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

Abstract

This application discloses a K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, its hybridoma cell line, and its applications. The sequence information of the K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody is shown in SEQ NO:1 to SEQ NO:2. As shown in NO:48, the accession numbers of the hybridoma cell lines are CCTCC:C2023296, CCTCC:C2023311, CCTCC:C2023312, CCTCC:C2023313, CCTCC:C2023314 and CCTCC:C2024339, respectively. The K47 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application can specifically bind to the capsular polysaccharide of Klebsiella pneumoniae, which is beneficial for the typing of Klebsiella pneumoniae in clinical practice. In both in vivo and in vitro animal experiments, it has been shown to have excellent effects in the prevention and treatment of K47 Klebsiella pneumoniae 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 new generation Klebsiella pneumoniae drugs.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and in particular relates to a K47 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. K47 Klebsiella pneumoniae is typically associated with more severe infections in clinical settings, particularly in immunocompromised patients.

[0004] Therefore, there is a need in this field to develop a K47 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 K47 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 K47-5B9-F8, with the accession number CCTCC NO: C2023314. The hybridoma cell line is used to prepare the aforementioned Klebsiella pneumoniae capsular polysaccharide monoclonal antibody.

[0008] Thirdly, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 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 K47-15D7-B10, with the accession number CCTCC NO: C2023312. The hybridoma cell line is used to prepare the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody described in the third aspect.

[0011] Fifthly, this application provides a Klebsiella pneumoniae capsular polysaccharide monoclonal antibody, wherein the Klebsiella pneumoniae is K47 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: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 K47-18G6-B2, with the accession number CCTCC NO: C2023311. The hybridoma cell line is used to prepare the 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 K47 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 K47-6E2, with accession number CCTCC NO: C2023296, 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 K47 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 K47-20C10-B8, with the accession number CCTCC NO: C2023313. The hybridoma cell line 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 K47 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 K47-4H8, with the accession number CCTCC:C2024339, 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 pharmaceutical composition comprising any one of the preceding Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies, 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.

[0024] In a fourteenth aspect, this application provides a kit for detecting Klebsiella pneumoniae type K47, 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.

[0025] In a fifteenth aspect, this application provides the use of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody described in any one of the preceding statements in the preparation of a therapeutic and / or diagnostic product for Klebsiella pneumoniae 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.

[0026] Capsular polysaccharides are important toxicological factors of Klebsiella pneumoniae. This application uses capsular polysaccharides coupled with carrier proteins to enhance immunogenicity, and prepares K47 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.

[0027] The beneficial effects of this application are as follows:

[0028] The monoclonal antibody against Klebsiella pneumoniae capsular polysaccharide provided in this application can specifically bind to the capsular polysaccharide of Klebsiella pneumoniae type K47, which is beneficial for the clinical typing of Klebsiella pneumoniae. In vivo and in vitro animal experiments have demonstrated its excellent efficacy in the prevention and treatment of Klebsiella pneumoniae type K47 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

[0029] Figure 1 This is a graph showing the detection results of the titer level of the K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application using the indirect ELISA method in Example 2.

[0030] Figure 2 This is a graph showing the flow cytometry results of the binding of monoclonal antibody K47-20C10-B8 to capsular Klebsiella pneumoniae HVKP4 and non-capsular Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout) in Example 4.

[0031] Figure 3 This is a graph showing the flow cytometry analysis results of the binding of monoclonal antibody K47-18G6-B2 to capsular Klebsiella pneumoniae HVKP4 and non-capsular Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout) in Example 4.

[0032] Figure 4 The in vitro bactericidal curves of K47-5B9-F8 and K47-15D7-B10 against Klebsiella pneumoniae HVKP4 strain are shown in Example 5.

[0033] Figure 5 The survival curve of the K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody prophylaxis test provided in this application is shown in Example 6. Detailed Implementation

[0034] 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 in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. The reagents involved in the examples below are commercially available reagents in the art unless otherwise specified.

