FPV-neutralizing antibody and application thereof

Abstract

Provided are an FPV-neutralizing antibody and an application thereof. The provided FPV-neutralizing monoclonal antibody has strong neutralizing activity against the FPV virus, and has important research and application value in dealing with the specific prevention, treatment and diagnosis of diseases caused by the FPV virus.

Description

FPV neutralizing antibody and application TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to FPV neutralizing antibody and application. BACKGROUND

[0002] Feline panleukopenia virus (FPV), also known as feline parvovirus or feline distemper virus, belongs to the parvovirus family and is a single-stranded linear DNA virus. FPV infection is characterized by rapid leukopenia, diarrhea, hemorrhagic enteritis, sudden biphasic high fever, dehydration and vomiting. It is the most pathogenic and widest range of parvovirus. The virus infection has a high incidence and a mortality rate of up to 90%, causing significant harm and economic loss to the global pet industry.

[0003] The preliminary diagnosis is made through the clinical manifestations of cats, and the comprehensive diagnosis is made by combining blood routine test (blood routine), blood biochemistry, SAA concentration, fecal colloidal gold test, FPV fluorescence quantification and medical imaging examination. The clinical treatment of feline distemper includes symptomatic treatment (including clearing heat and relieving pain, anti-inflammatory, anti-emetic, anti-diarrhea, etc.), fluid replacement to correct acid-base balance, antibiotics to prevent secondary infection, and immune enhancement (polypeptide, ribose and other small molecule auxiliary cell immune stimulation).

[0004] FPV neutralizing antibody plays an important role in the treatment of feline panleukopenia. When a cat is infected with feline distemper virus, injection of FPV neutralizing antibody can help the body quickly clear the virus and reduce the damage of the virus to the cat. It has been reported that the use of FPV neutralizing antibody in combination with the selection of antiviral drugs, active symptomatic treatment including antibacterial and anti-inflammatory, hemostasis, anti-emesis, white blood cell enhancement, and maintenance of body fluid energy can significantly improve the cure rate of FPV.

[0005] However, there are not many monoclonal antibodies with broad-spectrum neutralizing ability against FPV in the art, and the clinical manifestations are uneven. FPV VP2 protein is one of the main structural proteins of feline panleukopenia virus (FPV), and its key role is to induce the production of antibodies in the body, especially the reported FPV neutralizing antibody induced by VP2 protein. Therefore, it is still a problem to be solved in the art to further develop antibodies with neutralizing performance using the VP2 protein of FPV.

[0006] SUMMARY

[0007] Therefore, the present application provides FPV neutralizing antibody and application.

[0008] The present application provides an FPV antibody or antigen binding molecule,

[0009] the heavy chain CDR1 has an amino acid sequence as set forth in SEQ ID NO: 1;

[0010] the heavy chain CDR2 has an amino acid sequence as set forth in SEQ ID NO: 2 or 7;

[0011] the heavy chain CDR3 has an amino acid sequence as set forth in SEQ ID NO: 3 or 8;

[0012] the light chain CDR1 has an amino acid sequence as set forth in SEQ ID NO: 4 or 9;

[0013] the light chain CDR2 has an amino acid sequence as set forth in SEQ ID NO: 5 or 10;

[0014] the light chain CDR3 has an amino acid sequence as set forth in SEQ ID NO: 6 or 11;

[0015] or, the CDR regions thereof have sequences with 1 or more amino acids substituted, deleted, added and / or replaced based on the amino acid sequences as set forth above; or have sequences with 80% or more identity to the amino acid sequences as set forth in any one of the above. The 80% or more identity means 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity.

[0016] The FPV antibody provided by the application is a neutralizing monoclonal antibody, which has strong neutralizing activity against FPV-SD2018 strain, and has important research and application value in diagnosis, prevention and treatment of diseases caused by FPV virus, especially FPV-SD2018 strain.

[0017] In some embodiments, the heavy chain CDR1-3 of the FPV antibody or antigen binding molecule has the amino acid sequences as set forth in SEQ ID NOs: 1-3 in sequence, and the light chain CDR1-3 thereof has the amino acid sequences as set forth in SEQ ID NOs: 4-6 in sequence.

[0018] In other embodiments, the heavy chain CDR1-3 of the FPV antibody or antigen binding molecule has the amino acid sequences as set forth in SEQ ID NOs: 1, 7-8 in sequence, and the light chain CDR1-3 thereof has the amino acid sequences as set forth in SEQ ID NOs: 9, 10 or 11 in sequence.

[0019] In some embodiments, the antibody or antigen binding molecule as set forth above,

[0020] the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 28 or 30,

[0021] The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 29 or 31,

[0022] Alternatively, the heavy chain variable region and / or the light chain variable region has a sequence of 1 or more amino acids substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown before; or has a sequence with 80% or more identity to the amino acid sequence as shown in any one of the preceding. The 80% or more identity means 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity.

