Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, hybridoma cell strain thereof and application thereof

Abstract

The application discloses K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies, hybridoma cell strains thereof and application of the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies, sequence information of the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies is shown as SEQ NO:1-SEQ NO:56, preservation numbers of the hybridoma cell strains are CCTCC:C2023365, CCTCC:C2023366, CCTCC:C2023367, CCTCC:C2024340, CCTCC:C2024341, CCTCC:C2024342 and CCTCC:C2024343 respectively, the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided by the application can be specifically combined with capsular polysaccharide of Klebsiella, are favorable for typing of Klebsiella pneumoniae in clinic, and can prove excellent effects of the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies in preventing and treating infection of Klebsiella pneumoniae in in-vivo and in-vitro tests, and the application provides a way for solving problems of Klebsiella pneumoniae infection and multiple drug resistance, has remarkable significance for preventing, diagnosing and treating Klebsiella pneumoniae infection, and has important value for developing a new generation of Klebsiella pneumoniae drugs.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and in particular relates to a K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, its hybridoma cell line and its application. Background Technology

[0002] Klebsiella pneumoniae is a capsule-encapsulated Gram-negative bacterium (G-). It primarily colonizes the digestive and respiratory tracts. When the body's resistance is lowered, it can cause lung infections, urinary tract infections, and even bloodstream infections. It is also one of the most common bacteria causing respiratory infections.

[0003] Klebsiella pneumoniae infections have become a serious global public health problem. Statistics show that the mortality rate of Klebsiella pneumoniae infection is extremely high; the mortality rate of bloodstream infections caused by Klebsiella pneumoniae is 20% to 30%, while the mortality rate of Klebsiella pneumoniae bacteremia complicated by pneumonia can reach over 50%. Treatment of Klebsiella pneumoniae mainly relies on antibiotics, but in recent years, due to the widespread use of various antibacterial drugs, Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases has developed resistance to almost all available β-lactams (including carbapenems). In the past 10 years, the rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has increased dramatically worldwide. Capsular polysaccharide is the main virulence factor of Klebsiella pneumoniae and an important protective antigen for the treatment or prevention of Klebsiella pneumoniae infection. Based on the different structures of capsular polysaccharides, Klebsiella pneumoniae can be divided into at least 77 serotypes; capsular polysaccharides are also known as K antigens or K types. Different K serotypes exhibit varying resistance profiles and differ in geographical and clinical contexts, potentially leading to different epidemiological patterns in hospital-acquired infections and thus directly impacting the choice of clinical treatment. Among them, K64 serotype K, despite fewer reported cases, can still cause serious infections, especially in immunocompromised individuals. Compared to other serotypes, K64 may exhibit unique pathogenic characteristics in certain situations. While K64 may have a different antibiotic resistance profile and may exhibit specific pathogenicity in certain environments or populations, clinical research on K64 Klebsiella pneumoniae is limited.

[0004] Therefore, there is a need in this field to develop a K64 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody with high specificity, good affinity, and strong binding force, which would be beneficial for the prevention and control of drug-resistant Klebsiella pneumoniae and its clinical application. Summary of the Invention

[0005] To address the above technical problems, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae. The sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:1 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:2 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0006] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:3, SEQ ID:4, and SEQ ID:5, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:3, SEQ ID:4, and SEQ ID:5; and the complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:6, SEQ ID:7, and SEQ ID:8, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:6, SEQ ID:7, and SEQ ID:8.

[0007] Secondly, this application provides a hybridoma cell line named K64-1, with the accession number CCTCC NO: C2023365. The hybridoma cell line is used to prepare the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the first aspect.

[0008] Thirdly, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae. The sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:9 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:10 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0009] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:11, SEQ ID:12 and SEQ ID:13, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one of SEQ ID:11, SEQ ID:12 and SEQ ID:13. The complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:14, SEQ ID:15 and SEQ ID:16, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one of SEQ ID:14, SEQ ID:15 and SEQ ID:16.

[0010] Fourthly, this application provides a hybridoma cell line named K64-23, with the accession number CCTCC NO: C2023366. The hybridoma cell line is used to prepare the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the third aspect.