[0035] The following is information on strain preservation:

[0036] The hybridoma cell lines K47-5B9-F8 (CCTCC NO:C2023314), K47-20C10-B8 (CCTCC NO:C2023313), K47-15D7-B10 (CCTCC NO:C2023312), K47-18G6-B2 (CCTCC NO:C2023311), and K47-6E2 (CCTCC NO:C2023296) provided in this application were all deposited at the China Center for Type Culture Collection on October 17, 2023.

[0037] The hybridoma cell line K47-4H8 (CCTCC:C2024339) provided in this application was deposited at the China Center for Type Culture Collection on September 25, 2024.

[0038] Location of the collection: Wuhan University, Wuhan City, Hubei Province.

[0039] Collection name and collection number:

[0040] Hybridoma cell line K47-5B9-F8, CCTCC NO: C2023314.

[0041] Hybridoma cell line K47-20C10-B8, CCTCC NO: C2023313.

[0042] Hybridoma cell line K47-15D7-B10, CCTCC NO: C2023312.

[0043] Hybridoma cell line K47-18G6-B2, CCTCC NO: C2023311.

[0044] Hybridoma cell line K47-6E2, CCTCC NO: C2023296.

[0045] Hybridoma cell line K47-4H8: Hybridoma cell line K47-6E2, CCTCC:C2024339.

[0046] In Example 5 of this application, the negative control antibody light chain variable region (VL) sequence used (as shown in SEQ ID NO:49) is as follows:

[0047] QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPRTFGGGTKLEIK

[0048] The negative control antibody heavy chain variable region (VH) sequence used (as shown in SEQ ID NO:50):

[0049] QVQLQQPGAELVRPGASVKLSCKASGYTFTTYWMNWIKQRPEQGLEWIGRIDPYDSETHYSQKFKDKAILTVDKSSSTAYMQLSSSLTSEDSAVYYCARGYANGAFVYWGQGTLVTVSA

[0050] Example 1: Preparation of monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

[0051] 1) Establishment of a Klebsiella pneumoniae K47 capsular polysaccharide monoclonal antibody cell line

[0052] The capsular polysaccharide extracted from K47 Klebsiella pneumoniae (GDMCC No: 62131) after fermentation was chemically coupled with a carrier protein (a non-toxic variant of diphtheria toxoid CRM197) to obtain a K47 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). K47 capsular polysaccharide-protein conjugate was 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, five monoclonal cell lines were obtained: hybridoma cell line K47-5B9-F8 (CCTCC NO:C2023314), hybridoma cell line K47-20C10-B8 (CCTCC NO:C2023313), hybridoma cell line K47-15D7-B10 (CCTCC NO:C2023312), hybridoma cell line K47-18G6-B2 (CCTCC NO:C2023311), and hybridoma cell line K47-6E2 (CCTCC NO:C2023311). The study used two hybridoma cell lines, NO:C2023296 and K47-4H8 (CCTCC:C2024339), and their purity was verified by sequencing.

[0053] The sequence information of six Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies is as follows (SEQ NO:1~SEQ NO:48 in sequence):

[0054] K47-5B9-F8-Light chain variable region (VL) sequence:

[0055] MDFQVQIFSLLLISVTVIVSCGEIVLTQSPTTLAASPGEKITITCSASSSISSNYLHWYQQK

[0056] PGFSPKLLIYRTSNLASGLPARFSGSGSGTSYSLTIGTMEAEDVATYFCQQGGGIPRITFG

[0057] SGTKLEIK

[0058] K47-5B9-F8 heavy chain variable region (VH) sequence:

[0059] MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVR

[0060] QPPGKALEWLGFIRNKANGYTTEYTASVKGRFTISRDNSQSILYLQMNTLRAEDSATYY

[0061] CAREAYRFDGAMDYWGQGTSVTVSS

[0062] The complementarity-determining region 1 (LCDR1) sequence of the K47-5B9-F8 light chain:

