Antibodies or antigen-binding fragments bound to KPC enzymes and their applications

Abstract

This invention provides an antibody or antigen-binding fragment thereof that binds to KPC enzyme and its application, relating to the technical field of monoclonal antibodies. The antibody or antigen-binding fragment that binds to KPC enzyme has a light chain CDR as shown in Seq_1-3, or as shown in Seq_11-13; and / or a heavy chain CDR as shown in Seq_4-6, or as shown in Seq_14-16. This antibody or antigen-binding fragment that binds to KPC enzyme exhibits high specificity and high affinity, alleviating the technical problem of poor efficacy of existing monoclonal antibodies that bind to KPC enzyme.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to an antibody or antigen-binding fragment thereof that binds to KPC enzyme and its applications. Background Technology

[0002] KPC-producing bacteria have spread widely globally, causing resistance to almost all β-lactam antibiotics, including carbapenems, penicillins, cephalosporins, and aztreonam. Furthermore, the types of KPC-producing strains are increasing, posing a potential threat of hospital-acquired infection outbreaks and epidemics. Therefore, KPC-producing bacterial infections are a serious problem that deserves the high attention of microbiologists and clinicians. Since the discovery of KPC-1 in Caroline, USA, KPC-producing bacteria have been reported in various countries and regions, showing a general trend of global dissemination. Multiple mutants of KPC exist, with epidemiological studies indicating that KPC-2 and KPC-3 are the main subtypes, present in almost all reported countries.

[0003] The KPC-1 enzyme exhibits high resistance to imipenem and meropenem. β-lactamase activity against imipenem and meropenem is inhibited in the presence of clavulanic acid; it is also resistant to cephalosporins and aztreonam. The KPC-1 gene is carried by a non-binding plasmid of approximately 50 kb. The KPC-1 enzyme is a novel carbapenem-hydrolyzing β-lactamase belonging to the Bush2f group (class A). Carbapenem resistance in Klebsiella pneumoniae 1534 is primarily mediated by the KPC-1 enzyme, and alterations in porin expression may also play a role. The KPC-2 enzyme differs from the KPC-1 enzyme by one amino acid change, S174G, and is also classified as belonging to the Bush2f group (class A). The KPC-2 enzyme, derived from *Salmonella enterica* and *Klebsiella pneumoniae*, exhibits 98% homology in its DNA sequence with plasmids from *Salmonella*. Therefore, this plasmid can bind to and transmit KPC-2 within *Enterobacteria*. The KPC-2 enzyme can induce carbapenem resistance independently, without the loss of porin. Its gene is encoded by a transferable 70kb plasmid located on a transposon, making it highly transmissible. The KPC-3 enzyme, encoded by a 75kb plasmid, is transferable via binding. The KPC3 enzyme is a 293-amino acid peptide chain encoded by an 882bp base, differing from the KPC-1 enzyme by two bases. It can be transferred and bound via electroporation. All KPC conjugates are more sensitive to carbapenems than the original strains. Both KPC-1 and KPC-3 enzymes require binding to the porin ompK35; its loss leads to carbapenem resistance.

[0004] According to CLSIM 100S30, the clinical detection of KPC-producing bacteria mainly relies on routine antimicrobial susceptibility testing, such as the broth dilution method, disk diffusion method, and concentration gradient method. These methods are challenging for detecting KPC-producing bacteria for the following reasons: KPC-producing bacteria only show reduced sensitivity to carbapenems, not complete resistance; they are easily identified as ESBL-sensitive strains when tested positive for ESBLs; and the inoculation effect of KPC-producing bacteria can affect the MIC of imipenem, leading to false positives. Currently, most studies use a modified Hodge test to detect carbapenemases, but this cannot further confirm whether the bacteria are KPC-producing. Besides the CarbaNP method, other methods are primarily culture-based, taking 3-7 days, and are complex to operate, requiring specialized technical personnel. They are ineffective for difficult-to-culture or slow-growing bacteria, failing to meet clinical needs. The CarbaNP method requires special reagents, some of which need to be prepared in-house, has a short shelf life, and low sensitivity. Currently, molecular biology methods are widely considered the gold standard for detecting KPC-producing bacteria. However, PCR testing requires specialized equipment, qualified personnel, and is costly per test, thus it is not routinely performed in clinical laboratories. Accurate detection of KPC-producing bacteria remains a significant challenge for clinical laboratories.

[0005] Monoclonal antibodies are highly homogeneous antibodies produced from a single B cell clone that target only a specific antigenic epitope. They are usually prepared using hybridoma cells. Based on cell fusion technology, sensitized B cells capable of secreting specific antibodies are fused with myeloma cells with unlimited proliferative capacity to form a B cell hybridoma. After culturing the cell population, a specific antibody against a single antigenic epitope, i.e., a monoclonal antibody, can be prepared.

[0006] The primary purpose of specific antibody detection is to assist in clinical diagnosis. In some diseases, it also serves as an indicator for observing treatment efficacy and prognosis. Furthermore, specific antibody detection holds special and important significance in drug resistance and infectious disease epidemiological surveys. Antibody immunological detection offers the following advantages: high specificity (using specific monoclonal antibodies for single cytokine detection); simple and rapid operation (no cell line dependence, thus eliminating the need for maintenance culture, increasing operability, and facilitating widespread adoption and screening); relatively fewer influencing factors that are easy to control; good reproducibility; and ease of standardization. Therefore, monoclonal antibodies that specifically bind to KPC enzymes are currently in demand in the market.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide an antibody or antigen-binding fragment thereof that binds to KPC enzymes, thereby alleviating the technical problem of poor efficacy of monoclonal antibodies that bind to KPC enzymes in the prior art.

