mRNA molecule encoding bispecific antibody against GPC3 and CD3

Abstract

The present invention relates to the field of biomedicine, and in particular to the field of RNA drugs. Specifically, the present invention provides an mRNA molecule encoding a bispecific antibody against GPC3 and CD3 and a lipid nanoparticle (LNP) comprising the mRNA molecule. The present invention also provides a medical use of the mRNA molecule and the LNP.

Description

mRNA molecules encoding bispecific antibodies against GPC3 and CD3 Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to the field of RNA drugs. Specifically, this invention provides an mRNA molecule encoding a bispecific antibody against GPC3 and CD3, and lipid nanoparticles (LNPs) comprising said mRNA molecule. This invention also provides pharmaceutical uses for said mRNA molecule and said LNPs.

[0002] Background of the Invention

[0003] GPC3 (Glypican-3) is a cell surface glycoprotein primarily involved in cell proliferation, differentiation, migration, and apoptosis. GPC3 is expressed in the liver and kidneys of fetuses, but is almost absent in healthy human tissues. However, it is highly expressed in 70-80% of hepatocellular carcinoma (HCC) tissues and is also expressed in small amounts in other tumors such as ovarian clear cell carcinoma, melanoma, and lung squamous cell carcinoma. Although the function of GPC3 is not fully understood, numerous studies have shown that it is closely related to the occurrence, metastasis, and prognosis of HCC. Therefore, GPC3 has become a novel target for HCC diagnosis and immunotherapy. Immunotherapy targeting GPC3 protein mainly includes monoclonal antibodies, antibody-drug conjugates, tumor vaccines, chimeric antigen receptor T cells (CAR-T), and bispecific antibodies. Currently, there are no GPC3-targeted drugs on the market globally. Among these, bispecific antibodies targeting GPC3 and CD3 can form an immune synapse between T cells and tumor cells, recruiting T cells to the tumor site, activating the T cell immune mechanism, and mediating T cell killing of GPC3-overexpressing tumor cells. As a powerful extension of monoclonal antibody therapy, several bispecific antibody projects are undergoing preclinical and clinical research, showing promising development prospects.

[0004] In recent years, the rapid development of mRNA technology and lipid nanoparticle (LNP) delivery systems has made mRNA therapy a promising candidate for the prevention and treatment of various diseases, with a very broad range of applications, including infectious disease vaccines, protein replacement therapy, tumor immunotherapy, cell reprogramming, and gene editing.

[0005] mRNA therapy can achieve higher protein expression efficiency and longer protein expression time, and has significant advantages over DNA therapy, including: (1) mRNA begins to translate proteins after entering the cytoplasm, without needing to enter the cell nucleus to perform its function. This process is more efficient than DNA, which requires the cell nucleus to transcribe into mRNA. (2) Unlike DNA and viral vectors, mRNA cannot insert into the genome and can only express encoded proteins transiently, resulting in a lower risk of insertional mutagenesis. (3) mRNA is easy to synthesize in vitro, relatively inexpensive, and can be applied quickly. (4) mRNA can theoretically express any protein and can be used to treat almost any disease. The current combination of mRNA technology and AI has brought new possibilities for the research and development and application of mRNA drugs. For example, AI can be used to predict RNA secondary structure and protein structure; AI can also be used to analyze mRNA, enhance its stability or improve its translation efficiency through sequence optimization, increase protein yield, and enable more efficient design and production of related drugs. In the future, this combination will play an increasingly important role in the fields of medicine and biology, promoting the development of scientific research and medical treatment.

[0006] Invention Summary

[0007] This invention provides at least the following embodiments:

[0008] Implementation Scheme 1. An mRNA molecule comprising a coding sequence (CDS) of a bispecific antibody against GPC3 and CD3, wherein the bispecific antibody comprises an scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprising VH CDR1 shown in SEQ ID NO:17, VH CDR2 shown in SEQ ID NO:18, and VH CDR3 shown in SEQ ID NO:19, and the light chain variable region of the scFv against GPC3 comprising CDR1 shown in SEQ ID NO:20, VL CDR2 shown in SEQ ID NO:21, and VL CDR3 shown in SEQ ID NO:22; preferably, the scFv against GPC3 comprises the heavy chain variable region shown in SEQ ID NO:23 and the light chain variable region shown in SEQ ID NO:24; preferably, the bispecific antibody against GPC3 and CD3 is in the form of BiTE.

[0009] Implementation Scheme 2. The mRNA molecule of Implementation Scheme 1, wherein the scFv targeting GPC3 comprises the amino acid sequence shown in SEQ ID NO:25.

[0010] Implementation Scheme 3. The mRNA molecule of Implementation Scheme 1 or 2, wherein the bispecific antibody further comprises a CD3-targeting scFv, for example, the CD3-targeting scFv comprises the amino acid sequence shown in SEQ ID NO:26.

[0011] Implementation Scheme 4. The mRNA molecule of any one of Implementation Schemes 1-3, wherein the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3.

[0012] Implementation Scheme 5. The mRNA molecule of any one of Implementation Schemes 1-4, wherein the coding sequence of the bispecific antibody is selected from SEQ ID NO:4-8, preferably, the coding sequence of the bispecific antibody is shown in SEQ ID NO:4 or SEQ ID NO:7, more preferably, the coding sequence of the bispecific antibody is shown in SEQ ID NO:4.

