CN117070523A - mRNA (messenger ribonucleic acid) for encoding A33R protective antibody and application thereof - Google Patents

mRNA (messenger ribonucleic acid) for encoding A33R protective antibody and application thereof Download PDF

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CN117070523A
CN117070523A CN202310652517.8A CN202310652517A CN117070523A CN 117070523 A CN117070523 A CN 117070523A CN 202310652517 A CN202310652517 A CN 202310652517A CN 117070523 A CN117070523 A CN 117070523A
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mrna
antibody
seq
pseudouridine
construct
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秦成峰
迟航
邓永强
赵锁群
陈如意
索星星
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Academy of Military Medical Sciences AMMS of PLA
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Academy of Military Medical Sciences AMMS of PLA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Abstract

The invention relates to mRNA encoding A33R protective antibody and application thereof; belongs to the field of biotechnology. The mRNA provided by the invention consists of a light chain mRNA construct of the monoclonal antibody mab22 for resisting the vaccinia virus EEV surface protein A33R and a heavy chain mRNA construct of the monoclonal antibody mab22 for resisting the vaccinia virus EEV surface protein A33R, and the accurate synthesis of the monoclonal antibody mab22 for resisting the vaccinia virus EEV surface protein A33R in an organism is realized by delivering an mRNA molecule for encoding a protective antibody into the organism. After the mRNA is inoculated to a BALB/c mouse, an antibody with neutralizing activity can be generated in the mouse body, and the mRNA is proved to have application in preparing anti-orthopoxvirus infection medicines, can be used before and after exposure, has the effects of disease prevention and treatment, and has great development potential. The medicament may be presented in the form of a pharmaceutical composition or a kit.

Description

mRNA (messenger ribonucleic acid) for encoding A33R protective antibody and application thereof
Technical Field
The invention relates to mRNA for encoding A33R protective antibody and application thereof, in particular to mRNA molecule for encoding anti-vaccinia virus A33R protein specific antibody and application thereof in preparing anti-orthopoxvirus infection medicine; belongs to the field of biotechnology.
Background
Orthopoxvirus (Orthopoxvirus) is classified in the subfamily of Poxviridae (Poxviridae) Chordopoxvirinae (chord), members of which include a variety of poxviruses that can infect humans and mammals. Among them, there are 4 types of currently known orthopoxviruses that are pathogenic to humans: smallpox virus, vaccinia virus and monkey poxvirus, and the 4 viruses have genomic homology of greater than 90% and have cross protection. Along with the increasing number of cases and the increasing spread of the distribution range, along with the confirmation of interpersonal transmission and community transmission, the world health organization announces monkey pox epidemic situation as an international public health event of interest, and the orthopoxvirus infection starts to draw close attention in the field of global public health. Currently, no batch of specific anti-orthopoxvirus drugs are available in China, mainly for symptomatic support and treatment of complications. It is necessary to develop specific drugs that are safe and efficient to effectively address the threat of potential orthopoxvirus infection.
The neutralizing antibody has strong targeting property and high specificity, can be directly combined with virus surface protein to block virus infection, can play a protective role before and after the people are exposed to the virus, and is an effective means for preventing and treating infectious diseases. However, traditional protein antibody drug development often depends on cell culture, and has long production cycle, complex process and high cost. Compared with protein, the physical and chemical characteristics of nucleic acid are relatively simple, easy to prepare and purify and low in cost. The messenger ribonucleic acid (mRNA) molecule for encoding the specific antibody is introduced into a living body through a delivery system, and the corresponding antibody is expressed by taking a body cell as a bioreactor so as to exert the biological function of the antibody, so that the heterogeneity and the misfolding of the antibody caused by abnormal modification of the in-vitro expression and translation of the antibody can be effectively reduced, the complex in-vitro production and purification processes of the antibody can be effectively avoided, and the rapid and large-scale production can be easily realized.
mRNA vaccines are used for disease prevention by delivering mRNA molecules encoding protective antigens into the body, and expressing the corresponding antigens in the body to stimulate the body to produce specific humoral immune responses to produce antibodies, and often require at least 2 immunizations to achieve the desired antibody levels. Unlike mRNA vaccines, mRNA antibodies are produced directly by delivering mRNA molecules encoding protective antibodies into the body, and can be used both before and after exposure, with both disease prevention and treatment effects.
Orthopoxviruses exist in 2 different forms of infectious viral particles during replication: extracellular enveloped viruses (Extracellular enveloped virion, EEV) and intracellular mature viruses (Intracellular mature virion, IMV). These surface structural proteins are mainly responsible for viral adhesion, entry, packaging, replication, etc., and are also the major proteins that activate the host humoral immune response. Taking vaccinia virus as an example, its EEV surface protein a33R plays a key role in the transmission of virus particles among cells, and has become one of the important targets for vaccine development. It was also found that vaccinia virus A33R was highly homologous to monkey pox virus A35R, smallpox virus A36R, vaccinia virus A34R, and cross-protected from each other.
