Synergistic monoclonal antibody based on plasmodium falciparum antigen PfRH5 and application of synergistic monoclonal antibody in malaria treatment medicine

Abstract

The invention relates to the technical field of biological medicine, and discloses a synergistic monoclonal antibody based on a plasmodium falciparum antigen PfRH5 and application of the synergistic monoclonal antibody in malaria treatment medicine, the synergistic monoclonal antibody is P27-2, the heavy chain amino acid sequence of the synergistic monoclonal antibody is shown as SEQ ID NO: 2, and the light chain amino acid sequence of the synergistic monoclonal antibody is shown as SEQ ID NO: 3. The epitope of the monoclonal antibody for recognizing a PfRH5 antigen is a middle region of a PfRH5'kite type 'structure, Beta-strands 1 and 2, Helix 1, Helix 2 and Helix 5 are involved, and the specific recognition sequences are Q146-H148, N156-K167 and I176-Y185. The monoclonal antibody P27-2 provided by the invention can obviously enhance the inhibition effect of other multiple anti-PfRH5 antibodies on the growth of plasmodium falciparum in the erythrogenous stage, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 and its application in malaria treatment drugs. Background Technology

[0002] Malaria is a serious global infectious disease caused by Plasmodium parasites, primarily transmitted to humans through the bite of infected female Anopheles mosquitoes. Of the five types of Plasmodium parasites that infect humans, Plasmodium falciparum is the most deadly, particularly prevalent in Africa, accounting for over 90% of malaria cases in 2023. After infecting the human body, the Plasmodium sporozoites first enter the body, develop and multiply in the liver, and are then released into the bloodstream. Some are phagocytosed by phagocytes, while others invade red blood cells and multiply, leading to massive destruction of red blood cells and subsequently causing a series of severe symptoms such as fever, anemia, splenomegaly, and kidney damage. In areas with high prevalence of falciparum malaria, children under 5 years old, pregnant women, HIV / AIDS patients, and individuals without malaria immunity are highly susceptible to severe malaria. Without timely or proper treatment, these infections often lead to death. Despite significant global efforts in malaria control over the years, malaria remains a serious public health problem, ranking alongside tuberculosis and AIDS as one of the world's three major infectious diseases. Currently, antimalarial drugs, mosquito control, and malaria vaccines are the main means of malaria prevention and control. However, the emergence of drug-resistant Plasmodium strains and Anopheles mosquitoes globally poses a significant challenge to malaria control. The invasion of erythrocytes by Plasmodium merozoons is the core mechanism of malaria pathogenesis, a process dependent on the interaction between merozoite ligands and host receptors. Plasmodium falciparum merozoons utilize multiple ligand families, such as the erythrocyte binding-like proteins (EBL) family and the reticulocyte binding-like proteins (RBL) family, to invade erythrocytes. PfRH5, a new member of the RBL family of Plasmodium falciparum, is located in the rod-shaped body at the apex of the merozoite and is a key protein discovered in recent years involved in merozoite invasion of erythrocytes. Its importance is multifaceted: First, PfRH5 binds to its ligands in numerous strains of Plasmodium falciparum, while previously discovered pathways mediating erythrocyte invasion were often only effective against a few strains; second, studies have shown that PfRH5-based vaccines can induce resistance against different Plasmodium species in night monkeys. Therefore, PfRH5 is considered a key target for conquering Plasmodium falciparum and is hailed as the universal key to unlocking this "Achilles' heel" of Plasmodium falciparum. However, current research on PfRH5 still faces many challenges in practical application. While some vaccine studies on PfRH5 have been conducted, their protective efficacy is unsatisfactory; for example, the most advanced PfRH5 vaccine, RH5.1 / AS01B, showed only moderate protective efficacy in clinical trials. In the field of monoclonal antibodies, although research has focused on developing monoclonal antibodies against PfRH5 to inhibit the invasion of Plasmodium merozoons, no monoclonal antibody has yet emerged that can significantly enhance efficacy and be widely used in clinical treatment. Polyclonal antibodies, due to their complex composition, have many limitations in clinical treatment and are not suitable for clinical use.