[0009] A second objective of this invention is to provide biomaterials, reagents, kits, and their applications related to antibodies or antigen-binding fragments of the KPC enzyme that bind to the above-mentioned KPC enzyme, in order to improve existing methods for detecting KPC enzymes.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] According to one aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to a KPC enzyme is provided, comprising a light chain variable region and / or a heavy chain variable region;

[0012] The light chain variable region has a light chain CDR composed of CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_1, Seq_2, and Seq_3, respectively.

[0013] Alternatively, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in Seq_11, Seq_12, and Seq_13, respectively;

[0014] The heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_4, Seq_5, and Seq_6, respectively.

[0015] Alternatively, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_14, Seq_15, and Seq_16, respectively.

[0016] According to one aspect of the present invention, the present invention also provides a composition comprising the antibody or antigen-binding fragment thereof that is conjugated to the KPC enzyme and a label; wherein the label is coupled to the antibody or antigen-binding fragment thereof; or, the label and the antibody or antigen-binding fragment thereof are packaged separately.

[0017] According to one aspect of the present invention, a biomaterial is provided, the biomaterial comprising a nucleic acid fragment, a carrier, or a host cell;

[0018] The nucleic acid fragment is selected from (a1) or (a2): (a1) DNA or RNA encoding the antibody or its antigen-binding fragment described above; (a2) a nucleic acid fragment complementary to the nucleic acid fragment defined in (a1); the vector includes the nucleic acid fragment; the host cell is transformed by the vector, or the nucleic acid is integrated into the genome of the host cell.

[0019] According to one aspect of the present invention, the present invention also provides a method for producing an antibody or antigen-binding fragment thereof that binds to a KPC enzyme, comprising expressing the antibody or antigen-binding fragment thereof that binds to the KPC enzyme using the aforementioned host cells.

[0020] According to one aspect of the present invention, the present invention also provides a reagent or kit for detecting KPC enzymes, comprising the above-described antibody or its antigen-binding fragment, or the above-described composition.

[0021] According to one aspect of the present invention, the present invention also provides the use of the above-described antibodies or antigen-binding fragments thereof that bind to KPC enzymes, biological materials, production methods, compositions, or reagents or kits for detecting KPC enzymes in the detection of KPC enzymes for non-diagnostic and therapeutic purposes or in the detection of KPC enzyme-producing microorganisms for non-diagnostic and therapeutic purposes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention obtains the conserved sequences of KPC enzymes (KPC-1 to KPC-82) through NCBI sequence alignment, and then purifies them using E. coli expression to obtain high-purity proteins. Hybridoma cells are prepared by immunizing New Zealand white rabbits with KPC antigen protein and obtaining spleen cells that secrete antibodies binding to KPC. Hybridoma cells capable of secreting highly specific antibodies are selected through screening. Sequencing yields light chain CDRs as shown in Seq_1–3, or Seq_11–13; and heavy chain CDRs as shown in Seq_4–6, or Seq_14–16.

[0024] The CDR region sequence of the KPC enzyme-binding antibody or its antigen-binding fragment provided by this invention is derived from rabbits and has the ability to specifically bind to different subtypes of KPC enzyme. Antibodies or antigen-binding fragments possessing the aforementioned CDR sequence exhibit high specificity and affinity, demonstrating superior performance in various aspects. Therefore, they are suitable as immunodiagnostic reagents for in vitro diagnosis of KPC enzymes or microorganisms that secrete KPC enzymes, achieving a titer exceeding 1:1,280,000. In particular, the different antibodies formed by the aforementioned light chain CDR and heavy chain CDR in certain combinations can pair well and can be applied to immunoassay methods based on the formation of antibody-antigen-antibody complexes, such as serving as the primary and secondary antibodies in a double-antibody sandwich ELISA; or as the labeled antibody and the antibody coating the detection area in an immunochromatographic detection card, etc.

[0025] The KPC-binding antibodies or their antigen-binding fragments provided by this invention, along with reagents or kits prepared from related biological materials, can be used for early typing of drug-resistant strains, guiding clinical medication and assisting in clinical infection control and treatment. Reagents or kits containing antibodies or their antigen-binding fragments with the aforementioned CDR sequence can rapidly and accurately determine the degree of bacterial resistance in patients by qualitatively or semi-quantitatively detecting KPC enzymes in bacterial samples isolated from patients or positive blood cultures, and rapidly detecting the presence of KPC enzymes in bacterial samples, providing more options for clinical methods to guide medication. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 The SDS-PAGE electrophoresis results of antibodies 1 and 2 prepared in the embodiments of the present invention are shown below.

[0028] Figure 2 The results of the affinity activities of antibodies 1 and 2 prepared in the embodiments of the present invention for KPC enzyme were detected by ELISA.

[0029] Figure 3 The reaction results of antibody 1 prepared in this embodiment of the invention with KPC, NDM, VIM, IMP and OXA-48 enzyme;

[0030] Figure 4The reaction results of antibody 2 prepared in the embodiments of the present invention with KPC, NDM, VIM, IMP and OXA-48 enzyme are shown. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0033] According to one aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to a KPC enzyme is provided, said antibody or antigen-binding fragment comprising at least one light chain variable region or one heavy chain variable region, or both light chain variable regions and heavy chain variable regions.