[0023] Experiments show that on the basis of the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody provided in the application, one or more amino acids can be added, deleted, replaced and the like by conventional protein engineering methods to obtain a conservative variant or a fragment thereof, which can still neutralize the FPV-SD2018 strain. For example, the variable regions of the two antibodies in the embodiments of the application have 82.6% identity, both of which can specifically bind to the FPV virus and have good neutralization performance.

[0024] In the application, the light chain type of the antibody or antigen binding molecule is Lambda, and the subtype is IgG 1.

[0025] Experiments show that on the basis of the full-length monoclonal antibody provided in the application, one or more amino acids can be added, deleted, replaced and the like by conventional protein engineering methods, and under the condition of 94% or more identity, the obtained antibody can still neutralize the FPV virus.

[0026] Further, the application also provides a nucleic acid encoding the antibody or antigen binding molecule as described before.

[0027] The nucleic acid in the application is a nucleic acid encoding the CDR region of the antibody as described before, or a nucleic acid encoding the variable region, or a nucleic acid encoding the variable region and the constant region of the antibody, which is not limited in the application.

[0028] Further, the application also provides a plasmid vector containing the nucleic acid as described before.

[0029] In the application, the plasmid vector is a mammalian cell vector, a prokaryotic expression vector or a shuttle vector, which is not limited in the application.

[0030] The application also provides a host with the nucleic acid integrated into the genome or transformed or transfected with the plasmid vector. In some embodiments, the host provided in the application is a HEK293F cell.

[0031] The present application also provides a method for preparing the antibody or antigen binding molecule, comprising culturing the host to obtain a culture containing the antibody or antigen binding molecule.

[0032] Further, the present application also provides a labeled antibody comprising the antibody or antigen binding molecule and a labeling substance, wherein the labeling substance is a chemical label or a biological label.

[0033] In some embodiments, the chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope, and / or a colloidal indicator; and the biological label is biotin, avidin, or an enzyme label.

[0034] In some specific embodiments,

[0035] the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, beta-galactosidase-anti-beta-galactosidase conjugate;

[0036] the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye;

[0037] the chemiluminescent indicator is selected from one or more of acridinium ester, acridinium sulphonamide and derivatives thereof, luminol, isoluminol, isoluminol isothiocyanate and derivatives thereof, N-(4-aminobutyl)-N-ethylisoluminol, 4,5-diaminobenzohydrazide, or aminobutylethylbenzhydrazide;

[0038] the isotope is selected from one or more of 125 I, 131 I, 124 I, 3 H, 14 C, 111 In, 89 Zr, or 32 P;

[0039] the colloidal indicator is selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

[0040] Further, the present application also provides a combination comprising the antibody or antigen binding molecule and a medium; the medium is selected from colloidal gold, enzyme-labeled plate, magnetic beads or latex microspheres. In the present application, the connection between the antibody and the medium can be through amide bond or through the streptavidin-biotin system, and the present application does not limit this.

[0041] Further, the present application also provides the use of any one of a) to g) in the preparation of a product for detecting, preventing and / or treating FPV virus.

[0042] a) the antibody or antigen binding molecule as described above;

[0043] b) the nucleic acid as described above;

[0044] c) the plasmid vector as described above;

[0045] d) the host as described above;

[0046] e) the culture or the purified antibody prepared by the preparation method as described above;

[0047] f) the labeled antibody as described above;

[0048] g) the combination as described above.

[0049] Further, the present application also provides a product for preventing and / or treating FPV virus, and the raw material for the preparation of the product comprises any one of a) to g) as described above.

[0050] a) the antibody or antigen binding molecule as described above;

[0051] b) the nucleic acid as described above;

[0052] c) the plasmid vector as described above;

[0053] d) the host as described above;

[0054] e) the culture or the purified antibody prepared by the preparation method as described above;

[0055] f) the labeled antibody as described above;

[0056] g) the combination as described above.

[0057] In some embodiments, the product comprises an adjuvant and the antibody or antigen binding molecule as described above;

[0058] or comprises an adjuvant and the culture or the purified antibody prepared by the preparation method as described above.

[0059] In some embodiments, the product further comprises other drugs for preventing and / or treating FPV viral infection.

[0060] The drugs for preventing and / or treating FPV viral infection include cat triple vaccine, recombinant cat interferon, Cervonin, metoclopramide, Bayovac, feline distemper inhibitory protein, liver strong, giant peptide, barbital, ceftriaxone sodium, ascites, adrenocorticotropic hormone, montmorillonite powder, etc.

