Mouse-cat chimeric anti-cat CD20 antibody and application thereof

Abstract

The invention provides a mouse-cat chimeric anti-cat CD20 antibody and an application thereof. According to the mouse-cat chimeric anti-cat CD20 antibody, variable regions of a heavy chain and a light chain of a mouse CD20 antibody and constant regions of cat IgG1 heavy chain and cat Igkappa light chain sequences are selected, and the heavy chain and the light chain of the antibody obtained after assembly respectively comprise amino acid sequences shown as SEQ ID NO.1-4. The amino acid sequence of the mouse-cat chimeric anti-cat CD20 antibody is subjected to codon optimization, expression and purification, the obtained anti-cat CD20 antibody is subjected to indirect ELISA (enzyme-linked immunosorbent assay) test, and the anti-cat CD20 antibody is proved to have antibody biological activity. After verification by a model mouse, it can be found that the mouse-cat chimeric anti-cat CD20 antibody provided by the invention can effectively relieve the swelling degree of arthritic paws, and is expected to play a role in the process of treating cat rheumatoid arthritis in the future.

Description

Technical Field

[0001] The present invention relates to the field of veterinary antibody preparation, and particularly relates to a murine-cat chimeric anti-cat CD20 antibody and its application. Background Art

[0002] Rheumatoid arthritis is a chronic, progressive, erosive, and immune-mediated polyarticular disease, and its exact cause and pathogenesis are unknown. Rheumatoid arthritis is common in elderly dogs and cats. The initial symptoms generally include listlessness, some fever, and loss of appetite. As the disease progresses, the synovium thickens and hypertrophies, forming a vascularized granulation tissue, which interferes with the nutrition of cartilage from synovial fluid, causing cartilage necrosis, eroding the subchondral bone, producing local osteolysis, and causing the joint surface to collapse. In severe cases, it can involve joint ligaments and tendons. In the later stage, the articular cartilage is further eroded, the surrounding tissues of the joint are more severely damaged, leading to ligament rupture, joint deformity, and lameness in cats. Because the pain level during the onset fluctuates, the lameness symptoms are also sometimes mild and sometimes severe, recurring repeatedly, with joint stiffness, joint swelling, and often involving several joints.

[0003] There is no good treatment method for rheumatoid arthritis, and it can only relieve pain and improve the quality of life. Currently, the treatment methods mainly focus on relieving pain with prescription painkillers, non-steroidal anti-inflammatory drugs, etc. However, non-steroidal anti-inflammatory drugs have damage to the liver and kidneys. The diseased cats need to take medicine frequently, and the pet owners also need to take the pets to detect liver and kidney functions every month, increasing the burden on the diseased cats and pet owners.

[0004] There are a large number of CD20-positive cells in the lymphocytes of the synovial tissue of rheumatoid arthritis patients. CD20 is an antigen molecule expressed on the surface of B cells, which can directly act on B cells by regulating transmembrane calcium ion flow and plays an important regulatory role in B cell proliferation and differentiation. Anti-CD20 antibodies can specifically bind to the CD20 antigen on the surface of B cells, clear B lymphocytes by directly inducing apoptosis and mediating the mechanism of antibody-dependent cell-mediated cytotoxicity, effectively reduce the number of abnormally activated B cells, thereby inhibiting the immune response, relieving the symptoms of rheumatoid arthritis, and delaying the progression of the disease. Therefore, anti-CD20 antibodies have good curative effects on autoimmune diseases such as rheumatoid arthritis.

[0005] Currently, CD20 monoclonal antibodies are widely used in the field of human antibody drugs. Rituximab has achieved good curative effects in the treatment of rheumatoid arthritis and has high safety. However, the R & D personnel have ignored the demand for CD20 antibodies in the veterinary field. Compared with the two, due to the differences in body size and metabolic rate, as well as the cross-reactivity of antibodies in different animals, the field of animal CD20 antibody preparation is still relatively lacking.