[0013] Implementation Scheme 6. An mRNA molecule of any one of Implementation Schemes 1-5, wherein the mRNA molecule further comprises a 5'UTR and / or a 3'UTR.

[0014] Implementation Scheme 7. The mRNA molecule of Implementation Scheme 6, wherein the 5'UTR contains the nucleotide sequence shown in SEQ ID NO:1.

[0015] Implementation Scheme 8. The mRNA molecule of Implementation Scheme 6 or 7, wherein the 3'UTR contains the nucleotide sequence shown in SEQ ID NO:2.

[0016] Implementation Scheme 9. An mRNA molecule of any one of Implementation Schemes 1-8, wherein the mRNA molecule further comprises a poly(A) sequence.

[0017] Implementation Scheme 10. The mRNA molecule of Implementation Scheme 9, wherein the poly(A) sequence comprises about 20 to about 500 adenine nucleotides (A), preferably, the poly(A) sequence comprises 120 adenine nucleotides (A).

[0018] Implementation Scheme 11. An mRNA molecule of any one of Implementation Schemes 1-10, wherein the mRNA molecule comprises a nucleotide sequence selected from SEQ ID NO:9-13, preferably, the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9 or SEQ ID NO:12, more preferably, the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9.

[0019] Implementation Scheme 12. An mRNA molecule of any one of Implementation Schemes 1-11, wherein the mRNA molecule further comprises a 5' cap structure, for example, the 5' cap structure is a Cap1 cap structure.

[0020] Implementation Scheme 13. An mRNA molecule of any one of Implementation Schemes 1-12, wherein the mRNA molecule may further comprise at least one modifying nucleotide, preferably, the modifying nucleotide being selected from pseudouridine, N1-methyl-pseudouridine, 5-methylcytidine, or combinations thereof.

[0021] Implementation Scheme 14. An mRNA molecule of any one of Implementation Schemes 1-13, wherein the mRNA molecule may further include a microRNA binding site.

[0022] Implementation Scheme 15. An mRNA molecule of any one of Implementation Schemes 1-14, wherein the mRNA molecule is chemically synthesized or obtained by in vitro transcription.

[0023] Implementation Scheme 16. A lipid nanoparticle (LNP) comprising an mRNA molecule of any one of Implementation Schemes 1-15, wherein the mRNA molecule is contained in a lipid.

[0024] Implementation Scheme 17. A pharmaceutical composition comprising an mRNA molecule of any one of Implementation Schemes 1-15 or an LNP of any one of Implementation Schemes 15-16, and a pharmaceutically acceptable carrier.

[0025] Implementation Scheme 18. A pharmaceutical composition comprising an mRNA encoding a coding sequence (CDS) of a bispecific antibody polypeptide against GPC3 and CD3, the pharmaceutical composition being administered via in vivo injection to treat a tumor; preferably, the mRNA comprising the coding sequence of a bispecific antibody polypeptide against GPC3 and CD3 is selected from the mRNA molecules of any one of Implementation Schemes 1-15.

[0026] Implementation Scheme 19. Use of the mRNA molecule of any one of Implementation Schemes 1-15 or the LNP of Implementation Scheme 16 or the pharmaceutical composition of any one of Implementation Schemes 17-18 in the preparation of a medicament for use in subjects and / or prevention of GPC3-related diseases such as malignant tumors.

[0027] Implementation Scheme 20. A method for treating and / or preventing GPC3-related diseases, such as malignant tumors, in a subject, the method comprising administering to the subject an effective amount of an mRNA molecule of any one of Implementation Schemes 1-15, an LNP of Implementation Scheme 16, or a pharmaceutical composition of any one of Implementation Schemes 17-18.

[0028] Implementation Scheme 21. The use of Implementation Scheme 19 or the method of Implementation Scheme 20, wherein the GPC3-related disease is a tumor that highly expresses GPC3, such as liver cancer such as hepatocellular carcinoma (HCC), lung cancer such as squamous cell carcinoma of the lung (SqCC), gastric cancer, ovarian cancer such as clear cell ovarian cancer, melanoma, or pediatric embryonal tumor.

[0029] Brief description of the attached diagram

[0030] Figure 1. Expression of sequence-optimized mRNA encoding GPC3×CD3 bispecific antibody in cell supernatant.

[0031] Figure 2. The correlation between antibody concentration and activity in cells and the expression of sequence-optimized mRNA encoding GPC3×CD3 bispecific antibody.

[0032] Figure 3. Killing effect of GPC3×CD3 bispecific antibody in cells.

[0033] Figure 4. Expression of 4#GPC3×CD3 bispecific antibody mRNA in animals.

[0034] Figure 5. Antitumor effect of 4#GPC3×CD3 bispecific antibody mRNA.

[0035] Invention Details

[0036] I. Definition

[0037] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields.

[0038] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0039] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0040] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0041] When the term “comprising” is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activities described in this invention.

[0042] Sequence identity between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the online alignment tool EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm with default parameters. Sequence identity can be along the full length of a given sequence.

[0043] The terms “mRNA” or “messenger RNA” are used interchangeably in this article and refer to single-stranded ribonucleic acid molecules that carry genetic information and guide protein synthesis within cells. mRNA typically includes a 5' UTR, a protein-coding sequence (CDS), and a 3' UTR. mRNA may also include a poly(A) sequence at the 3' end and a 5' cap. mRNA is generally linear; however, the term can also encompass covalently closed circular RNA molecules.

