Ribonucleic acid (RNA) compositions targeting claudin 18.2

Abstract

The present disclosure provides RNA techniques for targeting a claudin 18.2 polypeptide. In some embodiments, such RNA techniques may be used to treat diseases associated with positive expression of claudin-18.2. For example, in some embodiments, such RNA techniques may be used to treat claudin-18.2 positive cancers, including, for example, but not limited to, bile duct cancer, ovarian cancer, gastric cancer, gastroesophageal cancer, pancreatic cancer. In some embodiments, such RNA techniques may be used in combination therapy (e.g., in combination with chemotherapeutic agents). The disclosure also provides RNA backbones comprising specific sequences located upstream and / or downstream of the coding sequence.

Description

Background Art

[0001] Cancer is the second leading cause of death globally and is expected to cause an estimated 9.6 million deaths in 2018 (Bray et al. 2018). In general, with few exceptions (e.g., germ cells and some carcinoids), once solid tumors metastasize, the 5-year survival rate rarely exceeds 25%.

[0002] Recent advances in conventional treatments such as chemotherapy, radiotherapy, surgery, and targeted and immunotherapies have improved outcomes in patients with advanced solid tumors. In the past several years, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved eight checkpoint inhibitors (an anti-CTLA-4 pathway monoclonal antibody ipilimumab and seven antibodies targeting programmed death receptor / ligand [PD / PD-L1], which include atezolizumab, avelumab, durvalumab, nivolumab, cemiplimab, and pembrolizumab) for the treatment of patients with multiple cancer types (primarily solid tumors). These approvals have greatly changed the landscape of cancer treatment. However, certain cancers, such as pancreatic adenocarcinoma or metastatic biliary cancer, still do not benefit from existing treatments, including immunotherapy. Summary of the Invention

[0003] The poor prognosis of certain cancers such as pancreatic cancer and cholangiocarcinoma types highlights the need for additional treatment options.

[0004] The present disclosure particularly provides insights and techniques for treating cancer, particularly cancers associated with Claudin-18.2 (CLDN-18.2) expression. In some embodiments, the present disclosure provides techniques for treating cancers selected from pancreatic cancer, gastric cancer or gastroesophageal cancer, cholangiocarcinoma, ovarian cancer, and the like. In some embodiments, the present disclosure provides techniques for administering treatment to locally advanced tumors. In some embodiments, the present disclosure provides techniques for treating unresectable tumors. In some embodiments, the techniques provided provide techniques for treating metastatic tumors. Thus, for example, in some embodiments, the treatment provided can be administered to a subject or population of subjects having or at risk of having cancer (e.g., cancers selected from pancreatic cancer, gastric cancer or gastroesophageal cancer, biliary tract cancer, ovarian cancer, and / or additional cancers involving one or more pancreatic, gastric, gastroesophageal, biliary tract, and / or ovarian tumors), which can be or include one or more locally advanced tumors, one or more unresectable tumors, and / or one or more metastatic tumors.

[0005] The present disclosure particularly provides the insight that Claudin-18.2 (CLDN-18.2) represents a particularly useful tumor-associated antigen that can be targeted by treatment. Without being bound by any particular theory, the present disclosure points out that the tissue expression pattern of CLDN-18.2 (including its particularly limited expression in non-cancerous tissues) can contribute to its usefulness as a target as described herein. To date, no treatment targeting CLDN-18.2 has been approved for any cancer indication.

[0006] Zolbetuximab (development code IMAB362) is a monoclonal antibody targeting Claudin-18 isoform 2 and is being investigated for the treatment of gastrointestinal adenocarcinoma and pancreatic tumors (Türeci et al. 2019).

[0007] The present disclosure also provides the insight that in some embodiments, as described herein, treatment targeting CLDN-18.2 can usefully involve administering RNA encoding an antibody agent targeting CLDN-18.2 (e.g., ssRNA, e.g., mRNA). Further, the present disclosure provides the particular insight that delivery of RNA via lipid nanoparticles targeting hepatocytes can be a particularly beneficial strategy for delivering such antibody agents.

[0008] The present disclosure also provides the insight that the RiboMab format (e.g., as in Figure 13as shown herein), and in particular the RNA sequences and sequence elements described herein can be particularly useful for delivering RNA (e.g., ssRNA, e.g., mRNA) of a CLDN-18.2 targeting agent (e.g., a CLDN-18.2 targeting antibody agent) as described herein.

[0009] The present disclosure particularly provides the insight that administering RNA (e.g., ssRNA, e.g., mRNA) encoding a CLDN-18.2 targeting agent and in particular a CLDN-18.2 targeting antibody agent and specifically IMAB362 can represent a particularly desirable strategy for treating CLDN-18.2 targeting. Without wishing to be bound by any particular theory, the present disclosure proposes that such a delivery mode can achieve one or more improvements, such as effective administration with a reduced incidence of TEAE (e.g., frequency and / or severity) and / or an improved relationship between the potency level and the TEAE level (e.g., an improved therapeutic window), as compared to those observed when administering the corresponding (e.g., encoded) protein (e.g., antibody) agent itself. In particular, the present disclosure teaches that such improvements can be achieved particularly by delivering IMAB362 via administering RNA (e.g., ssRNA, e.g., mRNA) encoding IMAB362.

[0010] In some embodiments, the present disclosure particularly provides the insight that mRNA encoding an antibody agent (e.g., IMAB362) or a functional portion thereof (which is a lipid nanoparticle (LNP) / or formulated with a lipid nanoparticle for intravenous (IV) administration) can be taken up by target cells (e.g., hepatocytes) to effectively produce a therapeutically relevant plasma concentration of the encoded antibody agent (e.g., IMAB362), e.g., as Figure 14 shown for the CLDN-18.2 targeting RiboMab described therein.

[0011] In some embodiments, the present disclosure utilizes RiboMab as a CLDN-18.2 targeting agent. In some embodiments, such a RiboMab is an antibody agent encoded by mRNA, e.g., engineered for minimal immunogenicity and / or formulated in a lipid nanoparticle (LNP).

[0012] In addition, the present disclosure particularly provides the insight that the ability of an antibody agent targeting CLDN-18.2, as described herein, to induce antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while harnessing the immune system of a subject can enhance the cytotoxic effects of chemotherapy and / or other anti-cancer treatments. In some embodiments, such combination therapies can prolong progression-free and / or overall survival, e.g., relative to individual therapies administered alone and / or relative to another suitable reference.

[0013] Without wishing to be bound by a particular theory, the present disclosure has observed that certain chemotherapeutic agents, such as gemcitabine, oxaliplatin, and 5-fluorouracil, show an upregulation of existing CLDN-18.2 expression levels in pancreatic cancer cell lines; furthermore, no increase in de novo expression of these agents was observed in CLDN-18.2-negative cell lines. See, e.g., Türeci et al. (2019) “Characterization of zolbetuximab in pancreatic cancer models” In Oncoimmunology 8(1), pp.e1523096.

[0014] The present disclosure particularly provides the insight that a treatment targeting CLDN-18.2, as described herein, can be particularly useful and / or effective when administered to a tumor (e.g., tumor cells, a subject suspected and / or detected with such a tumor and / or tumor cells, etc.) characterized by (e.g., having been determined to show and / or expected or predicted to show) an elevated expression and / or activity of CLDN-18.2 in tumor cells (e.g., which may be due to or have been due to exposure to one or more chemotherapeutic agents). Indeed, the present disclosure particularly teaches that the provided treatment targeting CLDN-18.2, as described herein (e.g., administering RNA, and more particularly, mRNA encoding a CLDN-18.2-targeting antibody agent), can provide a synergistic treatment when administered in combination with one or more CDLN18.2 enhancers (e.g., one or more specific chemotherapeutic agents) (e.g., to a subject who has received and / or is receiving or otherwise exposed to the one or more CDLN18.2 enhancers (e.g., one or more specific chemotherapeutic agents)). Thus, in some embodiments, a treatment targeting CLDN-18.2, as described herein, in combination with other anti-cancer agents expected and / or shown to upregulate CLDN-18.2 expression in tumor cells can be useful.

[0015] In some aspects, provided herein are pharmaceutical compositions that target CLDN-18.2. In some embodiments, such pharmaceutical compositions comprise: (a) at least one RNA (e.g., ssRNA) that comprises one or more coding regions encoding an antibody agent (“CLDN-18.2 targeting antibody agent”) that binds to a Claudin-18.2 (CLDN-18.2) polypeptide (e.g., preferentially binds to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 (CLDN18.1) polypeptide); and (b) lipid nanoparticles; wherein at least one single-stranded RNA is encapsulated within at least one lipid nanoparticle. In some embodiments, such pharmaceutical compositions can comprise and / or deliver one or more RNAs that encode an antibody that binds to a CLDN-18.2 polypeptide (e.g., preferentially binds to a CLDN-18.2 polypeptide relative to a CLND18.1 polypeptide). In some embodiments, such pharmaceutical compositions can comprise and / or deliver one or more RNAs that encode an antigen-binding fragment that binds to a CLDN-18.2 polypeptide (e.g., preferentially binds to a CLDN-18.2 polypeptide relative to a CLND18.1 polypeptide).

[0016] In some embodiments, an antibody agent that targets CLDN-18.2 (and can be encoded by an RNA such as ssRNA, e.g., an mRNA as described herein) specifically binds to the first extracellular domain (ECD1) of the CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an epitope of ECD1 that is exposed in cancer cells.

[0017] In some embodiments, at least one RNA (e.g., ssRNA, e.g., mRNA) encodes the variable heavy chain (V H ) domain and the variable light chain (V L ) domain of a CLDN-18.2 targeting antibody agent. In some embodiments, such V H domains and V L domains of a CLDN-18.2 targeting antibody agent can be encoded by a single RNA construct; or, in some embodiments, they can be encoded by at least two separate RNA constructs. For example, in some embodiments, an RNA as used herein comprises two or more coding regions that comprise at least a heavy chain coding region encoding the V H domain of the antibody agent; and at least a light chain coding region encoding the V L domain of the antibody agent. In some alternative embodiments, the pharmaceutical composition can comprise: (i) a first RNA that comprises at least a V Hthe heavy chain coding region of the domain; and (ii) a second RNA that comprises at least the V L light chain coding region of the domain.

[0018] In some embodiments, the heavy chain coding region may further encode a constant heavy chain (C H ) domain; and / or the light chain coding region may further encode a constant light chain (C L ) domain. For example, in some embodiments, the heavy chain coding region may encode the V H domain, C H1 domain, C H2 domain, and C H3 domain of an antibody agent in the form of immunoglobulin G (IgG); and / or the light chain coding region may encode the V L domain and C L domain of an antibody agent in the form of IgG. In some embodiments, the antibody agent in the form of IgG is IgG1.

[0019] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full-length heavy chain of zolbetuximab or Claudiximab. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full-length light chain of zolbetuximab or Claudiximab.

[0020] In some embodiments, the RNA encoding a CLDN-18.2-targeting antibody agent may comprise a secretion signal coding region. In some embodiments, such a secretion signal coding region allows the CLDN-18.2-targeting antibody agent encoded by one or more RNAs to be secreted after being translated by cells (e.g., present in a subject to be treated), thus generating a plasma concentration of the biologically active CLDN-18.2-targeting antibody agent.