[0006] At present, there are no related antibody drugs targeting the CD20 target for the treatment of rheumatoid arthritis in the domestic and foreign pet markets. Therefore, developing a murine-cat chimeric anti-cat CD20 antibody to treat feline rheumatoid arthritis and slow down the disease course can not only improve the quality of life of cats and relieve the pressure on pet owners, but also add new products to the field of pet monoclonal antibodies. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a murine-cat chimeric anti-cat CD20 antibody and its application.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a murine-cat chimeric anti-cat CD20 antibody, and the heavy chain of the murine-cat chimeric anti-cat CD20 antibody contains the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.3;

[0010] The light chain of the murine-cat chimeric anti-cat CD20 antibody contains the amino acid sequence shown in SEQ ID NO.2 and / or SEQ ID NO.4.

[0011] In the present invention, the variable regions of the heavy chain and light chain of the mouse CD20 antibody, and the constant regions of the cat IgG1 heavy chain and cat Igκ light chain sequences are selected, and they are assembled into a new antibody sequence and synthesized and expressed; specifically, in the present invention, the mouse CD20 antibody heavy chain sequence in NCBI (truncated from the amino acids encoded by the serial number MA814136.1, specifically shown in SEQ ID NO.1), the light chain sequence (truncated from the amino acids encoded by the serial number MA814137.1, specifically shown in SEQ ID NO.2) are selected, and their variable region sequences are selected; according to the cat IgG1 heavy chain sequence in NCBI (truncated from the amino acids encoded by the serial number AB016711.1, specifically shown in SEQ ID NO.3), the cat Igκ light chain sequence (truncated from the amino acids encoded by the serial number KY795158.1, specifically shown in SEQ ID NO.4), their constant regions are selected; the cat light chain and heavy chain constant regions are assembled with the light chain and heavy chain variable regions of the mouse CD20 antibody.

[0012] The specific sequences are shown in Table 1 below:

[0013] Table 1

[0014]

[0015]

[0016] As a preferred technical solution of the present invention, the heavy chain of the murine-cat chimeric anti-cat CD20 antibody comprises any one of the amino acid sequences shown in (I), (II) or (III):

[0017] (I) The amino acid sequence shown in SEQ ID NO.5;

[0018] (II) An amino acid sequence having ≥98% similarity to the amino acid sequence shown in SEQ ID NO.5;

[0019] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.5;

[0020] SEQ ID NO.5 is as follows:

[0021] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSATTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0022] As a preferred technical solution of the present invention, the light chain of the murine-cat chimeric anti-cat CD20 antibody comprises any one of the amino acid sequences shown in (IV), (V) or (VI):

[0023] (IV) The amino acid sequence shown in SEQ ID NO.6;

[0024] (V) An amino acid sequence having ≥98% similarity to the amino acid sequence shown in SEQ ID NO.6;

[0025] (VI) An amino acid sequence obtained by modifying, substituting, deleting, or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.6.

[0026] SEQ ID NO.6 is as follows:

[0027] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE

[0028] After the obtained sequence was expressed and purified, and confirmed by an indirect ELISA test, the obtained antibody sequence has antibody biological activity. Therefore, a new murine-cat chimeric anti-cat CD20 antibody with biological activity is provided in the present invention.

[0029] In addition, during the synthesis and assembly of SEQ ID NO.1 - 4 in the present invention, to meet the requirements of subsequent experiments, restriction enzyme cleavage sites may need to be inserted at appropriate positions to facilitate sequence replacement; therefore, in the present invention, restriction enzyme cleavage sites are inserted between the variable region and the constant region of SEQ ID NO.5 and SEQ ID NO.6, and the amino acid sequences after the insertion of the restriction enzyme cleavage sites are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0030] SEQ ID NO.7 is as follows:

[0031] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA ASTTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (The underlined part is the amino acid translation of the cleavage site)

[0032] SEQ ID NO.8 is as follows:

[0033] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTV RT DAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE (The underlined part is the amino acid translation of the cleavage site)

[0034] In addition to SEQ ID NO.7 and SEQ ID NO.8, other types of cleavage sites can be inserted into the chimeric antibody sequences shown in the present invention without affecting the amino acid structure.