[0011] Fifthly, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae. The sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:17 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:18 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0012] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:19, SEQ ID:20, and SEQ ID:21, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:19, SEQ ID:20, and SEQ ID:21. The complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:22, SEQ ID:23, and SEQ ID:24, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:22, SEQ ID:23, and SEQ ID:24.

[0013] In a sixth aspect, this application provides a hybridoma cell line named K64-24, with the accession number CCTCC NO: C2023367, which is used to prepare Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the fifth aspect.

[0014] In a seventh aspect, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:25 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:26 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0015] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:27, SEQ ID:28, and SEQ ID:29, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:27, SEQ ID:28, and SEQ ID:29; and the complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:30, SEQ ID:31, and SEQ ID:32, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:30, SEQ ID:31, and SEQ ID:32.

[0016] Eighthly, this application provides a hybridoma cell line named K64-3, with accession number CCTCC NO: C2024340, which is used to prepare Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the seventh aspect.

[0017] Ninthly, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:33 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:34 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0018] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:35, SEQ ID:36, and SEQ ID:37, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:35, SEQ ID:36, and SEQ ID:37. The complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:38, SEQ ID:39, and SEQ ID:40, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:38, SEQ ID:39, and SEQ ID:40.

[0019] In a tenth aspect, this application provides a hybridoma cell line named K64-5, with the accession number CCTCC NO: C2024341, which is used to prepare Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the ninth aspect.

[0020] In one aspect, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae, and the sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:41 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:42 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0021] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:43, SEQ ID:44, and SEQ ID:45, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:43, SEQ ID:44, and SEQ ID:45. The complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:46, SEQ ID:47, and SEQ ID:48, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:46, SEQ ID:47, and SEQ ID:48.

[0022] In the twelfth aspect, this application provides a hybridoma cell line named K64-8, with the accession number CCTCC NO: C2024342, which is used to prepare the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the eleventh aspect.

[0023] In a thirteenth aspect, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K64 type Klebsiella pneumoniae. The sequence of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is as shown in SEQ NO:49 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it. The amino acid sequence of the heavy chain variable region is as shown in SEQ NO:50 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to it.

[0024] Specifically, the complementary determining region of the light chain variable region comprises three sequences, as shown in SEQ ID:51, SEQ ID:52, and SEQ ID:53, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:51, SEQ ID:52, and SEQ ID:53. The complementary determining region of the heavy chain variable region comprises three sequences, as shown in SEQ ID:54, SEQ ID:55, and SEQ ID:56, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one of SEQ ID:54, SEQ ID:55, and SEQ ID:56.

[0025] In the fourteenth aspect, this application provides a hybridoma cell line named K64-27, with accession number CCTCC NO: C2024343, which is used to prepare Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in the fifteenth aspect.

[0026] In a fifteenth aspect, this application provides a pharmaceutical composition comprising any one of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies described above, wherein the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody can bind to Klebsiella pneumoniae capsular polysaccharide target antigens and non-Klebsiella pneumoniae capsular polysaccharide target antigens, and the pharmaceutical composition further comprises any one or more combinations of pharmaceutically acceptable carriers, excipients or diluents.

[0027] In a sixteenth aspect, this application provides a kit for detecting Klebsiella pneumoniae type K64, the kit comprising the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described above, and further comprising any one or more combinations of positive control, negative control, antibody diluent, chromogenic solution, stop solution, blocking solution or washing solution.

[0028] In a seventeenth aspect, this application provides the use of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody described in any of the preceding statements in the preparation of a therapeutic and / or diagnostic product for Klebsiella pneumoniae type K64 infection, the disease comprising one or more symptoms caused by Klebsiella infection, including pneumonia, urinary tract infection, sepsis / bacteremia / septicemia, neonatal sepsis I bacteremia / septicemia, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, pulmonary infection, purulent liver abscess, lung abscess, cellulitis, necrotizing myofascitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, or spondyloarthritis.