[0034] Light chain variable region:

[0035] The light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3.

[0036] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below:

[0037] CDR-L1:RASQSVRSNLA(Seq_1);

[0038] CDR-L2:GASTRAT(Seq_2);

[0039] CDR-L3:QQYNTWPPLT(Seq_3).

[0040] The preferred amino acid sequence of the light chain variable region containing the light chain CDR composed of the above-mentioned CDR-L1 (Seq_1), CDR-L2 (Seq_2), and CDR-L3 (Seq_3) is as shown in Seq_7:

[0041] TGETTQAPASLSFSLGEEATLSCRASQSVRSNLAWYQQKAEQVPRLLQHGASTRATGVPVRFSGTGDGTDFTLTISSLEPEDAAVYYCQQYNTWPPLTFGGGTKVEIK(Seq_7).

[0042] Alternatively, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown below:

[0043] CDR-L1:RSSQSLLHSNGYNYLD(Seq_11);

[0044] CDR-L2:LGSHRAS(Seq_12);

[0045] CDR-L3:MQALQRRT(Seq_13).

[0046] The preferred amino acid sequence of the light chain variable region containing the light chain CDRs composed of CDR-L1 (Seq_11), CDR-L2 (Seq_12), and CDR-L3 (Seq_13) is shown in Seq_17:

[0047] DVVLTQTPLSLPVSLGDQASISCRSSQSLLHSNGYNYLDWYLQKPGQSPKLPIYLGSHRASGKPDRFSGSGSGTDFTLKISRVEAEDLGVYYCMQALQRRTFGQGTKVEIK(Seq_17).

[0048] In some optional embodiments, the N-terminus of the aforementioned light chain variable region further contains a signal peptide, the amino acid sequence of which is:

[0049] MDMRVPAQLLGLLLLWLSGARC(Seq_21).

[0050] Heavy chain variable region:

[0051] The heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3.

[0052] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown below:

[0053] CDR-H1:NYAMH(Seq_4);

[0054] CDR-H2:AIRSNGGGTYYANSVKG(Seq_5);

[0055] CDR-H3:DSGGPVREWYFDL(Seq_6).

[0056] The preferred amino acid sequence of the heavy chain variable region containing the heavy chain CDR composed of the above-mentioned CDR-H1 (Seq_4), CDR-H2 (Seq_5), and CDR-H3 (Seq_6) is shown in Seq_9:

[0057] EVQLVESGSGLVQPGGSLKLSCAASVFTFSNYAMHWVVQTPDKRLELVAAIRSNGGGTYYANSVKGRFTISRDDNAKNTLYDQMSSLKSEDMAVYYCARDSGGPVREWYFDLWGRGTLVTVSS.

[0058] Alternatively, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in Seq_14, 15, and 16, respectively:

[0059] CDR-H1:SYYVH(Seq_14);

[0060] CDR-H2:LINPSGGSTRYAQKFQG(Seq_15);

[0061] CDR-H3:DYGSVATGDFYFNY(Seq_16).

[0062] The preferred amino acid sequence of the heavy chain variable region containing the heavy chain CDR composed of the above-mentioned CDR-H1 (Seq_14), CDR-H2 (Seq_15), and CDR-H3 (Seq_16) is shown in Seq_19:

[0063] QVQLQQSGPELVQPGASVKISCKAKGYTFTSYYVHWVKQRPYQGLEWIGLINPSGGSTRYAQKFQGKATLTSDTSSDTAYMQLSSLTSEDSAVYFCARDYGSVATGDFYFNYWGQGTLVTVSS(Seq_19).

[0064] In some optional embodiments, the N-terminus of the aforementioned heavy chain variable region further contains a signal peptide, the amino acid sequence of which is:

[0065] MDWTWRFLFVVAAATGVQS(Seq_22).

[0066] In some preferred embodiments, the antibody or its antigen-binding fragment that binds to the KPC enzyme contains both a light chain variable region and a heavy chain variable region. When both light chain and heavy chain variable regions are present, the light chain CDR and heavy chain CDR are preferably antibody 1 or antibody 2.

[0067] Antibody 1:

[0068] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, which make up the CDR of the light chain variable region, are shown in Seq_1, Seq_2, and Seq_3, respectively.

[0069] Meanwhile, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3, which make up the heavy chain variable region CDR, are shown in Seq_4, Seq_5, and Seq_6, respectively.

[0070] In this combination, a preferred embodiment is that the amino acid sequence of the light chain variable region is as shown in Seq_7 or Seq_23, while the amino acid sequence of the heavy chain variable region is as shown in Seq_9 or Seq_24. The light chain variable region shown in Seq_23 is the amino acid sequence shown in Seq_7 with the signal peptide shown in Seq_21 fused to its N-terminus; the heavy chain variable region shown in Seq_24 is the amino acid sequence shown in Seq_9 with the signal peptide shown in Seq_22 fused to its N-terminus.

[0071] Antibody 2:

[0072] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, which constitute the light chain variable region CDR, are shown in Seq_11, Seq_12, and Seq_13, respectively; and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3, which constitute the heavy chain variable region CDR, are shown in Seq_14, Seq_15, and Seq_16, respectively.