[0061] The present application also provides a method for preventing and / or treating FPV viral infection, which comprises administering the product as described above to a subject.

[0062] The administration mode of the drugs includes, but is not limited to, oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, or non-gastrointestinal administration, such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intrathecal injection, intraventricular injection, sternal injection or intracranial injection; or by means of an external implant reservoir. Preferably, the administration mode is oral administration and / or aerosol inhalation.

[0063] In the present application, when the product comprises other drugs for preventing and / or treating FPV viral infection, two or more drugs can be administered simultaneously or sequentially, and the present application does not limit this.

[0064] For example, the product is administered before contacting the virus, and the administration is advanced for no more than 3 years, preferably no more than 1 year, more preferably no more than 6 months, more preferably no more than 3 months, more preferably no more than 1 month, more preferably no more than 15 days, and more preferably no more than 7 days.

[0065] For example, the product is administered after contacting the virus, and the administration is advanced for no more than 3 years, preferably no more than 1 year, more preferably no more than 6 months, more preferably no more than 3 months, more preferably no more than 1 month, more preferably no more than 15 days, more preferably no more than 7 days, more preferably no more than 3 days, and more preferably no more than 1 day.

[0066] In the present application, when the product comprises antibodies and other therapeutic agents, the administration frequency of two or more drugs can be the same or different, and the present application does not limit this. The administration interval of two or more drugs is no more than 6 hours, preferably no more than 5 hours, more preferably no more than 4 hours, even more preferably no more than 3 hours, more preferably no more than 2 hours, and most preferably no more than 1 hour, and / or is no more than 6 hours, preferably no more than 5 hours, more preferably no more than 4 hours, even more preferably no more than 3 hours, more preferably no more than 2 hours, and most preferably no more than 1 hour after administration of other therapeutic agents.

[0067] Further, the present application also provides a reagent for detecting FPV, and the raw material for preparing the reagent includes any one of the following a) to g):

[0068] a) the antibody or antigen binding molecule as described above;

[0069] b) the nucleic acid as described above;

[0070] c) the plasmid vector as described above;

[0071] d) the host as described above;

[0072] e) the culture or purified antibody prepared by the preparation method as described above;

[0073] f) the labeled antibody as described above;

[0074] g) the combination as described above.

[0075] The reagent as described above specifically includes the antibody or antigen binding molecule as described above; or the culture or purified antibody prepared by the preparation method as described above; and further includes an ELISA detection reagent, a western blot detection reagent and / or a colloidal gold detection test paper.

[0076] The present application also provides a method for detecting FPV virus, which includes detecting a sample by using the reagent as described above.

[0077] The FPV neutralizing monoclonal antibody of the present application has strong neutralizing activity to FPV virus, and has important research and application value in specific prevention, treatment and diagnosis of diseases caused by FPV virus. Moreover, the antibody having more than 82.6% identity with the variable region of the antibody of the present application and more than 94% identity with the full length of the antibody also has good specificity and neutralizing effect. BRIEF DESCRIPTION OF DRAWINGS

[0078] Fig. 1 is a serum neutralizing antibody titer;

[0079] Fig. 2 is an ELISA determination result of the antibody binding with FPV VP2, wherein a is the OD 450 of 32 antibodies expressed in a small amount in the first round binding with FPV VP2 protein, and b is the OD 450 of 13 antibodies expressed in a large amount in the second round binding with FPV VP2 protein;

[0080] Fig. 3 is a SDS-PAGE diagram of 13 antibodies expressed;

[0081] Fig. 4 is a neutralizing activity of monoclonal antibody;

[0082] Fig. 5 is an EC 50a is the EC of antibody 40A 50 b is the EC of antibody 3B 50 c is the EC of antibody 35B 50 ;

[0083] Figure 6 is a graph of the number of white blood cells in each group of animals. DETAILED DESCRIPTION

[0084] The present application discloses FPV neutralizing antibodies and applications, and those skilled in the art can refer to the content herein to improve the process parameters as appropriate to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0085] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have meanings that are commonly understood by those of ordinary skill in the art.

[0086] In this application, the term "and / or", describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent: A exists alone, A and B exist together, and B exists alone. Wherein A, B can be singular or plural.

[0087] In this application, the terms "include", "contain" and "have" are used interchangeably, and are intended to mean the inclusiveness of the scheme, meaning that the scheme can have other elements in addition to the listed elements. It should also be understood that the use of "include", "contain" and "have" in this text also provides a scheme of "consisting of" or "is".

[0088] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items.

[0089] In the present application, "antibody" refers to whole antibodies and any antigen binding fragment thereof ("antigen binding portion") or single chains. A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Chimeric or humanized antibodies are also encompassed by the antibodies according to the present application.