[0021] Those skilled in the art will recognize the emerging field of nucleic acid therapeutics and, in addition, RNA (e.g., ssRNA, e.g., mRNA) therapeutics (see, e.g., mRNA encoding proteins and / or cytokines). Certain features of RNA (e.g., ssRNA, e.g., mRNA) therapeutic technologies and / or delivery systems can be utilized in various embodiments of the techniques provided herein. For example, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include one or more modified nucleotides (e.g., but not limited to pseudouridine), nucleosides, and / or linkages. As an alternative or supplement, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and / or translation efficiency. As an alternative or supplement, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include a specific combination of at least two 3’UTR sequences (e.g., a combination of a sequence element of the amino-terminal enhancer of split RNA and a sequence derived from mitochondrially encoded 12S RNA). As an alternative or supplement, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include a ‘5UTR sequence derived from human α-globin mRNA. As an alternative or supplement, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include a 5’ cap analogue, e.g., for co-transcriptional capping. As an alternative or supplement, in some embodiments, RNA (e.g., ssRNA, e.g., mRNA) can include a secretion signal coding region (e.g., a human secretion signal coding sequence) with reduced immunogenicity such that the encoded antibody agent is expressed and secreted. In some embodiments, RNA can be formulated in or with one or more delivery vehicles (e.g., nanoparticles such as lipid nanoparticles, etc.). As an alternative or supplement, in some embodiments, RNA can be formulated in or with liver-targeting lipid nanoparticles (e.g., cationic lipid nanoparticles).

[0022] In some embodiments, the RNA encoding a CLDN-18.2 targeting antibody agent may comprise at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, 3' untranslated region (UTR), 5' UTR, a cap structure for co-transcriptional capping of mRNA, a polyadenine (poly A) tail, and any combination thereof. For example, in some embodiments, the RNA (e.g., the first RNA and / or the second RNA) independently comprises, in a 5' to 3' direction: (a) a 5' UTR; (b) a secretion signal coding region; (c) an antibody chain coding region; (d) a 3' UTR; and (e) a polyA tail. In some embodiments, the polyA tail coding region comprised in the RNA is a modified polyA sequence or comprises a modified polyA sequence.

[0023] In some embodiments, the RNA encoding a CLDN-18.2 targeting antibody agent may comprise a 5' cap.

[0024] In some embodiments, the RNA encoding a CLDN-18.2 targeting antibody agent may comprise at least one modified ribonucleotide. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of the RNA may be replaced by a modified ribonucleotide. In some embodiments, such a modified ribonucleotide may be pseudouridine or comprise pseudouridine.

[0025] In which the pharmaceutical composition comprises a first RNA encoding a variable heavy chain (V H ) domain (e.g., the heavy chain of a CLDN-18.2 targeting antibody agent) of a CLDN-18.2 targeting antibody agent and a variable light chain (V L)In some embodiments of the second RNA of the domain (such as the light chain of a CLDN-18.2 targeting antibody agent), such first RNA and second RNA may be present in a molar ratio of about 1.5:1 to about 1:1.5. In some embodiments, such first RNA and second RNA may be present in a molar ratio of about 1.30, about 1.29, about 1.28, about 1.27, about 1.26, about 1.25, about 1.24, about 1.23, about 1.22, about 1.21, about 1.20, about 1.19, about 1.18, about 1.17, about 1.16, about 1.15, about 1.14, about 1.13, about 1.12, about 1.11, about 1.10, about 1.09, about 1.08, about 1.07, about 1.06, about 1.05, about 1.04, about 1.03, about 1.02, about 1.01, about 1.00, about 0.99, about 0.98, about 0.97, about 0.96, about 0.95, about 0.94, about 0.93, about 0.92, about 0.91, about 0.90, about 0.89, about 0.88, about 0.87, about 0.86, about 0.85, about 0.84, about 0.83, about 0.82, about 0.81 or about 0.80. In some embodiments, such first RNA and second RNA may be present in a weight ratio of 3:1 to 1:1. In some embodiments, such first RNA and second RNA may be present in a weight ratio of about 2:1. In some embodiments, such first RNA and second RNA may be present in a weight ratio of about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1 or about 1.2:1.

[0026] In some embodiments, the RNA content of the pharmaceutical compositions described herein (such as one or more RNAs encoding a CLDN-18.2 targeting antibody agent) is present at a concentration of 0.5 mg / mL to 1.5 mg / mL.

[0027] In some embodiments, the lipid nanoparticles provided in the pharmaceutical compositions described herein are liver-targeting lipid nanoparticles. In some embodiments, the lipid nanoparticles provided in the pharmaceutical compositions described herein are cationic lipid nanoparticles. In some embodiments, the average size of the lipid particles provided in the pharmaceutical compositions described herein may be about 50 to 150 nm.

[0028] In some embodiments, the lipids forming the lipid nanoparticles comprise: polymer-conjugated lipids, cationic lipids, and neutral lipids. In some such embodiments, the polymer-conjugated lipids are present at about 1 mol% to 2.5 mol% of the total lipids; the cationic lipids are present at 35 mol% to 65 mol% of the total lipids; and the neutral lipids are present at 35 mol% to 65 mol% of the total lipids.

[0029] A variety of lipids (which include, for example, polymer-conjugated lipids, cationic lipids, and neutral lipids) are known in the art and can be used herein to form lipid nanoparticles, such as lipid nanoparticles that target specific cell types (e.g., hepatocytes). In some embodiments, the polymer-conjugated lipids included in the pharmaceutical compositions described herein can be PEG-conjugated lipids (e.g., 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide or derivatives thereof). In some embodiments, the cationic lipids included in the pharmaceutical compositions described herein can be ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-ethylhexanoate) or derivatives thereof. In some embodiments, the neutral lipids included in the pharmaceutical compositions described herein can be phospholipids or derivatives thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol, or comprise phospholipids or derivatives thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol.

[0030] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, e.g., in some embodiments, which can enhance the stability of such compositions under certain conditions. For example, in some embodiments, the pharmaceutical compositions may further comprise cryoprotectants (e.g., sucrose) and / or aqueous buffer solutions, which may comprise one or more salts (e.g., sodium salts) in some embodiments.

[0031] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more active agents other than RNAs (e.g., ssRNA, e.g., mRNA) encoding CLDN-18.2 targeting agents (e.g., antibody agents). For example, in some embodiments, such other active agents can be chemotherapeutic agents or comprise chemotherapeutic agents. Exemplary chemotherapeutic agents can be chemotherapeutic agents suitable for treating pancreatic cancer or comprise chemotherapeutic agents suitable for treating pancreatic cancer.

[0032] In some embodiments, the pharmaceutical compositions described herein can be taken up by target cells for the production of encoded CLDN-18.2-targeted antibody agents at therapeutically relevant plasma concentrations. In some embodiments, such pharmaceutical compositions described herein can induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells).

[0033] Accordingly, another aspect of the present disclosure relates to methods of using the pharmaceutical compositions described herein. For example, one aspect provided herein relates to a method comprising administering the provided pharmaceutical composition to a subject having a CLDN-18.2-positive solid tumor. Examples of CLDN-18.2-positive solid tumors are, but are not limited to, biliary tract tumors, gastric tumors, gastroesophageal tumors, ovarian tumors, pancreatic tumors, and tumors that express or exhibit a certain level of CLDN-18.2 polypeptide. In some embodiments, a CLDN-18.2-positive tumor can be characterized in that ≥50% of the tumor cells show a ≥2+ CLDN-18.2 protein staining intensity, as evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tumor tissue from the subject to be administered. In some embodiments, a subject having a CLDN-18.2-positive solid tumor can have a locally advanced, unresectable, or metastatic tumor. In some embodiments, a subject having a CLDN-18.2-positive solid tumor may have received pre-treatment sufficient to increase the level of CLDN-18.2 such that his / her solid tumor is characterized as a CLDN-18.2-positive solid tumor.

[0034] In some embodiments, the pharmaceutical compositions described herein can be administered as a single therapy. In some embodiments, the pharmaceutical composition can be administered as part of a combination therapy comprising such pharmaceutical composition and a chemotherapeutic agent. Accordingly, in some embodiments, a subject receiving the provided pharmaceutical composition has received a chemotherapeutic agent. In some embodiments, a chemotherapeutic agent is administered to a subject receiving the provided pharmaceutical composition such that such subject receives both as a combination therapy. In some embodiments, the provided pharmaceutical composition and the chemotherapeutic agent can be administered simultaneously or sequentially. For example, in some embodiments, the chemotherapeutic agent can be administered after (e.g., at least four hours after) the administration of the provided pharmaceutical composition.

[0035] In some embodiments, the techniques provided herein can be used to treat CLDN-18.2 positive pancreatic tumors. In some embodiments involving administering the provided pharmaceutical composition to a subject having a CLDN-18.2 positive pancreatic tumor, such a subject can receive such provided composition as a single therapy or as part of a combination therapy comprising such provided pharmaceutical composition and a chemotherapeutic agent suitable for treating pancreatic tumors. In some embodiments, such chemotherapeutic agent can be gemcitabine and / or paclitaxel (e.g., nab-paclitaxel) or comprise gemcitabine and / or paclitaxel (e.g., nab-paclitaxel). In some embodiments, such chemotherapeutic agent can be FOLFIRINOX or comprise FOLFIRINOX, which is a combination of cancer drugs comprising: folinic acid (FOL), fluorouracil (F), irinotecan (IRIN), and oxaliplatin (OX).

[0036] In some embodiments, the techniques provided herein can be used to treat CLDN-18.2 positive biliary tract tumors. In some embodiments involving administering the provided pharmaceutical composition to a subject having a CLDN-18.2 positive biliary tract tumor, such a subject can receive such provided composition as a single therapy or as part of a combination therapy comprising such provided pharmaceutical composition and a chemotherapeutic agent suitable for treating biliary tract tumors. In some embodiments, such chemotherapeutic agent can be gemcitabine and / or cisplatin or comprise gemcitabine and / or cisplatin.

[0037] The pharmaceutical compositions and methods described herein can be applicable to subjects of any age having a CLDN-18.2 positive solid tumor. In some embodiments, the subject having a CLDN-18.2 positive solid tumor is an adult subject.

[0038] The pharmaceutical compositions described herein can be administered to subjects in need thereof by suitable methods known in the art. For example, in some embodiments, the provided pharmaceutical composition can be administered by intravenous injection to a subject having a CLDN-18.2 positive solid tumor.

[0039] The dosage of the pharmaceutical compositions described herein can vary with many factors, including for example but not limited to the weight of the subject to be treated, the type and / or stage of cancer, and / or single or combination therapy. In some embodiments, the pharmaceutical compositions described herein are administered to a subject with CLDN-18.2 positive solid tumors in at least one or more (which includes, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more) dosing cycles. In some embodiments, each dosing cycle can be a three-week dosing cycle. In some embodiments, the pharmaceutical compositions described herein are administered at at least one dose per dosing cycle. In some embodiments, a dosing cycle involves administering a set number and / or pattern of doses; in some embodiments, a dosing cycle involves administering a set cumulative dose, for example, over a specific time period, and optionally via multiple doses, which can be administered, for example, at set intervals and / or according to a set pattern. In some embodiments, each dose or cumulative dose of the pharmaceutical compositions described herein can comprise one or more RNAs encoding a CLDN-18.2 targeting antibody agent (whether encoded by a single RNA or two or more RNAs), in an amount in the range of 0.1 mg / kg to 5 mg / kg of the weight of the subject to be administered.