[0035] In addition, the inventors also tried to assemble using other sequences or other methods in the experiments;

[0036] 1. Using the heavy chain and light chain variable regions of the anti-CD20 monoclonal antibody disclosed in CN103173457A;

[0037] 2. Using the heavy chain and light chain variable regions disclosed in CN114773474A;

[0038] 3. Using the heavy chain and light chain variable regions disclosed in CN104640881A;

[0039] The constant regions of the feline IgG1 heavy chain sequence (extracted from the sequence number AB016711.1, specifically shown as SEQ ID NO.3) and the feline Igκ light chain sequence (extracted from the sequence number KY795158.1, specifically shown as SEQ ID NO.4) in NCBI were assembled, and the resulting antibody sequence could not be normally expressed or did not have biological activity. The inventor speculated that during the protein expression process, due to the interaction between internal amino acids, the antibody structure was disordered, resulting in abnormal synthesis or failure to exert biological functions.

[0040] Meanwhile, in the selection of the constant regions of the heavy chain and the light chain, the inventor selected IgG1 from the heavy chain subtypes (IgG1, IgG2a, IgG2b, IgG3); and selected Igκ from the light chain subtypes (Igκ, Igλ). Through experiments, it was found that the IgG1 subtype is not only the first choice in human antibodies but also preferred in the present invention. At the same time, the light chain subtype Igκ is also applicable to the present invention.

[0041] As a preferred technical solution of the present invention, the murine-feline chimeric anti-feline CD20 antibody is further linked to the human IL-2 signal peptide, preferably linked to the human IL-2 signal peptide at the N-terminus.

[0042] The amino acid sequence of the human IL-2 signal peptide (SEQ ID NO.9) is:

[0043] MYRMQLLSCIALSLALVTNS.

[0044] In the second aspect, a gene sequence encoding the murine-feline chimeric anti-feline CD20 antibody as described in the first aspect.

[0045] As a preferred technical solution of the present invention, the gene sequence is further linked to the gene sequence of the human IL-2 signal peptide, and the gene sequence of the human IL-2 signal peptide (SEQ ID NO.10) is:

[0046] ATGTATCGAATGCAACTACTAAGTTGCATCGCTCTGTCCTTGGCCCTCGTGACAAACTCT;

[0047] As a preferred technical solution of the present invention, the gene sequence is further linked to a Kozak sequence.

[0048] In the present invention, the gene sequence is optimized according to the codons of CHO cells. After optimization, the gene sequence encoding the heavy chain is as shown in SEQ ID NO.11, and the gene sequence encoding the light chain is as shown in SEQ ID NO.12.

[0049] SEQ ID NO.11 (The underlined part represents the Kozak sequence, and the italic part represents the human IL-2 signal peptide):

[0050]

[0051]

[0052]

[0053] SEQ ID NO.12 (The underlined part represents the Kozak sequence, and the italic part represents the human IL-2 signal peptide):

[0054]

[0055] In the third aspect, an expression vector contains the gene sequence described in the second aspect.

[0056] In the fourth aspect, a pharmaceutical composition includes the murine-cat chimeric anti-cat CD20 antibody described in the first aspect, the gene sequence described in the second aspect, or the expression vector described in the third aspect.

[0057] In the fifth aspect, use of the murine-cat chimeric anti-cat CD20 antibody described in the first aspect, the gene sequence described in the second aspect, or the expression vector described in the third aspect in the preparation of a drug for treating feline rheumatoid arthritis.

[0058] The numerical ranges described in the present invention not only include the above-listed point values, but also include any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] The present invention provides a murine-cat chimeric anti-cat CD20 antibody and its application. The antibody uses the variable regions of the heavy and light chains of a murine antibody and the constant regions of the feline IgG1 heavy chain and feline Igκ light chain sequence as the antibody backbone to form a new murine-cat chimeric anti-cat CD20 antibody. This antibody can be normally synthesized and expressed, can bind to the CD20 antigen, and has biological activity. After verification using model mice, it can be found that the murine-cat chimeric anti-cat CD20 antibody provided by the present invention can effectively relieve the swelling degree of the arthritic paw, and is expected to play an important role in the treatment of feline rheumatoid arthritis in the future. Description of the Drawings

[0061] Figure 1It is the SDS-PAGE detection result diagram; among them, lane 1: non-reduced CD20 antibody; 2: non-reduced cell supernatant; 3: reduced cell supernatant; 4: reduced CD20 antibody; M: protein Marker.

[0062] Figure 2 It is the HPLC detection result diagram.