[0090] In the present application, "antigen binding molecule" refers to a molecule that specifically binds to an antigen. For example, the antigen binding molecule includes, but is not limited to, an antibody or an antibody mimetic. "Antibody mimetic" refers to a biomolecule that mimics the structure and function of a natural antibody by non-natural synthetic methods. For example, the antibody mimetic includes, but is not limited to, an affibody, an affitin, an affilin, a designed ankyrin repeat protein (DARPin), a nucleic acid aptamer, or a Kunitz-type domain peptide.

[0091] In the present application, "neutralizing antibody" is a soluble protein secreted by adaptive immune response cells. After a virus invades the human body, the immune cells secrete neutralizing proteins into the blood, which bind to viral particles in the blood and prevent the virus from infecting cells and destroying viral particles.

[0092] In the present application, the skilled person can refer to Current Protocols in Molecular Biology (Ausubel) in particular. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to designate one of the 20 common L-amino acids.

[0093] In the present application, "identity" can be calculated by determining the percent "identity" of two amino acid sequences or two nucleic acid sequences, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0094] In the present application, "nucleic acid" encompasses any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually denoted 5' to 3'.

[0095] In the present application, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. The nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both the sense and the antisense strand, as well as single- and double-stranded forms. Also, the nucleic acid molecules described herein can contain naturally-occurring or non-naturally-occurring nucleotides. Examples of non-naturally-occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. The nucleic acid molecule also encompasses DNA and RNA molecules which are suitable as vectors for the direct expression of the antibodies of the present application in vitro and / or in vivo, e.g. in a host or patient. Such DNA (e.g. cDNA) or RNA (e.g. mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce the antibody in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0096] As used herein, "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0097] As used herein, "host" or "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cell and progeny of the transformed cell that are not identical to the parent cell in having the same genotypic characteristics but that can include mutations. Mutant progeny that have the same functional or biological activity as screened or selected for in the originally transformed cell are included herein.

[0098] As used herein, "prevention" or "preventing" includes prophylaxis and / or treatment. "Treatment" refers to a surgical or therapeutic treatment, whose purpose is to prevent, slow down (reduce), or halt an undesired physiological change or pathological condition in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those who have a condition or disease and those in which a condition or disease is to be prevented or prevented. When referring to the terms slow down, reduce, diminish, palliate, alleviate, and the like, the meaning also includes elimination, disappearance, nonoccurrence, and the like.

[0099] As used herein, "product" includes a vaccine for preventing FPV, a drug for treating FPV, and a reagent for detecting FPV. The vaccine includes the antibody and the adjuvant as described above. The drug includes the antibody and the excipient as described above. The reagent for detecting FPV includes the antibody and the related reagent such as a detection buffer as described above.

[0100] As used herein, the administration dose of the "product" is not less than the effective dose. If the drug includes the antibody and other therapeutic agent as described above, the administration dose of the other therapeutic agent is less than the effective dose, or not less than the effective dose; and the administration dose of the antibody as described above is less than the effective dose, or not less than the effective dose.

[0101] In the present application, the effective dose includes "therapeutically effective amount" or "prophylactically effective amount", which refers to an amount sufficient to treat or prevent FPV but low enough to avoid serious side effects (at a reasonable benefit / risk ratio) within the scope of reasonable medical judgment. The prophylactic or therapeutic effective amount of a drug will vary depending on the specific drug selected (e.g. considering the potency, effectiveness and half-life of the drug), the selected route of administration, the disease being prevented or treated, the severity of the disease being prevented or treated, the age, size, weight and physical condition of the patient being prevented or treated, the medical history of the patient being prevented or treated, the duration of prevention or treatment, the nature of concurrent therapy, the desired prophylactic or therapeutic effect, and the like, but can still be routinely determined by one skilled in the art.

[0102] In the present application, the object of prevention and treatment is a mammal, including canine and / or feline. For example: cats, tigers, leopards, lions, cheetahs, lynxes, etc.

[0103] The sequences involved in the embodiments of the present application include:

[0104] FPV VP2 protein amino acid sequence

[0105] The present application is further illustrated below in conjunction with the examples:

[0106] Example 1 Preparation of FPV monoclonal antibody

[0107] The preparation process of the FPV monoclonal antibody of the present example is as follows:

[0108] (1) Preparation of FPV monoclonal antibody

[0109] a) Immunization of New Zealand rabbits with FPV VP2 protein

[0110] The semi-finished inactivated VP2 protein (amino acid sequence as SEQ ID NO. 32) preserved in the laboratory was diluted to 150 μg / ml, and then mixed with an equal amount (V:V) of Q adjuvant and placed in a vortex shaker for 30 minutes. Three New Zealand rabbits were subcutaneously injected with the inactivated VP2 protein with adjuvant, 1 ml per rabbit (containing 75 μg VP2 protein), and the same dose and route was used for the second injection 28 days later. Three New Zealand rabbits were subcutaneously injected with the inactivated VP2 protein with adjuvant, 1 ml per rabbit (containing 75 μg VP2 protein), and the same dose and route was used for the second injection 28 days later.