[0063] The complementarity-determining region 2 (LCDR2) sequence of the SASSSISSNYLH K47-5B9-F8 light chain:

[0064] The complementarity-determining region 3 (LCDR3) sequence of the RTSNLAS K47-5B9-F8 light chain:

[0065] The complementarity-determining region 1 (HCDR1) sequence of the QQGGGIPRIT K47-5B9-F8 heavy chain:

[0066] The complementarity-determining region 2 (HCDR2) sequence of the DYYMN K47-5B9-F8 heavy chain:

[0067] FIRNKANGYTTEYTASVKG K47-5B9-F8-heavy chain complementarity-determining region 3 (HCDR3) sequence:

[0068] EAYRFDGAMDY K47-15D7-B10-Light Chain Variable Region (VL) Sequence:

[0069] MDFQVQIFSLLLISVTVIVSNGEIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQ

[0070] KPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPRITF

[0071] GSGTKLEIK

[0072] K47-15D7-B10 heavy chain variable region (VH) sequence:

[0073] MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWFRQ

[0074] PPGKALEWLGFIRNKANGYTIEYTPSVQGRFTISRDNSQSILYLHMITLRTEDSATYYCA

[0075] REAYRFDGAMDYWGQGTSVTVSS

[0076] The complementarity-determining region 1 (LCDR1) sequence of the K47-15D7-B10 light chain:

[0077] The complementarity-determining region 2 (LCDR2) sequence of the SASSSISSNYLH K47-15D7-B10 light chain:

[0078] The complementarity-determining region 3 (LCDR3) sequence of the RTSNLAS K47-15D7-B10 light chain:

[0079] The complementarity-determining region 1 (HCDR1) sequence of the QQGSSIPRIT K47-15D7-B10 heavy chain:

[0080] The complementarity-determining region 2 (HCDR2) sequence of the DYYMN K47-15D7-B10 heavy chain:

[0081] FIRNKANGYTIEYTPSVQG K47-15D7-B10-heavy chain complementarity-determining region 3 (HCDR3) sequence:

[0082] EAYRFDGAMDY K47-18G6-B2-Light Chain Variable Region (VL) Sequence:

[0083] MSPAQFLVLLLFWIPASRGDVVLTQTPLSLPVSFGDQVSISCRSSQSLVNSYGITYLSWY

[0084] LHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPW

[0085] TFGGGTKLEIK

[0086] K47-18G6-B2 heavy chain variable region (VH) sequence:

[0087] MEWTGIFILSVTAGVHSQVQLQQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRP

[0088] GHGLEWIGDFYPGSGSTYYNEKFKGKATLTADKSSSTAYMQVSSLTSEDAAVYFCTRK

[0089] GFYGTSGRYFAYWGQGTTLTVSS

[0090] The complementarity-determining region 1 (LCDR1) sequence of the K47-18G6-B2 light chain:

[0091] The complementarity-determining region 2 (LCDR2) sequence of the K47-18G6-B2-light chain:

[0092] The complementarity-determining region 3 (LCDR3) sequence of the GISNRFS K47-18G6-B2-light chain:

[0093] The complementarity-determining region 1 (HCDR1) sequence of the LQGTHQPWT K47-18G6-B2 heavy chain:

[0094] The complementarity-determining region 2 (HCDR2) sequence of the NYWLG K47-18G6-B2 heavy chain:

[0095] The complementarity-determining region 3 (HCDR3) sequence of the heavy chain: DFYPGSGSTYYNEKFKG K47-18G6-B2-

[0096] KGFYGTSGRYFAY K47-6E2-Light chain variable region (VL) sequence:

[0097] MDFQVQIFSLLLISVTVMVSNGEIVLTQSPTTMAASPGEKITITCSASSSVRSNYLHWYQ

[0098] QKPGCSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSMPRI

[0099] TFGSGTKLEIN

[0100] K47-6E2-heavy chain variable region (VH) sequence:

[0101] MNFGLSWVFLVTLLNGIQCEVKLVESGGGLVLPGGSLRLSCTTSGFTFTDYYMNWVRQ

[0102] PPGKALEWLGFIRNKANGYTTDYNASVKGRFTISRDNSQSILYLQMNTLRAEDSASYY

[0103] CARESYRYDGAMDYWGQGTSVTVSS

[0104] The complementarity-determining region 1 (LCDR1) sequence of the K47-6E2 light chain:

[0105] The complementarity-determining region 2 (LCDR2) sequence of the SASSSVRSNYLH K47-6E2-light chain:

[0106] The complementarity-determining region 3 (LCDR3) sequence of the RTSNLAS K47-6E2-light chain:

[0107] The complementarity-determining region 1 (HCDR1) sequence of the QQGSSMPRIT K47-6E2 heavy chain:

[0108] The complementarity-determining region 2 (HCDR2) sequence of the DYYMN K47-6E2 heavy chain:

[0109] FIRNKANGYTTDYNASVKG K47-6E2-Complementarity Determinant Region 3 (HCDR3) Sequence:

[0110] ESYRYDGAMDY K47-20C10-B8-Light Chain Variable Region (VL) Sequence:

[0111] MESHSQVFIFLLFWIPVSRGDILLTQSPAILSVSPGERVSFSCRASQSIGRSIHWYQQRTN

[0112] GSPRLLIKYASESIYGIPSRFSGSGSGTDFTLSINSVESEDIAAYYCQQSYNWPRTFGGGT

[0113] KLEIK

[0114] K47-20C10-B8 heavy chain variable region (VH) sequence:

[0115] MNFGLSWVFLVALLNGVQCQVQLVETGGGLVRPGNSLNLSCITSGFTFSNYRLHWLRQ

[0116] PPGKGLEWLAVIAVKSDNFGAIYADSVKGRFTISRDDSRSSVYLQMNRLREEDTATYYC

[0117] VRAGVSFFDYWGQGTSLTVSS

[0118] The complementarity-determining region 1 (LCDR1) sequence of the K47-20C10-B8 light chain:

[0119] The complementarity-determining region 2 (LCDR2) sequence of the RASQSIGRSIH K47-20C10-B8-light chain:

[0120] The complementarity-determining region 3 (LCDR3) sequence of the YASESIY K47-20C10-B8 light chain:

[0121] QQSYNWPRT

[0122] The complementarity-determining region 1 (HCDR1) sequence of the K47-20C10-B8 heavy chain:

[0123] NYRLH

[0124] The complementarity-determining region 2 (HCDR2) sequence of the K47-20C10-B8 heavy chain:

[0125] VIAVKSDNFGAIYADSVKG

[0126] The complementarity-determining region 3 (HCDR3) sequence of the K47-20C10-B8 heavy chain:

[0127] AGVSFFDY

[0128] K47-4H8-Light chain variable region (VL) sequence:

[0129] MSPTQFLVLLLFWIPASRGDVVTQTPLSLPVSFGDQVSISCRSSQSLANSYGNTYLSW

[0130] YLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP

[0131] WTFGGGTKLEIK

[0132] K47-4H8-heavy chain variable region (VH) sequence:

[0133] MGWSVVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQ

[0134] RPGHGLEWIGDIYPGGDKTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSGVYFCAR

[0135] KGYYGSNGRYFAYWGQGTTLTVSS

[0136] The complementarity-determining region 1 (LCDR1) sequence of the K47-4H8-light chain:

[0137] RSSQSLANSYGNTYLS

[0138] The complementarity-determining region 2 (LCDR2) sequence of the K47-4H8-light chain:

[0139] GISNRFS

[0140] The complementarity-determining region 3 (LCDR3) sequence of the K47-4H8-light chain:

[0141] LQGTHQPWT

[0142] The complementarity-determining region 1 (HCDR1) sequence of the K47-4H8 heavy chain:

[0143] NYWLG

[0144] The complementarity-determining region 2 (HCDR2) sequence of the K47-4H8 heavy chain:

[0145] DIYPGGDKTYYNEKFKG

[0146] The complementarity-determining region 3 (HCDR3) sequence of the K47-4H8 heavy chain:

[0147] KGYYGSNGRYFAY

[0148] 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 for use in this application and in one or more claims that may be included herein.