[0040] Another aspect of the present disclosure relates to certain improvements to methods of delivering a CLDN-18.2 targeting antibody agent in a subject for cancer treatment, the method comprising administering the provided pharmaceutical composition to a cancer subject. In some embodiments, the pharmaceutical compositions described herein can achieve one or more improvements, such as effective administration with reduced TEAE (e.g., frequency and / or severity) and / or an improved relationship between the potency level and the TEAE level (e.g., improved therapeutic window), as compared to those observed when administering the corresponding (e.g., encoded) protein (e.g., antigen) agent itself. In particular, the present disclosure teaches that such improvements can be achieved in particular by delivering IMAB362 via administration of RNA encoding IMAB362 (e.g., ssRNA, e.g., mRNA).

[0041] Methods of producing a CLDN-18.2 targeting antibody agent are also within the scope of the present disclosure. In some embodiments, a method of producing a CLDN-18.2 targeting antibody agent comprises administering to a cell a composition comprising at least one RNA (e.g., an RNA described herein) comprising one or more coding regions encoding a CLDN-18.2 targeting antibody agent such that such a cell expresses and secretes the CLDN-18.2 targeting antibody agent encoded by such an RNA. In some embodiments, the cell to be administered or targeted is a hepatocyte or comprises hepatocytes.

[0042] In some embodiments, the cells are present in a cell culture.

[0043] In some embodiments, the cells are present in a subject. In some such embodiments, the pharmaceutical compositions described herein can be administered to a subject in need thereof. In some embodiments, such a pharmaceutical composition can be administered to a subject such that a CLDN-18.2 targeting antibody agent is produced at a therapeutically relevant plasma concentration. In some embodiments, the therapeutically relevant plasma concentration is sufficient to mediate cancer cell death by antibody-dependent cell cytotoxicity (ADCC). For example, in some embodiments, the therapeutically relevant plasma concentration is from 0.3 to 28 μg / mL.

[0044] The present disclosure also particularly provides a method of characterizing one or more characteristics of an RNA or a composition thereof, wherein the RNA encodes part or all of an antibody agent. In some embodiments, the method comprises the steps of: determining one or more characteristics of an antibody agent expressed by at least one mRNA introduced into a cell, wherein such at least one mRNA comprises one or more characteristics of at least one or more RNAs, the RNA comprising a coding region encoding an antibody agent that binds to a claudin-18.2 (CLDN-18.2) polypeptide (e.g., preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide relative to a claudin-18.1 polypeptide), wherein such one or more characteristics comprise: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to CLDN-18.2; (iii) the potency of the antibody agent to mediate target cell death by ADCC; and (iv) the potency of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC).

[0045] In some embodiments, provided herein is a method of characterizing a pharmaceutical composition targeting CLDN-18.2. Such a method comprises the steps of: (a) contacting a cell with at least one of the compositions or pharmaceutical compositions described herein (which encodes part or all of a CLDN-18.2 targeting antibody agent); and detecting the antibody agent produced by the cell. In some embodiments, the cell can be a hepatocyte or comprise hepatocytes.

[0046] In some embodiments, such methods may further comprise determining one or more characteristics of an antibody agent expressed by one or more of the RNAs described herein, wherein such one or more characteristics comprise: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to the CLDN-18.2 polypeptide; (iii) the potency of the antibody agent to mediate target cell death via ADCC; and (iv) the potency of the antibody agent to mediate target cell death via complement-dependent cytotoxicity (CDC). In some embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more of the RNAs described herein may comprise comparing such characteristics of a CLDN-18.2-targeting antibody agent to the characteristics of a reference CLDN-18.2-targeting antibody.

[0047] In some embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more of the RNAs described herein may comprise assessing that the protein expression level of the antibody agent is higher than a threshold level. For example, in some embodiments, the threshold level corresponds to a therapeutically relevant plasma concentration.

[0048] In some embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more of the RNAs described herein may comprise assessing the binding of the antibody agent to the CLDN-18.2 polypeptide. In some embodiments, such binding assessment may comprise determining the binding of the antibody agent to the CLDN-18.2 polypeptide relative to the binding of the antibody agent to the CLDN18.1 polypeptide. In some embodiments, such binding assessment may comprise determining that the binding preference profile of the antibody agent is at least comparable to the binding preference profile of a reference CLDN-18.2-targeting antibody. For example, in some embodiments, the reference CLDN-18.2-targeting antibody is zolbetuximab or claudiximab.

[0049] In some embodiments, if an antibody agent expressed by one or more of the RNAs described herein comprises the following characteristics, the provided method of characterizing a pharmaceutical composition or its components targeting CLDN-18.2 may further comprise characterizing the antibody agent as a CLDN-18.2-targeting antibody agent: (a) the protein level of the antibody agent expressed by the cell is higher than a threshold level; (b) the antibody agent preferentially binds to CLDN-18.2 relative to CLDN18.1; and (c) mediates killing of at least 50% of target cells (e.g., cancer cells) via ADCC and / or CDC.

[0050] In some embodiments, if the characteristics of the test antibody are at least comparable to those of zolbetuximab or clausimab, the provided method of characterizing a pharmaceutical composition or its components that target CLDN-18.2 may further include characterizing an antibody agent expressed by one or more RNAs described herein as a zolbetuximab- or clausimab-equivalent antibody.

[0051] In some embodiments of the step involving determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein, such a step may include determining one or more of the following characteristics: · Whether the cell expresses a CLDN-18.2-targeting antibody agent encoded by at least one RNA when evaluated after 48 hours of contact or administration; · Whether the antibody agent expressed by the cell binds preferentially to the CLDN-18.2 polypeptide relative to the CLDN18.1 polypeptide; · Whether the antibody agent expressed by the cell exhibits target specificity for CLDN-18.2 that is comparable to that of a reference CLDN-18.2-targeting monoclonal antibody, as observed in a flow cytometry binding assay; · Whether, when evaluated after incubating immune effector cells (e.g., PBMC cells) with CLDN-18.2-positive cells or CLDN-18.2-negative control cells for 48 hours in the presence of the antibody agent, it is the CLDN-18.2-positive cells rather than the control cells that are lysed; · Whether the antibody agent expressed by the cell exhibits an ADCC profile of the targeted CLDN-18.2-positive cells that is at least comparable to that observed in the case of a reference CLDN-18.2-targeting monoclonal antibody at the same concentration; and · Whether, when evaluated after incubating CLDN-18.2-positive cells or CLDN-18.2-negative control cells with human serum for 2 hours in the presence of the antibody agent, it is the CLDN-18.2-positive cells rather than the control cells that are lysed.

[0052] In some embodiments, the cells used in the provided methods of characterizing a pharmaceutical composition or its components that target CLDN-18.2 are present in vivo, such as in a subject (e.g., a mammalian subject, such as a non-human mammalian subject, such as a mouse or monkey subject). In some such embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more of the RNAs described herein may include determining the antibody level in one or more tissues of such a subject. In some embodiments, if the composition or pharmaceutical composition described herein is characterized as a CLDN-18.2-targeting antibody agent, such a characterization method may further include administering such a composition or pharmaceutical composition to a group of animal subjects each bearing a human CLDN-18.2-positive xenograft tumor to determine anti-tumor activity.

[0053] Also included within the scope of the present disclosure are manufacturing methods, which include the following steps: (A) determining one or more characteristics of an RNA or a composition thereof that encodes part or all of an antibody agent, the one or more characteristics selected from: (i) the length and / or sequence of the RNA; (ii) the integrity of the RNA; (iii) the presence and / or location of one or more chemical moieties in the RNA; (iv) the degree of expression of the antibody agent when the RNA is introduced into a cell; (v) the stability of the RNA or its composition; (vi) the level of the antibody agent in a biological sample from an organism into which the RNA has been introduced; (vii) the binding specificity of the antibody agent expressed by the RNA, optionally to CLDN-18.2 and optionally relative to CLDN18.1; (viii) the potency of the antibody agent to mediate target cell death via ADCC; (ix) the potency of the antibody agent to mediate target cell death via complement-dependent cytotoxicity (CDC); (x) the identity and amount / concentration of lipids within the composition; (xi) the size of lipid nanoparticles within the composition; (xii) the polydispersity of lipid nanoparticles within the composition; (xiii) the amount / concentration of RNA within the composition; (xiv) the degree of encapsulation of the RNA within the lipid nanoparticles; and (xv) combinations thereof; (B) comparing such one or more characteristics of the RNA or its composition to the characteristics of an appropriate reference standard; and (C) (i) If the comparison indicates that the RNA or its composition meets or exceeds the reference standard, one or more additional steps of the RNA or its composition are designated for manufacture and / or distribution; or (ii) If the comparison indicates that the RNA or its composition does not meet or does not exceed the reference standard, alternative actions are taken.

[0054] In some embodiments of the manufacturing method, when evaluating an RNA (e.g., the RNA described herein) and one or more characteristics of the RNA meet or exceed the appropriate reference standard, such RNA is designated for formulation, which in some embodiments involves formulation with lipid particles as described herein.

[0055] In some embodiments of the manufacturing method, when evaluating a composition comprising an RNA (e.g., the RNA described herein) and one or more characteristics of the composition meet or exceed the appropriate reference standard, such composition is designated for release and / or distribution of the composition.

[0056] In some embodiments of the manufacturing method, when the RNA (e.g., the RNA described herein) is designated for formulation, and / or the composition comprising the RNA (e.g., the RNA described herein) is designated for release and / or distribution of the composition, such method may further include administering the formulation and / or composition to a group of animal subjects bearing human CLDN-18.2 positive xenograft tumors, respectively, to determine the anti-tumor activity.

[0057] The present disclosure also provides a method for determining a dosing regimen of a pharmaceutical composition targeting CLDN-18.2. For example, in some embodiments, such method includes the following steps: (A) administering a pharmaceutical composition (e.g., the pharmaceutical composition described herein) to a subject with a CLDN-18.2 positive solid tumor under a pre-determined dosing regimen; (B) regularly monitoring or measuring the tumor size of the subject over a period of time; (C) evaluating the dosing regimen based on the tumor size measurement. For example, if the reduction in tumor size after administering the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is not treatment-related, the dose and / or dosing frequency may be increased; or if the reduction in tumor size after administering the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is treatment-related, but adverse effects (e.g., toxic effects) are shown in the subject, the dose and / or dosing frequency may be decreased. If the reduction in tumor size after administering the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is treatment-related and no adverse effects (e.g., toxic effects) are shown in the subject, no change is made to the dosing regimen.

[0058] In some embodiments, such a method of determining a dosing regimen for a pharmaceutical composition targeting CLDN-18.2 can be carried out in groups of animal subjects (e.g., mammalian non-human subjects) each bearing a human CLDN-18.2 positive xenograft tumor. In some such embodiments, if, after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein), less than 30% of the animal subjects exhibit a reduction in tumor size and / or the degree of reduction in tumor size exhibited by the animal subjects is not treatment-related, the dose and / or dosing frequency can be increased; or if the reduction in tumor size after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is treatment-related, but significant adverse effects (e.g., toxic effects) are shown in at least 30% of the animal subjects, the dose and / or dosing frequency can be decreased. If the reduction in tumor size after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is treatment-related and no significant adverse effects (e.g., toxic effects) are shown in the animal subjects, no change is made to the dosing regimen.