[0073] In this combination, a preferred embodiment is that the amino acid sequence of the light chain variable region is as shown in Seq_17 or Seq_25, while the amino acid sequence of the heavy chain variable region is as shown in Seq_19 or Seq_26. The light chain variable region shown in Seq_25 is the amino acid sequence shown in Seq_17 with the signal peptide shown in Seq_21 fused to its N-terminus; the heavy chain variable region shown in Seq_26 is the amino acid sequence shown in Seq_19 with the signal peptide shown in Seq_22 fused to its N-terminus.

[0074] The aforementioned antibodies 1 and 2 not only exhibit good specificity and binding ability to KPC enzymes independently, but they can also form paired antibodies for use in detection methods based on the principle of antibody-antigen-antibody complex formation, such as double-antibody sandwich ELISA or immunochromatographic assay cards. In some optional embodiments, antibody 1 / antibody 2 is coated on a solid-phase carrier as the primary antibody, and the corresponding antibody 2 / antibody 1 is linked to a label as the secondary antibody, such as horseradish peroxidase or alkaline phosphatase; or, antibody 1 / antibody 2 is labeled with a label, and the corresponding antibody 2 / antibody 1 is used as the antibody coating the detection area of ​​the immunochromatographic assay card. Antibodies 1 and 2 may optionally be either complete antibodies or antigen-binding fragments, respectively.

[0075] As is well known in the art, the binding specificity and affinity of antibodies are primarily determined by the CDR sequence. Based on mature and well-known existing technologies, amino acid sequences in non-CDR regions can be easily altered to obtain variants with similar biological activities. The monoclonal antibody variants of this invention, possessing a CDR sequence identical to the aforementioned CDR sequence, exhibit similar biological activity because they have the exact same CDR sequence as the KPC enzyme-binding antibody described in this invention.

[0076] The antigen-binding fragment is an antibody fragment with the same specificity as the parent antibody, and may be, for example, one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibodies, and the smallest antibody recognition unit. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0077] These functional fragments typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art, based on the description herein, infer that the antigen-binding fragments of the present invention can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0078] The antigen-binding fragment can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as an automated peptide synthesizer; or by expressing a gene encoding the above-mentioned functional fragment in a host cell.

[0079] The antibodies or antigen-binding fragments of the KPC enzyme provided by this invention, excluding the CDR region, are derived from one or more species including, but not limited to, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, dairy cows, pigs, horses, monkeys, and humans.

[0080] When the KPC enzyme antibody is a complete antibody molecule, its antibody type can be, for example, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Given that those skilled in the art are familiar with the CDR region or the variable regions of the light and heavy chains of an antibody, they can use conventional methods in the art to obtain different types of antibodies, such as fusing a variable region gene with a corresponding heavy chain or heavy chain constant region coding gene and expressing the fusion protein in a host cell to obtain different types of antibodies.

[0081] In some preferred embodiments, the antibody comprises a sequence of a constant region of any one of rabbit antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0082] In some preferred embodiments, the amino acid sequence of the light chain constant region of the antibody is shown in Seq_31; the amino acid sequence of the heavy chain constant region of the antibody is shown in Seq_32.

[0083] According to another aspect of the present invention, the present invention also provides a composition comprising the aforementioned antibody or antigen-binding fragment thereof that binds to the KPC enzyme and a label; wherein the label is coupled to the antibody or antigen-binding fragment thereof; or, the label and the antibody or antigen-binding fragment thereof are packaged separately and then conjugated before use. The label includes, but is not limited to, one or more of the following: enzymes, latex particles, fluorescent molecular labels, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver, colloidal carbon, biotin, or streptavidin.

[0084] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Antibodies or antigen-binding fragments of KPC labeled with alkaline phosphatase or horseradish peroxidase can be used as secondary antibodies in chemiluminescent enzyme-linked immunosorbent assay (ELISA) methods and kits.

[0085] According to another aspect of the invention, the invention also provides a biomaterial comprising a nucleic acid fragment, a vector, or a host cell.

[0086] Nucleic acid fragments: selected from (a1) or (a2):

[0087] (a1) It encodes an antibody or antigen-binding fragment thereof that binds to KPC enzyme; in some preferred embodiments, the nucleic acid fragment is DNA containing a DNA fragment encoding a light chain variable region and / or a DNA fragment encoding a heavy chain variable region.

[0088] Preferably, the nucleotide sequence of the DNA fragment encoding the light chain variable region is as shown in Seq_8, Seq_18, Seq_27, or Seq_28; the sequences shown in Seq_27 and Seq_28 are the sequences obtained by linking the sequences shown in Seq_8 and Seq_18 to encode the signal peptide fragment, respectively.

[0089] Preferably, the nucleotide sequence of the DNA fragment encoding the variable region of the heavy chain is as shown in Seq_10, Seq_20, Seq_29 or Seq_30; the sequences shown in Seq_29 and Seq_30 are the sequences obtained by linking the sequences shown in Seq_10 and Seq_20 to the sequences encoding the signal peptide fragment.

[0090] Preferably, the nucleotide sequence of the DNA fragment encoding the light chain constant region is shown in Seq_33.

[0091] Preferably, the nucleotide sequence of the DNA fragment encoding the heavy chain constant region is shown in Seq_34.

[0092] (a2) Nucleic acid fragments that are complementary to the nucleic acid fragments defined in (a1).