[0044] As used herein, the "5'UTR" generally refers to the sequence from the 5' end of an mRNA molecule to the translation initiation codon, which recruits the ribosome complex and initiates mRNA translation. The 5'UTR regulates post-transcriptional modifications, translation initiation complex formation, and stability by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The term "5'UTR" also encompasses the ribosome entry site (IRES) sequence, particularly for circular RNA molecules.

[0045] As used in this article, "3'UTR" refers to the sequence between the stop codon of the polypeptide coding sequence in mRNA and the poly(A) sequence. The 3'UTR can regulate mRNA translation by interacting with mRNA-binding proteins, miRNAs, and other organisms. The sequence and structural features of the 3'UTR can affect mRNA stability, ribosome scanning, and the formation of translation termination complexes, thereby influencing protein expression levels.

[0046] Poly(A) sequences typically contain multiple adenine nucleotides. The addition of a poly(A) sequence contributes to mRNA stability and transport, prevents its degradation, and plays an important role in post-transcriptional modifications. A poly(A) sequence can be a continuous chain of pure adenine nucleotides, or it can be a variant containing non-adenine nucleotides, as long as its function is equivalent to the conventional poly(A) sequence, providing similar biological functions as the natural poly(A) sequence, such as affecting mRNA stability, translation efficiency, or ribosome binding. Known poly(A) sequences include the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants may differ in nucleotide composition but are functionally considered equivalent to the conventional poly(A) sequence.

[0047] As used in this article, the "5' cap" for RNA includes the 5' cap structure present on native mRNA and its analogues. The 5' cap structure on native mRNA refers to the methylated guanosine monophosphate linked to the 5' terminal nucleotide of RNA via pyrophosphate, forming a 5',5'-triphosphate linkage. There are generally three types of 5' caps (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as Cap0, Cap1, and Cap2, respectively. Cap0 indicates that the ribose of the terminal nucleotide is unmethylated, Cap1 indicates that the ribose of the terminal nucleotide is methylated, and Cap2 indicates that the ribose of both terminal nucleotides are methylated.

[0048] Methods for capping mRNA molecules are known in the art. The 5' cap structure of the mRNA molecule can be added via an enzymatic reaction after the mRNA molecule has been obtained through chemical synthesis or in vitro transcription (e.g., using a commercially available kit containing a vaccinia capping enzyme and a 2'-O-methyltransferase for the mRNA cap structure). However, capped mRNA can also be produced by directly incorporating a capped nucleotide analog as the first nucleotide into the transcript during in vitro transcription.

[0049] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0050] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain, containing an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), the CDR being part of the antigen-binding site domain.

[0051] As used herein, “hypervariant region,” “HV,” “complementarity-determining region,” and “CDR” and “antibody CDR” are interchangeably used to refer to one of the multiple portions within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3. The three CDRs in the variable region are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbers (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J.Mol.Biol. 196: 901-917).

[0052] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0053] The term "bispecific antibody" refers to an antibody or antigen-binding fragment that recognizes two different antigenic determinants. These antigenic determinants can be different antigenic determinants on the same antigen or different antigenic determinants on different antigens. An example of a bispecific antibody is the so-called BiTE. A BiTE (Bispecific T cell Engager) is a bispecific antibody that connects the antigen-binding region of an antibody targeting a target cell-specific antigen to the antigen-binding region of an antibody targeting CD3 via a linker. It acts as a "bridge" between T cells and target cells, mediating the T cell's targeted recognition and specific killing of the target cell. The target cell is, for example, a tumor cell.

[0054] "Lipid nanoparticles" refer to particles containing lipid components and having a nanoscale size.

[0055] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0056] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0057] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0058] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0059] As used in this article, the term "object" refers to mammals, such as humans.

[0060] II. mRNA molecules encoding bispecific antibodies against GPC3 and CD3

[0061] In one aspect, the present invention provides an mRNA molecule comprising a coding sequence (CDS) of a bispecific antibody against GPC3 and CD3, wherein the bispecific antibody comprises an scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprises VH CDR1 shown in SEQ ID NO:17, VH CDR2 shown in SEQ ID NO:18, and VH CDR3 shown in SEQ ID NO:19, and the light chain variable region of the scFv against GPC3 comprises CDR1 shown in SEQ ID NO:20, VL CDR2 shown in SEQ ID NO:21, and VL CDR3 shown in SEQ ID NO:22.

[0062] In some preferred embodiments, the scFv for GPC3 includes the heavy chain variable region shown in SEQ ID NO:23 and the light chain variable region shown in SEQ ID NO:24.

[0063] In some preferred embodiments, the bispecific antibody against GPC3 and CD3 is in the form of BiTE.

[0064] In some embodiments, the scFv targeting GPC3 comprises the amino acid sequence shown in SEQ ID NO:25.

[0065] In some embodiments, the bispecific antibody further comprises a CD3-targeting scFv containing the amino acid sequence shown in SEQ ID NO:26.

[0066] In some embodiments, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:3.