Antibodies with cross-neutralizing activity against a33R have been reported in the prior art. Researchers at the medical institute of army infectious diseases have further constructed mRNA antibodies based on the gene sequence encoding human-chimpanzee chimeric antibody C6C against the A33R protein, which can be raised in vivo after intramuscular injection into rabbits, but with low serum neutralizing antibody levels, are unlikely to provide challenge protection against poxvirus infection, and do not achieve the desired technical effect. The results of this study demonstrate the unpredictability of the technical effects of developing mRNA antibodies based on existing antibody sequences using mRNA technology. Currently, there are no reports on mRNA molecules encoding anti-a 33R protein antibodies that can provide protection against orthopoxvirus infection.
Disclosure of Invention
To overcome the drawbacks of the prior art, it is an object of the present invention to provide an mRNA encoding an a33R protective antibody, which is an mRNA encoding an anti-a 33R protein antibody having anti-orthopoxvirus infection activity, and which is capable of expressing a protective monoclonal antibody against an a33R protein in vivo and secreting the same into the blood circulation after being delivered into an organism, thereby protecting the organism from orthopoxvirus infection.
It is a second object of the present invention to provide the use of an mRNA encoding an a33R protective antibody for the preparation of a medicament against an orthopoxvirus infection.
In order to achieve the purpose of the invention, the following technical scheme is provided.
An mRNA encoding an a33R protective antibody, the mRNA consisting of construct a and construct B:
construct A is a light chain mRNA construct encoding monoclonal antibody mab22 to vaccinia virus EEV surface protein A33R (abbreviated as mRNA-mab 22-LC) comprising the following elements in sequence, in sense strand, 5 '. Fwdarw.3':
a 5' cap structure, a 5' non-coding region (5 ' UTR), a human antibody kappa light chain signal peptide, an antibody mab22 light chain, a 3' non-coding region (3 ' UTR), and a poly A sequence (poly A);
construct B is a heavy chain mRNA construct encoding the monoclonal antibody mab22 against the vaccinia virus EEV surface protein A33R (abbreviated as mRNA-mab 22-HC) comprising the following elements in sequence, in sense strand, i.e., 5 '. Fwdarw.3':
a 5' cap structure, a 5' non-coding region (5 ' UTR), a human antibody kappa light chain signal peptide, an antibody mab22 heavy chain, a 3' non-coding region (3 ' UTR), and a poly A sequence.
Wherein the nucleotide sequence of the 5' UTR is:
CUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGC；
the sequence table is shown as SEQ ID NO: 3.
The amino acid sequence of the humanized antibody kappa light chain signal peptide is as follows:
MELGLSWIFLLAILKGVQC；
the sequence table is shown as SEQ ID NO: 4.
The nucleotide sequence of the humanized antibody kappa light chain signal peptide is as follows:
AUGGAGCUGGGACUGAGCUGGAUUUUUCUGCUGGCCAUUCUGAAGGGGGUGCAGUGC；
the sequence table is shown as SEQ ID NO: shown at 5.
The amino acid sequence of the antibody mab22 light chain is:
AIVMTQSPATLSVSPGERATLSCRASQSVSSTLAWYQQKPGQAPRLLIYGASTKATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYNNWPPLLTFGGGTKVDIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
wherein CDR1, CDR2, CDR3 are underlined; the sequence table is shown as SEQ ID NO: shown at 6.
The nucleotide sequence of the antibody mab22 light chain is:
GCCAUUGUGAUGACACAAAGCCCUGCUACUCUCAGCGUGUCCCCCGGCGAGAGGGCCACACUGAGCUGCCGGGCCAGCCAGUCCGUGAGCAGCACACUGGCCUGGUACCAGCAGAAGCCCGGCCAGGCCCCCCGGCUGCUGAUCUACGGGGCCUCCACAAAGGCCACCGGCAUUCCCGCCCGGUUCAGCGGGAGCGGGAGCGGGACAGAGUUCACCCUGACCAUCAGCAGCCUGCAGUCUGAGGACUUCGCCGUGUACUACUGCCAGCACUACAACAACUGGCCCCCCCUGCUGAC CUUCGGGGGCGGCACCAAGGUGGAUAUCAAGACAGUGGCCGCCCCCUCCGUGUUCAUCUUCCCCCCCAGUGAUGAGCAGCUGAAGUCCGGCACAGCCAGCGUGGUGUGCCUGCUGAACAACUUCUACCCCAGGGAGGCCAAGGUGCAGUGGAAGGUCGACAACGCCCUGCAGAGCGGGAACAGCCAGGAGAGCGUGACCGAGCAGGAUAGCAAGGAUAGCACAUACAGCCUGUCCUCCACCCUGACACUGAGCAAGGCCGACUACGAGAAGCACAAGGUGUACGCCUGCGAGGUGACACACCAGGGGCUGAGCAGCCCCGUGACAAAGAGCUUCAACAGGGGCGAGUGC；
wherein CDR1, CDR2, CDR3 are underlined; in the sequence listing, SEQ ID NO: shown at 7.
The amino acid sequence of the antibody mab22 heavy chain is:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNSGMHWVRQAPGKGLEWVAVIWFDGTNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVPCGGDCYSGYLQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
wherein CDR1, CDR2, CDR3 are underlined; the sequence table is shown as SEQ ID NO: shown at 8.