[0059] The present disclosure particularly provides, in particular a composition or pharmaceutical formulation, comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein (i) the coding region described in (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO:16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO:16, and (ii) the coding region described in (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:17. In some embodiments, the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4. The present disclosure also provides a composition or pharmaceutical formulation, comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:18 or 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:18 or 20. In some embodiments, the RNA, such as each RNA, comprises a 3'UTR, the 3'UTR comprising the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:22. In some embodiments, the RNA, such as each RNA, comprises a 3'UTR, the 3'UTR comprising the nucleotide sequence of SEQ ID NO:19 or 21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:19 or 21. The present disclosure also provides a composition or pharmaceutical formulation, comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the RNA, such as each RNA, comprises a 5'UTR and / or a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:18 or 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:18 or 20, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO:19 or 21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:19 or 21. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:18 or 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:18 or 20, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO:19 or 21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:19 or 21. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:18 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:18, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO:19 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:19. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:21. In some embodiments, the RNA, such as each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:18, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO:19. In some embodiments, the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20 and the 3' UTR comprising the nucleotide sequence of SEQ ID NO: 21. In some embodiments, (a)(i) the coding region described therein comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and (ii) the coding region described therein comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and / or (b) the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4. In some embodiments, (i) the coding region described therein comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 16, and (ii) the coding region described therein comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule. In some embodiments, at least 90% is at least 95%, 96%, 97%, 98%, 99%. In some embodiments, the antibody agent preferentially binds to CLDN-18.2 relative to Claudin-18.1 (CLDN-18.1). In some embodiments, the antibody agent binds to the first extracellular domain (ECD1) of CLDN-18.2. In some embodiments, the antibody agent binds to an epitope of ECD1 of CLDN-18.2 that is exposed in cancer cells. In some embodiments, the antibody agent that binds to CLDN-18.2 comprises two binding arms, wherein each binding arm comprises a heavy chain of the antibody agent that binds to CLDN-18.2 and a light chain of the antibody agent that binds to CLDN-18.2. In some embodiments, the antibody agent is IgG1. In some embodiments, the IgG1 is human IgG1. In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form a binding domain that binds to CLDN-18.2. In some embodiments, the first polypeptide chain comprises the variable domain (VH) of the heavy chain of the antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)). In some embodiments, the VH(CLDN-18.2) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH(CLDN-18.2) comprises CDR1, CDR2, and CDR3 that contain the sequences shown in SEQ ID NO:5, 6, and 7, respectively. In some embodiments, the second polypeptide chain comprises the variable domain (VL) of the light chain of the antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)). In some embodiments, the VL(CLDN-18.2) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:15. In some embodiments, the VL(CLDN-18.2) comprises CDR1, CDR2, and CDR3 that contain the sequences shown in SEQ ID NO:8, 9, and 10, respectively. In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 and contains CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NO: 14 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 and contains CDR1, CDR2, and CDR3 having the amino acid sequence of SEQ ID NO: 15 (VL(CLDN-18.2)). In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 and contains CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 and contains CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively (VL(CLDN-18.2)). In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 and contains the amino acid sequence of SEQ ID NO: 14 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 and contains the amino acid sequence of SEQ ID NO: 15 (VL(CLDN-18.2)). In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)), wherein the VH(CLDN-18.2) interacts with the VL(CLDN-18.2) to form a binding domain that binds to claudin-18.2 (CLDN-18.2). In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), a constant domain 1 (CH1) of the heavy chain of the antibody agent, a constant domain 2 (CH2) of the heavy chain of the antibody agent, and a constant domain 3 (CH3) of the heavy chain of the antibody agent. In some embodiments, the VH(CLDN-18.2), CH1, CH2, and CH3 are present in the first polypeptide chain in the form of immunoglobulin G (IgG). In some embodiments, the second polypeptide chain comprises a variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) and a constant domain (CL) of the light chain of the antibody agent. In some embodiments, the VL(CLDN-18.2) and CL are present in the second polypeptide chain in the form of IgG. In some embodiments, CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain independently comprises a secretion signal, wherein the secretion signal is preferably located at the N-terminus of the first polypeptide chain and the second polypeptide chain. In some embodiments, the secretion signal of the first polypeptide chain and / or the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the coding region described in (i) comprises the nucleotide sequence of SEQ ID NO:16, and the coding region described in (ii) comprises the nucleotide sequence of SEQ ID NO:17. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the RNA, such as each RNA, comprises a poly-A sequence. In some embodiments, the poly-A sequence is an interrupted sequence of A nucleotides. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises the nucleotide sequence A x -L-A y or consists of the nucleotide sequence A x -L-A y wherein A x is a sequence of at least 20 A nucleotides, A y is a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides that may comprise nucleotides other than A. In some embodiments, the poly-A sequence comprises the nucleotide sequence of SEQ ID NO:23 or consists of the nucleotide sequence of SEQ ID NO:23. In some embodiments, the composition or pharmaceutical formulation contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 25 or 27. In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the composition or pharmaceutical formulation comprises: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27. The present disclosure also provides a composition or pharmaceutical formulation comprising: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27. The present disclosure also provides a composition or pharmaceutical formulation comprising: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) the nucleotide sequence of SEQ ID NO: 25. The present disclosure also provides a composition or pharmaceutical formulation comprising: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the RNA, such as each RNA, comprises a modified nucleoside in place of uridine. In some embodiments, the RNA, such as each RNA, comprises a modified nucleoside in place of every uridine. In some embodiments, the modified nucleoside is pseudouridine (ψ) and / or N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA, such as each RNA, comprises a 5' cap. In some embodiments, the RNA, such as each RNA, comprises a 5' cap m 2 7，3’-O Gppp(m 1 2’-O )ApG. In some embodiments, the RNA, such as each RNA, is single-stranded RNA. In some embodiments, the RNA, such as each RNA, is mRNA. In some embodiments, the RNA, such as each RNA, is formulated in lipid nanoparticles (LNPs), such as each RNA is co-formulated in lipid nanoparticles (LNPs). In some embodiments, the lipids forming the lipid nanoparticles comprise cationic lipids, polymer-conjugated lipids; and neutral lipids. In some embodiments, a. the cationic lipid is present at 35 mol% to 65 mol% of the total lipids; b. the polymer-conjugated lipid is present at about 1 mol% to 2.5 mol% of the total lipids; and c. the neutral lipid is present at 35 mol% to 65 mol% of the total lipids. In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate). In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (such as 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide). In some embodiments, the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. In some embodiments, the average size of the lipid nanoparticles is about 50 to 150 nm. In some embodiments, the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate), 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients. In some embodiments, the pharmaceutical formulation is a kit. In some embodiments, the RNA, such as each RNA, and optionally the particle-forming component are in separate vials. In some embodiments, the pharmaceutical preparation further comprises instructions for the use of the composition or pharmaceutical preparation for treating or preventing cancer. The present disclosure also provides the composition or pharmaceutical preparation described herein for use in medicine. In some embodiments, the use in medicine comprises therapeutic or prophylactic treatment of a disease or disorder. In some embodiments, the therapeutic or prophylactic treatment of the disease or disorder comprises treating or preventing cancer. In some embodiments, the cancer comprises CLDN-18.2 positive cancer. In some embodiments, the cancer comprises CLDN-18.2 positive solid tumor. In some embodiments, the cancer comprises CLDN-18.2 positive pancreatic cancer. In some embodiments, the cancer comprises CLDN-18.2 positive gastric cancer. In some embodiments, the cancer comprises CLDN-18.2 positive biliary tract tumor. In some embodiments, the cancer comprises locally advanced, unresectable or metastatic cancer that is CLDN-18.2 positive. In some embodiments, the therapeutic or prophylactic treatment of the disease or disorder further comprises administering an additional treatment. In some embodiments, the additional treatment comprises one or more selected from the following: (i) surgery to excise, resect or debulk the tumor, (ii) radiotherapy, and (iii) chemotherapy. In some embodiments, the additional treatment comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the composition or pharmaceutical preparation described herein is for administration to a human. In some embodiments, the composition or pharmaceutical preparation described herein is for intravenous administration. The present disclosure also provides a method of treating cancer in an object, which comprises administering the composition described herein to the object. In some embodiments, the cancer comprises CLDN-18.2 positive cancer. In some embodiments, the cancer comprises CLDN-18.2 positive solid tumor. In some embodiments, the cancer comprises CLDN-18.2 positive pancreatic cancer. In some embodiments, the cancer includes CLDN-18.2 positive gastric cancer. In some embodiments, the cancer includes CLDN-18.2 positive biliary tract tumors. In some embodiments, the cancer includes locally advanced, unresectable or metastatic cancers that are CLDN-18.2 positive. In some embodiments, the methods described herein further comprise administering an additional treatment. In some embodiments, the additional treatment includes one or more selected from the following: (i) surgery to excise, resect or debulk the tumor, (ii) radiotherapy, and (iii) chemotherapy. In some embodiments, the additional treatment includes administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the subject is a human. In some embodiments, the composition is administered intravenously. The present disclosure also provides the composition described herein for use in the methods described herein. In some embodiments, after a polypeptide chain expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) is expressed, the polypeptide chain is secreted into the bloodstream as a fully assembled antibody and / or as a functional antibody. A fully assembled antibody is a tetramer composed of two pairs of identical polypeptide chains, each pair having a light chain and a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2). A functional antibody is an antibody having the expected biological activity of an antibody, such as binding to the target of the antibody and / or recruiting and / or stimulating the immune system (e.g., ADCC), for example, to the same or a similar level as the corresponding antibody expressed in vitro. In some embodiments, the composition or pharmaceutical formulation described herein is used to introduce the RNA into hepatocytes and express the polypeptide chain encoded by the RNA in the hepatocytes. In some embodiments, the composition or pharmaceutical formulation described herein is used for systemic delivery of the polypeptide chain. In some embodiments, the composition or pharmaceutical formulation described herein is used for systemic delivery of the polypeptide chain after the polypeptide chain is expressed in hepatocytes. The present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprising: (a) administering the composition described herein such that the RNA is introduced into hepatocytes; and (b) Express the polypeptide chain encoded by the RNA in the hepatocytes. The present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprising: (a) Administering the composition described herein such that the RNA is introduced into hepatocytes; and (b) Expressing the polypeptide chain encoded by the RNA in the hepatocytes, wherein, after expression, the polypeptide chain is secreted into the bloodstream. The present disclosure also provides a method for systemically delivering an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprising: (a) Administering the composition described herein such that the RNA is introduced into hepatocytes; and (b) Expressing the polypeptide chain encoded by the RNA in the hepatocytes, wherein, after expression, the polypeptide chain is secreted into the bloodstream. In some embodiments, the administration is parenteral administration. In some embodiments, the administration is intravenous administration.