[0067] In some embodiments, the coding sequence of the bispecific antibody is selected from SEQ ID NO:4-8. In some preferred embodiments, the coding sequence of the bispecific antibody is shown in SEQ ID NO:4 or SEQ ID NO:7. In some further preferred embodiments, the coding sequence of the bispecific antibody is shown in SEQ ID NO:4.

[0068] In some implementations, the mRNA molecule also includes a 5'UTR and / or a 3'UTR.

[0069] Various available 5'UTRs and / or 3'UTRs are known in the art. Those skilled in the art can determine the 5'UTR and / or 3'UTRs suitable for this invention.

[0070] In some embodiments, the 5'UTR contains the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the 3'UTR contains the nucleotide sequence shown in SEQ ID NO:2.

[0071] In some implementations, the mRNA molecule also includes a poly(A) sequence.

[0072] In some embodiments of the invention, the poly(A) sequence comprises from about 20 to about 500 (e.g., consecutive) adenine nucleotides (A), for example, about 25, about 50, about 100, about 150, about 175, about 200, about 300, about 400, or about 500 (e.g., consecutive) adenine nucleotides (A). In some preferred embodiments, the poly(A) sequence comprises 120 (e.g., consecutive) adenine nucleotides (A). The addition of the poly(A) sequence contributes to the stability and transport of mRNA, prevents its degradation, and plays an important role in post-transcriptional modification. The poly(A) sequence can be a continuous chain of pure adenine nucleotides, but it can also be a variant containing non-adenine nucleotides, as long as its function is equivalent to that of the conventional poly(A) sequence, i.e., it can provide biological functions similar to those of the natural poly(A) sequence, such as affecting mRNA stability, translation efficiency, or ribosome binding. Known poly(A) sequences include the human growth hormone (hGH) poly(A) sequence and the simian virus 40 (SV40) poly(A) sequence. These variants may differ in nucleotide composition but are considered functionally equivalent to the conventional poly(A) sequence.

[0073] In some embodiments, the mRNA molecule comprises a nucleotide sequence selected from SEQ ID NO:9-13. In some preferred embodiments, the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9 or SEQ ID NO:12. In some further preferred embodiments, the mRNA molecule comprises the nucleotide sequence shown in SEQ ID NO:9.

[0074] In some embodiments, the mRNA molecule further includes a 5' cap structure. In some embodiments of the present invention, the 5' cap structure is a Cap1 cap structure.

[0075] In some embodiments, the mRNA molecule of the present invention may further comprise at least one nucleotide modification. The at least one nucleotide modification includes, but is not limited to, cytidine modification, uridine modification, or adenosine modification. In some embodiments, the at least one nucleoside modification includes, but is not limited to, 5-methylcytosine (m5C), N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5molU).

[0076] In some embodiments, the mRNA molecule is chemically synthesized. In some embodiments, the RNA molecule is obtained through in vitro transcription.

[0077] Furthermore, the mRNA molecules involved in this invention possess a certain degree of stability, tolerating a certain degree of insertion of additional sequences (such as microRNA binding sites) without affecting their translational ability or the stability of the mRNA molecules. In some embodiments, the additional sequences (such as microRNA binding sites) may be inserted into the 3'UTR. The microRNA binding site includes a full-length microRNA inverse complementary sequence (exemplary length may be 19-25 nt) or an inverse complementary sequence of its seed sequence (exemplary length may be 7-8 nt).

[0078] In one aspect, the present invention provides a nucleic acid vector containing the coding sequence of the mRNA molecule of the present invention. In some embodiments, the nucleic acid vector is used to generate the mRNA molecule of the present invention.

[0079] As used herein, a "vector" refers to a segment of DNA extracted from a virus, plasmid, or cell of a higher organism, into which a foreign DNA fragment may be inserted or has been inserted for cloning and / or expression purposes. In some embodiments, the vector can be stably maintained in the organism. A vector may contain, for example, an origin of replication, a selection marker or reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, granules, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc.

[0080] In some embodiments, the nucleic acid vector further comprises an RNA polymerase promoter sequence operatively linked to the coding sequence of the mRNA molecule. The operatively linked promoter allows for in vivo and / or in vitro transcription of the RNA molecule. The promoter is, for example, a T7 RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0081] In some embodiments, the nucleic acid vector is a plasmid vector. In some embodiments, the nucleic acid vector contains a restriction endonuclease site, such as an IIS-type restriction endonuclease site, on the 3' flanking of the coding sequence of the mRNA molecule. Suitable restriction endonucleases include, but are not limited to, BsmBI, BsaI, and SapI. The restriction endonuclease site can be used to linearize the nucleic acid vector for in vitro transcription.

[0082] Methods for obtaining mRNA molecules from nucleic acid vectors through in vitro transcription are known in the art, for example, in vitro transcription can be performed using commercially available kits.

[0083] III. LNP, pharmaceutical compositions, disease treatment and / or prevention

[0084] The present invention also provides a lipid nanoparticle (LNP) comprising the mRNA molecule of the present invention, wherein the mRNA molecule is contained within lipids. In some embodiments, the mRNA molecule is encapsulated within lipids.

[0085] Various methods and materials for constructing LNPs containing specific nucleic acid molecules such as mRNA molecules are known in the art, and all of them can be applied to this invention.

[0086] The present invention also provides a pharmaceutical composition comprising the mRNA molecule of the present invention or the LNP of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition is intended for the treatment and / or prevention of GPC3-related diseases such as malignancies in subjects.

