Pharmaceutical composition

Abstract

The object of the present invention is to provide an active ingredient for a systemically administered mRNA pharmaceutical that expresses an antibody specific to the V antigen protein or its homologous protein of Gram-negative bacteria, and a systemically administered mRNA pharmaceutical using the same. [Solution] mRNA containing an antibody coding region encoding a specific scFv antibody against the V antigen protein or its homologous protein of pathogenic Gram-negative bacteria is useful as an active ingredient in mRNA pharmaceuticals for systemic administration.

Description

Technical Field

[0001] The present invention relates to a nucleic acid medicine used for passive immunotherapy against Gram-negative bacteria.

Background Art

[0002] With the advent of advanced medicine or aging, many fatal infectious diseases caused by multi-drug resistant bacteria have been reported. Gram-negative bacteria are frequent causative agents of ventilator-associated pneumonia in intensive care patients, opportunistic infections in immunocompromised patients or the elderly, etc. In recent years, due to the rapid progress of high-level multi-drug resistance of Gram-negative bacteria, the development of antibacterial therapeutic agents that do not rely on antibacterial drugs has been strongly demanded.

[0003] Pathogenic Gram-negative bacteria such as Pseudomonas aeruginosa directly contact target eukaryotic cells and use a special secretion apparatus called a type III secretion system (TTSS) to directly transfer protein toxins into the cytoplasm of target cells. As reported in Non-Patent Documents 1 to 4, etc., the existence of this TTSS has been confirmed as a highly homologous system in many pathogenic Gram-negative bacteria.

[0004] The injection pathway of toxins into the target cytoplasm by TTSS is established by connecting the V antigen protein that forms a pentamer complex at the tip of the needle-like structure of TTSS and the translocon composed of PopB / PopD in the target cell membrane. And in Non-Patent Document 5, it has been clarified that a specific antibody against the V antigen protein of Pseudomonas aeruginosa can block the passage of toxins through the translocon, suggesting the effectiveness of an antibody medicine targeting this V antigen protein against Pseudomonas aeruginosa infections.

[0005] The advantages of typical antibody drugs include not only their specific binding to target molecules, but also their longer half-lives in the blood compared to other biopharmaceuticals. Non-patent document 6 shows that in metabolic studies using IgG, F(ab')2, Fab', or scFv of the tumor-associated glycoprotein 72 antibody CC49 in mice, the plasma and systemic clearance of scFv was extremely rapid. Non-patent document 7 reports on the blood half-lives of antibodies, stating that in pharmacokinetic studies using CD1 mice, the half-lives were 7 hours in the α phase and 222 hours in the β phase for IgG, 2.8 hours in the α phase and 140 hours in the β phase for scDb-Fc, and 2.5 hours in the α phase and 104 hours in the β phase for scFv-Fc, compared to 0.4 hours in the α phase and 1.3 hours in the β phase for scDb and 0.2 hours in the α phase and 0.6 hours in the β phase for scFv.

[0006] In other words, the smaller the antibody fragment size, the shorter its half-life in the blood, and the half-life is extremely short, especially for scFv. Therefore, at least an Fc domain is essential to achieve a certain degree of blood retention, and the IgG type is necessary to achieve sufficient blood retention.

[0007] Furthermore, sufficient blood retention of antibodies is a critical factor in obtaining the pharmacological effects of antibodies.

[0008] For example, Non-Patent Document 6, mentioned above, describes a tumor targeting study using four types of radiolabeled CC49 (scFv, Fab', F(ab')2, IgG) in an LS-174T human colon cancer xenograft model. It shows that scFv and Fab' have significantly lower tumor binding rates to injectable doses compared to F(ab')2 and IgG. Furthermore, Non-Patent Document 5, mentioned above, discloses that intravenous administration of IgG or F(ab')2 as antibodies against Pseudomonas aeruginosa V antigen protein (anti-PcrV antibodies) resulted in insufficient protection against Pseudomonas aeruginosa infection with intravenous administration of F(ab')2, ultimately leading to sepsis and bacteremia. This document mentions the possibility of an Fc-dependent mechanism other than TTSS blockade contributing to protection against Pseudomonas aeruginosa infection, and the fact that F(ab')2 is cleared from the circulatory system much faster than IgG, concluding that IgG is superior as an intravenous therapeutic agent.

[0009] In fact, the blood half-life is a hurdle for the practical application of pharmaceuticals, and all antibody drugs developed so far have used antibodies with an IgG-type structure.

[0010] On the other hand, mRNA drugs are medicines that treat or prevent illness by administering mRNA that codes for a pharmacologically effective protein, causing the cytoplasm to produce that protein. Drug discovery strategies for mRNA drugs, as opposed to antibody drugs, are also being considered, and the usefulness of antibody-expressing mRNA has actually been demonstrated in the treatment or prevention of infectious diseases and cancer. mRNA drugs can be manufactured at a lower cost than antibody drugs, and their sequences can be rapidly modified in response to antigenic mutations in pathogens, offering the advantage of efficiently developing new drugs in a short period of time, and research and development are becoming increasingly active worldwide. It should be noted that mRNA drugs, like nucleic acid drugs, are designed to express full-length antibodies, i.e., IgG, in the body. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Rumbaugh KP et al. Kluwer Academic Publishers, 2003; 183-199. [Non-Patent Document 2] Sawa T et al. Anesthesiol Clin North America 2004; 22: 591-606. [Non-Patent Document 3] Kipnis E et al. Med Mal Infect 2006; 36: 78-91. [Non-Patent Document 4] Sato H et al. Front. Microbiol. 2011; 2 Article 142 [Non-Patent Document 5] Shime N, Sawa T, et al. J Immunol. 2001; 167(10):5880-6 [Non-Patent Document 6] Cancer Res. 1991 Dec 1;51(23 Pt 1):6363-71. [Non-Patent Document 7] MAbs. 2016 Jan; 8(1): 120-128. [Overview of the project] [Problems that the invention aims to solve]

[0012] It is desirable that drugs that treat infections caused by Gram-negative bacteria through the mechanism of TTSS blockade be developed as mRNA drugs. Furthermore, it is desirable that such mRNA drugs be designed as systemically administered drugs that can be administered using simple and minimally invasive procedures and that can target many organs.