[0060] The present disclosure also particularly provides the following items: 1. A pharmaceutical composition, comprising: a. At least one single-stranded RNA comprising one or more coding regions encoding an antibody agent that preferentially binds to a Claudin-18.2 polypeptide relative to a Claudin-18.1 polypeptide; and b. A lipid nanoparticle; wherein the at least one single-stranded RNA is encapsulated within at least one of the lipid nanoparticles. 2. The pharmaceutical composition according to item 1, wherein the antibody agent specifically binds to the first extracellular domain (ECD1) of the Claudin-18.2 polypeptide. 3. The pharmaceutical composition according to item 2, wherein the antibody agent specifically binds to an epitope of ECD1 exposed in cancer cells. 4. The pharmaceutical composition according to any one of items 1 to 3, wherein the antibody agent is an antibody or an antigen-binding fragment thereof or comprises an antibody or an antigen-binding fragment thereof. 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the at least one single-stranded RNA encodes both: the variable heavy chain (V ) domain of the antibody agent; and the variable light chain (V H ) domain of the antibody agent. L ) domain. 6. The pharmaceutical composition according to item 5, wherein the at least one single-stranded RNA is a first single-stranded RNA, which comprises a heavy-chain coding region encoding at least the V H domain of the antibody agent; and a. wherein the first single-stranded RNA further comprises a light-chain coding region encoding at least the V L domain of the antibody agent; or b. wherein the pharmaceutical composition further comprises a second single-stranded RNA, which comprises a light-chain coding region encoding at least the V L domain of the antibody agent. 7. The pharmaceutical composition according to item 6, wherein the heavy-chain coding region further encodes a constant heavy chain (C H ) domain; and / or the light-chain coding region further encodes a constant light chain (C L ) domain. 8. The pharmaceutical composition according to item 6, wherein the heavy-chain coding region encodes the V H domain, C H1 domain, C H2 domain and C H3 domain of the antibody agent in the form of immunoglobulin G (IgG); and / or the light-chain coding region encodes the V L domain and C L domain of the antibody agent in the form of IgG. 9. The pharmaceutical composition according to item 8, wherein the IgG is IgG1. 10. The pharmaceutical composition according to any one of items 6 to 9, wherein the heavy-chain coding region consists of or comprises a nucleotide sequence encoding the full-length heavy chain of zolbetuximab or clausimab. 11. The pharmaceutical composition according to any one of items 6 to 9, wherein the light-chain coding region consists of or comprises a nucleotide sequence encoding the full-length light chain of zolbetuximab or clausimab. 12. The pharmaceutical composition according to any one of items 6 to 11, wherein the first single-stranded RNA and / or the second single-stranded RNA each independently comprises a secretion signal coding region. 13. The pharmaceutical composition according to any one of items 6 to 12, wherein the first single-stranded RNA and / or the second single-stranded RNA each independently comprises at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). 14. The pharmaceutical composition according to item 13, wherein the at least one non-coding sequence element comprises a 3' untranslated region (UTR), a 5' UTR, a cap structure for co-transcriptional capping of mRNA, and / or a polyadenine (polyA) tail. 15. The pharmaceutical composition according to any one of items 6 to 14, wherein the first single-stranded RNA comprises, in the 5' to 3' direction: a. A 5' UTR coding region; b. A secretion signal coding region; c. A heavy chain coding region; d. A 3' UTR coding region; and e. A polyA tail coding region. 16. The pharmaceutical composition according to any one of items 6 to 15, wherein the second single-stranded RNA comprises, in the 5' to 3' direction: a. A 5' UTR coding region; b. A secretion signal coding region; c. A light chain coding region; d. A 3' UTR coding region; and e. A polyA tail coding region. 17. The pharmaceutical composition according to item 14 or 15, wherein the polyA tail is a modified polyA sequence or comprises a modified polyA sequence. 18. The pharmaceutical composition according to any one of items 6 to 16, wherein the first single-stranded RNA and / or the second single-stranded RNA comprises a 5' cap. 19. The pharmaceutical composition according to any one of items 6 to 18, wherein the first single-stranded RNA and / or the second single-stranded RNA comprises at least one modified ribonucleotide. 20. The pharmaceutical composition according to item 19, wherein the modified ribonucleotide comprises pseudouridine. 21. The pharmaceutical composition according to any one of items 6 to 20, wherein the at least one single-stranded RNA comprises the first single-stranded RNA and the second single-stranded RNA. 22. The pharmaceutical composition according to any one of items 6 to 21, wherein the first single-stranded RNA and the second single-stranded RNA are present in a weight ratio of 3:1 to 1:1. 23. The pharmaceutical composition according to any one of items 1 to 22, wherein the lipid nanoparticle is a liver-targeting lipid nanoparticle. 24. The pharmaceutical composition according to any one of items 1 to 23, wherein the lipid nanoparticle is a cationic lipid nanoparticle. 25. The pharmaceutical composition according to item 24, wherein the lipids forming the lipid nanoparticle comprise: - Polymer-conjugated lipids; - Cationic lipids; and - Neutral lipids. 26. The pharmaceutical composition according to item 25, wherein: a. The polymer-conjugated lipid is present at about 1 mol% to 2.5 mol% of the total lipids; b. The cationic lipid is present at 35 mol% to 65 mol% of the total lipids; and c. The neutral lipid is present at 35 mol% to 65 mol% of the total lipids. 27. The pharmaceutical composition according to item 25 or 26, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide). 28. The pharmaceutical composition according to any one of items 25 to 27, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-ethylhexyl) dicaprylate. 29. The pharmaceutical composition according to any one of items 25 to 28, wherein the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. 30. The pharmaceutical composition according to any one of items 1 to 29, wherein the average size of the lipid nanoparticles is about 50 to 150 nm. 31. The pharmaceutical composition according to any one of items 1 to 30, which further comprises a cryoprotectant (e.g., sucrose). 32. The pharmaceutical composition according to any one of items 1 to 31, which comprises an aqueous buffer solution. 33. The pharmaceutical composition according to item 32, wherein the aqueous buffer solution contains sodium ions. 34. The pharmaceutical composition according to any one of items 1 to 33, which further comprises a chemotherapeutic agent. 35. The pharmaceutical composition according to item 34, wherein the chemotherapeutic agent is a chemotherapeutic agent suitable for the treatment of pancreatic cancer. 36. The pharmaceutical composition according to any one of items 1 to 35, wherein the at least one single-stranded RNA is present at a concentration of 0.5 mg / mL to 1.5 mg / mL. 37. A method comprising administering to a subject having a CLDN-18.2 positive solid tumor the pharmaceutical composition according to any one of items 1 to 36. 38. The method according to item 37, wherein the CLDN-18.2 positive tumor is a pancreatic tumor. 39. The method according to item 37, wherein the CLDN-18.2 positive tumor is a gastric tumor. The method according to item 37, wherein the CLDN-18.2 positive tumor is a biliary tract tumor. The method according to any one of items 37 to 40, wherein the CLDN-18.2 positive solid tumor is locally advanced, unresectable or metastatic. The method according to any one of items 37 to 41, wherein the subject has received pre-treatment sufficient to increase the level of CLDN-18.2 such that the solid tumor suffered by the subject is characterized as a CLDN-18.2 positive solid tumor. The method according to any one of items 37 to 42, wherein the CLDN-18.2 positive tumor is characterized in that ≥50% of the tumor cells show a CLDN-18.2 protein staining intensity of ≥2+, as evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tumor tissue from the subject. The method according to any one of items 37 to 43, wherein the pharmaceutical composition is administered as a single therapy. The method according to any one of items 37 to 44, wherein the pharmaceutical composition is administered as part of a combination therapy comprising the pharmaceutical composition and a chemotherapeutic agent. The method according to any one of items 37 to 45, wherein the subject has received the chemotherapeutic agent. The method according to item 45, further comprising administering the chemotherapeutic agent to the subject such that the subject receives the combination therapy. The method according to item 47, wherein the chemotherapeutic agent is administered at least four hours after administering the pharmaceutical composition. The method according to any one of items 45 to 48, wherein for a subject with a CLDN-18.2 positive pancreatic tumor, the chemotherapeutic agent is gemcitabine and / or paclitaxel (e.g., nab-paclitaxel) or comprises gemcitabine and / or paclitaxel (e.g., nab-paclitaxel). The method according to any one of items 45 to 48, wherein for a subject with a CLDN-18.2-positive pancreatic tumor, the chemotherapeutic agent is FOLFIRINOX or comprises FOLFIRINOX. The method according to any one of items 45 to 48, wherein for a subject with a CLDN-18.2 positive biliary tract cancer, the chemotherapeutic agent is gemcitabine and / or cisplatin or comprises gemcitabine and / or cisplatin. The method according to any one of items 37 to 51, wherein the subject is an adult subject. 53. The method according to any one of items 37 to 52, wherein the administration is by intravenous injection. 54. The method according to any one of items 37 to 53, wherein the pharmaceutical composition is administered in at least one, at least two, at least three or more dosing cycles. 55. The method according to item 54, wherein the pharmaceutical composition is administered as one or more doses per dosing cycle. 56. The method according to item 55, wherein each dosing cycle is a three-week dosing cycle. 57. The method according to item 55 or 56, wherein the one or more doses comprise the at least one single-stranded RNA in the range of 0.1 mg / kg to 5 mg / kg of the body weight of the subject. 58. In a method of delivering a CLDN-18.2 targeting antibody to a subject for cancer treatment, the improvement comprises administering to the subject the pharmaceutical composition according to any one of items 1 to 36. 59. A method of producing a CLDN-18.2 targeting antibody, which comprises administering to a cell the pharmaceutical composition according to any one of items 1 to 35, such that the cell expresses and secretes the CLDN-18.2 targeting antibody encoded by the at least one single-stranded RNA in the pharmaceutical composition. 60. The method according to item 59, wherein the cell is a hepatocyte. 61. The method according to item 59 or 60, wherein the cell is in a subject. 62. The method according to item 61, wherein the CLDN-18.2 targeting antibody is produced at a therapeutically relevant plasma concentration. 63. The method according to item 62, wherein the therapeutically relevant plasma concentration is sufficient to mediate cancer cell death by antibody-dependent cell cytotoxicity (ADCC). 64. The method according to item 63, wherein the therapeutically relevant plasma concentration is 0.3 to 28 μg / mL. 65. A method comprising the following steps: Determining one or more characteristics of an antibody agent expressed by at least one mRNA introduced into a cell, wherein the at least one mRNA comprises one or more characteristics of at least one or more single-stranded RNAs, the single-stranded RNA comprising a coding region encoding an antibody agent that preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide relative to a claudin-18.1 polypeptide, wherein the one or more characteristics comprise: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to CLDN-18.2; (iii) the potency of the antibody agent to mediate target cell death by ADCC; and (iv) the potency of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC). 66. A method of characterizing a pharmaceutical composition targeting CLDN-18.2, the method comprising the steps of: contacting a cell with at least one pharmaceutical composition according to any one of items 1 to 35; and detecting an antibody agent produced by the cell. 67. The method according to item 66, further comprising determining one or more characteristics of the antibody agent, wherein the one or more characteristics comprise: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to a CLDN-18.2 polypeptide; (iii) the potency of the antibody agent to mediate target cell death by ADCC; and (iv) the potency of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC). 68. The method according to any one of items 65 to 67, wherein the cell is a hepatocyte. 69. The method according to item 65 or 67, wherein the determining step comprises comparing one or more characteristics of the antibody agent with the characteristics of a reference CLDN-18.2 targeting antibody. 70. The method according to any one of items 65 and 67 to 69, wherein the determining step comprises assessing that the protein expression level of the antibody agent is higher than a threshold level. 71. The method according to item 70, wherein the threshold level is a level sufficient to induce ADCC. 72. The method according to any one of items 65 and 67 to 71, wherein the determining step comprises assessing the binding of the antibody agent to a CLDN-18.2 polypeptide. 73. The method according to item 72, wherein the assessing comprises determining the binding of the antibody agent to a CLDN-18.2 polypeptide relative to the binding of the antibody agent to a CLDN18.1 polypeptide. 74. The method according to item 72 or 73, wherein the evaluation includes determining that the binding preference profile of the antibody agent is at least comparable to the binding preference profile of a reference CLDN-18.2 targeting antibody. 75. The method according to item 69 or 74, wherein the reference CLDN-18.2 targeting antibody is zolbetuximab or claudiximab. 76. The method according to any one of items 65 to 75, further characterizing the antibody agent as a CLDN-18.2 targeting antibody agent if the antibody agent comprises the following characteristics: a. The protein level of the antibody agent expressed by the cell is higher than the threshold level sufficient to induce ADCC; b. The antibody agent binds to CLDN-18.2 preferentially relative to CLDN18.1; and c. Mediate killing of at least 50% of the target cells through ADCC and / or CDC. 77. The method according to item 76, further characterizing the antibody agent as a zolbetuximab or claudiximab equivalent antibody if the characteristics of the antibody are at least comparable to the characteristics of zolbetuximab or claudiximab. 78. The method according to any one of items 65 to 77, wherein the target cell is a cancer cell. 79. The method according to items 65 and 66 to 78, wherein the determining step includes determining whether the cell expresses an anti-CLDN18-2 antibody agent encoded by the at least one single-stranded RNA when evaluated after 48 hours of contact. 80. The method according to items 65 and 66 to 79, wherein the determining step includes determining one or more of the following characteristics: - Whether the antibody agent expressed by the cell binds to CLDN-18.2 polypeptide preferentially relative to CLDN18.1 polypeptide; - Whether the antibody agent expressed by the cell exhibits comparable target specificity for CLDN-18.2 as observed in the case of a reference CLDN-18.2 targeting monoclonal antibody in a flow cytometry binding assay; - Whether, when evaluated after incubating immune effector cells (e.g., PBMC cells) with CLDN-18.2 positive cells or CLDN-18.2 negative control cells in the presence of the antibody agent for 48 hours, it is the CLDN-18.2 positive cells rather than the control cells that are lysed; - Whether the antibody agent expressed by the cell exhibits an ADCC profile targeting CLDN-18.2 positive cells that is at least comparable to that observed in the case of a reference CLDN-18.2 targeting monoclonal antibody at the same concentration; and - When evaluated after incubating CLDN-18.2 positive cells or CLDN-18.2 negative control cells with human serum in the presence of the antibody agent for 2 hours, whether it is the CLDN-18.2 positive cells rather than the control cells that are lysed. 