[0087] The present invention also provides the use of the mRNA molecule of the present invention or the LNP of the present invention in the preparation of a medicament for use in and / or prevention of GPC3-related diseases such as malignant tumors in subjects.

[0088] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0089] The actual dose level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response to a specific subject, composition, and route of administration, without toxicity to the subject. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention applied, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the subject receiving treatment, and similar factors known in the medical field.

[0090] An "effective amount" of the mRNA molecule or LNP of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumors, an "effective amount" of the mRNA molecule or LNP of the present invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system for predicting the efficacy of treatment against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays known to those skilled in the art. An effective amount of the mRNA molecule or LNP of the present invention can reduce tumor size or otherwise alleviate symptoms in the subject, such as prevention and / or treatment of metastasis or recurrence. Those skilled in the art can determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

[0091] The mRNA molecules, LNPs, or pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the mRNA molecules or LNPs of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0092] The present invention also provides a method for treating and / or preventing GPC3-related diseases, such as malignant tumors, in a subject, the method comprising administering an effective amount of the mRNA molecule of the present invention or the LNP of the present invention or the pharmaceutical composition of the present invention to the subject.

[0093] GPC3-related diseases that can be treated and / or prevented by the mRNA molecule of the present invention, the LNP of the present invention, the pharmaceutical composition of the present invention, or the method of the present invention are tumors that highly express (as opposed to healthy tissue) GPC3, including but not limited to liver cancer such as hepatocellular carcinoma (HCC), lung cancer such as squamous cell carcinoma of the lung (SqCC), gastric cancer, ovarian cancer such as clear cell ovarian cancer, melanoma, or pediatric embryonal tumors.

[0094] In some embodiments, the mRNA molecule of the present invention, the LNP of the present invention, the pharmaceutical composition of the present invention, or the method of the present invention can be used in combination with chemotherapeutic agents, immune checkpoint inhibitors, antibodies targeting other tumor-specific antigens, or radiotherapy.

[0095] There are no particular limitations on the chemotherapeutic agents, immune checkpoint inhibitors, or antibodies that target other tumor antigens that can be used in combination with the mRNA molecule of the present invention, the LNP of the present invention, the pharmaceutical composition of the present invention. Examples of the chemotherapeutic agents, immune checkpoint inhibitors, and antibodies targeting other tumor antigens include, but are not limited to: ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, enoxabin, capecitabine, carmoflurane, cladribine, gemcitabine, cytarabine, tegafur, tegafur-uracil, TS-1, deoxyfluorouridine, nerabine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate, irinotecan, etoposide, eribulin, sobuzosen, docetaxel, paclitaxel, vinorelbine, vincristine, vindesine, actinomycin D, arubicin, amrubicin, idarubicin, epirubicin, doxorubicin, daunorubicin, doxorubicin. Pirarubicin, Bleomycin, Pyromycin, Mitomycin C, Mitoxantrone, Oxaliplatin, Carboplatin, Cisplatin, Nedaplatin, Anastrozole, Exemestane, Ethinylestradiol, Chlormadinone, Goserelin, Tamoxifen, Dexamethasone, Bicalutamide, Toremifene, Flutamide, Prednisolone, S-estradiol, Mitotan, Methyltestosterone, Leuprorelin, Letrozole, Methylprogesterone acetate, Ti... Imoxicillin, imatinib, everolimus, erlotinib, gefitinib, sunitinib, cetuximab, sorafenib, dasatinib, tamibarbitine, trastuzumab, retinoic acid, perumumab, bevacizumab, bortezomib, lapatinib, atezolizumab, pembrolizumab, tislelizumab, camrelizumab, sugemalimab, and nivolumab, etc.

[0096] The mRNA molecules, LNPs, or pharmaceutical compositions of the present invention, along with the chemotherapeutic agents, immune checkpoint inhibitors, or antibodies targeting other tumor antigens, can be administered all at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0097] The combined dosage of the mRNA molecule of the present invention, the LNP of the present invention, or the pharmaceutical composition of the present invention, and the chemotherapeutic agent, immune checkpoint inhibitor, or antibody targeting other tumor antigens in the pharmaceutical composition of the present invention is not particularly limited. As mentioned above, the dosage of the antibody of the present invention can be determined by referring to the dosage when the antibody is used alone. The chemotherapeutic agent, immune checkpoint inhibitor, and antibody targeting other tumor antigens can be used according to the dosage specified in their respective pharmaceutical descriptions or can be reduced (taking into account the combination effect with the antibody of the present invention).

[0098] The mRNA molecule of the present invention, the LNP of the present invention, or the pharmaceutical composition of the present invention may also be combined with radiotherapy, for example, by administering ionizing radiation to a subject earlier, during, and / or later than the administration of the antibody or pharmaceutical composition of the present invention. Example

[0099] The present invention can be further understood by referring to the specific embodiments described herein. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, those skilled in the art will recognize that various modifications and variations can be made to the present invention without departing from its spirit; therefore, such modifications and variations also fall within the scope of the present invention.