[0111] Fourteen days after the second immunization, the sera were collected to determine the neutralizing antibody titers against FPV (Figure 1). The serum samples were inactivated at 56°C for 30 min, and then serially diluted in EMEM medium to 1:2048. Four replicates were performed for each sample, and 50 μL / well was mixed with an equal volume of the FPV-SD2018 strain diluted to the working concentration (100 TCID50 / 0.1 mL). Negative serum control, positive serum control, unneutralized virus control, and normal cell control were also set up. The negative serum was diluted to the same degree as the sample serum, and the positive serum was diluted to a neutralizing antibody concentration of 1:128. The mixture was incubated at 37°C for 1 h with shaking. CRFK cells with a well-grown monolayer were washed three times with PBS (0.01 M, pH = 7.4), digested with 3% trypsin solution, and dispersed in EMEM medium containing 2% newborn calf serum. The cell suspension (cell content of about 105 / mL) was added to each test well at 100 μL / well, and incubated at 37°C in a 5% CO2incubator for 7 days. CPE was observed and recorded daily.

[0112] When the neutralizing antibody titer was ≥1:1024, the New Zealand rabbits were euthanized, and the spleens were removed for lymphocyte isolation.

[0113] b) Antigen-specific memory B cell sorting and reverse transcription

[0114] The spleen cell suspension was rinsed once with PBS containing 10% FBS, centrifuged, and the supernatant was discarded. The cells were resuspended in PBS containing 10% FBS. 10 μg of BV421 was incubated with the FPV VP2 protein at room temperature for 30 min in the dark for antigen-specific cell labeling. Each of 10 μg of FITC-anti-rabbit CD4, FITC-anti-rabbit CD8, APC-Cy7-anti-rabbit IgM, and PE-anti-rabbit IgG was mixed with the BV421-chimeric antibody and strain mixture, which was then added to the spleen cell suspension and incubated on ice for 1 h. A negative control was set up without the BV421-chimeric antibody and strain mixture. 20 mL of PBS containing 10% FBS was added for rinsing, and centrifugation was performed at 1000 rpm for 10 min. The supernatant was discarded, and the step was repeated once. The cells were resuspended in 3 mL of PBS containing 10% FBS and passed through a 200-μm filter, and then placed on the BD flow cytometer. Before being placed on the machine, 20 μL of PerCP-Cy5-5-7-AAD dye was added per mL of cell suspension. The live cells in the spleen cells that were CD4-, CD8-, IgM-, IgG+, and chimeric antibody+were sorted by flow cytometry for single antigen-specific B cells. The RNA of the rabbit-derived spleen cells was reverse transcribed into cDNA using the newbeibio R202-02 reverse transcription kit.

[0115] c) Nested PCR amplification of antibody gene variable region

[0116] Two rounds of amplification were performed for the antibody gene heavy chain variable region (VH) and light chain variable region (VLK), respectively. First, 5 μL of cDNA was used as a template for the first round of amplification using KOD One TM PCR Master Mix for the first round of amplification of VH and VLK, specifically, primers 1-4 for amplifying the heavy chain variable region (VH); primers 5-8 for amplifying the Kappa chain variable region (VLK). The second round of PCR was performed using nested primers with enzyme cutting sites, with the first round of product as a template, specifically, primers 9-26 for amplifying the heavy chain variable region (VH); primers 27-40 for amplifying the light chain variable region (VLK).

[0117] The amplification products were verified by 1.5% agarose gel electrophoresis, and pairs that simultaneously amplified the heavy chain variable region and the light chain variable region were selected for the next vector construction. Among the 96 single cells, 32 wells amplified paired antibody genes, which were used for subsequent small-scale expression.

[0118] Table 1 Primers used for antibody gene amplification (5'-3') Note: The underlined capital letter part is the homologous arm

[0119] d) Construction of transient eukaryotic expression vector

[0120] The obtained 32 rabbit monoclonal antibody VH and VLK genes were respectively constructed into pTT5 plasmids with rabbit IgG heavy chain and light chain skeletons, obtaining 32 single-antibody corresponding pTT5-rVH-CH and pTT5-rVL-CL plasmids, and using HEK293F eukaryotic expression system to obtain small-scale expressed antibodies, followed by ELISA.