[0003] Based on this, we provide a highly efficient, safe, and effective monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 that can effectively inhibit malaria parasite infection. Its application in malaria treatment drugs has extremely important practical significance and clinical value, and is expected to provide a new and effective means for the prevention and control of malaria. Summary of the Invention

[0004] In view of this, the present invention proposes an enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 and its application in malaria treatment drugs, aiming to solve at least one of the problems in the background art.

[0005] This invention proposes an enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5, wherein the enhanced monoclonal antibody is P27-2, and its heavy chain amino acid sequence is shown in SEQ ID NO: 2, and its light chain amino acid sequence is shown in SEQ ID NO: 3.

[0006] Preferably, the monoclonal antibody recognizes the epitopes of the PfRH5 antigen in the middle region of the PfRH5 "kite-shaped" structure, involving Beta-strands 1, 2 and Helix 1, Helix 2, Helix 5, specifically recognizing the sequences Q146-H148, N156-K167, and I176-Y185.

[0007] This invention also provides a method for preparing the enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 described in the above technical solution, comprising the following steps: Step 1: Prepare a PfRH5 protein-based mRNA vaccine, wherein the mRNA vaccine is PfRH5 mRNA encapsulated in lipid nanoparticles after optimization and modification of its nucleotide sequence. Step 2: Immunize animals with the mRNA vaccine and obtain B lymphocytes from the spleen of the immunized animals; Step 3: Fuse the B lymphocytes from the spleen of the immunized animal with myeloma cells, and screen for positive hybridoma cells that can secrete antibodies against PfRH5 protein. Step 4: The positive hybridoma cells that can secrete antibodies against PfRH5 protein are cultured in a monoclonal manner to screen and obtain hybridoma cell lines that can secrete monoclonal antibody P27-2; Step 5: Culture the hybridoma cell line that can secrete monoclonal antibody P27-2, and purify to obtain monoclonal antibody P27-2.

[0008] Preferably, the preparation of the PfRH5 protein-based mRNA vaccine specifically involves: linearizing the DNA template using BspQ I enzyme, and obtaining mRNA with a 5' cap and a 3' poly A tail through co-transcription and capping; the lipid nanoparticles are composed of cationic lipids, phosphatidylcholine, cholesterol lipids, and PEG lipids in a mass ratio of 50:10:38.5:1.5.

[0009] The present invention also provides a malaria treatment drug comprising the enhanced monoclonal antibody P27-2 as described in claim 1 or 2.

[0010] Preferably, it also contains at least one other antimalarial parasite antibody, said other antimalarial parasite antibody being an antibody against infection of Plasmodium falciparum during its erythrocytic stage.

[0011] Preferably, the other antimalarial antibodies include at least one of P20-2, P09, P13, P23, P24, P05-3, and P22.

[0012] The present invention also provides a composition for enhancing the neutralizing capacity of antimalarial parasite antibodies, comprising the synergistic monoclonal antibody P27-2 as described in claim 1 or 2.

[0013] Preferably, it further comprises at least one antimalarial parasite antibody, said antimalarial parasite antibody including antibodies with neutralizing capacity and antibodies without neutralizing capacity.

[0014] The present invention also provides the use of the malaria treatment drug described in the above technical solution or the composition for enhancing the neutralizing ability of antimalarial parasite antibodies in the preparation of malaria treatment drugs.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Although the monoclonal antibody P27-2 provided by the present invention has no neutralizing ability, it can significantly enhance the inhibitory effect of other anti-PfRH5 antibodies on the growth of Plasmodium falciparum in the erythrocytic stage, providing a new and effective means for the treatment of malaria. It has important clinical value, especially in the case of the increasing resistance of traditional antimalarial drugs.