81. The method according to any one of items 66 to 80, wherein the cells are present in a subject (e.g., a mouse or monkey subject). 82. The method according to item 81, wherein the one or more characteristics include the antibody level in one or more tissues of the subject. 83. The method according to any one of items 66 to 82, further comprising: If the pharmaceutical composition is characterized as CLDN-18.2 targeting, administering the pharmaceutical composition to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor to determine anti-tumor activity. 84. A manufacturing method, the method comprising the steps of: (A) Determining one or more characteristics of a single-stranded RNA (ssRNA) or a composition thereof, the ssRNA encoding part or all of an antibody agent, the one or more characteristics selected from: (i) The length and / or sequence of the ssRNA; (ii) The integrity of the ssRNA; (iii) The presence and / or position of one or more chemical moieties of the ssRNA; (iv) When the ssRNA is introduced into a cell, the degree of expression of the antibody agent; (v) The stability of the ssRNA or its composition; (vi) The antibody agent level in a biological sample from an organism into which the ssRNA has been introduced; (vii) The binding specificity of the antibody agent expressed by the ssRNA, optionally binding to CLDN-18.2 and optionally relative to CLDN18.1; (viii) The potency of the antibody agent to mediate target cell death by ADCC; (ix) The potency of the antibody agent to mediate target cell death by complement-dependent cytotoxicity (CDC); (x) The identity and amount / concentration of lipids within the composition; (xi) The size of lipid nanoparticles within the composition; (xii) The polydispersity of lipid nanoparticles within the composition; (xiii) The amount / concentration of ssRNA within the composition; (xiv) The degree of encapsulation of the ssRNA within the lipid nanoparticles; and (xv) Their combination; (B) Comparing one or more characteristics of the ssRNA or its composition with the characteristics of a suitable reference standard; and (C) (i) If the comparison indicates that the ssRNA or its composition meets or exceeds the reference standard, one or more additional steps for designating the ssRNA or its composition for manufacture and / or distribution; or (ii) If the comparison indicates that the ssRNA or its composition does not meet or does not exceed the reference standard, taking alternative actions. 85. The method according to item 84, wherein the ssRNA is evaluated and one or more of the additional steps described in step (C)(i) is or includes formulating at least the ssRNA. 86. The method according to item 84 or 85, wherein the composition is evaluated and the composition comprises lipid nanoparticles, and one or more of the additional steps described in step (C)(i) is or includes releasing and distributing the composition. 87. The method according to item 85, further comprising administering the formulation to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor to determine anti-tumor activity. 88. A method for determining a dosing regimen of a drug composition targeting CLDN-18.2, the method comprising the steps of: (A) Administering the drug composition according to any one of items 1 to 35 to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor under a pre-determined dosing regimen; (B) Regularly measuring the tumor size of the animal subjects; (C) (i) If the reduction in tumor size after administering the drug composition is not treatment-related, increasing the dose and / or dosing frequency; or (ii) If the reduction in tumor size after administering the drug composition is treatment-related and shows toxic effects in at least 30% of the animal subjects, reducing the dose and / or dosing frequency; or (iii) If the reduction in tumor size after administering the drug composition is treatment-related and no toxic effects are shown in the animal subjects, making no changes to the dosing regimen. The present disclosure also provides the insight that the 3'-terminal region of mRNA is a very sensitive and special region in terms of translation ability and the function of mRNA. Both in vitro and in vivo results indicate that single nucleotide substitutions upstream of the poly(A) tail affect the translation ability and function of mRNA. Accordingly, the present disclosure also particularly provides a composition or pharmaceutical preparation comprising an RNA, wherein the RNA comprises: (i) a coding sequence encoding a polypeptide, (ii) a 3'UTR sequence, (iii) a poly-A sequence, and (iv) a nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, which comprises the sequence CUXGAGCUAGC, wherein X is C, A or U. In some embodiments, the nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence comprises the sequence CUCGAGCUAGC. In some embodiments, the RNA comprises, in the 5'→3' direction, the coding sequence encoding the polypeptide, the 3'UTR sequence, the nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, and the poly-A sequence. In some embodiments, the 3'UTR sequence comprises the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:22. In some embodiments, the RNA comprises a 3'UTR, which comprises the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36. In some embodiments, the RNA comprises a 3'UTR, which comprises the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37. In some embodiments, the poly-A sequence is an interrupted sequence of A nucleotides. In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises the nucleotide sequence A x -L-A y or consists of the nucleotide sequence A x -L-Ay comprises, wherein A x is a sequence of at least 20 A nucleotides, A y is a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides that may contain nucleotides other than A. In some embodiments, the poly-A sequence comprises the nucleotide sequence of SEQ ID NO:23 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:23, or consists of the nucleotide sequence of SEQ ID NO:23 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:23. In some embodiments, the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20. In some embodiments, the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4AACUAGU, wherein X1 is any nucleotide, preferably A or C, X2 is any nucleotide, preferably A or C, X3 is any nucleotide, preferably C, U or G, and X4 is A or is absent. In some embodiments, the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, wherein X1 is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U. In some embodiments, the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU. In some embodiments, the RNA comprises a 5' UTR that comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU. In some embodiments, the RNA comprises a 5' UTR that comprises the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20. In some embodiments, the RNA comprises a 5' UTR that comprises the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20. In some embodiments, the RNA comprises a 5' UTR that comprises the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:38. In some embodiments, the RNA comprises the following: a 5' UTR that comprises the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide that comprises the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5' UTR that comprises the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide that comprises the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, comprising the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:36. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence encoding the polypeptide, comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence of the polypeptide, comprising the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:36. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, comprising the nucleotide sequence of SEQ ID NO:36. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of positions 1 to 298 of SEQ ID NO:36; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of SEQ ID NO:36. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of positions 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of positions 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:37. In some embodiments, the RNA comprises the following: a 5’UTR that comprises the nucleotide sequence of positions 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, that comprises the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, which comprises the nucleotide sequence of positions 1 to 295 of SEQ ID NO:37; and a poly-A sequence. In some embodiments, the RNA comprises the following: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, which comprises the nucleotide sequence of SEQ ID NO:37. In some embodiments, at least 90% is at least 95%, 96%, 97%, 98% or 99%. In some embodiments, the RNA comprises two or more coding sequences encoding two or more polypeptides. In some embodiments, the polypeptide encoded by the coding sequence is an antibody or a polypeptide chain thereof, such as an antibody that binds to CLDN-18.2 or a polypeptide chain thereof. In some embodiments, the antibody that binds to CLDN-18.2 or a polypeptide chain thereof is as described herein. However, the polypeptide encoded by the coding sequence can be any polypeptide, including but not limited to pharmaceutically active polypeptides and peptides, particularly those described herein. In some embodiments, the RNA does not encode a polypeptide that binds to Claudin-6 (CLDN-6) and / or CD3. In some embodiments, the RNA does not encode one or more polypeptide chains of an agent that binds to Claudin-6 (CLDN-6) and / or CD3. In some embodiments, the RNA does not encode a cytokine. In some embodiments, the RNA does not encode IL2 and / or IL7. In some embodiments, the RNA does not encode a polypeptide that binds to HIV. In some embodiments, the RNA does not encode one or more polypeptide chains of an agent that binds to HIV. In some embodiments, the RNA does not encode a polypeptide that binds to Claudin-18.2 (CLDN-18.2). In some embodiments, the RNA does not encode one or more polypeptide chains of an agent that binds to Claudin-18.2 (CLDN-18.2). In some embodiments, the RNA encodes an antibody or an antibody-like molecule. In some embodiments, the RNA comprises at least two, e.g., two RNA molecules, and at least one of the RNA molecules, e.g., all of the RNA molecules, comprises a 5'UTR, 3'UTR, 3'UTR sequence, poly-A sequence, and / or a nucleotide sequence linking the 3'UTR sequence and the poly-A sequence as defined herein. In some embodiments, the RNA contains: (i) an RNA comprising a coding sequence encoding a first polypeptide chain that comprises a heavy chain of an antibody agent, and (ii) an RNA comprising a coding sequence encoding a second polypeptide chain that comprises a light chain of an antibody agent. In some embodiments, the RNA in (i) is a first RNA molecule and the RNA in (ii) is a second RNA molecule. In some embodiments, the antibody agent binds to Claudin-18.2 (CLDN-18.2). In some embodiments, the antibody agent that binds to CLDN-18.2 is as described herein. In some embodiments, the coding sequence encoding the first polypeptide chain that comprises the heavy chain of the antibody agent that binds to CLDN-18.2 and the coding sequence encoding the second polypeptide chain that comprises the light chain of the antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the first polypeptide chain that comprises the heavy chain of the antibody agent that binds to CLDN-18.2 and the second polypeptide chain that comprises the light chain of the antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces uridine. In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces every uridine. In some embodiments, the modified nucleoside is pseudouridine (ψ) and / or N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap. In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap m 2 7，3’-O Gpp(m 1 2’-O )ApG. In some embodiments, the RNA, e.g., each RNA, is a single-stranded RNA. In some embodiments, the RNA, such as each RNA, is mRNA. In some embodiments, the RNA, such as each RNA, is formulated in a lipid nanoparticle (LNP), such as each RNA is co-formulated in a lipid nanoparticle (LNP). In some embodiments, the lipids forming the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid. In some embodiments, a. the cationic lipid is present at 35 mol% to 65 mol% of the total lipids; b. the polymer-conjugated lipid is present at about 1 mol% to 2.5 mol% of the total lipids; and c. the neutral lipid is present at 35 mol% to 65 mol% of the total lipids. In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate). In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (such as 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide). In some embodiments, the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. In some embodiments, the average size of the lipid nanoparticles is about 50 to 150 nm. In some embodiments, the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate), 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients. In some embodiments, the pharmaceutical formulation is a kit. In some embodiments, the RNA, such as each RNA, and optionally the particle-forming components are in separate vials. In some embodiments, the composition or pharmaceutical formulation is for intravenous administration. In some embodiments, the composition or pharmaceutical formulation is for introducing the RNA into hepatocytes and expressing a polypeptide encoded by the RNA in the hepatocytes. In some embodiments, the composition or pharmaceutical formulation is for systemic delivery of the polypeptide. In some embodiments, the composition or pharmaceutical formulation is for systemic delivery of the polypeptide after expression of the polypeptide in hepatocytes. The present disclosure also particularly provides a method for expressing a polypeptide in a subject, the method comprising: (a) administering a composition as described herein such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes. The present disclosure also particularly provides a method for expressing a polypeptide in a subject, the method comprising: (a) administering a composition as described herein such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes, wherein, after expression, the polypeptide is secreted into the bloodstream. The present disclosure also particularly provides a method for systemic delivery of a polypeptide in a subject, the method comprising: (a) administering a composition as described herein such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes, wherein, after expression, the polypeptide is secreted into the bloodstream. In some embodiments, the administration is parenteral administration. In some embodiments, the administration is intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1Shows the expression of the CLDN-18.2 targeting antibody (RiboMab01) encoded by two RNAs encoding the heavy and light chains of the CLDN-18.2 targeting antibody, respectively, in primary human hepatocytes and CHO-K1 cells. (Figure A) Primary human hepatocytes were lipofected with a composition containing two or more RNAs encoding the heavy and light chains of the CLDN-18.2 targeting antibody (RB_RMAB01) at 0.22 to 55.50 μg / mL. (Left) ELISA analysis of RiboMab01 concentration 48 hours after transfection. (Right) Western blot analysis of the cell culture supernatant from the indicated lipofection. Recombinant purified IMAB362 was used as a reference for Western blot analysis. The analysis was performed under non-reducing conditions and with an HRP-conjugated anti-human antibody. A mixture of Fcγ fragment-specific and anti-κ light chain-specific antibodies was used to detect full-length IgG, free heavy chain (HC), and free light chain (LC). The supernatant of untransfected primary human hepatocytes was used as a mock control. (Figure B) CHO-K1 cells were lipofected with 2.00 to 182.00 ng / mL RB_RMAB01. The RiboMab01 concentration measured by ELISA 48 hours after transfection is shown. Error bars are the standard error of the mean (n = 3).