The nucleotide sequence of the antibody mab22 heavy chain is:
CAAGUGCAGCUGGUGGAGAGCGGCGGCGGCGUGGUGCAGCCCGGCCGGAGCCUGCGGCUCAGCUGCGCCGCCAGCGGGUUCACCUUCAGCAACAGCGGGAUGCACUGGGUCCGGCAGGCCCCCGGCAAGGGGCUGGAGUGGGUGGCCGUGAUCUGGUUCGACGGCACCAACAAGUACUACGCCGACAGCGUGAAGGGCAGGUUCACAAUCAGCAGGGAUAACUCCAAGAACACCCUGUACCUGCAGAUGAACAGCCUGCGGGCCGAGGACACAGCCGUGUACUACUGCGCCAGGGU GCCCUGCGGGGGCGAUUGCUACUCCGGCUACCUGCAGCACUGGGGCCAGGGCACACUCGUGACCGUGUCCUCCGCAUCCACAAAGGGCCCCUCCGUGUUUCCCCUGGCCCCCAGCAGCAAGAGCACAAGCGGGGGGACCGCCGCCCUCGGGUGCCUGGUCAAGGAUUACUUCCCCGAGCCCGUGACCGUGUCUUGGAACUCCGGCGCCCUGACCUCCGGGGUCCACACCUUCCCCGCCGUCCUGCAGAGCUCCGGGCUGUACAGCCUGAGCAGCGUGGUGACAGUGCCCAGCUCCAGCCUGGGGACCCAGACAUACAUUUGCAACGUGAACCACAAGCCCUCAAACACCAAGGUGGAUAAGCGGGUGGAGCCCAAGAGCUGCGACAAGACCCACACAUGCCCCCCCUGCCCUGCCCCCGAGGCCGCCGGGGGCCCUUCAGUGUUCCUGUUCCCCCCCAAGCCUAAGGAUACACUGUACAUUACCAGGGAGCCCGAGGUCACAUGCGUGGUGGUGGACGUGAGCCACGAGGACCCCGAGGUGAAGUUCAACUGGUACGUCGAUGGGGUGGAGGUGCACAACGCCAAGACAAAGCCCAGGGAGGAGCAGUACAACAGCACCUACCGGGUGGUGAGCGUGCUGACAGUGCUGCACCAGGACUGGCUGAACGGGAAGGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCAGCCCCCAUUGAAAAGACCAUCUCUAAGGCCAAGGGGCAGCCCAGGGAGCCCCAGGUGUACACACUGCCACCCAGCCGGGAGGAGAUGACCAAGAACCAGGUGAGCCUGACCUGCCUGGUGAAGGGGUUCUACCCCAGCGAUAUCGCCGUGGAGUGGGAGAGCAAUGGCCAGCCCGAGAACAACUACAAGACAACACCCCCUGUGCUGGAUAGCGACGGGAGCUUCUUCCUGUACUCCAAGCUGACAGUGGAUAAGUCCAGGUGGCAGCAGGGGAACGUGUUCAGCUGCUCCGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGUCCCUGAGCCUGUCUCCCGGCAAG；
wherein CDR1, CDR2, CDR3 are underlined; the sequence table is shown as SEQ ID NO: shown at 9.
The CDR analysis of the invention is completed according to the http:// www.abysis.org/analysis/sequence_input/key_analysis.
The nucleotide sequence of the 3' UTR is:
UAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCACUAGU；
the sequence table is shown as SEQ ID NO: shown at 10.
According to the demand of different mRNAs, different 5 'cap structures, such as m, can be flexibly added at the 5' end of the mRNAs 7 GpppG、m 2 7 , 3’-O GpppG、m 7 Gppp (5') N1 and m 7 Gppp(m 2’-O ) At least one of N1.
In some preferred embodiments of the invention, the 5' cap structure is m 7 GpppG。
“m 7 G "represents a 7-methylguanosine cap nucleoside," ppp "represents a triphosphate bond between the 5 '-end of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the most 5' -end nucleotide," G "represents a guanosine, and" m "is 7 "represents methyl at the 7-position of guanine," m 2’-O "represents a methyl group at the 2' -O position of the nucleotide.
The poly A sequence may comprise 50 to 400 adenylates, preferably 100 to 160 adenylates.
Some or all of the uracil and/or cytosine in the mRNA is chemically modified to increase the stability of the mRNA in vivo.
The chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA with.
Wherein the substance replacing uracil is selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine and 2' -O-methyl-uridine, preferably pseudouridine, N1-methyl-pseudouridine or N1-ethyl-pseudouridine, further preferably N1-methyl-pseudouridine; and/or the number of the groups of groups,
the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosines in the mRNA with 5-methylcytosines.