[0100] Example 1: Optimization design of mRNA sequence encoding GPC3×CD3 bispecific antibody molecule

[0101] UTR combinations (SEQ ID NO: 1-2) reported to effectively improve translation efficiency were selected for sequence design and screening. These UTR combinations were derived from the Modern mRNA1273 expression vector, complete sequence (GenBank: OR134578.1). Highly active GPC3×CD3 bispecific antibody molecules were screened, and their amino acid sequences were optimized (SEQ ID NO: 3). Natural language processing techniques and RNA secondary structure prediction models were used to assign weights and identify stable mRNA sequences. For expression efficiency, the Codon Adaptation Index (CAI) was used for optimization to improve mRNA translation efficiency. For stability, the Minimum Free Energy (MFE) was used to assess and optimize mRNA stability, extending the half-life of mRNA molecules in cells. A batch of mRNA sequences potentially achieving high protein yields were screened, resulting in candidate sequences encoding GPC3×CD3 bispecific antibodies (Seq ID NO: 4-8, corresponding to candidate sequences #4 to #8, respectively).

[0102] Example 2: Preparation of candidate mRNA molecules encoding GPC3×CD3 bispecific antibodies

[0103] A nucleic acid fragment containing a T7 promoter, encoding a GPC3×CD3 bispecific antibody (SEQ ID NO: 9-13), and a restriction endonuclease cleavage site of type IIS was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid#101362). The mRNA molecule transcribed by the vector contained the 5'UTR shown in SEQ ID NO: 1, the 3'UTR shown in SEQ ID NO: 2, and a polyA tail.

[0104] The obtained vector was linearized and in vitro transcribed using T7 RNA polymerase to produce mRNA molecules, with a 5'-cap structure added simultaneously. The 5'-cap structure, through co-transcriptional capping, incorporates a cap analog as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with the Cap1 structure. The resulting mRNA molecules were purified and resuspended in water. Quality control of the mRNA molecules was performed using an Agilent 5200 fragment analyzer to check the mRNA molecule length and integrity (values ​​derived from the area under the curve of the expected length fragment), which met the requirements.

[0105] Example 3: Expression of Optimized mRNA Encoding GPC3×CD3 Bispecific Antibody in Cells

[0106] 293T cells at 1×10 5 The cells were seeded at a density of 0.5 μg / well in 24-well plates and incubated overnight at 37°C with 5% CO2. The next day, mRNA was transfected using Lipofectamine MessengerMAX (Invitrogen) at a dose of 0.5 μg / well. After 24 hours of transfection, the supernatant was collected. The negative control consisted of untransfected wells. The concentration of bispecific antibody protein in the cell supernatant was detected by ELISA: GPC3-Fc (Nearshore Technology) was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well. The plates were incubated overnight at 4°C. The next day, the plates were washed with ELISA washing buffer (Sigma), and then 200 μL of ELISA blocking buffer (Solepro) was added to each well. The plates were blocked at room temperature for 2 hours and then washed with ELISA washing buffer. The standard curve was prepared by diluting the GPC3-CD3 bispecific antibody standard protein with blank culture medium at a concentration of 250 ng / mL as the highest dose well. This was serially diluted six times. Blank culture medium diluent was added to the blank wells, followed by cell supernatant. The plates were incubated at room temperature for 2 hours, and then washed with ELISA washing buffer. The detection antibody (Biotinylated Human CD3 epsilon Protein (His Tag, ultra-sensitivity, primary amine labeling)) (Acro) was diluted to 0.2 μg / mL with sample diluent, and 100 μL was added to each well. The plates were incubated at room temperature for 90 minutes, washed with ELISA washing buffer, and 100 μL of TMB chromogenic solution (Solepro) was added to each well. The reaction was allowed to proceed for 15 minutes at room temperature, followed by 100 μL of top solution (Solepro). The plate was then measured using a microplate reader at OD 450 nm.

[0107] Figure 1 shows that the mRNAs encoding the GPC3×CD3 bispecific antibody (numbers 4 and 7) after sequence optimization were detectably expressed in the cell supernatant, with number 4 showing a higher expression level. The expression levels for each group are shown in Table 1 below.

[0108] Table 1. Expression levels of the optimized mRNA encoding the GPC3×CD3 bispecific antibody in cells.

[0109] Example 4: Sequence optimization of GPC3×CD3 bispecific antibody mRNA expression in cells; Correlation between antibody concentration and activity.

[0110] 293T cells at 2.5 × 10 5Seeds were placed in 24-well plates at a density of 1 / 2 well and incubated overnight at 37°C with 5% CO2. The following day, cells were transfected with 100 ng, 300 ng, and 500 ng of #4 mRNA using Lipofectamine MessengerMAX (Invitrogen) at a dose of 1.5 μL / well. The transfection supernatant was collected 24 hours after transfection and centrifuged at 2000g for 5 min at 4°C to remove cell debris before being used for cell viability assay. On the same day as transfection, Hep3B cells were simultaneously seeded at a density of 2 × 10⁶ cells / well. 4 Seeds were planted at a density of 1×10⁶ cells / well in white 96-well plates and incubated overnight at 37°C with 5% CO₂. On the third day, the cells were removed from the culture medium and cultured at 1×10⁶ cells / well. 5 Add 50 μL of Jurkat CD3-BsAb Reporter Cell Line to each well, followed by 50 μL of cell transfection supernatant serially diluted 10 times from the original solution. Incubate together for 6 hours. Detect chemiluminescence values ​​using a microplate reader.