[0013] Therefore, the present invention aims to provide an active ingredient for a systemically administered mRNA pharmaceutical that expresses an antibody specific to the V antigen protein or its homologous protein of Gram-negative bacteria, and a systemically administered mRNA pharmaceutical using the same. [Means for solving the problem]

[0014] When mRNA drugs are administered systemically, they produce antibodies (therapeutic antibodies) in the organs where they accumulate, and these antibodies are eliminated through internal clearance, similar to antibody drugs. As has been reported in various studies, it is common knowledge that an Fc domain is necessary in the structure of therapeutic antibodies to ensure their retention in the bloodstream and to obtain effective therapeutic effects, and in practice, there were no options other than IgG. Furthermore, it has been reported that even F(ab')2 does not produce effective therapeutic effects when administered systemically, and single-chain antibodies scFv, which are even smaller molecules, have not even been considered as a therapeutic antibody structure due to their extremely short blood half-life.

[0015] mRNA drugs are expected to be more effective than antibody drugs because they continuously produce therapeutic antibodies in the body, but like nucleic acid drugs, they are designed to express IgG. scFv has an extremely short half-life in the blood, so it has not been considered and has been overlooked in the design of mRNA drugs.

[0016] The inventors designed mRNA, which is an active ingredient of an mRNA pharmaceutical for systemic administration, to express scFv, and surprisingly found that a strong anti-gram-negative bacterium effect can be obtained by systemic administration. Surprisingly, the anti-gram-negative bacterium effect of the mRNA pharmaceutical designed to express scFv was superior to that of the mRNA pharmaceutical designed to express Fc-fused scFv. This result overturned the common sense of antibody drug discovery that the Fc domain is necessary to ensure blood retention and obtain an effective therapeutic effect. Without being bound by theory, it is considered that the high tissue permeability of scFv contributed to such a specific effect. That is, although scFv has poor blood retention, by being mRNAized and produced in the body, the poor blood retention is compensated, and the high tissue permeability greatly contributes to the anti-gram-negative bacterium effect. This effect is a unique effect due to the selection of scFv as the structure of the antibody expressed in the mRNA pharmaceutical and was extremely unexpected.

[0017] The present invention was completed by further studies based on these findings. That is, the present invention provides inventions in the following aspects.

[0018] Item 1. A pharmaceutical composition for systemic administration containing, as an active ingredient, mRNA containing an antibody coding region encoding a specific scFv antibody against a V antigen protein of a pathogenic gram-negative bacterium or its homologous protein. Item 2. The pharmaceutical composition according to Item 1, wherein the pathogenic gram-negative bacterium is Pseudomonas aeruginosa, Yersinia pestis, Bordetella pertussis, Escherichia coli, Salmonella, Shigella, Aeromonas hydrophila, Legionella, Mycobacterium avium, pathogenic Escherichia coli, Vibrio parahaemolyticus, Vibrio harveyi, Burkholderia pseudomallei, or Chlamydia. Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the pathogenic gram-negative bacterium is Pseudomonas aeruginosa. Item 4. The pharmaceutical composition according to any one of Items 1 to 3, wherein the V antigen protein or its homologous protein is PcrV, LcrV, LssV, AcrV, VcrV, Vp1659, IpaD, SipD, SeeB, BipD, CT584, EspA, or Bsp22. Item 5. The pharmaceutical composition according to any one of Items 1 to 4, wherein the V antigen protein or its homologous protein is PcrV. Item 6. The pharmaceutical composition according to any one of Items 1 to 5, wherein the mRNA is carried by lipid nanoparticles. Item 7. The pharmaceutical composition according to any one of Items 1 to 6, wherein the systemic administration is intravenous administration or intramuscular administration. Item 8. The pharmaceutical composition according to any one of Items 1 to 7, which is used for the prevention or treatment of pathogenic Gram-negative bacterial infections. Item 9. The pharmaceutical composition according to Item 8, wherein the infection is pneumonia, acute lung injury, or sepsis. Item 10. An mRNA comprising an antibody coding region encoding a specific scFv antibody against the V antigen protein of a pathogenic Gram-negative bacterium or its homologous protein. Item 11. The mRNA according to Item 10, which has a tissue plasminogen activator signal sequence at the 5' end of the coding region.

Advantages of the Invention

[0019] According to the present invention, there are provided an active ingredient of an mRNA pharmaceutical for systemic administration that expresses an antibody specific to the V antigen protein of Gram-negative bacteria or its homologous protein, and an mRNA pharmaceutical for systemic administration using the same.