[0016] (2) The monoclonal antibody described in this invention recognizes a novel epitope of PfRH5, which provides a new idea and foundation for vaccine design based on RH5 as a target and antibody combination therapy, and helps to address the drug resistance risk caused by the single antigen recognition site of existing antibodies.

[0017] (3) The PfRH5 antigen prepared by the present invention using mRNA vaccine technology can better maintain its natural conformation and function, thereby inducing the production of antibodies that are closer to the natural infection state, thus ensuring the quality and effectiveness of the screened antibodies.

[0018] (4) The pharmaceutical composition containing the monoclonal antibody described in this invention can be used in combination with other antimalarial parasite antibodies, which can significantly improve the therapeutic effect and expand the range of options for malaria treatment. Attached Figure Description

[0019] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings: Figure 1 This is a schematic diagram of the inhibition rate results in Example 3 of the present invention; Figure 2 This is a schematic diagram of the mass spectrometry analysis results for identifying the P27-2 antibody epitope in this invention. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0021] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention provides an enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5, wherein the enhanced monoclonal antibody is P27-2, and its heavy chain amino acid sequence is shown in SEQ ID NO: 2, and its light chain amino acid sequence is shown in SEQ ID NO: 3.

[0026] The SEQ ID NO: 2 is specifically: VHSQVQLQQPGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKATLTADKSSSTAYMEFRSLTSEDSAVYFCTCNEITGIYY FDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC; The SEQ ID NO: 3 is specifically: VHSDVQITQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPRTFGG GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0027] In this invention, the monoclonal antibody recognizes the epitopes of the PfRH5 antigen as the middle region of the PfRH5 "kite-shaped" structure, involving Beta-strands 1, 2 and Helix 1, Helix2, Helix 5, specifically recognizing the sequences Q146-H148, N156-K167, and I176-Y185.

[0028] The sequences Q146-H148, N156-K167, and I176-Y185 described in this invention are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Specifically, SEQ ID NO: 4 is: QYH; Specifically, SEQ ID NO: 5 is: NIANSIDILQEK; The SEQ ID NO: 6 is specifically: IPHYTFLDY.

[0029] This invention also provides a method for preparing the enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 described in the above technical solution, comprising the following steps: Step 1: Prepare a PfRH5 protein-based mRNA vaccine, wherein the mRNA vaccine is PfRH5 mRNA encapsulated in lipid nanoparticles after optimization and modification of its nucleotide sequence. Step 2: Immunize animals with the mRNA vaccine and obtain B lymphocytes from the spleen of the immunized animals; Step 3: Fuse the B lymphocytes from the spleen of the immunized animal with myeloma cells, and screen for positive hybridoma cells that can secrete antibodies against PfRH5 protein. Step 4: The positive hybridoma cells that can secrete antibodies against PfRH5 protein are cultured in a monoclonal manner to screen and obtain hybridoma cell lines that can secrete monoclonal antibody P27-2; Step 5: Culture the hybridoma cell line that can secrete monoclonal antibody P27-2, and purify to obtain monoclonal antibody P27-2.

[0030] The preferred preparation method is: Step 1: Preparation of a PfRH5 protein-based mRNA vaccine, wherein the mRNA vaccine is PfRH5 mRNA with optimized nucleotide sequence and modified and encapsulated in lipid nanoparticles; wherein, the preparation of PfRH5 mRNA includes linearizing the DNA template with BspQ I enzyme, and obtaining mRNA with a 5' cap and a 3' poly A tail by co-transcription and capping; the composition ratio of lipid nanoparticles is cationic lipid: phosphatidylcholine: cholesterol lipid: PEG lipid = 50:10:38.5:1.5; the expression of PfRH5 protein is verified by 293T cell transfection and Western blotting.