[0063] Figure 3 It is the curve graph of the body weight change of each group of mice in Example 3.

[0064] Figure 4 It is the curve graph of the AI score change of each group of mice in Example 3.

[0065] Figure 5 It is the curve graph of the change of the paw swelling degree score of each group of mice in Example 3. Detailed implementation mode

[0066] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0067] Unless otherwise specified, the experimental methods and technical means used are all conventional methods and means in the art.

[0068] Example 1 Preparation of murine-cat chimeric anti-cat CD20 antibody

[0069] 1. Optimization and synthesis of sequences

[0070] According to the mouse CD20 heavy chain sequence in NCBI (the amino acids encoded by the truncated sequence number MA814136.1, specifically shown as SEQ ID NO.1), and the light chain sequence (the amino acids encoded by the truncated sequence number MA814137.1, specifically shown as SEQ ID NO.2), select its variable region sequence;

[0071] According to the cat IgG1 heavy chain sequence in NCBI (the amino acids encoded by the truncated sequence number AB016711.1, specifically shown as SEQ ID NO.3), and the cat Igκ light chain sequence (the amino acids encoded by the truncated sequence number KY795158.1, specifically shown as SEQ ID NO.4), select its constant region.

[0072] The light chain and heavy chain constant regions of the cat are linked to the light chain and heavy chain variable regions of the mouse CD20 antibody. Without changing the amino acid sequence, it is further optimized according to the codon preference of CHO cells. To enhance protein expression, a human IL-2 signal peptide is added to the 5' end of the sequence, and a Kozak sequence is added before the signal peptide. A cleavage site is inserted between the constant region and the variable region. The resulting amino acid sequences are shown in SEQ ID NO.7 - 8; the resulting nucleic acid sequences are shown in SEQ ID NO.11 - 12.

[0073] 2. Experimental procedures

[0074] (1) Synthesize the sequence into the pcDNA3.4 vector through XbaⅠ and EcoRⅤ.

[0075] (2) Transfection: Passage one day in advance at 2.5 - 3×10 6 cells / ml, and perform transfection 18 - 22 hours later (the cell density is expected to reach 4 - 6×10 6 cells / ml).

[0076] Prepare 1 EP tube, add culture medium (calculated according to 0.08 mL of culture medium per mL of cells), add plasmid to the culture medium (added according to the ratio of 1.5 μg of plasmid per mL of cells), and let it stand at room temperature for 5 min. Add PEI to the plasmid (plasmid:PEI = 1.5:7), mix well, and let it stand at room temperature for 20 min.

[0077] Add the DNA-PEI mixture to the cells, shaking the cell flask while adding. After 24 h, add 10% MetaCell CHOTransFeed and 1% MetaCell Titer Enhancer, and add 10% MetaCell CHOTransFeed again after 120 h.

[0078] Observe the cell viability, and harvest the supernatant by centrifugation after 10 - 12 days.

[0079] (3) Purify using a Protein A affinity chromatography column, and then use a G25 desalting column to perform solvent replacement on the concentrated protein. The solvent is replaced with 20 mM PBS to obtain the mouse-cat chimeric anti-cat CD20 antibody.

[0080] 3. Identification

[0081] (1) SDS-PAGE detection

[0082] Take 5 μg of the purified mouse-cat chimeric anti-cat CD20 antibody for SDS-PAGE detection.

[0083] The sample was analyzed by Coomassie brilliant blue staining after SDS-PAGE. The results showed that a single band was visible around 180 KD for the non-reducing antibody, and single bands were visible at 55 KD and 25 KD for the reducing antibody, indicating that the murine-cat chimeric anti-cat CD20 antibody could be expressed in eukaryotic cells. The results are shown in Figure 1 .

[0084] (2) HPLC detection

[0085] The purified murine-cat chimeric anti-cat CD20 antibody was subjected to HPLC detection to determine the purity of the purified CD20 antibody. The results showed that the purity of the murine-cat chimeric anti-cat CD20 antibody could reach 99.67%. The detailed results are shown in Figure 2 .