Brief Description of the Drawings

[0020] [Figure 1] Shows the time course of the increase in anti-PcrV titer after therapeutic administration (intravenous injection or intramuscular injection) of the mRNA of Example 1 to Pseudomonas aeruginosa pneumonia mice according to Test Example 1. The horizontal axis indicates the time after administration. [Figure 2A]Test Example 2 demonstrates the inhibitory effect on acute lung injury (suppression of pulmonary edema 24 hours after infection) against lung infection caused by lethal doses of Pseudomonas aeruginosa following prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice. [Figure 2B] Test Example 2 demonstrates the inhibitory effect on acute lung injury against lethal doses of Pseudomonas aeruginosa lung infection (inhibitory effect on the number of bacteria in the lungs 24 hours after infection) following prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice. [Figure 2C] Test Example 2 demonstrates the inhibitory effect on acute lung injury against lethal doses of Pseudomonas aeruginosa lung infection (inhibitory effect on pulmonary granulocyte peroxidase activity 24 hours after infection) following prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice. [Figure 3A] Test Example 2 demonstrates the inhibitory effect on pulmonary cytokine secretion against lung infection by lethal doses of Pseudomonas aeruginosa (inhibitory effect on interleukin-6 secretion 24 hours after infection) following prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice. [Figure 3B] Test Example 2 demonstrates the inhibitory effect on pulmonary cytokine secretion against lung infection by lethal doses of Pseudomonas aeruginosa (inhibitory effect on tumor necrosis factor TNF-α secretion 24 hours after infection) following prophylactic administration (intravenous or intramuscular) of mRNA from Example 1 to mice. [Figure 4A] This shows the survival rate of mice with Pseudomonas aeruginosa pneumonia after therapeutic administration (intravenous or intramuscular) of mRNA from Example 1 or Comparative Example 1, as shown in Test Example 3. [Figure 4B] This shows the survival rate of mice with Pseudomonas aeruginosa pneumonia after therapeutic administration (intravenous or intramuscular) of mRNA from Example 2 or Comparative Example 2, as in Test Example 3. [Figure 5] This shows the anti-PcrV antibody titer in bronchoalveolar lavage fluid 3 hours after therapeutic administration (intravenous injection) of mRNA from Example 1 or Comparative Example 1 to mice with Pseudomonas aeruginosa pneumonia, as shown in Test Example 3. [Modes for carrying out the invention]

[0021] The mRNA of the present invention is characterized by containing an antibody-coding region that encodes a specific scFv antibody against the V antigen protein or its homologous protein of pathogenic Gram-negative bacteria. Furthermore, the pharmaceutical composition of the present invention is characterized by containing mRNA containing an antibody-coding region that encodes a specific scFv antibody against the V antigen protein or its homologous protein of pathogenic Gram-negative bacteria as an active ingredient, and being used for systemic administration.

[0022] 1. mRNA 1-1. Antibody coding region 1-1-1. Pathogenic Gram-negative bacteria The pathogenic Gram-negative bacteria are not particularly limited and include, for example, Pseudomonas aeruginosa, Plague bacillus, Bordetella pertussis, Yersinia, Salmonella, Shigella, Aeromonas hydrophylla, bacteriovenus, Mycobacterium pseudotuberculosis, pathogenic Escherichia coli (enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enteroaggregative and adherent Escherichia coli (Eaec)), Vibrio parahaemolyticus, Vibrio haveyi, Melioidosis, Chlamydia, etc., with Pseudomonas aeruginosa being preferred. The mRNA and pharmaceutical composition of the present invention are particularly useful when the pathogenic Gram-negative bacteria are drug-resistant. Preferred examples of drug-resistant bacteria include carbapenem-resistant Pseudomonas aeruginosa and fluoroquinolone-resistant Salmonella.

[0023] 1-1-2. V antigen protein or its homologous protein (needle-tip protein) V antigen proteins or their homologous proteins are the proteins that constitute the tip portion of the needle-like structure of the type III secretory system (TTSS), i.e., needle-tip proteins. Examples of TTSS include Yop, Pop / Exo, Lop, Aop, Vop, Esp / Tir, Sop.Sip, and Ipa. Because TTSS have been confirmed to exist in a wide range of Gram-negative bacteria with very high homology, there are no particular restrictions on which V antigen proteins or their homologous proteins are used. Specific examples of V antigen proteins or their homologous proteins include PcrV [derived from: Pseudomonas aeruginosa, etc.], LcrV [derived from: Plague bacillus, Mycobacterium pseudotuberculosis, Yersinia enteritis, etc.], LssV [derived from: Bacillus oryzae, etc.], AcrV [derived from: Aeromonas hydrophylla, bacillus-causing bacteria], VcrV [derived from: Vibrio parahaemolyticus, Vibrio harveyi], Vp1659 [derived from: Vibrio parahaemolyticus], IpaD [derived from: Shigella, etc.], SipD, SeeB [derived from: Salmonella, etc.], BipD [derived from: Bacillus melioides], CT584 [derived from: Chlamydia], EspA [derived from: pathogenic Escherichia coli such as EPEC and EHEC], Bsp22 [derived from: Bordetella pertussis], etc., with PcrV being preferred.

[0024] 1-1-3.scFv antibody The scFv antibody encoded in the antibody coding region is a small antibody having a structure in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a peptide linker. In the present invention, the scFv antibody does not contain an Fc region (i.e., Fc-fused scFv antibodies are excluded). The amino acid sequences of VH and VL can be appropriately selected by a person skilled in the art based on common technical knowledge, depending on the V antigen protein or its homologous protein. Known sequences can be used as the amino acid sequences of VH and VL, or known sequences can be appropriately modified and used.

[0025] As a specific example, a specific scFv antibody against the V antigen protein of Pseudomonas aeruginosa may include, as complementarity-determining regions (CDRs), VH may contain VH CDR1 to VH CDR3 consisting of the following amino acid sequences, and VL may contain VL CDR1 to VL CDR3 consisting of the following amino acid sequences. VH CDR1: Amino acid sequence shown in SEQ ID NO: 1 VH CDR2: Amino acid sequence shown in SEQ ID NO: 2 VH CDR3: The amino acid sequence shown in SEQ ID NO: 3, or The amino acid sequence shown in Sequence ID No. 3, in which at least one of the following is introduced: substitution of the 13th amino acid residue with F, and substitution of the 15th amino acid residue with I, S, or Q (for example, Sequence ID No. 4 consists of an amino acid sequence in which the 15th amino acid residue is substituted with I). VL CDR1: Amino acid sequence shown in SEQ ID NO: 5 VL CDR2: Amino acid sequence shown in SEQ ID NO: 6 VL CDR3: The amino acid sequence shown in SEQ ID NO: 7, or The amino acid sequence shown in Sequence ID No. 7, wherein at least one of the following is introduced: a substitution of the second amino acid residue with Q, and a substitution of the fifth amino acid residue with G (for example, Sequence ID No. 8 consists of an amino acid sequence in which the substitution of the second amino acid residue with Q is introduced).