[0031] Step 2: Immunize animals with the mRNA vaccine prepared in Step 1. The animals are 6-8 week old BALB / c female mice. The immunization method is 3 immunizations, each with a dose of 10 μg, with an interval of 3 weeks. The immunization effect is determined by detecting the specific antibody titer in the serum by ELISA. Splenic B lymphocytes are obtained from the immunized animals.

[0032] Step 3: Mix the spleen B lymphocytes obtained in Step 2 with myeloma cells (such as SP2 / 0 myeloma cells) at a ratio of 5:1, induce fusion with PEG-1500, and culture in HAT selective medium for 10-14 days to screen for positive hybridoma cells that can secrete anti-PfRH5 protein antibodies.

[0033] Step 4: The positive hybridoma cells obtained in Step 3 were cultured as monoclonal cells using the limiting dilution method. Positive monoclonal hybridoma cell lines that secrete anti-PfRH5 protein antibodies were screened by ELISA. Then, monoclonal antibody P27-2 was obtained by screening through a growth inhibition assay. The growth inhibition assay included combining the candidate antibody with other anti-PfRH5 antibodies and detecting the inhibitory effect on the growth of Plasmodium falciparum in the erythrocytic stage. Monoclonal antibody P27-2 that can enhance the neutralizing ability of other antibodies was screened out.

[0034] Step 5: Culture the hybridoma cell line obtained in Step 4 and purify it to obtain the monoclonal antibody P27-2; RNA can also be extracted from the hybridoma cell line obtained in Step 4, and the antibody light and heavy chain variable region gene can be amplified by RT-PCR. After humanization, it can be constructed into an expression vector, transfected into 293T or CHO cells for recombinant antibody expression, and then purified by Protein A or G affinity chromatography to obtain the recombinant monoclonal antibody P27-2.

[0035] In this invention, the preparation of the mRNA vaccine based on PfRH5 protein specifically involves: linearizing the DNA template with BspQ I enzyme, and obtaining mRNA with a 5' cap and a 3' poly A tail through co-transcription and capping; the lipid nanoparticles are composed of cationic lipids, phosphatidylcholine, cholesterol lipids, and PEG lipids in a mass ratio of 50:10:38.5:1.5.

[0036] The sequence of the PfRH5 protein used in this invention is shown in SEQ ID NO: 1, specifically:

[0037] The present invention also provides a malaria treatment drug comprising the enhanced monoclonal antibody P27-2 as described in claim 1 or 2.

[0038] This invention also includes at least one other antimalarial parasite antibody, which is an antibody against infection of Plasmodium falciparum during its erythrocytic stage.

[0039] In this invention, the other antimalarial antibodies include at least one of P20-2, P09, P13, P23, P24, P05-3, and P22.