[0029] Capsular polysaccharides are important toxicological factors of Klebsiella pneumoniae. This application uses capsular polysaccharides coupled with carrier proteins to enhance immunogenicity, and prepares K64 type Klebsiella pneumoniae capsular polysaccharide-specific monoclonal antibodies using hybridoma technology. These antibodies have the characteristics of good specificity, high affinity, and high binding force to Klebsiella pneumoniae capsular polysaccharides.

[0030] The beneficial effects of this application are as follows:

[0031] The Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application can specifically bind to the capsular polysaccharide of Klebsiella pneumoniae type K64, which is beneficial for the clinical typing of Klebsiella pneumoniae. In vitro and in vivo animal experiments have demonstrated its excellent efficacy in the prevention and treatment of Klebsiella pneumoniae type K64 infection, providing a way to solve the problems of Klebsiella pneumoniae infection and multidrug resistance. It has significant implications for the prevention, diagnosis and treatment of Klebsiella pneumoniae infection and is of great value for the development of next-generation Klebsiella pneumoniae drugs. Attached Figure Description

[0032] Figure 1 This is a graph showing the detection results of the titer level of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application using the indirect ELISA method in Example 2.

[0033] Figure 2 This is a graph showing the flow cytometry results of the binding of K64 monoclonal antibody, negative control, and K64 type Klebsiella pneumoniae Y8 in Example 4.

[0034] Figure 3The in vitro bactericidal curves of monoclonal antibodies K64-1, K64-23, and K64-24 against a highly virulent strain of Klebsiella pneumoniae (number Y8) are shown in Example 5.

[0035] Figure 4 This is the survival curve of the K64-1 antibody prophylactic test in Example 6. Detailed Implementation

[0036] The technical solutions of this application will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] The following is information on strain preservation:

[0038] The hybridoma cell lines K64-1 (CCTCC NO:C2023365), K64-23 (CCTCC NO:C2023366), and K64-24 (CCTCC NO:C2023367) provided in this application were all deposited at the China Center for Type Culture Collection on November 28, 2023.

[0039] The hybridoma cell lines K64-3 (CCTCC NO:C2024340), K64-5 (CCTCC NO:C2024341), K64-8 (CCTCC NO:C2024342), and K64-27 (CCTCC NO:C2024343) provided in this application were all deposited at the China Center for Type Culture Collection on September 25, 2024.

[0040] Location of the collection: Wuhan University, Wuhan City, Hubei Province.

[0041] Collection name and collection number:

[0042] Hybridoma cell line K64-1: CCTCC NO: C2023365.

[0043] Hybridoma cell line K64-23: CCTCC NO: C2023366.

[0044] Hybridoma cell line K64-24: CCTCC NO: C2023367.

[0045] Hybridoma cell line K64-3: CCTCC NO: C2024340.

[0046] Hybridoma cell line K64-5: CCTCC NO: C2024341.

[0047] Hybridoma cell line K64-8: CCTCC NO: C2024342.

[0048] Hybridoma cell line K64-27: CCTCC NO: C2024343.

[0049] The negative control antibody light chain variable region (VL) sequence used in Examples 4 and 5 of this application (as shown in SEQ ID NO:57):

[0050] MESHSQVFIFLLFWIPVSRGDILLTQSPAILSVSPGERVSFSCRASQSIGRSIHWYQQRTN

[0051] GSPRLLIKYASESIYGIPSRFSGSGSGTDFTLSINSVESEDIAAYYCQQSYNWPRTFGGGT

[0052] KLEIK

[0053] The negative control antibody heavy chain variable region (VH) sequence used (as shown in SEQ ID NO:58):

[0054] MNFGLSWVFLVALLNGVQCQVQLVETGGGLVRPGNSLNLSCITSGFTFSNYRLHWLRQ

[0055] PPGKGLEWLAVIAVKSDNFGAIYADSVKGRFTISRDDSRSSVYLQMNRLREEDTATYYC

[0056] VRAGVSFFDYWGQGTSLTVSS

[0057] Example 1: Preparation of monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

[0058] 1) Establishment of a Klebsiella pneumoniae K64 capsular polysaccharide monoclonal antibody cell line

