C4, 28B5, 11C4+28B5, and 11C4-28B5 all exhibited neutralizing activity against LSDV fluorescent toxin, with 11C4-28B5 demonstrating superior neutralizing activity against LSDV fluorescent toxin, surpassing the neutralizing activity of any single monoclonal antibody or the combined effect of the two monoclonal antibodies. This result indicates that the bispecific antibody further enhanced the synergistic effect of the two antigen-binding epitopes, resulting in a breakthrough improvement in neutralizing efficacy.

[0288] 7. Evaluation of the in vitro blocking activity of monoclonal antibodies and bispecific antibodies

[0289] The test antibodies include bispecific antibody 11C4-28B5, control antibody 11C4, and control antibody 28B5. The test antibodies are serially diluted (from 0.002 mg / mL to 0.00000002 mg / mL by a 10-fold dilution). Take 50 μL of the serially diluted test antibody and 50 μL of the 10-fold dilution... 7.8 TCID 50LSDV virus solution (the virus is the LSDV used in the IFA experiment in the literature: Yu Haoyang, Li Haoxuan, Sun Yu, Geng Yujing, Yang Zhen, Wang Caixia, Zhao Xuejin, Zhang Zhou, Qiu Songyin, Wang Huiyu, Lin Xiangmei, Wu Shaoqiang, Feng Chunyan. Preparation and immunological identification of polyclonal antibody against bovine nodular dermatitis virus A33R [J / OL]. Chinese Journal of Animal Infectious Diseases. https: / / doi.org / 10.19958 / j.cnki.cn31-2031 / s.20250902.001) was incubated at room temperature for 10 min, then added to an ELISA plate pre-coated with the A27L recombinant protein prepared in Example 1, incubated at 37°C for 1 h, then washed 3 times with PBST, added 1:10000 diluted goat anti-human HRP secondary antibody, incubated at 37°C for 1 h, then washed 3 times with PBST, and developed with TMB. OD was read. 450nm Values ​​were used to plot the blocking rate curves of monoclonal antibodies 11C4 and 28B5 and bispecific antibody 11C4-28B5 against the virus. Figure 10 ), and calculate the IC50 of the three antibodies respectively. 50 Comparing the IC50 of the three antibodies 50 Value. Through calculation and comparison, it was found that the IC50 value of monoclonal antibody 11C4 was... 50 The IC50 concentration of monoclonal antibody 28B5 was 2.006 nmol / L. 50 The IC50 of the bispecific antibody 11C4-28B5 was 0.3122 nmol / L. 50 The concentration was 0.02445 nmol / L, and the IC50 of the bispecific antibody was... 50 The minimum value indicates that the bispecific antibody obtained by this invention can be effective even at low concentrations, greatly improving the effect of blocking viruses in vitro and showing good application prospects.

[0290] 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. Bovine nodular dermatitis virus antibody or antigen-binding fragment thereof, wherein the bovine nodular dermatitis virus antibody or antigen-binding fragment thereof comprises: The heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; And, having LCDR1, LCDR2 and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:

28.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 29, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 31; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 32, LCDR2 having the amino acid sequence DTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; Wherein, the CDR is defined according to the IMGT numbering system; or The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO:34, HCDR2 having the amino acid sequence shown in SEQ ID NO:35, and HCDR3 having the amino acid sequence shown in SEQ ID NO:36; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO:37, LCDR2 having the amino acid sequence shown in SEQ ID NO:38, and LCDR3 having the amino acid sequence shown in SEQ ID NO:33; Wherein, the CDR is defined according to the Kabat numbering system; or The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 39, HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; Wherein, the CDR is defined according to the Chothia numbering system; or The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 41, HCDR2 having the amino acid sequence shown in SEQ ID NO: 42, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 43; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 44, LCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; The CDR is defined according to the Contact numbering system.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain variable region of the bovine nodular dermatitis virus antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region; and / or The light chain variable region of the bovine nodular dermatitis virus antibody or its antigen-binding fragment also includes the framework region of the light chain variable region.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The framework region of the heavy chain variable region includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and / or The framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

28.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

28.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

28.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 27; and / or, the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO:

28.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region includes at least a portion of the heavy chain constant region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and / or The light chain constant region includes at least a portion of the light chain constant region derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and / or The heavy chain constant region includes heavy chain constant regions derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM immunoglobulins; and / or The light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

11. The antibody or its antigen-binding fragment according to claim 10, characterized in that, The heavy chain constant region includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO:52; and / or The light chain constant region includes an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:

54.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The heavy chain constant region includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO:52; and / or The light chain constant region includes an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:

54.