The nucleotide sequence of preferred construct a is:
CUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCGCCGCCACCAUGGAGCUGGGACUGAGCUGGAUUUUUCUGCUGGCCAUUCUGAAGGGGGUGCAGUGCGCCAUUGUGAUGACACAAAGCCCUGCUACUCUCAGCGUGUCCCCCGGCGAGAGGGCCACACUGAGCUGCCGGGCCAGCCAGUCCGUGAGCAGCACACUGGCCUGGUACCAGCAGAAGCCCGGCCAGGCCCCCCGGCUGCUGAUCUACGGGGCCUCCACAAAGGCCACCGGCAUUCCCGCCCGGUUCAGCGGGAGCGGGAGCGGGACAGAGUUCACCCUGACCAUCAGCAGCCUGCAGUCUGAGGACUUCGCCGUGUACUACUGCCAGCACUACAACAACUGGCCCCCCCUGCUGACCUUCGGGGGCGGCACCAAGGUGGAUAUCAAGACAGUGGCCGCCCCCUCCGUGUUCAUCUUCCCCCCCAGUGAUGAGCAGCUGAAGUCCGGCACAGCCAGCGUGGUGUGCCUGCUGAACAACUUCUACCCCAGGGAGGCCAAGGUGCAGUGGAAGGUCGACAACGCCCUGCAGAGCGGGAACAGCCAGGAGAGCGUGACCGAGCAGGAUAGCAAGGAUAGCACAUACAGCCUGUCCUCCACCCUGACACUGAGCAAGGCCGACUACGAGAAGCACAAGGUGUACGCCUGCGAGGUGACACACCAGGGGCUGAGCAGCCCCGUGACAAAGAGCUUCAACAGGGGCGAGUGCUAAGAAUUCUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCACUAGUAn；
wherein An represents a polyadenylation sequence (poly A), n represents the amount of adenylate; the nucleotide sequence of the construct A is shown in a sequence table as SEQ ID NO: 1.
The nucleotide sequence of construct a may also be selected from the group consisting of SEQ ID NO:1, or a nucleotide sequence having at least 70%, at least 80%, or at least 90% homology.
Preferred nucleotide sequences for construct B are:
CUUGUUCUUUUUGCAGAAGCUCAGAAUAAACGCUCAACUUUGGCGCCGCCACCAUGGAGCUGGGACUGAGCUGGAUUUUUCUGCUGGCCAUUCUGAAGGGGGUGCAGUGCCAAGUGCAGCUGGUGGAGAGCGGCGGCGGCGUGGUGCAGCCCGGCCGGAGCCUGCGGCUCAGCUGCGCCGCCAGCGGGUUCACCUUCAGCAACAGCGGGAUGCACUGGGUCCGGCAGGCCCCCGGCAAG
GGGCUGGAGUGGGUGGCCGUGAUCUGGUUCGACGGCACCAACAAGUAC
UACGCCGACAGCGUGAAGGGCAGGUUCACAAUCAGCAGGGAUAACUCC
AAGAACACCCUGUACCUGCAGAUGAACAGCCUGCGGGCCGAGGACACA
GCCGUGUACUACUGCGCCAGGGUGCCCUGCGGGGGCGAUUGCUACUCC
GGCUACCUGCAGCACUGGGGCCAGGGCACACUCGUGACCGUGUCCUCCG
CAUCCACAAAGGGCCCCUCCGUGUUUCCCCUGGCCCCCAGCAGCAAGAG
CACAAGCGGGGGGACCGCCGCCCUCGGGUGCCUGGUCAAGGAUUACUU
CCCCGAGCCCGUGACCGUGUCUUGGAACUCCGGCGCCCUGACCUCCGGG
GUCCACACCUUCCCCGCCGUCCUGCAGAGCUCCGGGCUGUACAGCCUGA
GCAGCGUGGUGACAGUGCCCAGCUCCAGCCUGGGGACCCAGACAUACA
UUUGCAACGUGAACCACAAGCCCUCAAACACCAAGGUGGAUAAGCGGG
UGGAGCCCAAGAGCUGCGACAAGACCCACACAUGCCCCCCCUGCCCUGC
CCCCGAGGCCGCCGGGGGCCCUUCAGUGUUCCUGUUCCCCCCCAAGCCU
AAGGAUACACUGUACAUUACCAGGGAGCCCGAGGUCACAUGCGUGGUG
GUGGACGUGAGCCACGAGGACCCCGAGGUGAAGUUCAACUGGUACGUC
GAUGGGGUGGAGGUGCACAACGCCAAGACAAAGCCCAGGGAGGAGCAG
UACAACAGCACCUACCGGGUGGUGAGCGUGCUGACAGUGCUGCACCAG
GACUGGCUGAACGGGAAGGAGUACAAGUGCAAGGUGUCCAACAAGGCC
CUGCCAGCCCCCAUUGAAAAGACCAUCUCUAAGGCCAAGGGGCAGCCCA
GGGAGCCCCAGGUGUACACACUGCCACCCAGCCGGGAGGAGAUGACCA
AGAACCAGGUGAGCCUGACCUGCCUGGUGAAGGGGUUCUACCCCAGCG
AUAUCGCCGUGGAGUGGGAGAGCAAUGGCCAGCCCGAGAACAACUACA
AGACAACACCCCCUGUGCUGGAUAGCGACGGGAGCUUCUUCCUGUACU
CCAAGCUGACAGUGGAUAAGUCCAGGUGGCAGCAGGGGAACGUGUUCA
GCUGCUCCGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGUC
CCUGAGCCUGUCUCCCGGCAAGUAAGAAUUCUAGGCUGGAGCCUCGGU
GGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUC
CUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCA
CUAGU
An；
wherein An represents a polyadenylation sequence (poly A), n represents the amount of adenylate; the nucleotide sequence of the construct B is shown in a sequence table as SEQ ID NO: 2.