[0059] The capsular polysaccharide extracted from K64 type Klebsiella pneumoniae (GDMCC No: 62132) after fermentation was chemically coupled with a carrier protein (a non-toxic variant of diphtheria toxoid CRM197) to obtain a K64 type capsular polysaccharide-protein conjugate. The method used was a commonly used polysaccharide activation method, such as the cyanogen bromide method (refer to US6375846B1), 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (EP0720485), and the periodic acid oxidation method (US4711779). K64 polysaccharide-protein conjugates were added to Freund's adjuvant, and SPF-grade BALB / c mice were immunized multiple times. When the immunotiter was greater than 1:300,000, the spleens were harvested and hybridoma technology was used. After PEG1500 fusion, indirect ELISA screening of positive cell supernatants, and multiple subcloning, three monoclonal cell lines were obtained: hybridoma cell lines K64-1 (CCTCC NO: C2023365), K64-23 (CCTCC NO: C2023366), K64-24 (CCTCC NO: C2023367), K64-3 (CCTCC NO: C2024340), K64-5 (CCTCC NO: C2024341), K64-8 (CCTCC NO: C2024342), and K64-27 (CCTCC NO: C2024342). NO:C2024343), and its purity was verified by sequencing.

[0060] The sequence information of the seven Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies is as follows (SEQ NO:1~SEQ NO:56 in order):

[0061] K64-1 light chain variable region (VL) sequence (starting from FR-L1):

[0062] QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPA

[0063] RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK

[0064] K64-1 heavy chain variable region (VH) sequence (starting from FR-H1):

[0065] EVKLVESGGGLVKPGGSLKLSCAGSGFTFSSYGMSWVRQTPDKRLEWVATISGGGSYT

[0066] YYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARNYGYDGHYYAMDYWGQ

[0067] GTSVTVSS

[0068] Complementarity-determining region 1 (LCDR1) sequence of the K64-1 light chain:

[0069] SASSSISYMH

[0070] The complementarity-determining region 2 (LCDR2) sequence of the K64-1 light chain:

[0071] DTSKLAS

[0072] The complementarity-determining region 3 (LCDR3) sequence of the K64-1 light chain:

[0073] HQRSSYPWT

[0074] The complementarity-determining region 1 (HCDR1) sequence of the K64-1 heavy chain:

[0075] The complementarity-determining region 2 (HCDR2) sequence of the K64-1 heavy chain in SYGMS:

[0076] The complementarity-determining region 3 (HCDR3) sequence of the K64-1 heavy chain:

[0077] NYGYDGHYYAMDY K64-23 light chain variable region (VL) sequence (starting from FR-L1):

[0078] QIVLTQSPAIMSASPGEKVTMTCSATSIVNYMHWYQQKSGTSPKRWIYDTSKLASGVPA

[0079] RFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRT K64-23 Heavy chain variable region (VH) sequence (starting from FR-H1):

[0080] QVQLQQPWSELVRPGASVKLPCKASGYTFTSYWMNWVKQRPEQGLEWIGRIDPYDG

[0081] GTHYSQNFMDRAILTVDRSSNTAYMQLNSLTSEDSAVYYCARGYANGAFVYWGQGTL

[0082] VTVSA

[0083] The complementarity-determining region 1 (LCDR1) sequence of the K64-23 light chain:

[0084] The complementarity-determining region 2 (LCDR2) sequence of the SATSIVNYMH K64-23 light chain:

[0085] The complementarity-determining region 3 (LCDR3) sequence of the DTSKLAS K64-23 light chain:

[0086] The complementarity-determining region 1 (HCDR1) sequence of the K64-23 heavy chain of QQWSSNPRT:

[0087] The complementarity-determining region 2 (HCDR2) sequence of the SYWMN K64-23 heavy chain:

[0088] The complementarity-determining region 3 (HCDR3) sequence of the K64-23 heavy chain:

[0089] GYANGAFVY K64-24 light chain variable region (VL) sequence (starting from FR-L1):