[0026] Examples of VH having CDR1-3 as shown in SEQ ID NOs: 1-3 include VH consisting of the amino acid sequence from positions 22-146 of the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 70% or more (preferably 80% or more, 90% or more, or 95% or more) sequence identity with the amino acid sequence excluding CDR1-3. Examples of VL having CDR1-3 as shown in SEQ ID NOs: 5-7 include VL consisting of the amino acid sequence from positions 162-269 of the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 70% or more (preferably 80% or more, 90% or more, or 95% or more) sequence identity with the amino acid sequence excluding CDR1-3.

[0027] Examples of VH having CDR1-3 as shown in SEQ ID NOs: 1, 2, and 4 include VH consisting of the amino acid sequence from positions 22 to 146 of the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having 70% or more (preferably 80% or more, 90% or more, or 95% or more) sequence identity with the amino acid sequence excluding CDR1-3. Examples of VL having CDR1-3 as shown in SEQ ID NOs: 5, 6, and 8 include VL consisting of the amino acid sequence from positions 162 to 269 of the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having 70% or more (preferably 80% or more, 90% or more, or 95% or more) sequence identity with the amino acid sequence excluding CDR1-3.

[0028] "Sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) from BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1. Furthermore, in the present invention, in amino acid sequences with less than 100% sequence identity, if a mismatched amino acid residue is substituted with another amino acid from the reference amino acid sequence, substitution with a similar amino acid (i.e., a conservative amino acid substitution) is preferable. Specifically, based on the properties of the amino acid side chain, the following classification has been established, and it is preferable to substitute with an amino acid belonging to the same classification. Basic amino acids: lysine, arginine, histidine Acidic amino acids: glutamic acid, aspartic acid Neutral amino acids: Glycine, Alanine, Serine, Threonine, Methionine, Cysteine, Phenylalanine, Tryptophan, Tyrosine, Leucine, Isoleucine, Valine, Glutamine, Asparagine, Proline

[0029] Furthermore, if the V antigen protein or its homologous protein undergoes an antigenic mutation, a person skilled in the art can appropriately modify the amino acid sequences of VH and VL in response to the antigenic mutation, based on common technical knowledge. For example, the sequences of VH and VL can be identified by creating a specific blocking monoclonal antibody against the needle-tip protein of pathogenic Gram-negative bacteria and cloning the antibody gene from the antibody-producing hybridoma cells.

[0030] Regarding peptide linkers, those skilled in the art can appropriately select sequences of highly mobile peptide linkers. For example, specific examples of peptide linkers include Whitlow linkers and glycine-serine linkers.

[0031] 1-2. Other regions, chemical modifications, and sequence optimization As long as the mRNA molecule contains the predetermined antibody coding region described above, improvements and / or optimizations to the mRNA drug can be applied, including the addition of any other region, the introduction of any chemical modification, and / or sequence optimization, without impairing the effects of the present invention.

[0032] A typical mRNA may include, in this order from the 5' end, a cap structure, a 5' UTR, an antibody coding region (ORF), a 3' UTR, and a poly(A) tail region. In other mRNA examples, at least one of the following can be introduced in the cap structure: a modification that eliminates the need for a cap and applies an IRES, or a modification that confers degradation resistance; at least one of the following can be introduced in the 5' UTR: a sequence modification that controls binding to molecules involved in mRNA transport or translation, a modification to suppress 5'-exonucleotic degradation, or the introduction of an IRES; at least one of the following can be introduced in the 3' UTR: a sequence modification that controls binding to molecules involved in mRNA transport or translation, or a sequence modification to suppress deadenylation; and / or, at least one of the following can be introduced in the poly(A) tail region: a change to a masked poly(A) tail, control of the poly(A) chain length for mRNA stabilization, or the introduction of a chemically modified nucleic acid to suppress deadenylation.

[0033] mRNA can have a tag sequence encoding a protein or peptide tag inserted at the 3' end of the antibody coding region. In the typical example above, the tag sequence can be inserted between the antibody coding region and the 3'UTR. A specific example of such a protein or peptide tag is a detection tag, such as FLAG. (R) Non-fluorescent protein tags or epitope tags such as tags, c-myc tags, hemagglutinin antigen tags (HA tags); tags for affinity purification, such as His tags and Strep tags. (R) Examples include tags, GST tags, etc. These tags may be used individually or in combination.

[0034] mRNA can contain a secretion signal sequence at the 5' end of the antibody coding region. In the typical example above, the secretion signal sequence can be included between the 5'UTR and the antibody coding region. The secretion signal sequence is not particularly limited as long as it encodes an amino acid sequence having secretion signaling activity. Specific examples of secretion signal sequences include various signal sequences derived from secretory proteins, such as tissue plasmogen activator, interleukin 2, human growth hormone (hGH), serum albumin preproprotein, Igκ light chain precursor, azulosidine preproprotein, cystatin S precursor, trypsinogen 2 precursor, potassium channel inhibitors, α-conotoxin lp1.3, α-conotoxin, α-galactosidase, cellulose, aspartate proteinase nepenthesin-1, acid chitinase, K28 preprotoxin, killer toxin zygocin precursor, and cholera toxin, with tissue plasmogen activator signal sequences (tPAss) being preferred.

[0035] The mRNA sequence of the antibody coding region may be designed by applying, as appropriate, at least one of the following: codon optimization to improve translation efficiency, or the introduction of chemically modified nucleic acids to improve resistance to nucleases.