[0111] The results are shown in Figure 2 and Table 2. The mRNA-encoded GPC3×CD3 bispecific antibody in the Jurkat CD3-BsAb Reporter system can initiate luciferase dose-dependent activity through activation of the intracellular NFAT signaling pathway. The expression dose and activity of the mRNA-encoded bispecific antibody protein are positively correlated.

[0112] Table 2. Activity values ​​of sequence-optimized GPC3×CD3 bispecific antibody mRNA in cells.

[0113] Example 5: Killing effect of GPC3×CD3 bispecific antibody in cells

[0114] Both Hepa1-6-hGPC3-Luc target cells and MC38-Luc target cells were used at a concentration of 2.5 × 10⁻⁶. 5 40 μL of PBMCs were seeded into 96-well plates at a density of 1.25 × 10⁶ / mL. 6 Inoculate 40 μL of the drug ERY974 and the bispecific antibody encoded by 4# mRNA into 96-well plates at a density of / mL. After serial dilution, add 20 μL of each to a 96-well plate. Finally, gently tap to mix thoroughly and incubate at 37°C with 5% CO2 for 48 hours. Detect the chemiluminescence value using a microplate reader.

[0115] The results are shown in Figure 3 and Table 3. The GPC3×CD3 bispecific antibody encoded by 4# mRNA can efficiently mediate the killing of GPC3-positive tumor cells in vitro.

[0116] Table 3. Killing effect of GPC3×CD3 bispecific antibody in cells.

[0117] Example 6: Expression of the sequence-optimized mRNA encoding the GPC3×CD3 bispecific antibody in animals.

[0118] Based on the expression levels of 293T cell transfection, the highest-expressing mRNA (4#) was selected for in vivo expression validation in animals. The mRNA was delivered using Lipid 5-LNP (Lipid 5:cholesterol:DSPC:DMG-PEG molar percentage = 50%:38.5%:10%:1.5%). Eight 8-week-old C57BL6 mice (Beijing Vital River Laboratory Animal Co., Ltd.) were divided into two groups of four. The specific dosing regimen is shown in the table below.

[0119] Table 4. Dosage regimens for in vivo expression in animals

[0120] The concentration of bispecific antibody protein in mouse serum samples and liver homogenate was detected by ELISA. For the ELISA method in cell supernatant, GPC3-Fc (Nearshore Technology) was diluted to 2 μg / mL with ELISA coating buffer, and 100 μL was added to each well of an ELISA plate. The plate was incubated overnight at 4°C. The next day, the plate was washed with ELISA washing buffer (Sigma), and then 200 μL of ELISA blocking buffer (Solepro) was added to each well. After blocking at room temperature for 2 hours, the plate was washed with ELISA washing buffer. For the standard curve, blank serum was diluted with diluent, and then GPC3×CD3 bispecific antibody standard protein was diluted with blank serum diluent. 250 ng / mL was used as the highest dose well, and the plate was serially diluted 6 times. Blank serum diluent was added to the blank wells, followed by serum samples. The plates were incubated at room temperature for 2 hours and then washed with ELISA washing buffer. The detection antibody, Biotinylated Human CD3 epsilon Protein (His Tag, ultra-sensitivity, primary amine labeling) (Acro), was diluted to 0.2 μg / mL with sample dilution buffer. 100 μL was added to each well and incubated at room temperature for 90 min. The plate was washed with ELISA washing buffer, and 100 μL of TMB chromogenic solution (Solepro) was added to each well. After reacting at room temperature for 15 min, 100 μL of stop solution (Solepro) was added. The OD was measured at 450 nm using a microplate reader. Figure 4 shows that the expression level of the sequence-optimized 4#GPC3×CD3 bispecific antibody mRNA was significantly increased in vivo compared to the control bispecific antibody mRNA (SEQ ID NO:16).

[0121] Example 7: Efficacy of sequence-optimized mRNA encoding GPC3×CD3 bispecific antibody in an allogeneic mouse tumor model

[0122] Eight to ten-week-old CD3EDG humanized mice (Southern Model Organisms) were selected, and Hepa1-6 mouse hepatocellular carcinoma cells overexpressing human GPC3 and luciferase (Biocytogen) were in situ inoculated into the livers of the mice. Animals were intraperitoneally injected with an anesthetic (Avorin). After complete anesthesia, the liver lobes were removed and laid flat on alcohol-soaked gauze. Hepa1-6 mouse hepatocellular carcinoma cells overexpressing human GPC3 and luciferase, resuspended in PBS, were inoculated at a concentration of 1×10⁻⁶. 6 The dose of 0.02 mL / mouse was slowly injected into the left lobe of the mouse liver. After removing the syringe, the bleeding was gently stopped by pressing with a cotton swab, and then the liver lobe was reinserted into the mouse. The mouse was sutured and placed on a heated table. After the mouse recovered, it was returned to its cage for continued feeding and daily observation. Seven days after cell inoculation, the mice were analyzed using luciferase in vivo imaging (Lumina II small animal in vivo imaging system, PerkinElmer). Based on the imaging fluorescence value and body weight, suitable mice were selected for grouping (the average tumor imaging signal intensity was approximately 2.3E+07p / s) and evenly distributed into 5 experimental groups, with 5 mice in each group. Drug administration began on the day after grouping, designated as D0. The specific drug administration regimen is shown in the table below:

[0123] Table 5. Dosing regimens for animal efficacy experiments

[0124] Lipid5-LNP was used to deliver the 4#GPC3×CD3 bispecific antibody mRNA to the liver of mice via tail vein injection. In vivo imaging and weight measurement were performed twice weekly after administration. Figure 5 shows that the bispecific antibody protein expressed by the 4# mRNA effectively inhibited tumor growth in a dose-dependent manner.