[0090] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQRPGSSPKPWIYRTSNLASGVPAR

[0091] FSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK K64-24 Heavy chain variable region (VH) sequence (starting from FR-H1):

[0092] EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAYMDWVRQSPEKGLEWVAEIRSKANN

[0093] HATLYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTPYYYGSSQFAYWGQGTL

[0094] VTVSA

[0095] The complementarity-determining region 1 (LCDR1) sequence of the K64-24 light chain:

[0096] The complementarity-determining region 2 (LCDR2) sequence of the SASSSVSYMY K64-24 light chain:

[0097] The complementarity-determining region 3 (LCDR3) sequence of the RTSNLAS K64-24 light chain:

[0098] The complementarity-determining region 1 (HCDR1) sequence of the K64-24 heavy chain of QQYHSYPLT:

[0099] The complementarity-determining region 2 (HCDR2) sequence of the DAYMD K64-24 heavy chain:

[0100] The complementarity-determining region 3 (HCDR3) sequence of the K64-24 heavy chain:

[0101] YYYGSSQFAY K64-3 light chain variable region (VL) sequence (starting from FR-L1):

[0102] QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPA

[0103] RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK K64-3 Heavy chain variable region (VH) sequence (starting from FR-H1):

[0104] EVKLVESGGGLVKPGGSLTLSCAASGSIFSTYGMSWVRQTPEKRLEWVATISGGGRYTY

[0105] YPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARNYGYDGHYYPMDYWGQG

[0106] TSVTVSS

[0107] The complementarity-determining region 1 (LCDR1) sequence of the K64-3 light chain:

[0108] The complementarity-determining region 2 (LCDR2) sequence of the SASSSISYMH K64-3 light chain:

[0109] The complementarity-determining region 3 (LCDR3) sequence of the DTSKLAS K64-3 light chain:

[0110] The complementarity-determining region 1 (HCDR1) sequence of the HQRSSYPWT K64-3 heavy chain:

[0111] The complementarity-determining region 2 (HCDR2) sequence of the TYGMS K64-3 heavy chain:

[0112] The complementarity-determining region 3 (HCDR3) sequence of the K64-3 heavy chain:

[0113] NYGYDGHYYPMDY K64-5 light chain variable region (VL) sequence (starting from FR-L1):

[0114] QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPV

[0115] RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK K64-5 Heavy chain variable region (VH) sequence (starting from FR-H1):

[0116] EVKLVESGGGLVKPGGSLELSCAASGFTFSTYGMSWVRQTPEKRLEWVATISGGGSYT

[0117] YYVDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARNYGYDGHYYSMDYWGQ

[0118] GTSVTVSS

[0119] The complementarity-determining region 1 (LCDR1) sequence of the K64-5 light chain:

[0120] The complementarity-determining region 2 (LCDR2) sequence of the SASSSISYMH K64-5 light chain:

[0121] The complementarity-determining region 3 (LCDR3) sequence of the DTSKLAS K64-5 light chain:

[0122] The complementarity-determining region 1 (HCDR1) sequence of the HQRSSYPWT K64-5 heavy chain:

[0123] The complementarity-determining region 2 (HCDR2) sequence of the TYGMS K64-5 heavy chain:

[0124] The complementarity-determining region 3 (HCDR3) sequence of the K64-5 heavy chain:

[0125] NYGYDGHYYSMDY K64-8 light chain variable region (VL) sequence (starting from FR-L1):

[0126] QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPA

[0127] RFSGSGSGTSYSLTISSMEAEDAATYYCHQRSNYPWTFGGGTKLEIK K64-8 Heavy chain variable region (VH) sequence (starting from FR-H1):

[0128] EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGYYT

[0129] YYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCGRNYGYDGHYYAMDYWGQ

[0130] GTSVTVSS

[0131] The complementarity-determining region 1 (LCDR1) sequence of the K64-8 light chain:

[0132] The complementarity-determining region 2 (LCDR2) sequence of the SASSSISYMH K64-8 light chain:

[0133] DTSKLAS

[0134] The complementarity-determining region 3 (LCDR3) sequence of the K64-8 light chain:

[0135] HQRSNYPWT

[0136] The complementarity-determining region 1 (HCDR1) sequence of the K64-8 heavy chain:

[0137] SYGMS

[0138] The complementarity-determining region 2 (HCDR2) sequence of the K64-8 heavy chain:

[0139] TISGGGYYTYYPDSVKG

[0140] The complementarity-determining region 3 (HCDR3) sequence of the K64-8 heavy chain:

[0141] NYGYDGHYYAMDY

[0142] K64-27 light chain variable region (VL) sequence (starting from FR-L1):

[0143] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPAR

[0144] FSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK

[0145] K64-27 heavy chain variable region (VH) sequence (starting from FR-H1):

[0146] EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAYMDWVRQSPEKGLEWVAEIRSKANN

[0147] HATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTPYYYGSSQFAYWGQGT

[0148] LVTVSA

[0149] The complementarity-determining region 1 (LCDR1) sequence of the K64-27 light chain:

[0150] SASSSVSYMY

[0151] The complementarity-determining region 2 (LCDR2) sequence of the K64-27 light chain:

[0152] RTSNLAS

[0153] The complementarity-determining region 3 (LCDR3) sequence of the K64-27 light chain:

[0154] QQYHSYPLT

[0155] The complementarity-determining region 1 (HCDR1) sequence of the K64-27 heavy chain:

[0156] DAYMD

[0157] The complementarity-determining region 2 (HCDR2) sequence of the K64-27 heavy chain:

[0158] EIRSKANNHATYYAESVKG

[0159] The complementarity-determining region 3 (HCDR3) sequence of the K64-27 heavy chain:

[0160] YYYGSSQFAY

[0161] As described in this application, the term "CDR" or "complementarity-determining region" refers to a discontinuous antigen-binding site found within the variable region of a heavy-chain or light-chain polypeptide. In this application, the amino acid sequences of the CDRs listed above are all as shown in accordance with Kabat's definition rules (the sequences in the claims of this invention are also as shown in Kabat's definition rules). However, it is well known to those skilled in the art that the CDR of an antibody can be defined in various ways. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J.Mal.Biol., 273:927-948 (1997); MacCallum et al, J.Mal.Biol.262:732-745 (1996); Abhinandan and Martin, Mal.Immunol., 45:3832-3839 (2008); Lefranc MPet These specific regions have been described in al., Dev. Comp. Jmmunol., 27:55-77 (2003); and Honegger and Plicikthun, J. Mal Biol., 309:657-670 (2001), where these definitions include overlaps or subsets of amino acid residues when compared with each other. However, any definition used to indicate the CDR of an antibody or transplanted antibody or its variants is included within the scope of the terminology defined and used in this application. Algorithms and interfaces for CDR prediction are known in the art, for example, as described in Abhinandan and Martin, Mal. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The entire contents of the references cited in this paragraph are incorporated herein by reference and are applicable to this application and one or more claims that may be included herein.

[0162] As used herein, “monoclonal antibody” refers to a group of homologous antibodies that participate in the highly specific recognition and binding of a single antigenic determinant or epitope, encompassing full-length and complete monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, “monoclonal antibody” refers to such antibodies prepared in any number of ways (including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals). In humans, this category includes IgG1, IgG2, IgG3, and IgG4. In mice, this category includes IgG1, IgG2a, IgG2b, and IgG3. It is known in the art that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The monoclonal antibody or antigen-binding fragment described in the claims of this invention comprises the Fc fragment of antibodies in the above-described categories.

[0163] 2) Preparation and purification of ascites fluid containing Klebsiella pneumoniae K64 capsular polysaccharide monoclonal antibody

[0164] The obtained monoclonal antibody cells were cultured and then injected intraperitoneally with 0.5 mL of a solution at a concentration of 1×10⁻⁶. 6 Cells per mL were introduced into BALB / c mice sensitized with paraffin oil 7 days in advance. After 7-10 days, obvious abdominal distension was observed in the mice, and the ascites fluid was collected and purified.