[0036] It is preferable that mRNA molecules have chemical modifications that suppress immunogenicity (such as suppression of inflammation at the administration site). A typical example of chemical modification is the substitution of uridine with chemically modified uridine. Examples of chemically modified uridines include pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine, with N1-methylpseudridine being preferred.

[0037] 2. Lipid nanoparticles The mRNA molecule is preferably supported on lipid nanoparticles. Lipid nanoparticles that can serve as carriers for nonviral drug delivery systems (DDS) are not particularly limited. Typical components of the lipid nanoparticles include ionized lipids, phospholipids, sterols, and PEGylated lipids, and preferably a combination of these four lipids.

[0038] Examples of ionized lipids include aminolipids that exhibit charge neutrality at physiological pH and protonate in acidic regions, specifically D-Lin-MC3-DMA, 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleyloxy-3-trimethylammoniumpropane chloride (DOTAP), 1,2-dioleyloxy-3-trimethylammoniumpropane chloride (DOTMA), 1,2-dioleyloxy-3-trimethylammoniumpropane chloride (DODAP), ( (4-Hydroxybutyl)azandiyl)di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl8-{(2-hydroxyethyl(SM-102)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate (ALC-0315 analog), C12-200, DLin-KC2-DMA, LP01 (BP-Lipid 215), L319, 306Oi10, COATSOME (R) SS-EC, COATSOME (R) Examples of commercially available products include SS-OP, and one or more types of aminolipids are selected from these.

[0039] The phospholipids are not particularly limited as long as they are lipids that stabilize lipid nanoparticles, but for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero Cello-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanol-sn-glycero-3-phosphoethanolamine (ME16.0 Examples include PE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, etc., and one or more of these lipid nanoparticles are selected.

[0040] The sterols are not particularly limited as long as they are lipids that stabilize lipid nanoparticles, but examples include cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol, and one or more of these sterols are selected.

[0041] PEGylated lipids are not particularly limited as long as they suppress interaction with proteins in plasma and extend their blood half-life through PEGylation, but examples include PEGylated phosphatidylethanolamine, PEGylated phosphatidic acid, PEGylated ceramide, PEGylated dialkylamine, PEGylated diacylglycerol, and PEGylated dialkylglycerol. More specifically, examples include 1,2-dimiristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG) and 1,2-dipalmi Toyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG), 1,2-dioleoyl-rac-glycero-3-methylpolyoxyethylene (DOG-PEG), 1,2-distearoyl-rac-glycero-3-(succinimidyloxycarbonyl)polyoxyethylene, 1,2-distearoyl-rac-glycero-3-(2'-maleimidoethyl)polyoxyethylene Examples of commercially available products include ethylene, methoxypolyethylene glycol oxy-N,N-ditetradecylacetamide (ALC-0159), and one or more of these PEGylated lipids are selected.

[0042] The combination ratio of the four types of lipids mentioned above is not particularly limited and can be determined appropriately by those skilled in the art, but examples include 35-65 mol% of ionized lipids, 5-15 mol% of phospholipids, 15-60 mol% of sterols, and 0.5-2 mol% of PEGylated lipids.

[0043] The loading ratio of mRNA to lipid nanoparticles is not particularly limited and can be determined appropriately by those skilled in the art, but the number of moles of nitrogen atoms contained in the ionized lipids constituting the lipid nanoparticles is, for example, 2 to 10 times, preferably 3 to 7 times, the number of moles of phosphate groups contained in the mRNA.

[0044] 3. Other ingredients The pharmaceutical composition of the present invention may, in addition to the above mRNA, preferably the above mRNA and the lipid nanoparticles supporting it, further contain or omit additives and / or bases depending on the formulation form, dosage form, and application, to the extent that they do not impair the effects of the present invention. Such additives and bases, whether present or absent, are not particularly limited to those that are pharmaceutically acceptable, and include, for example, adjuvants, excipients, binders, disintegrants, lubricants, isotonic agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, gelling agents, water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, water-soluble polymers, surfactants, metal soaps, lower alcohols, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV inhibitors, preservatives, thickeners, chelating agents, etc. These additives may be used individually or in combination of two or more. Furthermore, the content of these additives and bases is appropriately determined according to the type of additives and bases used, the formulation form of the pharmaceutical composition of the present invention, the dosage form, and the intended use.

[0045] 4. Dosage Form The pharmaceutical composition of the present invention is used for systemic administration. Systemic administration is a form of administration that assumes systemic circulation, that is, is intended for systemic effects. Specific examples of systemic administration include intravenous administration, intramuscular administration, subcutaneous administration, oral administration, oral administration, and rectal administration, with intravenous or intramuscular administration being preferred.

[0046] 5. Properties and Formulation The properties and formulation form of the pharmaceutical composition of the present invention can be appropriately determined according to the administration method, etc. Specific properties include liquid preparations and solid preparations (powder preparations, tablets, capsules, etc.). Specific formulation forms include injections when prepared for intravenous, intramuscular, or subcutaneous administration; internal preparations (enteric-coated capsules) when prepared for oral or intraoral administration; and suppositories when prepared for rectal administration, with injections being preferred.

[0047] 6.Applications The pharmaceutical composition of the present invention can be used for the prevention or treatment of pathogenic Gram-negative bacterial infections by systemic administration. Pathogenic Gram-negative bacteria are as described in "1-1-1. Pathogenic Gram-negative bacteria" above.

[0048] Specific examples of infections caused by pathogenic Gram-negative bacteria include pneumonia (Pseudomonas aeruginosa pneumonia, ventilator-associated pneumonia, aspiration pneumonia), acute lung injury, and sepsis, although these depend on the type of pathogenic Gram-negative bacteria.