[0086] Example 2 Identification of murine-cat chimeric anti-cat CD20 antibody

[0087] 1. Identification of antibody bioactivity by indirect ELISA

[0088] (1) Operating steps:

[0089] Coating: Dilute the purified CD20 antigen with the coating solution to a final concentration of 1.25 μg / ml, 100 μl / well, and coat overnight at 4°C;

[0090] Blocking: Block with 5% skim milk powder, 200 μl / well, and block at 37°C for 2 h;

[0091] Washing the plate: Wash the plate 5 times with PBST, 5 min each time;

[0092] Incubating the primary antibody: Take the purified anti-cat CD20 antibody and dilute it serially with PBS (2×, 4×, 8×, 16×), 100 μl / well; Use PBS as a blank control, 100 μl / well, and incubate in a 37°C incubator for 1.5 h.

[0093] Washing the plate: Wash the plate 5 times with PBST, 5 min each time;

[0094] Incubating the secondary antibody: Add HRP-labeled goat anti-cat IgG (diluted with PBS at a ratio of 1:2000), 100 μl / well, and incubate at 37°C for 1 h;

[0095] Washing the plate: Wash the plate 5 times with PBST, 5 min each time;

[0096] Color development: Add TMB to the color development solution in the dark, 100 μl / well, and develop color in the dark for 3 min.

[0097] Termination: Add 0.5 M H2SO4 to terminate the color development, 50 μl / well.

[0098] Reading: Immediately read the OD using a machine 450Value

[0099] (2) Identification results

[0100] The bioactivity of the purified murine-cat chimeric anti-cat CD20 antibody was identified. The results showed that the murine-cat chimeric anti-cat CD20 antibody had the bioactivity of binding to the CD20 antigen. The results are shown in Table 2 for details.

[0101] Table 2

[0102] Antibody concentration (μg / ml) OD value 300 2.967 150 2.892 75 2.813 37.5 2.726 18.75 1.846 9.375 1.165 4.6875 0.701 2.34375 0.459

[0103] Example 3 Mouse treatment experiment of murine-cat chimeric anti-cat CD20 antibody

[0104] 1. Model establishment

[0105] Mice are animal models with relatively high maturity and recognition, so they were selected as the experimental subjects. The mice were randomly divided into a CD20 antibody treatment group, a high-dose CD20 antibody treatment group, a model control group, and a negative (blank) control group. Bovine type II collagen solution was mixed with Freund's complete adjuvant (Freund's incomplete adjuvant was used for booster immunization) in equal volume and emulsified thoroughly in an ice bath.

[0106] The CD20 antibody treatment group and the model group were subcutaneously injected with collagen emulsion at the tail, 100 μl per mouse. The same day was defined as D0, and booster immunization was performed on D21. The control group was injected with physiological saline correspondingly.

[0107] 2. Drug administration for treatment

[0108] When the mice showed joint swelling and redness, the mice in the CD20 antibody treatment group were given the murine-cat chimeric anti-cat CD20 antibody by tail vein injection at a dose of 200 μg / g, and the control group was injected with physiological saline.

[0109] After 21 days of interval, the second drug administration was carried out according to the route and dose of the first drug administration.

[0110] 3. Observation of clinical symptoms and scoring criteria

[0111] The body weight and clinical symptoms of the mice were observed and recorded, and the mice were scored for arthritis index (AI). The AI scoring criteria are shown in Table 3:

[0112] Table 3

[0113] Score Performance 0 points Normal 1 point Erythema and slight swelling appear at the ankle joint 2 points Erythema and slight swelling appear from the ankle joint to the metatarsophalangeal joint or metacarpophalangeal joint 3 points Erythema and moderate swelling appear from the ankle joint to the metatarsophalangeal joint or metacarpophalangeal joint 4 points Erythema and severe swelling appear from the ankle joint to the toe joint or metacarpophalangeal joint

[0114] The sum of the limb scores of each mouse is the arthritis index score. The higher the arthritis index score, the more severe the clinical symptoms. The swelling degree of the arthritic paw of the mouse was scored. The scoring criteria were that each paw of the mouse was divided into an ankle joint and 5 finger joints, and one place of redness and swelling was counted as 1 point. The sum of the scores of the four limbs of each mouse was the swelling degree score of the arthritic paw. The higher the swelling degree score of the arthritic paw, the more severe the clinical symptoms.