[0121] Two types of vectors were constructed, respectively for expressing IgG1 heavy chain and light chain. The pTT5-rVH-CH plasmid containing the rabbit IgG constant region and the pTT5-rVL-CL plasmid containing the rabbit IgG constant region were double-digested with EcoRI and HindIII, and then ligated with the fragments amplified in step c) through homologous recombination, and then transformed into E. coli DH5a competent cells. The next day, the recombinant plasmid was extracted and directly used for small-scale expression in HEK293F cells.

[0122] e) Small-scale expression of HEK293F cells and ELISA verification

[0123] When the concentration of HEK293F cells reached 1 x 10 6The cells were transfected when the cell density reached 1 x 106 / mL and the cell viability was greater than 90%. The EP tube was added with 100 μL of Opti-MEM I reduced serum medium, 2 μg of heavy chain recombinant plasmid and 2 μg of light chain recombinant plasmid, and 12 μL of PEI transfection reagent, mixed and allowed to stand at room temperature for 20 minutes to form a DNA-PEI cationic nucleic acid transfection reagent complex, and the v / v ratio of the plasmid and PEI transfection reagent was ensured to be 1:3. The mixed solution was then added to the 24-well culture plate with cells, and the plate was incubated at 37°C in 5% CO2 and 200 rpm. After 3 days, the cells were collected by centrifugation at 4000 rpm for 20 minutes, and the supernatant was transferred to a 96-well deep plate and stored in a -20°C refrigerator or directly used for ELISA detection.

[0124] The ELISA plate strip was coated with 1 μg / mL of VP2 antigen, and after blocking, it was washed with PBST for 5 times. 100 μL of 1 μg / mL of each monoclonal antibody, 1 μg / mL of mouse anti-His-tag monoclonal antibody (negative control), and 3% casein (blank control) were added to the enzyme-labeled plate, and incubated at 37°C for 1 hour, washed 5 times, and 100 μL of HRP-goat anti-mouse IgG monoclonal antibody (secondary antibody) was added to the ELISA plate at a dilution ratio of 1:3000 with 3% casein sodium, and incubated at 37°C for 1 hour, washed 5 times, and TMB was used for color development, and the stop solution was added, and the absorbance value was read by the enzyme-labeled instrument at 450 nm. The OD450nm ratio of the test sample and the blank control group was calculated S / N. When S / N < 2.1, the monoclonal antibody cannot bind to the VP2 protein, and when S / N ≥ 2.1, the monoclonal antibody can bind to the VP2 protein. As shown in Figure 2a, among the 32 small amount of expressed monoclonal antibodies, 13 had OD 450 greater than 0.5, and entered the second round of large-scale expression.

[0125] f) Large-scale expression and purification of monoclonal antibodies

[0126] Thirteen monoclonal antibodies with OD450nm value greater than 0.5 after ELISA detection were selected for mass expression. After transfection, the cells were cultured in a 37°C shaking incubator with 8% CO2 at 125 rpm for 72-96 h, and the supernatant was collected by centrifugation. Protein A gravity column was used for purification, and the buffer was replaced with PBS. ELISA verification was performed again according to the procedure described in e). As shown in Fig. 1b, the absorbance of the 13 antibodies at 450 nm was greater than 0.5. SDS-PAGE was used to verify the above antibodies, and the results showed that the heavy chain of the antibody was stable at about 50 kDa, and the light chain was about 25 kDa. Nine antibodies with clear main bands and stable expression were selected for subsequent screening, numbered 2A, 32A, 40A, 45A, 3B, 13B, 22B, 25B, and 35B. After filtering the above antibodies through a 0.22 μm filter to remove bacteria, BCA was used for quantification, and the samples were stored at -80°C.

[0127] Example 2 Specificity detection of FPV monoclonal antibody

[0128] Each 1 μg / mL of feline plague virus FPV-SD2018 strain, feline herpes virus FHV-SD2018 strain and feline calicivirus FCV-SD2018 strain was coated on the plate at 4°C overnight. After blocking and washing with blocking solution, nine monoclonal antibodies expressed in Example 1 were added, and PBS was set as a negative control, 100 μL per well, incubated at 37°C for 1 h, washed, then goat anti-rabbit HRP was added, incubated and washed, and then TMB was used for color development after the reaction was terminated. The absorbance at 450 nm was read. According to the results shown in Table 2, it can be seen that the nine antibodies prepared in Example 1 only bind to FPV virus and have no cross reaction with feline herpes virus and feline calicivirus.