[0040] The specific sequences of the antimalarial parasite antibodies P20-2, P09, P13, P23, P24, P05-3, and P22 described in this invention are as follows: The heavy chain sequence of P20-2 is shown in SEQ ID NO: 7, specifically as follows: VHSQVQMKESGPGLVQPSQSLSITCTVSGFPLSGYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSITKDNSRSQVLFKMNSLQPNDTAIYYCARNHYYEYDWYFDVW GAGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC; The light chain sequence of P20-2 is shown in SEQ ID NO: 8, specifically as follows: VHSDVQITQSSSSSSVSLGDRVTITCRASEDIFNRLAWYQQKPGNAPRLLISGASSLESGVLSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWIIPYTFGGGTKLE LKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The heavy chain sequence of P09 is shown in SEQ ID NO: 9, specifically as follows: VHSEVKLVESGAELVRPGALVKLSCKGSGLNIKDYYIHWVKQRPEQGLEWIGWIDPENGKTIYKPKFQGKATFTADTSSNTAYLQLSSLTSEDSAVYYCSRNYDASMDYWGQGTTVTVSSA The light chain sequence of P09 is shown in SEQ ID NO: 10, specifically as follows: VHSNIQVIQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGLGQTSPSTSILWRRRMLQPITVSTLGSLHVRRGDQTGIK The heavy chain sequence of P13 is shown in SEQ ID NO: 11, specifically as follows: VHSQVQLKQSGAELVRPGALVKLSCKTSGFNIKDYYIHWVKQRPEQALEWIGWIDPENGNTIYNPEFQDKASFTADASSNTAYLQLSSLASEDAAVYYCARNYDASMDYWGQGTTLTVSSA The light chain sequence of P13 is shown in SEQ ID NO: 12, specifically as follows: VHSDIVMTQAPLTSSVTIGQPASISCKSSQSLLDSDGKTYFHWLLQRPGQSPKRLIYLVSKLDSGFPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLEIK The heavy chain sequence of P23 is shown in SEQ ID NO: 13, specifically as follows: VHSQVQMKESGAELVRPGALVKLSCKGSGLNIKDYYIHWVKQRPEQGLEWIGWIDPENGKTIYSPKFQGKATFTADTSSNAAYLQLSSLTSEDTAVYYCSRNYDASMDYWGQGTSVTVSSA The light chain sequence of P23 is shown in SEQ ID NO: 14, specifically as follows: VHSQIVLTQSPLTLSVTIGQPASISCKSSQSLLDTDGKTYFHWLLQRPGQSPQRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTRLEIK The heavy chain sequence of P24 is shown in SEQ ID NO: 15, specifically as follows: VHSEVMLVESGAELVRPGALVKLSCKGSGLNIKDYYIHWVKQRPEQGLEWIGWIDPENGKTIYSPKFQGKATFTADTPSNTAYLQLSSLTSEDTAVYYCSRNYDASMDYWGQGTMVTVSSA The light chain sequence of P24 is shown in SEQ ID NO: 16, specifically as follows: VHSDIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGTRLEIKRTVAAPSVFGTRLEIK The heavy chain sequence of P22 is shown in SEQ ID NO: 17, specifically as follows: VHSQIQLVQSGAELVRPGALVKLSCKGSGLNIKDYYIHWVKQRPEQGLEWIGWIDPENGKTIYSPKFQGKATFTADTSSNTAYLQLSSLTSEDTAVYYCSRNYDASMDYWGQGTTVTVSSA The light chain sequence of P22 is shown in SEQ ID NO: 18, specifically as follows: CTFRCCGDSTPLTLSVTIGQPASISCKSSQSPLDTDGKTYFHWLLQRPGQSPQRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELK The heavy chain sequence of P05-3 is shown in SEQ ID NO: 19, specifically as follows: VHSEVMLVESGAELVRPGALVKLSCKGSGLNIKDYYMHWVKQRPEQGLEWIGWIDPENGKTIYNPKFQGKATFTADTSSNTAYLQLSSLTSEDTAVYYCSRNYDASMDYWGQGTTLTVSSA The light chain sequence of P05-3 is shown in SEQ ID NO: 20, specifically as follows: VHSEIVLTQSPLTLSVTIGQPASISCKSSQSLLDTDGKTYFHWLLQRPGQSPQRLIYLVSKLDSGVPDRFTGSGSGTHFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLEIK.

[0041] The present invention also provides a composition for enhancing the neutralizing capacity of antimalarial parasite antibodies, comprising the synergistic monoclonal antibody P27-2 as described in claim 1 or 2.

[0042] In this invention, at least one antimalarial parasite antibody is also included, said antimalarial parasite antibody comprising antibodies with neutralizing capacity and antibodies without neutralizing capacity.

[0043] The present invention also provides the application of the malaria treatment drug described above or the composition for enhancing the neutralizing ability of antimalarial parasite antibodies in the preparation of malaria treatment drugs. The experimental materials used in the embodiments of this invention are as follows: 1. Experimental animals: 6-8 week old BALB / c female mice.