[0149] 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 fragments 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.

[0150] 2) Preparation and purification of ascites fluid containing Klebsiella pneumoniae K47 polysaccharide monoclonal antibody

[0151] 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.

[0152] 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 90%. After sterile filtration through a 0.22 μm filter membrane, the antibodies were stored in PBS at pH 7.0-7.4.

[0153] Example 2: Detection of titer levels of monoclonal antibodies against Klebsiella pneumoniae K47 capsular polysaccharide

[0154] After cell fusion, screening, and several subcloning processes, six monoclonal antibody cell lines were obtained. The ELISA results of the cell culture supernatant binding to K47 capsular polysaccharide are shown in Table 1 below.

[0155] Table 1

[0156] K47-5B9-F8 2.335 K47-15D7-B10 1.826 K47-18G6-B2 2.158 K47-6E2 2.632 K47-20C10-B8 1.987 K47-4H8 0.9188

[0157] 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 all six monoclonal antibodies have good binding affinity to K47 capsular polysaccharide.

[0158] Example 3: ELISA detection of binding specificity of monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

[0159] The specificity of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application in reaction with capsular polysaccharides of serotypes 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 K47 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody was added to react with the coated polysaccharides at a ratio of 50 μL per well, and incubated overnight. After washing the plates, a secondary antibody diluted 1:30,000 was added to each well at a ratio of 100 μL, and incubated for 2 hours. After washing, 1 mg / mL PNPP-Na chromogenic substrate was added to each well at a ratio of 100 μL, and incubated for 2 hours. The reaction was stopped by adding 3M NaOH to each well at a ratio of 50 μL, and the OD405 value was read using an ELISA reader.

[0160] The results of the binding of the six Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided in this application to Klebsiella pneumoniae capsular polysaccharides of different serotypes are shown in Table 2 below:

[0161] Table 2

[0162]

[0163]

[0164] As can be seen from Table 2 above, the six Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided in this application are specific for K47 capsular polysaccharide, but do not cross-react with capsular polysaccharides of other serotypes of Klebsiella pneumoniae.

[0165] Example 4: Flow cytometry detection of the binding of Klebsiella pneumoniae K47 capsular polysaccharide monoclonal antibody to Klebsiella pneumoniae K47.

[0166] Highly virulent Klebsiella pneumoniae strains HVKP4 and 19-249ΔwbaP (capsular polysaccharide expression gene knockout) were 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 culture was centrifuged at 8000 g for 10 min, and the precipitate was collected. The precipitate was washed twice with PBS and resuspended in 0.2 mL of PBS. After fixation with 4% paraformaldehyde for 10 min, the precipitate was washed and blocked with 2% BSA for 1 h. The precipitate was then incubated with K47 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 precipitate was resuspended in PBS and detected by flow cytometry.

[0167] Test results as follows Figure 2 As shown, Figure 2 To analyze the binding peaks of monoclonal antibody K47-20C10-B8 with capsular Klebsiella pneumoniae HVKP4 and non-capsular Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout) by flow cytometry, Figure 3 The diagram shows the binding peaks of monoclonal antibody K47-18G6-B2 with Klebsiella pneumoniae HVKP4 and bacterial strain 19-249ΔwbaP (capsular polysaccharide expression gene knockout) for flow cytometry analysis.

[0168] See Figure 2 It can be seen that monoclonal antibody K47-20C10-B8 can bind to the highly virulent Klebsiella pneumoniae strain HVKP4, but does not bind to Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout). This shows that monoclonal antibody K47-20C10-B8 can be used for the diagnosis and typing of clinical strains.