[0093] Vector: Includes the nucleic acid fragments described above. The vector may also include portions encoding other constituent elements, such as, but not limited to, encoding regulatory sequences or marker genes. The vector may be, for example, but not limited to, a prokaryotic expression vector, a eukaryotic expression vector, or an insect expression vector.

[0094] Host cell: The host cell is transformed with the above-mentioned vector to enable the above-mentioned vector to be cloned or expressed; or, the host cell genome integrates the nucleic acid fragment to express the antibody or antigen-binding fragment that binds to the KPC enzyme.

[0095] According to one aspect of the present invention, the present invention also provides a method for producing an antibody or antigen-binding fragment thereof that binds to a KPC enzyme, comprising expressing the antibody or antigen-binding fragment thereof that binds to the KPC enzyme using the aforementioned host cells. This method uses raw materials derived from stably expressed cell lines, eliminating the risk of raw material supply issues and offering stable performance and low cost. Furthermore, the product exhibits strong stability, minimal batch-to-batch variation, and is unaffected by cell line degradation.

[0096] According to another aspect of the present invention, the present invention also provides a reagent or kit comprising the antibody or antigen-binding fragment thereof that binds to KPC. Optional examples include, but are not limited to, kits for conventional detection methods in the art, such as ELISA kits, Western blotting kits, immunohistochemical kits, or immunochromatographic test strips. It is understood that the reagent may also contain reagents conventional in the art, such as, but not limited to, one or more lyophilization protectants, buffers, and solvents; the kit may also contain reagents or consumables conventional in the art, such as, but not limited to, buffer solutions, blocking solutions, secondary antibodies, chromogenic substances, labels and reaction substrates, solid-phase carriers, test strips, and their supporting components.

[0097] In some optional embodiments, the kit includes an immunochromatographic assay card with a labeled monoclonal antibody 1 / antibody 2 against KPC enzyme embedded in the sample conjugation pad and a monoclonal antibody 2 / antibody 1 against KPC enzyme coated on the detection line (T). If the test sample is positive, the KPC enzyme binds to the labeled antibody 1 / antibody 2 to form a complex. Under chromatography, the complex moves forward along the paper strip and is captured by the pre-coated KPC enzyme antibody 2 / antibody 1 when it passes the detection line (T), forming an immune complex and producing a detectable signal value. If the test sample is negative, no immune complex is formed, and no detectable signal value appears at the detection line. The label can be, for example, but not limited to, colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres, or quantum dots. Preferably, antibody 1 is embedded in the sample conjugation pad and antibody 2 is coated on the detection line.

[0098] According to one aspect of the present invention, the present invention also provides the use of the above-described antibodies or antigen-binding fragments thereof that bind to KPC enzymes, biological materials, production methods, compositions, or reagents or kits for detecting KPC enzymes in the detection of KPC enzymes for non-diagnostic and therapeutic purposes or in the detection of KPC enzyme-producing microorganisms for non-diagnostic and therapeutic purposes.

[0099] Antibodies or antigen-binding fragments of KPC enzymes, or combinations thereof, are used to prepare products for detecting KPC enzymes or KPC enzyme-producing microorganisms. These products can be used for early typing of drug-resistant strains, guiding clinical medication, and assisting in clinical infection control and treatment. They can also be used to quickly and accurately determine the degree of bacterial resistance in patients by qualitatively or semi-quantitatively detecting KPC enzymes in bacterial samples isolated from patients or positive blood culture samples, and to rapidly detect the presence of KPC enzymes in bacterial samples, which is used clinically to guide medication.

[0100] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0101] Example 1 Preparation of Antigen

[0102] Conserved sequences of KPC enzymes (KPC-1 to KPC-82) were obtained through NCBI (National Center for Biotechnology Information) sequence alignment. Using standard molecular biology techniques such as enzyme digestion and ligation, the expression plasmid pET-28a(+)-PM was constructed, and the recombinant vector was transformed into *E. coli* DH5α competent cells using the CaCl2 heat shock method. Positive clones were screened using LB medium containing 100 μg / mL ampicillin. *E. coli* cells were cultured using standard methods, and the plasmid was extracted for PCR identification to confirm the presence of the target gene. The extracted expression plasmid pET-28a(+)-PMAA was transformed into *E. coli* BL21(DE3) competent cells, plated on selective medium, and single colonies resistant to 100 μg / mL ampicillin were screened, followed by overnight liquid culture. 1 mL of overnight culture was inoculated into 200 mL of LB medium containing 100 μg / mL ampicillin and cultured with shaking until the logarithmic phase (OD600 at 0.5–0.6). IPTG (1 mmol / L) was added and the culture was induced at 16 °C for 3 h. The fermentation broth was purified by passing it through a nickel column and high-purity protein was obtained through prokaryotic gene expression.

[0103] Example 2 Animal Immunization

[0104] Select New Zealand white rabbits of suitable age and weighing approximately 1.5 kg, and house them in a standard animal facility for 3 days. If no abnormalities are observed, begin immunization: Add 100 μg of KPC enzyme antigen to 0.5 mL of autoclaved physiological saline, mix thoroughly using a micro vortex mixer, add 0.5 mL of Freund's complete adjuvant, and emulsify thoroughly by pushing and pulling with a syringe. Administer the emulsification via multiple subcutaneous injections on the back of the New Zealand white rabbits. A booster immunization is given two weeks later, followed by booster immunizations every week for a total of six immunizations. Starting from the third immunization, one week after each immunization, collect 200–500 μL of blood from the marginal ear vein of the rabbits to determine the titer and affinity. After the final immunization, harvest the spleen for cell fusion to prepare hybridoma cells.