[0044] 2. Cell lines: 293T cells, SP2 / 0 myeloma cells, and Plasmodium falciparum strains.

[0045] 3. Main reagents: PfRH5 DNA template, BspQ I enzyme, mRNA transcription-related reagents, lipid nanoparticle components (cationic lipids, phosphatidylcholine, cholesterol lipids, PEG lipids), PEG-1500, HAT selective medium, ELISA-related reagents, Protein A / G affinity chromatography column, RPMI 1640 medium, human serum, sodium bicarbonate, gentamicin, Percoll, Sorbitol, lactate dehydrogenase substrate, etc.

[0046] 4. Main instruments: microplate reader, centrifuge, HPLC-MS system, Orbitrap Fusion mass spectrometer, etc.

[0047] Example 1: Preparation and Immunization of mRNA Vaccine Based on PfRH5 Protein (1) Preparation of mRNA: PfRH5 DNA template was linearized by BspQ I enzyme and mRNA with 5' cap and 3' poly A tail was obtained by co-transcription and capping.

[0048] (2) Lipid nanoparticle encapsulation: lipid nanoparticles were prepared in the ratio of cationic lipid: phosphatidylcholine: cholesterol lipid: PEG lipid = 50:10:38.5:1.5, and the above mRNA was encapsulated in them.

[0049] (3) Expression verification: The high-efficiency expression of RH5 protein was confirmed by transfection with 293T cells and Western blotting.

[0050] (4) Animal immunization: 6-8 week old BALB / c female mice were immunized 3 times, 10 μg each time, with an interval of 3 weeks. The serum was detected by ELISA and the specific antibody titer was significantly increased.

[0051] Example 2: Screening and preparation of monoclonal antibody P27-2 (1) Hybridoma cell preparation: Take the mouse spleen B lymphocytes with the highest titer after immunization, mix them with SP2 / 0 myeloma cells at a ratio of 5:1, induce fusion with PEG-1500, culture in HAT selective medium for 10-14 days, and screen for positive hybridoma cells.

[0052] (2) Monoclonal culture and screening: Positive hybridoma cells were cultured using the limiting dilution method, and antigen-specific antibody secretion wells were screened by ELISA to obtain positive monoclonal cells.

[0053] (3) Antibody expression and purification: RNA was extracted from positive monoclonal cells, and the variable region gene of the antibody light and heavy chains was amplified by RT-PCR. After humanization, it was constructed into an expression vector, transfected into 293T cells for recombinant antibody production, and the target antibody was obtained by Protein A affinity chromatography purification.

[0054] (4) Growth inhibition test screening: The obtained antibodies were subjected to growth inhibition verification, and 10 representative monoclonal antibodies were screened out. Among them, P27-2 showed the effect of enhancing the neutralizing ability of other antibodies in the antibody combination experiment.

[0055] Example 3: Verification of P27-2's enhancement of the neutralizing ability of other antibodies (1) Antibody preparation: Monoclonal antibody IgG was purified using Protein G, and the antibody buffer was replaced with RPMI 1640. The concentration was measured for later use.

[0056] (2) Culture medium preparation: Freshly prepare 2× complete culture medium (containing 20% ​​human serum, 2× sodium bicarbonate, and 2× gentamicin).

[0057] (3) Synchronization of Plasmodium: Plasmodium falciparum was synchronized by the Percoll / Sorbitol method to obtain highly synchronized late-stage Plasmodium falciparum (38-42 h) for growth inhibition experiments.