The nucleotide sequence of construct B may also be selected from the group consisting of SEQ ID NO:2, or a nucleotide sequence having at least 70%, at least 80%, or at least 90% homology.
Use of an mRNA encoding an a33R protective antibody according to the invention for the preparation of a medicament against an orthopoxvirus infection.
The orthopoxvirus is orthopoxvirus, preferably at least one of vaccinia virus, monkey pox virus, vaccinia virus and smallpox virus.
The anti-orthopoxvirus infection medicament refers to a medicament for preventing infection of orthopoxvirus and/or a medicament for treatment after infection of orthopoxvirus, and the medicament can be in the form of a pharmaceutical composition or a kit.
A pharmaceutical composition comprising an mRNA encoding an a33R protective antibody according to the invention and a delivery vehicle, as a medicament against an orthopoxvirus infection.
Preferably the delivery vehicle is a nanoparticle.
Further, it is preferred that the delivery vehicle is a lipid nanoparticle, such as a nanolipid sphere (LNP).
Still further, it is preferred that the lipid nanoparticle have an average diameter of 50nm to 200nm and have a polydispersity coefficient value of less than 0.4.
A kit comprising an mRNA encoding an a33R protective antibody according to the invention and/or a pharmaceutical composition according to the invention as a medicament against an orthopoxvirus infection.
Advantageous effects
(1) The present invention provides an mRNA encoding an a33R protective antibody by incorporating frequent codons and/or codons with higher tRNA abundance by having an inventive molecular design; enriching guanine-cytosine content to increase steady-state mRNA levels in vitro and protein expression in vivo; focusing on the design of mRNA expression key elements such as selection of 5'UTR and 3' UTR, selection of signal peptide and the like, the accurate synthesis of the monoclonal antibody mab22 encoding the anti-vaccinia virus EEV surface protein A33R in an organism is realized.
(2) The invention provides an application of mRNA for encoding an A33R protective antibody, which can generate an antibody with neutralizing activity in a mouse body after the BALB/c mouse is inoculated with the mRNA, can protect the mouse from being infected by orthopoxvirus and has application in preparing medicines for resisting the orthopoxvirus infection.
(3) The invention provides mRNA for encoding A33R protective antibody and application thereof, and the mRNA antibody provided by the invention has subverted advantage compared with the traditional protein antibody which depends on mammalian cell in-vitro culture expression. mRNA antibody takes organism self cells as a biological factory for antibody production, so that the antibody post-translational modification in a natural state can be generated, the heterogeneity and the misfolding of the antibody caused by the antibody in-vitro expression and translation abnormal modification can be effectively reduced, and the complex in-vitro production and purification process of the antibody can be effectively avoided. In addition, the mRNA technology is used as a general technical platform, and compared with the traditional protein antibody, the preparation process of the mRNA antibody is simpler, the rapid and large-scale standardized production is easy to realize, and the storage and transportation are convenient.
(4) The invention provides a pharmaceutical composition, which is prepared by combining mRNA of the invention with a pharmaceutically acceptable carrier and selecting lipid nanoparticles for package delivery, so as to obtain a messenger ribonucleic acid (mRNA) antibody for resisting orthopoxvirus infection.
(5) The invention provides a kit, which comprises mRNA for encoding the A33R protective antibody and/or the pharmaceutical composition disclosed by the invention, and has wide application prospect as a pharmaceutical product for resisting orthopoxvirus infection.
Drawings
FIG. 1 is a schematic diagram of the construction strategy of the mRNA molecules encoding the A33R protective antibodies in example 1.
FIG. 2 shows the results of agarose gel electrophoresis identification of mRNA molecules encoding the A33R protective antibodies of example 1.
FIG. 3 shows the results of in vitro expression characterization of mRNA encoding the A33R protective antibody of example 2.
FIG. 4 shows the results of particle size measurement of mRNA preparation mRNA-mab22-LNP in example 3.
FIG. 5 is the serum neutralizing antibody titers of mice injected with mRNA preparation mRNA-mab22-LNP in example 4.
FIG. 6 shows the results of an experiment for challenge protection of mRNA preparation mRNA-mab22-LNP in example 5 on a mouse infection model.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples, but is not intended to limit the scope of the patent.
EXAMPLE 1 Synthesis of mRNA molecules encoding orthopoxvirus antibodies
(1) molecular design of mRNA
The amino acid sequences of the light and heavy chains of mab22, a monoclonal antibody directed against vaccinia virus EEV surface protein a33R, were designed to encode an a33R protective antibody, consisting of construct a and construct B, as shown in fig. 1:
construct A is a light chain mRNA construct encoding monoclonal antibody mab22 to vaccinia virus EEV surface protein A33R (abbreviated as mRNA-mab 22-LC) comprising the following elements in sequence, in sense strand, 5 '. Fwdarw.3':
a 5' cap structure, a 5' non-coding region (5 ' UTR), a human antibody kappa light chain signal peptide, an antibody mab22 light chain, a 3' non-coding region (3 ' UTR), and a poly A sequence (poly A);
construct B is a heavy chain mRNA construct encoding the monoclonal antibody mab22 against the vaccinia virus EEV surface protein A33R (abbreviated as mRNA-mab 22-HC) comprising the following elements in sequence, in sense strand, i.e., 5 '. Fwdarw.3':
a 5' cap structure, a 5' non-coding region (5 ' UTR), a human antibody kappa light chain signal peptide, an antibody mab22 heavy chain, a 3' non-coding region (3 ' UTR), and a poly A sequence.