[0062] Figure 2 Shows the specific target binding of RiboMab01 to CLDN-18.2. The targeted binding of RiboMab01 to CLDN-18.2 was determined by a flow cytometry binding assay, which was visualized using a fluorescently labeled antibody against the F(ab')2 fragment of human IgG (H+L). The diluted rows of CHO-K1 cell culture supernatant containing RiboMab01 (Figure A and B, left) or IMAB362 reference protein (Figure A and B, right) were incubated with 5×10 5 (Figure A) CLDN-18.2+ or (Figure B) CLDN18.1+ HEK293 transfectants.

[0063] Figure 3Shows high target-specific cell cytotoxicity mediated by in vitro-expressed RiboMab01. Cell culture supernatants containing RiboMab01 from CHO-K1 cells transfected with RB_RMAB01 liposomes were subjected to (Figure A) ADCC and (Figure B) CDC assays. (Figure A) For the ADCC assay, CLDN-18.2+ NUG-C4 transfectants were used as target cells, and CLDN-18.2-negative MDA-MB-231 cells were used as control cells. Human PBMCs from three different healthy donors were used as effector cells (E:T ratio 30:1). Target or control cells and effector cells were incubated for 48 hours with the indicated RiboMab01 and IMAB362 reference protein concentrations. Specific cell lysis as determined in a luciferase-based assay is shown. (Figure B) For the CDC assay, CLDN-18.2+ CHO-K1 transfectants (solid line) were used as target cells, and CLDN-18.2-negative CHO-K1 (dashed line) were used as control cells. Target and control cells were incubated for 2 hours with the indicated concentrations of human serum and RiboMab01. CDC as determined in a luciferase-based assay is shown. Error bars are standard error of the mean (n = 3).

[0064] Figure 4 Shows specific tumor cell lysis mediated by RiboMab01 generated in mice. Plasma was sampled 24 hours after the 5th injection from mice administered 5 repeated injections of 1 μg (≈0.04 mg / kg), 3 μg (≈0.10 mg / kg), 10 μg (≈0.40 mg / kg), and 30 μg (≈1.20 mg / kg) RB_RMAB01 or 80 μg (≈3.20 mg / kg) IMAB362 and used for in vitro ADCC assays based on luciferase. Plasma from untreated mice spiked with IMAB362 was used as assay reference. CLDN-18.2+ NUG-C4 transfectants were used as targets, and human PBMCs were used as effector cells. (Figure A) Shows ADCC of NUG-C4 cells mediated by RiboMab01 after incubation with 1% plasma for 48 hours. (Figure B) There was no non-specific lysis of target-negative MDA-MB-231 cells. Error bars are standard error of the mean (n = 3).

[0065] Figure 5RiboMab01 expressed by non-human primates mediates dose-dependent ADCC. Non-Human Primates (NHPs) received three repeated doses of 0.1, 0.4, or 1.6 mg / kg RB_RMAB01 once a week. Serum containing RiboMab01 from all monkeys sampled 24 hours (black bars) and 168 hours (white bars) after the first injection was used for luciferase-based ex vivo ADCC assays. CLDN-18.2+NUG-C4 transfectants were used as target cells. Human PBMCs from two different healthy donors (24 hours, donor 1; 168 hours, donor 2) were used as effector cells. (Figure A) shows ADCC of NUG-C4 cells mediated by RiboMab01 after 48 hours of incubation. (Figure B) shows non-specific lysis of target-negative MDA-MB-231 cells. Error bars are standard errors of the mean (n = 3). (Figure C) Serum from NHP No. 14 (1.6 mg / kg RB_RMAB01, RiboMab01 serum concentration 232 μg / mL) collected 48 hours after the third dose was used for luciferase-based ex vivo ADCC assays. CLDN-18.2+NUG-C4 transfectants (solid line) were used as target cells, and CLDN-18.2-negative MDA-MB-231 cells (dashed line) were used as control cells. Human PBMCs from a healthy donor were used as effector cells. ADCC of NUG-C4 cells mediated by serum containing RiboMab01 (red solid line) or recombinant IMAB362 reference protein (black solid line) is shown, with EC50 values of 66 pM and 151 pM, respectively. Red and black dashed lines represent weak non-specific lysis of MDA-MB-231 control cells. The incubation time was 48 hours. Error bars are standard errors of the mean (n = 3).

[0066] Figure 6 Systemic availability of RiboMab01 mediates tumor growth inhibition in vivo. Mice bearing subcutaneous CLDN-18.2+NCI-N87 xenograft tumors were given IV injections of 1 μg (≈0.04 mg / kg), 3 μg (≈0.10 mg / kg), 10 μg (≈0.40 mg / kg), and 30 μg (≈1.20 mg / kg) RB_RMAB01, 800 μg (≈32 mg / kg) IMAB362 reference protein, 30 μg (≈1.20 mg / kg) luciferase mRNA, or saline only on days 15, 22, 29, 36, 43, and 50 after tumor cell inoculation. Median tumor growth of the treatment and control groups is shown. Dashed lines indicate injections. Significance was calculated by two-way ANOVA. ns indicates not significant.

[0067] Figure 7Shows the concentration-time curve of RiboMab01 in mouse serum after a single administration. Balb / cJRj mice received a single IV injection of 1 μg (about 0.040 mg / kg), 3 μg (about 0.10 mg / kg), 10 μg (about 0.40 mg / kg), or 30 μg (about 1.20 mg / kg) of the RB_RMAB01 drug product and 40 μg (about 1.60 mg / kg) of the IMAB362 reference protein. Plasma was sampled at 6, 24, 96, 168, 264, 336, and 504 hours after administration. The concentration of RiboMab01 in plasma measured by ELISA is shown. Error bars are the standard error of the mean (n = 3).

[0068] Figure 8 Shows the concentration-time curve of RiboMab01 in rat serum after a single administration. RjHan:Wister rats received a single IV injection of 0.04, 0.10, 0.40, or 1.20 mg / kg of RB_RMAB01 and 3.60 mg / kg of the IMAB362 reference protein. Plasma was sampled at 2, 6, 8, 10, 22, 24, 27, 30, 48, 72, 96, 168, 216, 264, and 336 hours after administration. The concentration of RiboMab01 in plasma measured by ELISA is shown. Error bars are the standard error of the mean (n = 3).

[0069] Figure 9 Shows the kinetics of RB_RMAB1 expression in mice after weekly injections. On test days 1, 8, 15, 21, and 29, Balb / cJRj mice received an IV injection of 1 μg (about 0.04 mg / kg), 3 μg (about 0.10 mg / kg), 10 μg (about 0.40 mg / kg), and 30 μg (about 1.20 mg / kg) of RB_RMAB01 and 80 μg (about 3.2 mg / kg) of the IMAB362 reference protein. Plasma was sampled 24 hours before and 24 hours after dosing. The concentration of RiboMab01 in plasma measured by ELISA is shown. The dashed line indicates the injection. Error bars are the standard error of the mean (n = 3).

[0070] Figure 10Shows the kinetics of RB_RMAB01 expression after repeated dosing in NHP. NHP received IV injections of 0.1, 0.4, or 1.6 mg / kg RB_RMAB01 on test days 1, 8, and 15. Plasma was sampled at 6, 24, 48, 72, 96, and 168 hours after the 1st and 3rd doses, at 48, 72, and 168 hours after the 2nd dose, and at 264, 336, and 504 hours after the 3rd dose. The concentration of RiboMab01 in plasma measured by ELISA is shown. Error bars are standard error of the mean (n = 3).

[0071] Figure 11 Shows the liver targeting of LNP-formulated mRNA in vivo. Mice received a single IV injection of LNP-formulated firefly luciferase mRNA. Bioluminescence was monitored at 6, 24, 48, 72, and 144 hours after administration. (Figure A) Shows the bioluminescence images of (left) individual mice in the ventral position (n = 5) and (right) individual organs of mice #1 and 2 at 6 hours after administration. (Figure B) Shows the quantification of luciferase signal (photons / second) at all analyzed time points (n = 5 or 3, mean). LN represents lymph node.