[0058] (4) Growth inhibition assay (GIA): The GIA system was 40 μL / well, the volume of plasma was 1%, and the initial protozoan rate was 0.3% ± 0.1%. The purified IgG was diluted according to the concentration to ensure that the volume added to the experimental well was ≥ 5 μL. The final IgG concentration of P20-2, P09, P13, P23, P24, P05-3, P22 and R5.015 monoclonal antibodies in the experimental wells was 500 μg / mL, and the final IgG concentration of P05-1, P27-2 and R5.011 monoclonal antibodies was 200 μg / mL. RPMI 1640 was added to 20 μL. Then, 20 μL of Plasmodium falciparum suspension (formulation: 2×CM with a protozoan rate of 0.3% ± 0.1% and a volume of plasma of 2%) was added to each well. The experimental wells contained RH5 monoclonal antibody IgG, the blank experimental wells contained IgG purified from blank mouse serum, and the control wells contained no IgG. Three replicate wells were set up. After 40-48 h, centrifuge and discard the supernatant. Wash twice with 1×PBS. Add 120 μL of 1×PBS to each well, centrifuge at 1300 g for 6 minutes at 4℃, and discard 120 μL of supernatant. Add 120 μL of lactate dehydrogenase substrate to each well, incubate in the dark for 20 min, and detect the value at 650 nm using a microplate reader to calculate the inhibition rate.

[0059] The result is as follows Figure 1As shown, based on Figure 1 It can be seen that: P20-2 has a very strong neutralizing ability; P09 and P23 have no neutralizing ability; P13, P24, P05-3, and P22 have relatively weak neutralizing abilities; and the monoclonal antibody R5.015, which has no neutralizing ability in published literature (Alanine, Daniel GW et al. “Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.” Cell vol. 178,1 (2019): 216-228.e21). (doi:10.1016 / j.cell.2019.05.025) Eight antibodies were combined with P27-2 and P05-1 (which lack neutralizing ability) to inhibit the in vitro growth of Plasmodium erythrocytic stage. The results showed that P27-2 not only enhanced the inhibitory effect of the already neutralizing monoclonal antibody P20-2, but also significantly improved the inhibitory effect of antibodies with no or weak neutralizing ability. P05-1, as a control, did not exhibit this effect. In other words, P27-2 effectively enhances the neutralizing ability of various other antibodies.

[0060] Identification of P27-2 antibody epitope Competitive ELISA and deuterium-hydrogen exchange mass spectrometry analysis: Candidate monoclonal antibodies and control antibodies were labeled with biotin, and the epitope competition between the antibodies was analyzed by competitive ELISA; a deuterium-hydrogen exchange assay was performed, and the samples were analyzed by mass spectrometry. Specific experimental methods are as follows: The specific experimental method is as follows: (1) The candidate monoclonal antibody and two control antibodies, R5.004 and R5.016, were labeled with biotin. First, the RH5 protein and the unlabeled protein were incubated separately. Then, the biotin-labeled antibodies were added separately and color development was performed to analyze the epitope competition relationship between the antibodies.

[0061] (2) Add 2 μL of pfRH5 protein sample (40 μM) or pfRH5-antibody complex (40 μM) to 18 μL of D2O buffer (25 mM Tris-HCl, pD 8.5, 100 mM NaCl) for deuteration labeling. Then, at different time points of 0 s, 10 s, 100 s, 1000 s, and 10000 s, add 20 μL of termination buffer (8 Murea, 0.5 MTCEP, pH 3.0) to the above system to terminate the hydrogen-deuterium exchange reaction. Finally, add 60 μL of dilution buffer (1.5% FA, 50 mMTCEP) to the above system and freeze in liquid nitrogen at -80°C for subsequent use.

[0062] (3) During mass spectrometry experiments, the above samples were thawed on ice and then rapidly injected into an HPLC-MS system (Thermo Ultimate 3000) consisting of a protease digestion column (NovabioAssays, NBA2014002), a Trap column (ThermoFisher, 2100100), and a BioBasic C8 analytical column (BIOBASIC 8, 03-051-676). The peptides after enzyme digestion were captured by the Trap column and separated by the BioBasic C8 analytical column, then directly analyzed by electrospray ionization in the Orbitrap Fusion mass spectrometer (Thermo). Peptide sequence identification was performed using mass spectrometry on deuterium-free samples (samples at time 0 s). Mass spectrometry was conducted in tandem MS / MS mode (orbi / orbi). All MS / MS spectra were searched using the MASCOT algorithm with an FDR of 1% to obtain sequence identification for all peptides. Subsequent mass spectrometry analyses of deuterated samples at other time points were performed in Level 1 MS mode. (4) Data analysis: Mass spectrometry data are input into HD-Examiner v2.3 software for processing to determine the antigen-binding epitope of each antibody.