Wherein, the nucleotide sequence of the 5' UTR is shown in SEQ ID NO: 3.
The amino acid sequence of the humanized antibody kappa light chain signal peptide is shown as SEQ ID NO:4, the nucleotide sequence is shown as SEQ ID NO: shown at 5.
The amino acid sequence of the antibody mab22 light chain is shown in SEQ ID NO:6, the nucleotide sequence is shown as SEQ ID NO: shown at 7.
The amino acid sequence of the antibody mab22 heavy chain is shown in SEQ ID NO:8, the nucleotide sequence is shown as SEQ ID NO: shown at 9.
The nucleotide sequence of the 3' UTR is shown in SEQ ID NO: shown at 10.
The 5' cap structure is m 7 GpppG。
(2) Synthesis of mRNA: primers were designed based on the light and heavy chain nucleotide sequences of mab22, and the mRNA expression vector ABOP-028 (Zhang NN, et al, A Thermostable mRNA Vaccine against COVID-19) sequences, with the primer names and sequences shown in Table 1.
TABLE 1
Primer name Nucleotide sequence (5 '-3') Numbering device
AB028-F CATGCCATGGAGCTGGGACTGAGCTG SEQ ID NO：11
22LC-R CCGGAATTCTTAGCACTCGCCCCTGTT SEQ ID NO：12
22HC-R CCGGAATTCTTACTTGCCGGGAGACAGG SEQ ID NO：13
Wherein, AB028-F is used as an upstream primer and forms an AB028-F/22LC-R primer pair with a downstream primer 22LC-R, and is used for constructing a DNA transcription template plasmid pAB028-mab22-LC (light chain plasmid); AB028-F served as the upstream primer and constituted the AB028-F/22HC-R primer pair with the downstream primer 22HC-R, for constructing the DNA transcription template plasmid pAB028-mab22-HC (heavy chain plasmid).
The light and heavy chain DNA transcription template plasmids constructed above were linearized with BsaI restriction enzyme (purchased from NEB Co.) respectively to give linearized plasmids. Respectively taking linearized plasmids as templates, taking natural nucleoside triphosphates (ATP, CTP, UTP and GTP, purchased from Shanghai megadimension company) as substrates, and performing In Vitro Transcription (IVT) reaction by using T7 RNA polymerase (purchased from offshore company); adding DNase I (purchased from Northenan company) to digest the template DNA; mRNA was purified by adding 7.5M lithium chloride solution (available from Invitrogen corporation); 5 '-end capping of the obtained mRNA with vaccinia virus capping enzyme (10U/. Mu.L, purchased from offshore company), and cap structure methylation was accomplished with cap structure 2' -O-methyltransferase (50U/. Mu.L, purchased from offshore company); finally, 7.5M lithium chloride solution (purchased from Invitrogen) was added to purify the capped mRNA to obtain the light chain mRNA molecule (mRNA-mab 22-LC) of the monoclonal antibody mab22 encoding the anti-vaccinia virus EEV surface protein A33R, the nucleotide sequence of which is shown in SEQ ID NO:1, and a heavy chain mRNA molecule (mRNA-mab 22-HC) encoding a monoclonal antibody mab22 against vaccinia virus EEV surface protein A33R, the nucleotide sequence of which is set forth in SEQ ID NO: 2.
The results of the identification of mRNA-mab22-LC and mRNA-mab22-HC by 1% (W/V) agarose gel electrophoresis are shown in FIG. 2, and the single band size of the light and heavy mRNA molecules was expected, indicating successful purification of the obtained light and heavy mRNA molecules encoding the monoclonal antibody mab22 against the vaccinia virus EEV surface protein A33R.
Example 2 in vitro expression profiling of mRNA
BHK-21 cells (ATCC, # CCL-10) were transferred to 6-well plates 1 day before transfection, respectively, at a cell density of 1X 10 5 And/or holes. And when the cell growth in the hole reaches 70-80% of confluence, setting an experimental group and a control group respectively. The experimental group was transfected with Lipofectamine MessengerMAX liposome transfection reagent (available from Thermo company), BHK-21 cells were co-transfected with mRNA-mab22-LC and mRNA-mab22-HC in a molar ratio of 1:1, and after 6 hours, the transfected supernatants were replaced with Opti-MEM medium (available from Thermo company); control group the procedure was the same as in the experimental group except that mRNA-mab22-LC and mRNA-mab22-HC were not added.
Cell culture supernatants from the experimental and control groups were collected 24 hours after transfection, respectively, and centrifuged at 5000g to remove cell debris.
mu.L of the cell culture supernatant after removal of cell debris from the experimental group, 1. Mu.g of the mab22 protein antibody and 5 Xreduction protein loading buffer (purchased from Bio Inc.) were thoroughly mixed, and the mixture was boiled in a boiling water bath for 10 minutes to obtain an experimental histone sample.
mu.L of the control cell culture supernatant from which cell debris was removed was thoroughly mixed with a 5 Xreduction protein loading buffer (available from Bio Inc.), and the mixture was boiled in a boiling water bath for 10 minutes to obtain a control protein sample.