[0169] See Figure 3 The binding of monoclonal antibody K47-18G6-B2 to Klebsiella pneumoniae HVKP4 was detected. The results showed that monoclonal antibody K47-18G6-B2 could bind to capsulated strains, but not to Klebsiella pneumoniae 19-249ΔwbaP (capsular polysaccharide expression gene knockout). It can be seen that monoclonal antibody K47-18G6-B2 can be used for the diagnosis and typing of clinical strains.

[0170] Example 5: Bactericidal assay of monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

[0171] This embodiment illustrates the opsonization and phagocytosis bactericidal assay of the K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application, specifically: a carbapenem-resistant Klebsiella pneumoniae HVKP4 strain diluted 10... 5 CFU / mL was added at 10 μL / well to a 96-well cell culture plate. The serially diluted Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application was added at 20 μL / well to the above cell culture 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. 7 cells / mL, then add 1×10 7 Cell suspension at a concentration of 1 / mL was mixed with diluted complement at a volume ratio of 1:4. 50 μL of this mixture was added 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 and shaking for 45 min. 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.

[0172] like Figure 4The image shows the in vitro bactericidal curves of the K47 type Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies K47-5B9-F8 and K47-15D7-B10 against Klebsiella pneumoniae HVKP4 strain, provided in this application. Figure 4 It can be seen that K47-5B9-F8 and K47-15D7-B10 have good bactericidal effects against the highly virulent strain HVKP4 in vitro, with the highest bactericidal rates reaching 91.5% and 95.5%, respectively. These results show significant differences compared to the negative control antibody.

[0173] Example 6: Prophylactic test of monoclonal antibody against Klebsiella pneumoniae K47 capsular polysaccharide

[0174] This embodiment illustrates the protective test of the K47 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody provided in this application. Specifically, six Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies were administered intraperitoneally to five mice in each group, with each mouse receiving 0.25 mg. PBS was used as a negative control. Four hours later, the highly virulent K47 Klebsiella pneumoniae strain HVKP4 was injected at 8.5 × 10⁻⁶ mg. 5 The bacterial count of CFU was used to infect Balb / c mice (n=5) via intraperitoneal infection. The survival rate of the mice was observed 10 days after infection, and survival curves were plotted.

[0175] like Figure 5 The image shows the survival curves of the prophylactic test for Klebsiella pneumoniae capsular polysaccharide monoclonal antibodies provided in this application. It can be seen that 4 hours after administration of the six K47 capsular polysaccharide monoclonal antibodies and the control PBS, the amount of Klebsiella pneumoniae K47 (strain number HVKP4) was 8.5 × 10⁻⁶. 5 CFU challenge resulted in a 20% survival rate in the control group and a 100% survival rate in all five monoclonal antibody groups, which was statistically significant (p<0.0001).

[0176] 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 K47 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 shown in SEQ NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ NO:

2. The amino acid sequences of the complementarity-determining region of the light chain variable region of the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody are SEQ ID:3, SEQ ID:4, and SEQ ID:5; and the amino acid sequences of the complementarity-determining region of the heavy chain variable region are SEQ ID:6, SEQ ID:7, and SEQ ID:

8.

2. A hybridoma cell line, characterized in that, The hybridoma cell line is named K47-5B9-F8, and its preservation number is CCTCC NO:C2023314. The hybridoma cell line is used to prepare the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody of claim 1, and the pharmaceutical composition further comprises any one or more combinations of pharmaceutically acceptable carriers or diluents.

4. A kit for detecting Klebsiella pneumoniae type K47, characterized in that, The kit for detecting Klebsiella pneumoniae type K47 includes the Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1, and the kit for detecting Klebsiella pneumoniae type K47 also includes any 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 Klebsiella pneumoniae capsular polysaccharide monoclonal antibody as described in claim 1 in the preparation of therapeutic drugs and / or detection products for Klebsiella pneumoniae type K47 infection.

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