[0049] The pharmaceutical composition of the present invention is intended for use in patients at high risk of developing pathogenic Gram-negative bacterial infections, or patients who have developed pathogenic Gram-negative bacterial infections. Specifically, this includes immunocompromised patients, patients on mechanical ventilation, patients with cystic fibrosis, and elderly individuals.

[0050] The animal species to which the pharmaceutical composition of the present invention can be applied include mammals such as humans, monkeys, cattle, pigs, goats, sheep, rabbits, dogs, cats, rats, and mice, with humans being preferred. [Examples]

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0052] [1] Preparation of mRNA encoding anti-PcrV antibody [1-1] Antibody design Antibodies specific to the V antigen protein of Pseudomonas aeruginosa, either anti-PcrV scFv or anti-PcrV scFv-Fc, were designed as shown in Tables 1A, 2A, 3A, and 4A.

[0053] [1-1-1] Mouse anti-PcrV scFv (scFv-m166) (Table 1A) The sequence of mouse anti-PcrV scFv (scFv-m166) was designed based on previous reports on mouse-derived monoclonal anti-PcrV mAb166 IgG (Frank DW, The Journal of Infectious Diseases 186(1) 64-73, 2002, Sawa T, InTech, 2017). It was created by linking a heavy chain variable region (VH) containing CDR1-3 (as shown in SEQ ID NOs. 1-3) and a light chain variable region (VL) containing CDR1-3 (as shown in SEQ ID NOs. 5-7) using a glycine-serine linker (3x(Gly4Ser1)). Furthermore, a tissue plasminogen activator signal sequence (tPAss) was added to the N-terminus of the antibody region, and a cMyc tag and a histidine tag (6xHis) were added to the C-terminus.

[0054] [1-1-2] Mouse anti-PcrV scFv-Fc (scFv-m166-mFc) (Table 2A) The sequence of mouse anti-PcrV scFv-Fc(scFv-m166-mFc) was designed by adding a mouse Fc domain sequence to the C-terminus of the above-mentioned mouse anti-PcrV scFv(scFv-m166). In vivo, this mouse anti-PcrV scFv-Fc(scFv-m166-mFc) forms a dimeric protein (111.4 kDa) by forming disulfide bonds at two cysteine ​​residues in the CH2 domain.

[0055] [1-1-3] Human anti-PcrV scFv (scFv-h166) (Table 3A) The sequence of human anti-PcrV scFv(scFv-h166) was designed for human therapeutic use based on the mouse anti-PcrV scFv(scFv-m166) sequence described above, based on previous reports (Baer M, Infection and Immunity 77(3) 1083-90, 2009; Sawa T, InTech, 2017). In this modification, parts of the amino acid sequences of CDR3 in VH and CDR3 and FR4 in VL were substituted, and the tag was eliminated.

[0056] [1-1-4] Human anti-PcrV scFv-mFc (scFv-h166-mFc) (Table 4A) The sequence of human anti-PcrV scFv-mFc (scFv-h166-mFc) was designed by adding a mouse Fc domain sequence to the C-terminus of the above-mentioned human anti-PcrV scFv(scFv-h166). In vivo, this human anti-PcrV scFv-mFc (scFv-h166-mFc) forms a dimeric protein (107.8 kDa) by forming disulfide bonds at two cysteine ​​residues in the CH2 domain.

[0057] [1-2] mRNA design and synthesis For the expression of the antibodies shown in Tables 1A, 2A, 3A, and 4A, we designed and synthesized codon-optimized mRNAs, as shown in Tables 1B, 2B, 3B, and 4B, in which uridine was replaced with N1-methylpseudridine.

[0058] [1-2-1] Example 1 (scFv-m166 mRNA) (Table 1B) As mRNA for expressing mouse anti-PcrV scFv(scFv-m166), we designed mRNA consisting of the nucleotide sequence of Sequence ID No. 13 shown in Table 1B. As shown in Sequence ID No. 13, a Cap structure and a 5'UTR were added to the 5' end of the region encoding the mouse anti-PcrV scFv(scFv-m166) sequence (consisting of nucleotide sequences 78-956 of Sequence ID No. 13), which includes tPAss, cMyc tag, and histidine tag, and a 3'UTR and poly(A) were added to the 3' end.

[0059] [1-2-2] Comparative example 1 (scFv-m166-mFc mRNA) (Table 2B) As mRNA for expressing mouse anti-PcrV scFv-Fc (scFv-m166-mFc), we designed mRNA consisting of the nucleotide sequence of SEQ ID NO: 14 shown in Table 2B. As shown in SEQ ID NO: 14, a Cap structure and a 5'UTR were added to the 5' end of the region encoding the mouse anti-PcrV scFv-Fc (scFv-m166-mFc) sequence (consisting of nucleotide sequences 67-1611 of SEQ ID NO: 14), which includes tPAss, cMyc tags, and histidine tags, and a 5'UTR and poly(A) were added to the 3' end.

[0060] [1-2-3] Example 2 (scFv-h166 mRNA) (Table 3B) As mRNA for expressing human anti-PcrV scFv(scFv-h166), we designed mRNA consisting of the nucleotide sequence of SEQ ID NO: 15 shown in Table 3B. As shown in SEQ ID NO: 15, a Cap structure and a 5'UTR were added to the 5' end of the region encoding the human anti-PcrV scFv(scFv-h166) sequence containing tPAss (consisting of nucleotide sequences 67-882 of SEQ ID NO: 15), and a 3'UTR and poly(A) were added to the 3' end.

[0061] [1-2-4] Comparative example 2 (scFv-h166-mFc mRNA) (Table 4B) As mRNA for expressing human anti-PcrV scFv-mFc (scFv-h166-mFc), we designed mRNA consisting of the nucleotide sequence of SEQ ID NO: 16 shown in Table 4B. As shown in SEQ ID NO: 16, a Cap structure and a 5'UTR were added to the 5' end of the region encoding the human anti-PcrV scFv-mFc (scFv-h166-mFc) sequence containing tPAss (consisting of nucleotide sequences 67-1548 of SEQ ID NO: 16), and a 3'UTR and poly(A) were added to the 3' end.