Western Blot identification was performed on experimental histone samples (mRNA-mab 22) and control histone samples (mock) by 8% -20% polyacrylamide gel electrophoresis, using HRP-labeled goat anti-human IgG as the secondary anti-heavy chain (purchased from Zhongsequoyins, diluted 1:5000 (V/V) with PBS containing 2% nonfat dry milk), and HRP-labeled goat anti-human IgG as the secondary anti-light chain (purchased from abcam, diluted 1:10000 (V/V) with PBS containing 2% nonfat dry milk), and the successful expression of antibody heavy chains (about 50 kDa) and antibody light chains (about 25 kDa) was detected in BHK-21 cell supernatants by transfection of antibody mRNA as shown in FIG. 3.
EXAMPLE 3 nanolipid sphere encapsulation of mRNA molecules
mRNA-mab22-LC and mRNA-mab22-HC prepared in example 1 were mixed in a molar ratio of 1:1 to obtain mRNA stock solution encoding mab22 antibody. By NanoAssemblr Ignite + The LNP lipid nanoparticle drug manufacturing system rapidly mixes the mRNA stock solution and lipid auxiliary materials (purchased from Ebola Suzhou corporation) uniformly, encapsulates the mRNA in nanolipid spheres (LNP) and obtains an mRNA-LNP preparation; the mRNA-LNP preparation was further dialyzed overnight at 4℃in PBS solution (available from VIVICUM Co.), and then concentrated by ultrafiltration through a 100kDa ultrafiltration tube (available from Merck Millipore Co.), to obtain mRNA-mab22-LNP, whose particle size was measured by a Unchained Labs Stunner high-throughput concentration particle size analyzer, as shown in FIG. 4, and the particle size of the mRNA preparation thus obtained was approximately 74.91nm, which corresponds to the expected particle size range.
Example 4 identification of in vivo neutralization Activity of mRNA molecules
The experimental and control groups were set as follows:
experimental group (mRNA-mab 22-LNP): 1mg/kg of mRNA-mab22-LNP prepared in example 3 was injected into 6-8 week old BALB/c mice by tail vein, 6 mice were collected by blood sampling by retroorbital venous plexus after 24 hours, serum was isolated, and serum was inactivated at 56℃for 30 minutes for plaque reduction neutralization test (Plaque Reduction Neutralization Test, PRNT). BSC-1 cells (ATCC, # CCL-26) were transferred to 24-well plates, and experiments were performed the next time the cell density reached 90%; serum was diluted with HBSS (HBSS-1% BSA) containing 1% by volume of BSA in a 2-fold gradient, and diluted to eachThe following gradient: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640; EEV virus of vaccinia virus WR strain (Taxonomy ID: 696871) was diluted to 50 PFU/90. Mu.L with HBSS-1% BSA, and 10. Mu.L of young rabbit complement (from Soy Corp.) and 20. Mu.g/mL of a murine anti-monkey poxvirus M1R antibody (from Pujian Corp.) were added to obtain a virus dilution; mixing the virus diluent with the diluted gradient serum respectively in equal volume, incubating at 37deg.C for 1 hr, discarding cell culture solution in 24-well plate, adding 200 μl of the serum-virus mixture, and incubating at 37deg.C with 5% CO 2 Incubating for 2h under the condition; the supernatant from each well was discarded, 500. Mu.L of methylcellulose cap (2 XDMEM medium containing 1% by volume of methylcellulose and 4% by volume of inactivated FBS) was added, and the 24-well plate was placed at 37℃and 5% CO 2 Culturing in a cell incubator for 2 days; fixing with 4% paraformaldehyde solution (purchased from biosharp company) at room temperature for 30min, discarding methyl cellulose cover, adding crystal violet solution for dyeing for 10min, observing plaque morphology and calculating plaque number per hole; 50% neutralizing antibody titers (PRNT) were calculated according to the Karber method 50 )。
Control group (Placebo): empty Nanolipid Spheres (LNPs) without mRNA were injected into 6-8 week old BALB/c mice via tail vein, and the rest of the experimental group.
The experimental results of the experimental group and the control group are shown in fig. 5, in which the left side is the control group result and the right side is the experimental group result, and it is shown that the mRNA molecule of mab22 encoding the anti-vaccinia virus EEV surface protein a33R can produce an antibody with neutralizing activity after being delivered into mice.
EXAMPLE 5 evaluation of toxicity counteracting protective Effect
The experimental and control groups were set as follows:
experimental group (mRNA-mab 22-LNP): 1mg/kg of mRNA-mab22-LNP prepared in example 3 was injected into 6-8-week-old BALB/c mice, 6 mice in number, via tail vein. After 24 hours, the vaccinia virus (WR strain) was infected with the mice via the nasal drip route, the survival status of each group of mice was observed daily, the weights of the mice were weighed and recorded,
control group (Placebo): empty Nanolipid Spheres (LNPs) without mRNA were injected into 6-8 week old BALB/c mice via tail vein, and the rest of the experimental group.
The results of the weight change in mice are shown in FIG. 6, which shows that mRNA-mab22-LNP is effective in protecting mice against lethal vaccinia virus infection.