[0105] Example 3: Preparation and Screening of Hybridoma Cells

[0106] The prepared rabbit antiserum was tested for potency. If the potency was qualified, rabbit spleen was used for cell fusion to prepare a monoclonal hybridoma cell line. The method was as follows: Immunized New Zealand rabbits were sacrificed, and the spleen was removed under aseptic conditions. The spleen was washed once with cell culture medium, then crushed and passed through a stainless steel sieve. The resulting cells were centrifuged and washed twice with cell culture medium. SP2 / 0 myeloma cells in the logarithmic growth phase were mixed with spleen cells, washed once with cell culture medium without fetal bovine serum, centrifuged, and the supernatant was discarded. Polyethylene glycol solution was added, and the mixture was incubated at 37°C for 90 seconds. The reaction was terminated with cell culture medium without fetal bovine serum (FBS), followed by centrifugation. Cells were resuspended in HAT selection medium containing 20% ​​FBS and added to 96-well plates, where they were cultured at 37°C and 5.0% CO2. Cells in good growth condition in the 96-well plates were diluted with cell culture medium to 1–3 cells / mL and added to the plates. The plates were then incubated at 37°C and 5.0% CO2. Each cell line was numbered, and cell lines showing positive results in the culture supernatant were selected for further expansion to obtain hybridoma cell lines. The obtained hybridoma cells were screened using ELISA. Cell growth was observed on day 5 post-fusion, and the titer of the cell culture supernatant was detected using indirect ELISA on days 10–14. The hybridoma cells with the highest titer were expanded until 100% positivity was achieved, and the resulting hybridoma cell lines were cryopreserved in liquid nitrogen for later use.

[0107] Example 4: Isolation of antibody variable region gene from hybridoma cells using RT-PCR

[0108] After homogenizing the hybridoma cells screened in Example 3, RNA was extracted by adding cell lysis buffer. RNA was precipitated from the aqueous phase with isopropanol, washed by centrifugation to remove impurities, resuspended, and reverse transcribed to obtain cDNA. PCR was performed using specific primers from New Zealand rabbits, using hybridoma cell cDNA as a template to amplify the variable regions of the heavy and light chains of the antibody. A 50 μL system contained 5 μL of cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM of specific primers. PCR amplification was performed under the following conditions: pre-denaturation at 94℃ for 5 min; 35 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 50 s; 72℃ for 7 min. The obtained PCR products were identified by 1% agarose gel electrophoresis, the target fragment was recovered, and sequenced. The sequencing results were compared with the IMGT database (http: / / www.imgt.org / IMGT_vquest / vquest). The sequence information of antibody 1 and antibody 2 is shown below:

[0109] Antibody 1:

[0110] Light chain:

[0111] MDMRVPAQLLGLLLLWLSGARCTGETTQAPASLSFSLGEEATLSCRASQSVRSNLAWYQQKAEQVPRLLQHGASTRATGVPVRFSGTGDGTDFTLTISSLEPEDAAVYYCQQYNTWPPLTFGGGTKVEIK(Seq_23).

[0112] Positions 1-22 are the signal peptide; positions 23-45 are FR1; positions 46-56 are CDR-L1; positions 57-71 are FR2; positions 72-78 are CDR-L2; positions 79-110 are FR3; positions 111-120 are CDR-L3; and positions 121-130 are FR4.

[0113] Heavy chain:

[0114] MDWTWRFLFVVAAATGVQSEVQLVESGSGLVQPGGSLKLSCAASVFTFSNYAMHWVVQTPDKRLELVAAIRSNGGGTYYANSVKGRFTISRDNAKNTLYDQMSSLKSEDMAVYYCARDSGGPVREWYFDLWGRGTLVTVSS(Seq_24).

[0115] Positions 1-19 are signal peptides; positions 20-49 are FR1; positions 50-54 are CDR-H1; positions 55-68 are FR2; positions 69-85 are CDR-H2; positions 86-117 are FR3; positions 118-130 are CDR-H3; and positions 131-141 are FR4.

[0116] Antibody 2:

[0117] Light chain:

[0118] MDMRVPAQLLGLLLLWLSGARCDVVLTQTPLSLPVSLGDQASISCRSSQSLLHSNGYNYLDWYLQKPGQSPKLPIYLGSHRASGKPDRFSGSGSGTDFTLKISRVEAEDLGVYYCMQALQRRTFGQGTKVEIK(Seq_25).

[0119] Among them, positions 1-22 are signal peptides; positions 23-45 are FR1; positions 46-61 are CDR-L1; positions 62-76 are FR2; positions 77-83 are CDR-L2; positions 84-115 are FR3; positions 116-123 are CDR-L3; and positions 124-133 are FR4.

[0120] Heavy chain:

[0121] MDWTWRFLFVVAAATGVQSQVQLQQSGPELVQPGASVKISCKAKGYTFTSYYVHWVKQRPYQGLEWIGLINPSGGSTRYAQKFQGKATLTSDTSSDTAYMQLSSLTSEDSAVYFCARDYGSVATGDFYFNYWGQGTLVTVSS(Seq_26).