[0125] The sequence information involved in this application

Claims

1. An mRNA molecule comprising a coding sequence (CDS) for a bispecific antibody against GPC3 and CD3, wherein the bispecific antibody comprises a scFv against GPC3, the heavy chain variable region of the scFv against GPC3 comprises a VH CDR1 as set forth in SEQ ID NO: 17, a VH CDR2 as set forth in SEQ ID NO: 18, a VH CDR3 as set forth in SEQ ID NO: 19, the light chain variable region of the scFv against GPC3 comprises a CDR1 as set forth in SEQ ID NO: 20, a VL CDR2 as set forth in SEQ ID NO: 21, a VL CDR3 as set forth in SEQ ID NO: 22; preferably, the scFv against GPC3 comprises a heavy chain variable region as set forth in SEQ ID NO: 23 and a light chain variable region as set forth in SEQ ID NO: 24; preferably, the bispecific antibody against GPC3 and CD3 is in BiTE format.

2. The mRNA molecule of claim 1, wherein the scFv against GPC3 comprises an amino acid sequence as set forth in SEQ ID NO:

25.

3. The mRNA molecule of claim 1 or 2, wherein the bispecific antibody further comprises a scFv against CD3, for example, the scFv against CD3 comprises an amino acid sequence as set forth in SEQ ID NO:

26.

4. The mRNA molecule of any one of claims 1-3, wherein the bispecific antibody comprises an amino acid sequence as set forth in SEQ ID NO:

3.

5. The mRNA molecule of any one of claims 1-4, wherein the coding sequence of the bispecific antibody is selected from the group consisting of SEQ ID NOs: 4-8, preferably, the coding sequence of the bispecific antibody is set forth in SEQ ID NO: 4 or SEQ ID NO: 7, more preferably, the coding sequence of the bispecific antibody is set forth in SEQ ID NO:

4.

6. The mRNA molecule of any one of claims 1-5, wherein the mRNA molecule further comprises a 5’ UTR and / or a 3’ UTR.

7. The mRNA molecule of claim 6, wherein the 5’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO:

1.

8. The mRNA molecule of claim 6 or 7, wherein the 3’ UTR comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

9. The mRNA molecule of any one of claims 1-8, wherein the mRNA molecule further comprises a poly(A) sequence.

10. The mRNA molecule of claim 9, wherein the poly(A) sequence comprises about 20 to about 500 adenine nucleotides (A), preferably, the poly(A) sequence comprises 120 adenine nucleotides (A).

11. The mRNA molecule of any one of claims 1-10, wherein the mRNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9-13, preferably the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 12, more preferably the mRNA molecule comprises a nucleotide sequence as set forth in SEQ ID NO:

9.

12. The mRNA molecule of any one of claims 1-11, wherein the mRNA molecule further comprises a 5' cap structure, for example, the 5' cap structure is a Capl cap structure.

13. The mRNA molecule of any one of claims 1-12, wherein the mRNA molecule can further comprise at least one modified nucleotide, preferably the modified nucleotide is selected from the group consisting of pseudouridine, Nl-methyl-pseudouridine, 5-methylcytidine, or a combination thereof.

14. The mRNA molecule of any one of claims 1-13, wherein the mRNA molecule can further comprise a microRNA binding site.

15. The mRNA molecule of any one of claims 1-14, wherein the mRNA molecule is chemically synthesized, or is obtained by in vitro transcription.

16. A lipid nanoparticle (LNP) comprising the mRNA molecule of any one of claims 1-15, the mRNA molecule is comprised in a lipid.

17. A pharmaceutical composition comprising the mRNA molecule of any one of claims 1-15 or the LNP of any one of claims 15-16, and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising an mRNA encoding a coding sequence (CDS) of a bispecific antibody polypeptide against GPC3 and CD3, the pharmaceutical composition is administered by in vivo injection for treating a tumor; preferably the mRNA comprising a coding sequence of a bispecific antibody polypeptide against GPC3 and CD3 is selected from the group consisting of the mRNA molecule of any one of claims 1-15.

19. Use of the mRNA molecule of any one of claims 1-15 or the LNP of claim 16 or the pharmaceutical composition of any one of claims 17-18 in the manufacture of a medicament for treating and / or preventing a GPC3 related disease, such as a malignant tumor, in a subject.

20. A method of treating and / or preventing a GPC3 related disease, such as a malignant tumor, in a subject, the method comprising administering to the subject an effective amount of the mRNA molecule of any one of claims 1-15 or the LNP of claim 16 or the pharmaceutical composition of any one of claims 17-18.

21. The use of claim 19 or the method of claim 20, wherein the GPC3 related disease is a tumor that overexpresses GPC3, such as a liver cancer, e.g., hepatocellular carcinoma (HCC), a lung cancer, e.g., lung squamous cell carcinoma (SqCC), a gastric cancer, an ovarian cancer, e.g., ovarian clear cell, a melanoma, or a pediatric embryonal tumor.

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