13. The antibody or its antigen-binding fragment according to claim 12, characterized in that, The heavy chain constant region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:52; and / or The light chain constant region includes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO:

54.

14. The antibody or its antigen-binding fragment according to claim 13, characterized in that, The heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:52; and / or The light chain constant region includes the amino acid sequence shown in SEQ ID NO:

54.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, characterized in that, The bovine nodular dermatitis virus antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; and / or The bovine nodular dermatitis virus antibody or its antigen-binding fragment includes monoclonal antibodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv) or dsFv.

16. A multispecific antibody comprising two protein functional regions targeting antigens, wherein one protein functional region comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-15.

17. The multispecific antibody according to claim 16, characterized in that, The multispecific antibody is a bispecific antibody, which includes: a first protein functional region and a second protein functional region; The first protein functional region includes: The heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 5 includes HCDR1, HCDR2 and HCDR3; and the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 6 includes LCDR1, LCDR2 and LCDR3. The second protein functional region includes: The heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 27 includes HCDR1, HCDR2 and HCDR3; and the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 28 includes LCDR1, LCDR2 and LCDR3.

18. The multispecific antibody according to claim 17, characterized in that, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 8, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 9; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 10, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 29, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 31; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 32, LCDR2 having the amino acid sequence DTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; Wherein, the CDR is defined according to the IMGT numbering system; or The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 12, HCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO:34, HCDR2 having the amino acid sequence shown in SEQ ID NO:35, and HCDR3 having the amino acid sequence shown in SEQ ID NO:36; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO:37, LCDR2 having the amino acid sequence shown in SEQ ID NO:38, and LCDR3 having the amino acid sequence shown in SEQ ID NO:33; Wherein, the CDR is defined according to the Kabat numbering system; or The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 17, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 14; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 15, LCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 11; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 39, HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 36; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence shown in SEQ ID NO: 38, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 33; Wherein, the CDR is defined according to the Chothia numbering system; or The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 41, HCDR2 having the amino acid sequence shown in SEQ ID NO: 42, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 43; and / or VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 44, LCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; The CDR is defined according to the Contact numbering system.

19. The multispecific antibody according to claim 18, characterized in that, The heavy chain variable region of the first protein functional region also includes the framework region of the heavy chain variable region; and / or The heavy chain variable region of the second protein functional region also includes the framework region of the heavy chain variable region; and / or The light chain variable region of the first protein functional region also includes the framework region of the light chain variable region; and / or The light chain variable region of the second protein functional region also includes the framework region of the light chain variable region.

20. The multispecific antibody according to claim 19, characterized in that, The first protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6; and / or The second protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

28.

21. The multispecific antibody according to claim 20, characterized in that, The first protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 5; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6; and / or The second protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

28.

22. The multispecific antibody according to claim 21, characterized in that, The first protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6; and / or The second protein functional region includes: The heavy chain variable region (VH) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 27; and / or, the light chain variable region (VL) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

28.

23. The multispecific antibody according to claim 22, characterized in that, The first protein functional region includes: Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 5; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 6; and / or The second protein functional region includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 27; and / or, the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO:

28.

24. The multispecific antibody according to any one of claims 17-23, characterized in that, The first and second protein functional regions are directly connected or connected through a linker fragment; and / or The first protein functional region and the second protein functional region are independently one, two, or more; and / or The first and second protein functional regions are each independently immunoglobulin or antigen-binding fragments.

25. The multispecific antibody according to claim 24, characterized in that, The antigen-binding fragments include Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, or single-chain Fv (scFv).

26. The multispecific antibody according to claim 25, characterized in that, The antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv (scFv).

27. The multispecific antibody according to claim 24, characterized in that, The first protein functional region is an immunoglobulin, and the second protein functional region is an antigen-binding fragment; or, the first protein functional region is an antigen-binding fragment, and the second protein functional region is an immunoglobulin.

28. The multispecific antibody according to claim 27, characterized in that, The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain Fv.