[0165] Mouse ascites fluid was diluted with PBS, and crude purity was precipitated by adding 50% final concentration of ammonium sulfate. The crude purity was then obtained by Protein A affinity column chromatography and ion exchange chromatography to obtain monoclonal antibodies with a purity greater than 95%. After sterile filtration through a 0.22 μm filter membrane, the antibodies were stored in PBS at pH 7.0-7.4.

[0166] Example 2: Detection of titer levels of monoclonal antibodies against Klebsiella pneumoniae K64 capsular polysaccharide

[0167] After cell fusion, screening, and several subcloning processes, seven monoclonal antibody cell lines were obtained. The ELISA results of the cell culture supernatant binding to K64 capsular polysaccharide are shown in Table 1 below.

[0168] Table 1

[0169] Cell line number OD value K64-1 3.816 K64-23 1.315 K64-24 2.011 K64-3 3.227 K64-5 0.707 K64-8 3.362 K64-27 1.043

[0170] like Figure 1 As shown, the titer level of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application was detected by indirect ELISA. It can be seen that the supernatant of the seven cell strains all have good binding affinity to K64 capsular polysaccharide.

[0171] Example 3: ELISA detection of binding specificity of monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

[0172] The specificity of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application in reaction with capsular polysaccharides of types K47, K64, K19, K1, K2, K38, and K57 was evaluated by indirect ELISA. Klebsiella pneumoniae capsular polysaccharides of different serotypes were coated onto ELISA plates and incubated at 37°C for 3 hours. After blocking with 1% BSA for 1 hour, an appropriate amount of K64 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody was added to react with the coated polysaccharides, 50 μL per well, and incubated overnight. After washing the plates, AP-labeled goat anti-mouse secondary antibody was diluted 1:30,000 and added to each well, 100 μL per well. After incubation for 2 hours, the plates were washed, and then 1 mg / mL PNPP-Na chromogenic substrate was added to each well, 100 μL per well. After incubation for 2 hours, the reaction was stopped by adding 3M NaOH to 50 μL of each well, and the OD405 value was read using an ELISA reader.

[0173] The results of the binding of the seven K64 Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided in this application to Klebsiella pneumoniae capsular polysaccharides of different serotypes are shown in Table 2 below:

[0174] Table 2

[0175]

[0176]

[0177] As can be seen from Table 2 above, the seven K64 Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided in this application are specific for K64 capsular polysaccharide, but do not cross-react with capsular polysaccharides of other serotypes of Klebsiella pneumoniae.

[0178] Example 4: Flow cytometry detection of the binding of Klebsiella pneumoniae K64 capsular polysaccharide monoclonal antibody to Klebsiella pneumoniae K64.

[0179] Highly virulent K64-Serotype Klebsiella pneumoniae Y8 (Li, J.; Sheng, Y.; Ma, R.; Xu, M.; Liu, F.; Qin, R.; Zhu, M.; Zhu, X.; He, P. Identification of a Depolymerase Specific for K64-Serotype Klebsiella pneumoniae: Potential Applications in Capsular Typing and Treatment. Antibiotics 2021, 10, 144.) was inoculated into 5 ml of LB medium and cultured at 37°C and 250 rpm until the OD value reached 0.7. 0.2 mL of the bacterial suspension was centrifuged at 8000 g for 10 min, the precipitate was collected, washed twice with PBS, and resuspended in 0.2 mL of medium. In PBS, the cells were fixed with 4% paraformaldehyde for 10 min, washed, blocked with 2% BSA for 1 h, and then incubated with K64 capsular polysaccharide monoclonal antibody overnight. After washing, anti-mouse fluorescent antibody (brand - CST, catalog number - #4410S) was added and incubated in the dark for 1 h. After washing with PBS, the cells were resuspended in PBS and detected by flow cytometry.

[0180] After culturing the highly virulent Klebsiella pneumoniae strain Y8, the binding ability of it to the K64 monoclonal antibody was analyzed by flow cytometry. The results are as follows: Figure 2 As shown, seven K64 monoclonal antibodies were tested for binding to the highly virulent K64 Klebsiella pneumoniae strain Y8. The results showed that all seven K64 monoclonal antibodies could bind to Y8, while the negative control could not bind to Y8. This indicates that these seven K64 capsular polysaccharide monoclonal antibodies can be used for the diagnosis and typing of clinical strains.