[0062] [Table 1A] [Table 1B]

[0063] [Table 2A] [Table 2B]

[0064] [Table 3A] [Table 3B]

[0065] [Table 4A] [Table 4B]

[0066] [2] Loading (encapsulation) of mRNA molecules onto lipid nanoparticles (NLPs) Following a previously described method (Tockary, Proceedings of the National Academy of Sciences 120, e2214320120, 2023), the mRNA molecules of Examples 1 and 2 and Comparative Examples 1 and 2 obtained in [1] above were encapsulated in lipid nanoparticles. Specifically, an ethanol solution containing ALC-0315 (46.3 mol%), p1,2-distearoyl-sn-glycero-3-phosphocholine (9.4 mol%), cholesterol (42.7 mol%), and ALC-0159 (1.6 mol%) was mixed with an mRNA solution containing mRNA in citrate buffer (pH 3) using Nanoassembr Spark (Precision NanoSystems, Vancouver, Canada) so that the number of moles of nitrogen atoms in ALC-0135 was five times the number of moles of phosphate groups in mRNA, and nanoparticles were formed. Subsequently, the buffer was replaced with PBS to prepare an injectable preparation.

[0067] [3] Pseudomonas strains The Pseudomonas aeruginosa strain PA103 (MLST type: ST298, type III secretory toxin genotype exoS- / exoU+) was used as the infectious agent. Bacteria were frozen and spread onto tryptone soy agar, then cultured in tryptone soy broth with 10 mM nitrilotriacetic acid at 32°C for 13 hours in a shaking incubator. The cultured bacteria were centrifuged at 8,500 × g for 10 minutes, and the bacterial pellet was washed twice with physiological saline. 9 The solution was diluted to CFU / mL (determined by spectrophotometer).

[0068] [4] Mouse infection study design All animal experiments were conducted using male ICR mice (5 weeks old, 25g body weight). The study designs (purpose of administration to mice, administered formulation, and route of administration) are shown in Tables 5-7.

[0069] [Table 5]

[0070] [Table 6]

[0071] [Table 7]

[0072] After the experiment, mice were temporarily anesthetized by inhalation of the inhalation anesthetic sevoflurane, and bacterial inoculum (PA103, 1.0 × 10⁶) was administered. 6 CFU (60 μL) was slowly administered into the left lung lobe using a garbage needle (Improved Animal Feeding Needle, 24 G, Popper & Sons) inserted into the trachea via the oral cavity. Accurate needle insertion was confirmed by observing the movement of the solution in the syringe during the mouse's respiratory effort. In the prophylactic setting, the lungs were collected after 24 hours and the wet weight of the lungs was measured. The lungs were homogenized in sterile water in a sterile container. The lung homogenate was serially diluted and spread on sheep blood agar plates to assess bacterial counts. In the therapeutic setting, the survival rate of infected mice was monitored for one week.

[0073] Separately, tracheotomy was performed on mice 3 hours after intravenous injection, and euthanasia was carried out by deep inhalation of sevoflurane. A total of 2 mL of PBS was injected into the lungs using a catheter, and bronchoalveolar lavage fluid was collected using a vibrator. The recovery rate was approximately 50-70%. After centrifugation of the collected fluid at 1,000 rpm for 10 minutes, the supernatant was secured, and the anti-PcrV antibody titer was measured by the ELISA method described later.

[0074] [5] Measurement of anti-PcrV antibody titer by enzyme-linked immunosorbent assay (ELISA) Microwell plates (Nunc C96 Maxisorp, Thermo Fisher Scientific Inc.) were coated with recombined PcrV in coating buffer (1.5 μg / mL in coating solution, 0.05 M NaHCO3, pH 9.6) for 2 hours at 4°C. The plates were washed twice with phosphate-buffered saline (PBS) supplemented with 0.05% Tweem-20 (P9416, Sigma-Aldrich Co., St. Louis, MO, USA), 200 μL of 1% bovine serum albumin / PBS was added, and the plates were blocked overnight at 4°C. Samples (dilution ratios: 32-1024-fold) were applied to the plates at a ratio of 100 μL / well and incubated overnight at 4°C. Peroxidase-labeled anti-6xHis-IgG (HRP-66005, Protein Tech, Tokyo, Japan) was added at a dilution ratio of 1:60,000 and incubated at 37°C for 1 hour. After washing six times, the plates were incubated at room temperature for 30 minutes in 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (A3219, Sigma-Aldrich Co.). Next, 0.5 M H2SO4 (100 μL / well) was added to the plates, and the optical density (OD) at 450 nm was measured using a microplate reader (MTP-880Lab, Corona Electric Co., Ltd.). A dilution index of OD 450 nm > 0.15 was considered positive.

[0075] [6] Cytokines, myeloperoxidase activity, and lung histology The concentrations of IL-6 and TNF-α in lung homogenate and plasma were measured using an ELISA kit (BD OptEIA ELISA set, BD Bioscience). Myeloperoxidase activity in lung homogenate was measured using biochemical methods. For fixation of lung tissue, the lungs were perfused with 10% buffered formalin phosphate and embedded in paraffin. Lung tissue sections were stained with hematoxylin and eosin, and observed and imaged under a light microscope.

[0076] [7] Results [7-1] Results of Test Example 1 Figure 1 shows the time course of anti-PcrV titer elevation after therapeutic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice with Pseudomonas aeruginosa pneumonia, as in Test Example 1. Intravenous or intramuscular administration of the mRNA of Example 1 transfected muscle cells or hepatocytes with scFv-m166 mRNA in the mouse body, causing these non-immune cells to express and secrete scFv-m166 antibodies. As a result, the mice with Pseudomonas aeruginosa pneumonia acquired immunity to counteract the toxicity of Pseudomonas aeruginosa. Specifically, as shown in Figure 1, serum anti-PcrV antibody titers after intravenous or intramuscular administration of the mRNA of Example 1 to mice showed an increase 2 hours after administration, peaking at 8 hours after administration.