[0122] Among them, positions 1-19 are signal peptides; positions 20-49 are FR1; positions 50-54 are CDR-H1; positions 55-68 are FR2; positions 69-85 are CDR-H2; positions 86-117 are FR3; positions 118-131 are CDR-H3; and positions 132-142 are FR4.

[0123] Example 5: Construction, expression, and purification of monoclonal antibodies

[0124] Homologous recombination arms and signal peptide sequences were added to both ends of the antibody heavy chain variable region gene and the light chain variable region gene, respectively, using homologous recombination primers. The amino acid sequence of the heavy chain variable region signal peptide is shown in Seq_22, and the amino acid sequence of the light chain variable region signal peptide is shown in Seq_21. The expression plasmids containing the rabbit antibody heavy chain IgG1 constant region and the expression plasmid containing the rabbit antibody light chain IgG1 constant region were linearized using double enzyme digestion to generate homologous recombination arms. The amino acid sequence of the light chain constant region is shown in Seq_31, and the nucleotide sequence is shown in Seq_33; the amino acid sequence of the heavy chain constant region is shown in Seq_32, and the nucleotide sequence is shown in Seq_34. The variable region gene fragments with added homologous recombination arms and the linearized plasmids were ligated together by homologous recombination to construct complete light chain expression vectors and heavy chain expression vectors. The recombinant products were transformed into TOP10 E. coli competent cells for plasmid amplification.

[0125] The obtained monoclonal antibody heavy and light chain expression plasmids were added to Opti-Mem transfection medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was incubated at room temperature in the dark for 30 min, and then added to 293T cells. After incubation for 6 h, the transfection system was removed, and FreeStyle™ 293 expression medium was added. The expressed antibody supernatant was purified using the AKTA protein purification system and the affinity purification method (Protein A) to obtain the anti-KPC enzyme monoclonal antibody. The specific steps were as follows: (1) Centrifuge the expressed antibody supernatant at 2500×g at room temperature for 10 min to remove the precipitate; (2) Wash the affinity purification column containing Protein A thoroughly with 10 times the volume of binding buffer; (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min; (4) Wash the purification column thoroughly with 20 times the volume of binding buffer; (5) Use 0.1M The purification column was eluted with citrate buffer at pH 3.0 to 3.5 until the elution peak reached equilibrium. The pH was then adjusted to 7.0 with 1M Tris-HCl buffer at pH 9.0. (6) The purified monoclonal antibody was concentrated using a centrifugal column. PBS was used as the antibody storage buffer. Finally, the concentration of the concentrated antibody was determined using the BSA protein concentration detection method.

[0126] Example 6 Molecular weight determination

[0127] The molecular weight of monoclonal antibodies was identified by SDS-PAGE electrophoresis. 5 μg of sample was loaded into each lane, using a known molecular weight standard series as a reference. Electrophoresis was first performed at 90 V for 20 min, then at 140 V until all the indicator was elute. The gel was then removed, stained with Coomassie Brilliant Blue, and the molecular weight of the biological material was analyzed after staining. The SDS-PAGE electrophoresis image is shown below. Figure 1 As shown.

[0128] Example 1: Potency Measurement

[0129] The affinity activity (titer) of monoclonal antibodies for KPC enzyme was detected by ELISA. The main steps are as follows: (1) KPC antigen was diluted to 1 ng / μL with PBS and 100 μL was added to each well of a 96-well microplate. The plate was coated at 37°C for 2 h. (2) The supernatant was discarded and the plate was washed 3 times with 0.01 M PBST. Blocking buffer containing 3% BSA was prepared with PBST and 100 μL was added to each well. The plate was blocked at 37°C for 2 h. (3) The supernatant was discarded and the plate was washed 5 times with PBST. The purified and concentrated antibody was then added to the plate. Serial dilutions were performed, from 1:1000 to 1:2560000, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h. (4) The antibody diluent was discarded, and the cells were washed 6 times with PBST. Goat anti-rabbit IgG-HRP was diluted with 1:5000 blocking buffer, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h. (5) The secondary antibody diluent was discarded, and the cells were washed 6 times with PBST. TMB was added, 100 μL / well, and the mixture was incubated at 37°C for 15 min in the dark. (6) 50 μL of 1M dilute sulfuric acid was added to each well to stop the reaction, and the absorbance was measured at 450 nm. The results are as follows. Figure 2 As shown, the screened monoclonal antibodies have a strong binding ability to KPC enzyme, and the titer against KPC enzyme antigen reaches 1:1280000 (OD value > 0.5).

[0130] Comparison of Effect Example 2 with Existing Monoclonal Antibodies

[0131] The affinity (titer) of the KPC enzyme was detected using ELISA with antibodies 1, 2, natural antibodies, and the published murine monoclonal antibody (purchased from Zhuhai Bomei Biotechnology Co., Ltd.) prepared in the above examples, respectively. The specific steps were the same as in Example 1. The results are shown in Table 1, indicating that the monoclonal antibodies prepared in the above examples have increased affinity and enhanced biological activity compared with the prior art.