[0129] Table 2 Specificity determination of monoclonal antibody

[0130] Example 3 Neutralization activity determination of FPV monoclonal antibody against FPV-SD2018 strain

[0131] The starting concentration of the monoclonal antibody was 2 mg / mL, which was diluted 2-fold in series to 1:2048 in EMEM medium, and 4 wells of cells were added to each dilution. FPV-SD2018 strain was diluted to 100 TCID 50 / 0.1 mL, and the antibody and virus solution were mixed in equal amounts to a total of 50 μL, which was incubated at 37°C for 1 hour. 10 5CRFK cell culture fluid 100 μL, 37°C, 5% CO2 incubator for 7 days, record the cytopathic effect. The reciprocal of the highest dilution that protects 50% of the cells from cytopathic effect is the neutralizing antibody titer. The results show that the titers of 40A, 3B and 35B are 1:1024, the titers of 32A and 22B are 1:512; the titers of 2A, 45A, 13B and 25B are all ≤1:256. Therefore, the monoclonal antibodies 40A, 3B and 35B are screened for subsequent verification. As shown in Figure 4, the minimum antibody concentration required for 40A antibody to neutralize and inhibit FPV-SD2018 strain is 0.74 μg / mL, the minimum antibody concentration required for 3B antibody to neutralize and inhibit FPV-SD2018 strain is 0.98 μg / mL, and the minimum antibody concentration required for 35B antibody to neutralize and inhibit FPV-SD2018 strain is 1.47 μg / mL.

[0132] Example 4 EC of FPV monoclonal antibody against FPV VP2 protein 50 determination

[0133] The affinity of monoclonal antibodies 40A, 3B and 35B to antigen VP2 was determined by indirect ELISA method.

[0134] VP2 protein was diluted with PBS to 1 μg / mL, 100 μL per well to coat the ELISA plate, 4°C overnight. Wash the plate with PBST, block with 2% skim milk powder at 37°C for 2 h. Dilute monoclonal antibodies 40A, 3B and 35B by 12 gradients and add to the wells, incubate at 37°C for 1 h. Add HRP-labeled goat anti-rabbit IgG secondary antibody, incubate at 37°C for 1 h, develop with TMB, add 0.2 M sulfuric acid to stop the color development, and detect the absorbance at 450 nm wavelength in the enzyme label instrument (Tables 3-5).

[0135] The binding effect of antibodies 40A, 3B, 35B to antigen is shown in Figure 5, the dose-effect curve is successfully fitted with the regression model, the EC 50 of 40A binding to VP2 protein is 144.0 ng / mL, the EC 50 of 3B binding to VP2 protein is 201.1 ng / mL, and the EC 50 of 35B binding to VP2 protein is 173.3 ng / mL, indicating that the three antibodies are all active and have good affinity, and among them, the affinity of antibody 40A to antigen FPV VP2 protein is the best, so the FPV neutralizing monoclonal antibody 40A is selected for subsequent treatment test.

[0136] Table 3 EC of 40A binding to antigen VP2 50

[0137] Table 4 EC of 3B binding to antigen VP2 50

[0138] Table 5 EC of 35B binding to antigen VP2 50

[0139] Example 5 Therapeutic verification of FPV neutralizing monoclonal antibody 40A

[0140] Taking three adult cats infected with FPV as examples, the symptoms of depression, vomiting and low food intake appeared in the early stage of infection, the FPV neutralizing monoclonal antibody was diluted to 2 mg / mL, and was continuously injected at a dose of 1 mg / kg for two days. Blood samples were collected for 5 consecutive days to determine the blood routine and observe the number of white blood cells. As shown in Figure 6, the initial white blood cell counts of the three sick cats were all lower than the normal range (5.5 x 10 9 / L ~ 19.5 x 10 9 / L), after the first injection of neutralizing antibody, the white blood cell count showed an upward trend, and on the third day after injection, the white blood cell count of the sick cats was within the normal range. And with the increase of injection time, the mental state of the sick cats improved significantly, the vomiting symptoms disappeared, the diet was normal, and the activity increased. This shows that FPV neutralizing monoclonal antibody 40A can be used for the treatment of cat plague.

[0141] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. An FPV antibody or antigen-binding molecule, the heavy chain CDR1 has an amino acid sequence as shown in SEQ ID NO: 1; the heavy chain CDR2 has an amino acid sequence as shown in SEQ ID NO: 2 or 7; the heavy chain CDR3 has an amino acid sequence as shown in SEQ ID NO: 3 or 8; the light chain CDR1 has an amino acid sequence as shown in SEQ ID NO: 4 or 9; the light chain CDR2 has an amino acid sequence as shown in SEQ ID NO: 5 or 10; the light chain CDR3 has an amino acid sequence as shown in SEQ ID NO: 6 or 11; or the CDR region thereof has a sequence with 1 or more amino acids substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown above; or a sequence with 80% or more identity to the amino acid sequence as shown in any one of the above.