[0077] [7-2] Results of Test Example 2 The inhibitory effect of prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice in Test Example 2 on acute lung injury from lung infection by lethal doses of Pseudomonas aeruginosa is shown in Figure 2A (suppression of pulmonary edema 24 hours after infection), Figure 2B (suppression of intrapulmonary bacterial count 24 hours after infection), and Figure 2C (suppression of pulmonary granulocyte peroxidase activity 24 hours after infection). As shown in Figures 2A to 2C, prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice resulted in significantly lower levels of pulmonary edema, intrapulmonary bacterial count, and pulmonary granulocyte peroxidase activity 24 hours after infection compared to the luciferase mRNA administration group. In other words, it was confirmed that prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 significantly suppresses acute lung injury caused by Pseudomonas aeruginosa infection.

[0078] Furthermore, the inhibitory effect on intrapulmonary cytokine secretion against lung infection by lethal doses of Pseudomonas aeruginosa after prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice, as shown in Test Example 2, is illustrated in Figure 3A (inhibitory effect on interleukin-6 secretion 24 hours after infection) and Figure 3B (inhibitory effect on tumor necrosis factor TNF-α secretion 24 hours after infection). As shown in Figures 3A and 3B, prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 to mice resulted in significantly lower intrapulmonary cytokine secretion 24 hours after infection compared to the luciferase mRNA administration group. In other words, it was confirmed that prophylactic administration (intravenous or intramuscular) of the mRNA of Example 1 significantly suppressed pneumonia caused by Pseudomonas aeruginosa infection.

[0079] In the luciferase mRNA administration group, mice developed a lethal dose of Pseudomonas aeruginosa lung infection after administration, resulting in a mortality rate of approximately 90% within 24 hours. On the other hand, in the mRNA administration group of Example 1, even when mice developed a lethal dose of Pseudomonas aeruginosa lung infection after administration, the survival rate after 24 hours showed a dramatic improvement to 90-100%.

[0080] [7-3] Results of Test Example 3 Figures 4A and 4B show the survival rates of mice with Pseudomonas aeruginosa pneumonia after therapeutic administration (intravenous or intramuscular) of mRNA from Examples 1 and 2 or Comparative Examples 1 and 2, according to Test Example 3. As shown in Figures 4 and 4B, despite intravenous or intramuscular administration being systemic, the survival rate was significantly higher when the mRNA from Examples 1 and 2, which expresses scFv antibodies lacking Fc and exhibiting extremely poor retention in the body, was administered compared to the mRNA from Comparative Examples 1 and 2, which expresses antibodies with Fc that enhance retention in the body.

[0081] Figure 5 shows the anti-PcrV antibody titers in bronchoalveolar lavage fluid 3 hours after therapeutic administration (intravenous injection) of mRNA from Example 1 or Comparative Example 1 to mice with Pseudomonas aeruginosa pneumonia, as in Test Example 3. As shown in Figure 5, not only compared with the non-infected control group, but also compared with the group administered mRNA from Comparative Example 1 (scFv-m166-mFc / LNP) that expresses antibodies with Fc, it was confirmed that the antibody concentration in the alveolar space was significantly and remarkably higher in the group administered mRNA from Example 1 (scFv-m166 / LNP) that expresses scFv antibodies.

[0082] Furthermore, since TTSS is commonly present with high homology among pathogenic Gram-negative bacteria, the effectiveness of the mRNA of the present invention against Pseudomonas aeruginosa, as confirmed in Test Examples 1-3 above, can be reasonably inferred to be effective against other pathogenic Gram-negative bacteria as well.

Claims

1. A pharmaceutical composition for systemic administration containing, as an active ingredient, mRNA containing an antibody coding region that encodes a specific scFv antibody against the V antigen protein or its homologous protein of pathogenic Gram-negative bacteria.

2. The pharmaceutical composition according to claim 1, wherein the pathogenic Gram-negative bacteria are Pseudomonas aeruginosa, Plague bacillus, Bordetella pertussis, Yersinia, Salmonella, Shigella, Aeromonas hydrophylla, Bacillus subtilis, Mycobacterium pseudotuberculosis, pathogenic Escherichia coli, Vibrio parahaemolyticus, Vibrio haveyi, Glanders bacillus, or Chlamydia.

3. The pharmaceutical composition according to claim 1, wherein the pathogenic Gram-negative bacterium is Pseudomonas aeruginosa.

4. The pharmaceutical composition according to claim 1, wherein the V antigen protein or its homologous protein is PcrV, LcrV, LssV, AcrV, VcrV, Vp1659, IpaD, SipD, SeeB, BipD, CT584, EspA, or Bsp22.

5. The pharmaceutical composition according to claim 1, wherein the V antigen protein or its homologous protein is PcrV.

6. The pharmaceutical composition according to claim 1, wherein the mRNA is supported on lipid nanoparticles.

7. The pharmaceutical composition according to claim 1, wherein the systemic administration is intravenous or intramuscular administration.

8. The pharmaceutical composition according to claim 1, used for the prevention or treatment of the aforementioned pathogenic Gram-negative bacterial infection.

9. The pharmaceutical composition according to claim 8, wherein the infectious disease is pneumonia, acute lung injury, or sepsis.

10. mRNA containing an antibody-coding region that encodes a specific scFv antibody against the V antigen protein or its homologous protein of pathogenic Gram-negative bacteria.

11. The mRNA according to claim 10, wherein the 5' end of the coding region has a tissue plasmogen activator signal sequence.

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