[0181] Example 5: Bactericidal assay of monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

[0182] This embodiment illustrates the opsonization and phagocytosis bactericidal assay of the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application. Specifically, the K64 type highly virulent Klebsiella pneumoniae Y8 strain was diluted 10... 5 CFU / ml was added to 10 μL / well of a 96-well cell culture plate. K64 capsular polysaccharide monoclonal antibody, serially diluted, was added to 20 μL / well of the same plate. The cells and antibody were incubated at 700 rpm / min for 30 min. HL-60 cells differentiated from DMF were washed with HBSS buffer and adjusted to a concentration of 1×10⁻⁶. 7Cells / ml were added, and then 1×10⁷ cells / ml of cell solution was mixed with diluted complement at a 1:4 volume ratio. The mixture was added in 50 μL increments to each well of a 96-well cell culture plate. The 96-well plate was placed on a mixer and incubated in a CO₂ incubator at 37°C with 5% CO₂ for 45 min with shaking. After stopping phagocytosis, the cells were spotted onto blood agar plates and incubated overnight in a CO₂ incubator. The bactericidal rate of each antibody dilution was calculated.

[0183] like Figure 3 As shown, K64-type capsular polysaccharide monoclonal antibodies K64-1, K64-23, and K64-24 have good bactericidal effects against the highly virulent strain Y8 in vitro, with the highest bactericidal rates reaching 83.3% and 86.7%, respectively, which are significantly different from the negative control antibody.

[0184] Example 6: Prophylactic test of monoclonal antibody against Klebsiella pneumoniae K64 capsular polysaccharide

[0185] This embodiment illustrates the protective test of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application. Specifically, the K64-1 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody was administered intraperitoneally to 6 mice in each group, with each mouse receiving 0.025 mg. PBS was used as a negative control. Four hours later, the highly virulent Klebsiella pneumoniae strain Y8 was injected at 7.38 × 10⁻⁶ mg. 6 CFU was used to infect Balb / c mice (n=6) via intraperitoneal infection. The survival rate of the mice was observed 10 days after infection, and survival curves were plotted.

[0186] like Figure 4 As shown, the amount of Klebsiella pneumoniae 64 (strain number Y8) was 7.38 × 10⁻¹ 4 hours after administration of the monoclonal antibody K64-1 targeting the K64 capsular polysaccharide and the control PBS. 6 CFU challenge resulted in a 0% survival rate in the control group and a 100% survival rate in the monoclonal antibody group, which was statistically significant (p<0.05).

[0187] In summary, the above embodiments are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A monoclonal antibody against Klebsiella pneumoniae capsular polysaccharide, characterized in that, The Klebsiella pneumoniae is K64 type Klebsiella pneumoniae. The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO:

2. The sequences of complementarity-determining regions 1-3 of the light chain variable region of the monoclonal antibody are SEQ ID:3, SEQ ID:4, and SEQ ID:5, respectively; the sequences of complementarity-determining regions 1-3 of the heavy chain variable region of the monoclonal antibody are SEQ ID:6, SEQ ID:7, and SEQ ID:8, respectively.

2. A hybridoma cell line, characterized in that, The hybridoma cell line is named K64-1, with accession number CCTCC NO:C2023365. The hybridoma cell line is used to prepare the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1.

3. A pharmaceutical composition, characterized in that, The antibody comprises the K64 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1, and one or more combinations of a pharmaceutically acceptable carrier or diluent.

4. A kit for detecting K64 type Klebsiella pneumoniae, characterized in that, It includes the monoclonal antibody as described in claim 1, and one or more combinations of positive control, negative control, antibody diluent, chromogenic solution, stop solution, blocking solution or washing solution.

5. The use of the K64 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1 in the preparation of K64 Klebsiella pneumoniae infection treatment drugs and / or detection products.

Images