[0132] Table 1

[0133]

[0134] Example 3: Cross-reaction

[0135] Microplates were coated with KPC, NDM, VIM, IMP, and OXA-48 enzymes, respectively, with a coating volume of 50 ng per well. Monoclonal antibodies were diluted to 10 ng / mL and added to each well (100 μL). The plates were incubated at 37°C for 1 h. After washing, 100 μL of HRP-labeled goat anti-rabbit secondary antibody was added to each well, and the plates were incubated at 37°C for 0.5 h. After washing, TMB was added, and the plates were incubated at 37°C for 15 min before readings were taken. Results for antibody 1 are shown below. Figure 3 As shown, the results for antibody 2 are as follows: Figure 4 As shown, antibodies 1 and 2 do not cross-react with other types of carbapenemases, demonstrating high specificity.

[0136] Example 4: Antibody pairing verification

[0137] Two antibodies were used: Antibody 1 as the capture antibody and Antibody 2 as the labeling antibody (HRP-labeled). The capture antibody was also HRP-labeled as a control. The capture antibody was coated onto an antigen plate. First, serially diluted antigen was added, incubated, and then unbound antigen was washed away. Next, the labeling antibody was added, incubated, and then unbound labeling antibody was washed away. Finally, chromogenic buffer was added for color development. If color development occurred, it indicates that the labeling antibody specifically binds to the antigen, and the capture antibody and labeling antibody are a paired antibody pair. If color development did not occur, it indicates that the labeling antibody could not bind to the antigen and was eluted, and the capture antibody and labeling antibody are not a paired antibody pair. The results are shown in the table below, indicating that these two antibodies have the best ability to pair and bind to the antigen.

[0138] Table 2

[0139] KPC concentration (ng / mL) HRP-capture antibody Labeled antibody-HRP 100 0.124 2.084 50 0.061 1.102 25 0.033 0.646 12.5 0.023 0.343

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a KPC enzyme, characterized in that, comprises a light chain variable region and a heavy chain variable region; the antibody or antigen-binding fragment thereof is selected from (A) or (B): (A), the light chain variable region has light chain CDRs consisting of CDR-L1, CDR-L2, CDR-L3, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 are respectively as shown in Seq_1, Seq_2 and Seq_3; the heavy chain variable region has heavy chain CDRs consisting of CDR-H1, CDR-H2, CDR-H3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 are respectively as shown in Seq_4, Seq_5 and Seq_6; (B), the light chain variable region has light chain CDRs consisting of CDR-L1, CDR-L2, CDR-L3, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 are respectively as shown in Seq_11, Seq_12 and Seq_13; the heavy chain variable region has heavy chain CDRs consisting of CDR-H1, CDR-H2, CDR-H3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 are respectively as shown in Seq_14, Seq_15 and Seq_16.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the N-terminal of the light chain variable region further comprises a signal peptide; the amino acid sequence of the signal peptide of the light chain variable region is as shown in Seq_21.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the N-terminal of the heavy chain variable region further comprises a signal peptide; the amino acid sequence of the signal peptide of the heavy chain variable region is as shown in Seq_22. (A), the amino acid sequence of the light chain variable region is as shown in Seq_7 or Seq_23, and the amino acid sequence of the heavy chain variable region is as shown in Seq_9 or Seq_24. The type of the antibody is IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. The remaining part of the sequence of the antibody or antigen-binding fragment thereof, except for the CDR region, is derived from one or more of the following species: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human. The antibody comprises the sequence of any one of the constant regions of rabbit antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the light chain constant region of the antibody is as shown in Seq_31; the amino acid sequence of the heavy chain constant region of the antibody is as shown in Seq_32.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, The antigen-binding fragment comprises one or more of F(ab')2, Fab', Fab, Fv, scFv and dsFv.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-9 and a label; the label is coupled to the antibody or antigen-binding fragment thereof; or, the label is independently packaged with the antibody or antigen-binding fragment thereof, respectively.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, ​ 8. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, ​ 9. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, ​ 10. Composition, characterized in that, ​ 11. The composition of claim 10, wherein, The label comprises one or more of an enzyme, latex particles, fluorescent molecular label, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal silver, colloidal carbon, biotin or streptavidin; The enzyme comprises alkaline phosphatase or horseradish peroxidase.

12. A biomaterial, characterized in that, comprises a nucleic acid fragment, a vector or a host cell; The nucleic acid fragment is selected from (a1) or (a2): (a1), DNA or RNA encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-9; (a2), a nucleic acid fragment complementary to the nucleic acid fragment defined in (a1); The vector comprises the nucleic acid fragment; The host cell is transformed by the vector.

13. The biomaterial of claim 12, wherein, The nucleotide sequence of the DNA fragment encoding the light chain constant region is shown as Seq_33; The nucleotide sequence of the DNA fragment encoding the heavy chain constant region is shown as Seq_34.

14. A method for the production of an antibody or antigen binding fragment thereof that binds to a KPC enzyme, characterized in that, comprises expressing the antibody or antigen-binding fragment thereof that binds to KPC enzyme using the host cell according to claim 12.

15. A reagent or kit for detecting a KPC enzyme, characterized in that, comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the composition according to claim 10.

16. The reagent or kit of claim 15, wherein, The kit comprises an immunochromatographic test card; In the immunochromatographic test card, the label of the antibody is colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres or quantum dots.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, the composition according to claim 10, the biological material according to claim 12, the production method according to claim 14, or the reagent or kit for detecting KPC enzyme according to claim 15 in detecting KPC enzyme for non-diagnostic and therapeutic purposes or detecting microorganisms producing KPC enzyme for non-diagnostic and therapeutic purposes.

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