2. The antibody or antigen-binding molecule according to claim 1, characterized in that, the heavy chain CDR1-3 has the amino acid sequences as shown in SEQ ID NO: 1-3 in order, and the light chain CDR1-3 has the amino acid sequences as shown in SEQ ID NO: 4-6 in order; or the heavy chain CDR1-3 has the amino acid sequences as shown in SEQ ID NO: 1, 7-8 in order, and the light chain CDR1-3 has the amino acid sequences as shown in SEQ ID NO: 9, 10 or 11 in order.

3. The antibody or antigen-binding molecule according to claim 1 or 2, characterized in that, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 28 or 30, the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 29 or 31, or the heavy chain variable region and / or the light chain variable region has a sequence with 1 or more amino acids substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown above; or a sequence with 80% or more identity to the amino acid sequence as shown in any one of the above.

4. The antibody or antigen-binding molecule of any one of claims 1 to 3, wherein, the light chain type is Lambda, and the subtype is IgG 1.

5. A nucleic acid encoding the antibody or antigen-binding molecule according to any one of claims 1-4.

6. A plasmid vector containing the nucleic acid according to claim 5.

7. A host having the nucleic acid according to claim 5 integrated into its genome, or transformed or transfected with the plasmid vector according to claim 6.

8. A method of making the antibody or antigen binding molecule of any one of claims 1-4, comprising: culturing the host according to claim 7 to obtain a culture containing the antibody or antigen-binding molecule.

9. A labeled antibody comprising the antibody or antigen-binding molecule according to any one of claims 1-4, and a labeling substance, which is a chemical label or a biological label.

10. The labeled antibody according to claim 9, characterized in that, the chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope and / or a colloidal indicator; the biological label is biotin, avidin or an enzyme label.

11. The labeled antibody according to claim 10, characterized in that, the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, beta-galactosidase-anti-beta-galactosidase bridge; the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red or PerCP dye; the chemiluminescent indicator is selected from one or more of acridinium ester, acridinium sulphonamide and derivatives thereof, luminol, isoluminol, isoluminol isothiocyanate and derivatives thereof, N-(4-aminobutyl)-N-ethylisoluminol, 4,5-diaminobenzohydrazide or aminobutylethylbenzhydrazide; the isotopes are selected from 125 I, 131 I, 124 I, 3 H, 14 C, 111 In, 89 Zr or 32 one or more of P; the colloidal indicator is selected from one or more of colloidal gold, colloidal carbon or colloidal selenium.

12. A combination comprising the antibody or antigen binding molecule of any one of claims 1 to 4 and a medium; the medium is selected from colloidal gold, an enzyme label plate, magnetic beads or latex microspheres.

13. Use of any one of a) to g) below in the manufacture of a product for detecting, preventing and / or treating FPV virus: a) the antibody or antigen binding molecule of any one of claims 1 to 4; b) the nucleic acid of claim 5; c) the plasmid vector of claim 6; d) the host of claim 7; e) the culture or purified antibody produced by the method of claim 8; f) the labelled antibody of any one of claims 9 to 11; g) the combination of claim 12.

14. A product for preventing and / or treating FPV virus, the raw material for the manufacture of which comprises any one of a) to g) below: a) the antibody or antigen binding molecule of any one of claims 1 to 4; b) the nucleic acid of claim 5; c) the plasmid vector of claim 6; d) the host of claim 7; e) the culture or purified antibody produced by the method of claim 8; f) the labelled antibody of any one of claims 9 to 11; g) the combination of claim 12.

15. The product of claim 14, which comprises an adjuvant and the antibody or antigen binding molecule of any one of claims 1 to 4; or which comprises an adjuvant and the culture or purified antibody produced by the method of claim 8.

16. The product of claim 15, which further comprises another drug for preventing and / or treating FPV virus infection.

17. A reagent for detecting FPV, the raw material for the manufacture of which comprises any one of a) to g) below:

16. The product of claim 14, wherein, a) the antibody or antigen binding molecule of any one of claims 1 to 4; b) the nucleic acid of claim 5; c) the plasmid vector of claim 6; d) the host of claim 7; e) the culture or purified antibody produced by the method of claim 8; f) the labelled antibody of any one of claims 9 to 11; g) the combination of claim 12. c) the plasmid vector of claim 6; d) the host of claim 7; e) the culture or purified antibody prepared by the method of claim 8; f) the labeled antibody of any one of claims 9 to 11; g) the conjugate of claim 12.

18. The agent of claim 17, wherein which comprises the antibody or antigen binding molecule of any one of claims 1 to 4; or the culture or purified antibody prepared by the method of claim 8; which further comprises an ELISA detection reagent, a western blot detection reagent and / or a colloidal gold detection test paper.

19. A method for detecting a FPV virus, which comprises detecting a sample with the reagent of claim 17.

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