Because of the homology between orthopoxviruses and the antigenicity of the orthopoxviruses has been confirmed to be similar, antibodies produced by the induced organisms after viral infection can provide cross-immune protection, i.e. have the effect of resisting other orthopoxvirus infections. From the results of examples 4 and 5, it is understood by those skilled in the art that the mRNA molecule encoding the monoclonal antibody mab22 against vaccinia virus EEV surface protein A33R of the present invention is capable of producing protective antibodies having cross-neutralizing activity against other orthopoxviruses such as monkey pox virus, smallpox virus, vaccinia virus in organisms and is effective against infection by orthopoxviruses such as monkey pox virus, smallpox virus, vaccinia virus.

Claims (13)

1. An mRNA encoding an a33R protective antibody, characterized in that: the mRNA consists of construct a and construct B:
construct A is a light chain mRNA construct encoding monoclonal antibody mab22 to vaccinia virus EEV surface protein A33R, comprising the following elements in the 5 '. Fwdarw.3' direction:
a 5' cap structure, a 5' non-coding region, a humanized antibody kappa light chain signal peptide, an antibody mab22 light chain, a 3' non-coding region, and a polyadenylation sequence;
construct B is a heavy chain mRNA construct encoding monoclonal antibody mab22 to vaccinia virus EEV surface protein A33R, comprising the following elements in the 5 '. Fwdarw.3' direction:
a 5' cap structure, a 5' non-coding region, a humanized antibody kappa light chain signal peptide, an antibody mab22 heavy chain, a 3' non-coding region, and a polyadenylation sequence;
the nucleotide sequence of the 5' non-coding region is shown in SEQ ID NO:3 is shown in the figure;
the amino acid sequence of the humanized antibody kappa light chain signal peptide is shown as SEQ ID NO:4, the nucleotide sequence is shown as SEQ ID NO:5 is shown in the figure;
the amino acid sequence of the antibody mab22 light chain is shown in SEQ ID NO:6, the nucleotide sequence is shown as SEQ ID NO: shown in figure 7;
the amino acid sequence of the antibody mab22 heavy chain is shown in SEQ ID NO:8, the nucleotide sequence is shown as SEQ ID NO: shown as 9;
the nucleotide sequence of the 3' non-coding region is shown in SEQ ID NO:10 is shown in the figure;
some or all of the uracil and/or cytosine in the mRNA is chemically modified to increase the stability of the mRNA in vivo.
2. An mRNA encoding the a33R protective antibody according to claim 1, wherein: the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA with,
wherein the substance replacing uracil is selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-aza-uridine, dihydro-pseudouridine and 5-methoxy-uridine and 2' -O-methyl uridine;
and/or, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosines in the mRNA with 5-methylcytosines.
3. An mRNA encoding the a33R protective antibody according to claim 2, wherein: the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA with pseudouridine, N1-methyl pseudouridine or N1-ethyl pseudouridine.
4. An mRNA encoding the a33R protective antibody according to claim 1 or 2, characterized in that: the nucleotide sequence of the construct A is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the construct B is shown in SEQ ID NO: 2.
5. An mRNA encoding the a33R protective antibody according to claim 4, wherein: the nucleotide sequence of construct a is selected from the group consisting of SEQ ID NO:1, a nucleotide sequence having at least 70%, at least 80%, or at least 90% homology;
the nucleotide sequence of construct B is selected from the group consisting of SEQ ID NO:2, or a nucleotide sequence having at least 70%, at least 80%, or at least 90% homology.
6. An mRNA encoding the a33R protective antibody according to claim 1, wherein: the 5' cap structure is selected from m 7 GpppG、m 2 7 , 3’ -OGpppG、m 7 Gppp (5') N1 and m 7 Gppp(m 2’-O ) At least one of N1.
7. An mRNA encoding the a33R protective antibody according to claim 1, wherein: the 5' cap structure is m 7 GpppG。
8. An mRNA encoding the a33R protective antibody according to claim 1, wherein: the polyadenylation sequence comprises 50 to 400 adenylates.
9. An mRNA encoding the a33R protective antibody according to claim 1, wherein: the polyadenylation sequence comprises 100 to 160 adenylates.
10. A pharmaceutical composition characterized by: an mRNA encoding an a33R protective antibody according to any one of claims 1 to 9 and a delivery vehicle, said pharmaceutical composition being as an anti-orthopoxvirus agent.
11. A pharmaceutical composition according to claim 10, wherein: the delivery vehicle is a lipid nanoparticle.
12. The mRNA according to any one of claims 1 to 9, and the use of a pharmaceutical composition according to any one of claims 10 to 11 for the preparation of a medicament against an orthopoxvirus infection; the orthopoxvirus is orthopoxvirus, and the anti-orthopoxvirus infection drug is a drug for preventing from infecting orthopoxvirus and/or a drug for treating after infecting orthopoxvirus.
13. The use according to claim 12, characterized in that: the orthopoxvirus is at least one of vaccinia virus, monkey pox virus, vaccinia virus and smallpox virus.
CN202310652517.8A 2023-06-04 2023-06-04 mRNA (messenger ribonucleic acid) for encoding A33R protective antibody and application thereof Pending CN117070523A (en)

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