29. The multispecific antibody according to claim 28, characterized in that, When the first protein functional region is an immunoglobulin, the first protein functional region includes a heavy chain constant region and / or a light chain constant region; and / or The single chain Fv includes a heavy chain variable region, a light chain variable region, and a connecting segment for connecting the heavy chain variable region and the light chain variable region.

30. The multispecific antibody according to claim 29, characterized in that, The single-chain Fv has the following structure: NH2-VL-linker fragment-VH-COOH or NH2-VH-linker fragment-VL-COOH; and / or The single-chain Fv is connected via a linker fragment to the C-terminus of the heavy chain of the immunoglobulin, the N-terminus of the heavy chain of the immunoglobulin, or the C-terminus of CH1 of the immunoglobulin.

31. The multispecific antibody according to claim 30, characterized in that, The single-chain Fv is attached to the C-terminus of the heavy chain of the immunoglobulin via a linker fragment; and / or The number of immunoglobulins is 1, and the number of single-chain Fvs is 2.

32. The multispecific antibody according to claim 28, characterized in that, The bispecific antibody comprises a first polypeptide chain and a second polypeptide chain: The first polypeptide chain comprises: the heavy chain of the immunoglobulin, the linker fragment, and the single-chain Fv; the second polypeptide chain comprises: the light chain of the immunoglobulin.

33. The multispecific antibody according to claim 32, characterized in that, The first polypeptide chain comprises amino acids 19-733 of SEQ ID NO: 59 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 59; and The second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity with amino acid sequences of SEQ ID NO: 56 from amino acid positions 19 to 237.

34. The multispecific antibody according to claim 33, characterized in that, The first polypeptide chain comprises amino acids 19-733 of SEQ ID NO: 59 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 59; and The second polypeptide chain comprises an amino acid sequence having at least 85% sequence identity with amino acid sequences of SEQ ID NO: 56 from amino acid positions 19 to 237.

35. The multispecific antibody according to claim 34, characterized in that, The first polypeptide chain comprises amino acids 19-733 of SEQ ID NO: 59 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 59; and The second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with amino acid sequences of SEQ ID NO: 56 from amino acid positions 19 to 237.

36. The multispecific antibody according to claim 35, characterized in that, The first polypeptide chain comprises amino acids 19-733 of EQ ID NO: 59 or the amino acid sequence shown in SEQ ID NO: 59; and The second polypeptide chain includes the amino acid sequence from position 19 to 237 of SEQ ID NO: 56 or the amino acid sequence shown in SEQ ID NO:

56.

37. Biomaterials, including any one of n1)-n9): n1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-15 or the multispecific antibody as described in any one of claims 16-36; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) Cells comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-15 or the multispecific antibody as described in any one of claims 16-36; None of the cells described in n5)-n9) contain reproductive material.

38. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the multispecific antibody according to any one of claims 15-36, obtained by culturing the cells described in claim 37.

39. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-15 or a multispecific antibody as described in any one of claims 16-36; and a conjugation portion; The coupling portion is a detectable marker.

40. The coupling according to claim 39, characterized in that, The detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin.

41. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof as described in any one of claims 1-15, the multispecific antibody as described in any one of claims 16-36, the biomaterial as described in claim 37, or the conjugate as described in any one of claims 39-40; and a pharmaceutically acceptable carrier.

42. The pharmaceutical composition according to claim 41, characterized in that, The pharmaceutical composition further includes additional pharmaceutically active agents; and / or The additional pharmaceutically active agent is a drug for the prevention and / or treatment of bovine nodular dermatitis virus infection or the disease it causes.

43. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-15, the multispecific antibody according to any one of claims 16-36, the biomaterial according to claim 37, the conjugate according to any one of claims 39-40, or the pharmaceutical composition according to any one of claims 41-42.

44. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-15, the multispecific antibody according to any one of claims 16-36, the biomaterial according to claim 37, the conjugate according to any one of claims 39-40, or the pharmaceutical composition according to any one of claims 41-42 in any one of c1)-c4): c1) Prepare products for the diagnosis of bovine nodular dermatitis virus infection or diseases caused by it; c2) Prepare products for the prevention and / or treatment of bovine nodular dermatitis virus infection or diseases caused by it; c3) Prepare products for detecting the presence or level of bovine nodular dermatovirus or its A27L protein in samples; c4) Detect the presence or level of bovine nodular dermatovirus or its A27L protein in the sample.

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