[0063] The results are as follows Figure 2 As shown, based on Figure 2 It can be seen that the epitope targeted by P27-2 is the middle region of the "kite-shaped" structure of PfRH5, involving specific Beta-strands and Helix, with the specific identification sequences being Q146-H148, N156-K167, and I176-Y185.

[0064] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A potentiating monoclonal antibody based on the Plasmodium falciparum antigen PfRH5, characterized in that, The enhancing monoclonal antibody is P27-2, whose heavy chain amino acid sequence is shown in SEQ ID NO: 2 and whose light chain amino acid sequence is shown in SEQ ID NO:

3.

2. The enhancing monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody recognizes the epitopes of the PfRH5 antigen in the middle region of the PfRH5 "kite-shaped" structure, involving Beta-strands 1, 2 and Helix 1, Helix 2, Helix 5, specifically recognizing the sequences Q146-H148, N156-K167, and I176-Y185.

3. A method for preparing an enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 as described in any one of claims 1 to 2, characterized in that, Includes the following steps: Step 1: Prepare a PfRH5 protein-based mRNA vaccine, wherein the mRNA vaccine is PfRH5 mRNA encapsulated in lipid nanoparticles after optimization and modification of its nucleotide sequence. Step 2: Immunize animals with the mRNA vaccine and obtain B lymphocytes from the spleen of the immunized animals; Step 3: Fuse the B lymphocytes from the spleen of the immunized animal with myeloma cells, and screen for positive hybridoma cells that can secrete antibodies against PfRH5 protein. Step 4: The positive hybridoma cells that can secrete antibodies against PfRH5 protein are cultured in a monoclonal manner to screen and obtain hybridoma cell lines that can secrete monoclonal antibody P27-2; Step 5: Culture the hybridoma cell line that can secrete monoclonal antibody P27-2, and purify to obtain monoclonal antibody P27-2.

4. The method for preparing the enhanced monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 according to claim 3, characterized in that, The preparation of the PfRH5 protein-based mRNA vaccine specifically involves: linearizing the DNA template using BspQ I enzyme, and obtaining mRNA with a 5' cap and a 3' poly A tail through co-transcription and capping; the lipid nanoparticles are composed of cationic lipids, phosphatidylcholine, cholesterol lipids, and PEG lipids in a mass ratio of 50:10:38.5:1.

5.

5. A malaria treatment drug, characterized in that, It contains the enhancing monoclonal antibody P27-2 as described in claim 1 or 2.

6. The malaria treatment drug according to claim 5, characterized in that, It also contains at least one other antimalarial parasite antibody, which is an antibody against infection of Plasmodium falciparum during its erythrocytic stage.

7. The malaria treatment drug according to claim 5, characterized in that, The other antimalarial antibodies include at least one of P20-2, P09, P13, P23, P24, P05-3, and P22.

8. A composition for enhancing the neutralizing capacity of antimalarial parasite antibodies, characterized in that, It contains the enhancing monoclonal antibody P27-2 as described in claim 1 or 2.

9. The composition according to claim 8, characterized in that, It also contains at least one antimalarial parasite antibody, which includes antibodies with neutralizing capacity and antibodies without neutralizing capacity.

10. The use of the malaria treatment medicament of claim 5 or the composition of claim 8 for enhancing the neutralizing capacity of antimalarial parasite antibodies in the preparation of the malaria treatment medicament.

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