[0115] 4. Clinical symptom scores of mice in each group

[0116] Compared with the normal control group mice, the collagen-injected mice showed weight loss, listlessness, reduced food intake and other activities. On the 38th day after the first immunization, obvious redness and swelling appeared in the ankle joints of all collagen-injected mice in each group, and all the models were successfully established. Among them, the mice in the model group had gradually severe joint swelling, slow gait, difficulty in bearing weight, limited joint movement, and then slowly developed joint ankylosis, deformity and even dysfunction, and inconvenient movement.

[0117] The mice in the CD20 antibody treatment group were given drug treatment on the 38th day after the first immunization. The results are shown in Figure 3 、 Figure 4 and Figure 5 。

[0118] After the mice in the CD20 antibody treatment group received treatment, the joint swelling, spirit, food intake, activity and weight changes were alleviated compared with the model group, but there was no significant difference in the weight changes between the CD20 antibody treatment group and the model group. The changes in the body weights of the mice in each group are shown in Figure 3 。The joints of the healthy control group mice showed no redness and swelling. The collagen-injected mice gradually showed different degrees of joint inflammation about 5-6 weeks after the first collagen injection. About 6-8 weeks, the joints of the mice were significantly swollen and the scores increased. After drug treatment, the joint swelling degree of the mice in the CD20 antibody treatment group was reduced compared with the model group, and the score was lower than that of the model group mice; the arthritis symptom remission time of the mice in the CD20 antibody treatment group was earlier than that of the model group. The results are shown in Figure 4 、 Figure 5 。

[0119] Therefore, according to the clinical symptom scores of the mice in each group, it can be seen that the swelling degree of the mice treated with CD20 antibody is reduced and the symptoms subside faster, which proves that the CD20 antibody can be normally synthesized and expressed, can correctly act on the target antigen, and has good therapeutic effects.

[0120] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A murine-cat chimeric anti-cat CD20 antibody, characterized in that, The heavy chain of the murine-cat chimeric anti-cat CD20 antibody comprises the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.3; The light chain of the murine-cat chimeric anti-cat CD20 antibody comprises the amino acid sequence shown in SEQ ID NO.2 and / or SEQ ID NO.

4.

2. The murine-cat chimeric anti-cat CD20 antibody according to claim 1, wherein The heavy chain of the murine-cat chimeric anti-cat CD20 antibody comprises any one of the amino acid sequences shown in (I), (II) or (III): (I) The amino acid sequence shown in SEQ ID NO.5; (II) An amino acid sequence having ≥98% similarity to the amino acid sequence shown in SEQ ID NO.5; (III) An amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.5; The light chain of the murine-cat chimeric anti-cat CD20 antibody comprises any one of the amino acid sequences shown in (IV), (V) or (VI): (IV) The amino acid sequence shown in SEQ ID NO.6; (V) An amino acid sequence having ≥98% similarity to the amino acid sequence shown in SEQ ID NO.6; (VI) An amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.

6.

3. The murine-cat chimeric anti-cat CD20 antibody according to claim 1, wherein The murine-cat chimeric anti-cat CD20 antibody is linked to the human IL-2 signal peptide.

4. A gene sequence encoding the murine-cat chimeric anti-cat CD20 antibody according to any one of claims 1 to 3.

5. The gene sequence according to claim 4, wherein The gene sequence is further linked to the gene sequence of the human IL-2 signal peptide and / or the Kozak sequence.

6. The gene sequence according to claim 4 or 5, characterized in that, The gene sequence is optimized according to the CHO cell codons. After optimization, the gene sequence encoding the heavy chain is as shown in SEQ ID NO.11, and the gene sequence encoding the light chain is as shown in SEQ ID NO.

12.

7. An expression vector, characterized in that, Containing the gene sequence according to any one of claims 4 to 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the murine-cat chimeric anti-cat CD20 antibody according to any one of claims 1 to 3, the gene sequence according to any one of claims 4 to 6 or the expression vector according to claim 7.

9. Use of the murine-cat chimeric anti-cat CD20 antibody according to any one of claims 1 to 3, the gene sequence according to any one of claims 4 to 6 or the expression vector according to claim 7 in the preparation of a medicament for treating feline rheumatoid arthritis.

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