[0072] Figure 12 Shows some exemplary embodiments of RNA technologies and their applications that can be used to encode various antibody agent forms (“RiboMab”) and their formulations. (Figure A) The platform is suitable for providing RNA constructs encoding various antibody forms, which include, for example but not limited to, monospecific antibody IgG, bispecific antibody bi-(scFv) 2 and bispecific antibody Fab-(svFv) 2. (Figure B) In some embodiments, a therapeutic antibody such as IgG can be encoded by a purified mRNA comprising a modified ribonucleotide (e.g., uridine substituted with pseudouridine) mRNA and encapsulated in a lipid nanoparticle (mRNA / LNP). Such an mRNA construct can also include one or more non-coding sequence elements (e.g., to enhance RNA stability and / or translation efficiency). In some embodiments, exemplary non-coding sequence elements include, but are not limited to, a cap structure, 5'UTR, 3'UTR, polyadenylate tail, and any combination thereof. In some embodiments, the lipid nanoparticle can include a conjugated lipid (e.g., PEG-conjugated lipid), a cationic lipid, and a neutral helper lipid. Such an mRNA / LNP drug product formulation can be administered in vivo to an object such that the mRNA is translated in vivo to express the antibody. (Figure C) Somatic cells of a patient to whom the mRNA / LNP drug product formulation described herein is administered are capable of producing the active drug (e.g., IgG RiboMab) encoded by the mRNA. For example, in some embodiments, after IV injection, the mRNA / LNP encoding the antibody is internalized and translated by hepatocytes, resulting in a systemic plasma concentration of bioactive RiboMab. Abbreviations: A30L70, Poly(A) tail, measuring 100 adenosines eliminated by the linker at position 30; bi, bispecific; C, C-terminal; CDS, coding sequence; CH, constant heavy chain domain; CL, constant light chain domain; Fab, antigen-binding fragment; IgG, immunoglobulin G; LNP, lipid nanoparticle; m1Ψ, 1-methylpseudouridine; N, N-terminal; scFv, single-chain variable fragment; TAA, tumor-associated antigen; UTR, untranslated region; VH, variable heavy chain domain; VL, variable light chain domain.

[0073] Figure 13 are schematic diagrams of exemplary RNA constructs encoding the heavy chain (HC) and light chain (LC) of an antibody agent. As Figure 13 shown, such RNA constructs encoding HC and LC form an RNA composition (RB_RMAB01), which in some embodiments can be formulated into a lipid nanoparticle to form an RNA / LNP drug product formulation. Abbreviations: Poly A, polyadenine tail; CH, constant heavy chain domain; CL, constant light chain domain; Sec, secretion signal; UTR, untranslated region; VH, variable heavy chain domain; VL, variable light chain domain.

[0074] Figure 14 is a graph showing the dose-exposure correlation of RB_RMAB01 in cynomolgus monkeys at t max hours. Cynomolgus monkeys (n = 3) received IV injection of 0.1, 0.4, or 1.6 mg / kg RB_RMAB01. Depicted at C maxPlasma dose-dependent RiboMab01 concentration measured by ELISA here (mean, n = 3). The green line indicates the dose that can be administered to human subjects and its corresponding expected serum concentration.

[0075] Figure 15 Is an exemplary electropherogram of an exemplary RNA mixture that contains a first RNA encoding an antibody heavy chain (HC) and a second RNA encoding an antibody light chain (LC). The electropherogram shows two peaks for LC and HC respectively. A: Area under the peak, h: Peak height.

[0076] Figure 16 Shows the in vitro expression of anti-CLDN18.2 RiboMab. HEK293T / 17 cells were electroporated with mRNA encoding anti-CLDN18.2 RiboMab with the same backbone but different coding sequences. The anti-CLDN18.2 RiboMab concentration was measured by ELISA 48 hours after transfection. Error bars are the standard error of the mean (n = 2).

[0077] Figure 17 Shows anti-CLDN18.2 RiboMab exposure in mice after repeated RNA-LNP administration. Balb / cJRj mice received IV injections of 3 or 30 μg RNA-LNP twice a week, each RNA-LNP containing mRNA encoding the HC and LC of anti-CLDN18.2 RiboMab in the case of backbone A or backbone B respectively. Serum was sampled at the indicated time points after the first administration. The arithmetic mean (n = 3) and standard error of the anti-CLDN18.2 RiboMab concentration in serum measured by ELISA are shown. The limit of detection was 0.074 ng / mL. The downward arrows correspond to the first and second RNA-LNP injections. Luc-RNA-LNP was used as a negative control. ELISA = Enzyme-linked immunosorbent assay; Luc = Luciferase.

[0078] Figure 18Shows the cytotoxic activity of an anti-CLDN18.2 RiboMab encoded by RNA utilizing scaffolds A and B. Shown is ex vivo ADCC mediated by anti-CLDN18.2 RiboMab in mouse sera collected 24 hours after administration of RNA-LNP at the indicated doses. CLDN18.2-transduced NUGC-4 transfectants were used as target cells at an E:T ratio of 20:1 and human PBMCs from healthy donors were used as effector cells (upper panel). CLDN18.2-negative MDA-MB-231 was used as a negative control (lower panel). Target cells and effector cells were incubated for 24 hours. Results for control antibodies are shown in the right panel. Data are mean ± SD of three measurements per mouse. Ab = antibody; ADCC = antibody-dependent cytotoxicity; E:T ratio = effector cell to target cell ratio; M1 = mouse 1; PBMCs = peripheral blood mononuclear cells.

[0079] Figure 19 Shows that EPO mRNA transcribed from scaffold C in vivo is superior to EPO mRNA transcribed from scaffold A, but inferior to EPO mRNA derived from the scaffold B / scaffold D cassette.

[0080] Figure 20 Shows a comparison of the translation of mRNAs derived from scaffolds B, C, and D with different coding sequences and demonstrates that the performance differences between scaffold C and scaffold B / D are independent of the coding sequence. A, Firefly luciferase mRNAs in scaffolds B (●), C (■), and D (▲) were electroporated twice in hiDCs (solid and dashed lines). Bright-Glo assays were performed at the indicated times. B, eGFP mRNAs in scaffolds C (■) and D (▲) were electroporated twice in hiDCs (solid and dashed lines). Cells were harvested and eGFP expression was measured by FACS at the indicated times. C, Primary human hepatocytes were lipofected with hIL-18 mRNA in scaffolds B (●), C (■), and D (▲). Supernatants from transfected cells were collected at the indicated times and the presence of hIL-18 was measured by ELISA.

[0081] Figure 21 Shows the translation of firefly luciferase mRNAs derived from scaffolds B and C, which contain different nucleotides at position 9 upstream of polyA in hiDCs, and demonstrates that the 3’ UTR terminal sequence affects long-term translation in vitro. Firefly luciferase mRNAs from scaffolds B (Figure A) and C (Figure B) having A (◆), G (■), T (▲), or C (●) at position 9 upstream of the polyA sequence were electroporated in hiDCs in two separate experiments and luciferase expression was measured at the indicated time points.

[0082] Figure 22It is shown that the 3'UTR terminal sequence significantly affects long-term in vivo translation using scaffold B.

[0083] Figure 23 It is shown that the 3'UTR terminal sequence significantly affects long-term in vivo translation using scaffold D.

[0084] Figure 24 It is shown that the 5' sequence located upstream of the coding sequence also affects long-term in vivo translation.

[0085] Figure 25 It is shown that the combination of 5' sequence elements and 3' sequence elements affects long-term in vivo translation.

[0086] Figure 26 It is shown that for long-term in vivo translation, scaffold B performs significantly better than earlier scaffold versions (scaffolds A and G).

[0087] Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing some specific embodiments only and are not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0088] Hereinafter, the elements of the present disclosure will be described in more detail. These elements are listed together with some specific embodiments. However, it should be understood that they can be combined in any way and in any number to produce additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to the specifically described embodiments. This specification should be understood to support and cover embodiments that combine the specifically described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context otherwise requires, any arrangement and combination of all the elements described in this application should be considered to be disclosed by the specification of this application.

[0089] The use of any and all examples or exemplary language (e.g., "such as") provided herein is only intended to better illustrate the present disclosure and does not constitute a limitation on the scope of the present disclosure that is otherwise claimed. No language in this specification should be construed as indicating any unclaimed element necessary for the practice of the present disclosure.

[0090] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0091] Throughout the body of this specification, several documents are cited. Whether above or below, each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, guidelines, etc.) is hereby incorporated by reference in its entirety. Nothing in this text should be construed as an admission that the present disclosure is not entitled to antedate such disclosure. Certain Definitions

[0092] Indefinite Quantity Terms: Unless otherwise specified herein or clearly contradicted by context, indefinite quantity terms as used herein shall be construed to cover both singular and plural.

[0093] About or Approximately: As used herein, the term "about" or "approximately" when applied to one or more target values refers to a value similar to the reference value. Generally, those of ordinary skill in the art familiar with the context will understand the degree of relevant variation covered by "about" or "approximately" in the context. For example, in some embodiments, the term "about" or "approximately" may cover within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0094] Administration: As used herein, the term "administration" and variations thereof generally refer to the administration of a composition to a subject to effect delivery of an agent that is the composition or that is included in the composition to a target site or site to be treated. One of ordinary skill in the art will recognize the various routes that may be used to administer to a subject, such as a human, where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, percutaneous (which may be or include, for example, one or more of surface for dermal, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., intrahepatic), transmucosal, transnasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), transvaginal, transvitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the administration of a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses spaced in time) and / or periodic (e.g., separate doses separated by a common time period) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion) for at least a selected period of time.

[0095] And / or: As used herein, "and / or" is considered to be a specific disclosure of each of the two designated features or components with or without the other. For example, "X and / or Y" is considered to be a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were listed separately herein.

[0096] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may include one or more constant region sequences that are characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, as is known in the art, the antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or forms known or developed in the art for exploiting antibody structural and functional features in alternative presentations. For example, in some embodiments, the antibody agent used in accordance with the present disclosure is a form selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or groups thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g., ); small modular immunopharmaceuticals ("SMIPsTM"); single-chain or tandem diabodies VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; MicroProteins; and In some embodiments, the term "antibody" or "antibody agent" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. In some embodiments, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). In some embodiments, each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of VL are called LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into constant domains CH1, the hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxyl terminus: CH1, CH2, CH3). The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q. When used in the context of an antibody, the term "full-length" means that the antibody is not a fragment but contains all the domains of a particular isotype that are typically found in nature for that isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. As used herein, the term "Fab arm" or "arm" refers to a heavy chain-light chain pair and is used interchangeably herein with "half-molecule". In some embodiments, an antibody can lack covalent modifications (e.g., attachment of glycans) that it would have if produced naturally. In some embodiments, an antibody can comprise covalent modifications (e.g., attachment of glycans, payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.] or other side groups [e.g., polyethylene glycol, etc.]). In many embodiments, an antibody agent is a polypeptide or comprises a polypeptide whose amino acid sequence contains one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, an antibody agent is a polypeptide or comprises a polypeptide whose amino acid sequence contains at least one CDR that is substantially the same as a CDR present in a reference antibody (e.g., at least one heavy chain CDR and / or at least one light chain CDR).In some embodiments, the included CDR is substantially the same as the reference CDR because, compared to the reference CDR, its sequence is identical or contains 1 to 5 amino acid substitutions. In some embodiments, the included CDR is substantially the same as the reference CDR because it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because it shows at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because, compared to the reference CDR, at least one amino acid in the included CDR is deleted, added or substituted, but the included CDR has an amino acid sequence otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because, compared to the reference CDR, 1 to 5 amino acids in the included CDR are deleted, added or substituted, but the included CDR has an amino acid sequence otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because, compared to the reference CDR, at least one amino acid in the included CDR is substituted, but the included CDR has an amino acid sequence otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because, compared to the reference CDR, 1 to 5 amino acids in the included CDR are deleted, added or substituted, but the included CDR has an amino acid sequence otherwise identical to that of the reference CDR. In some embodiments, the antibody agent is a polypeptide or comprises a polypeptide, the amino acid sequence of which comprises structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or highly homologous to an immunoglobulin binding domain.

[0097] Antibody agents can be prepared by those skilled in the art using methods known in the art and commercially available services and kits. For example, methods for preparing monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Some other antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (August 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (February 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (September 30, 2013)).

[0098] Antibodies can be produced by standard techniques, such as by immunizing with a suitable polypeptide or a portion thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (such as a mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing the desired epitope (optionally haptenated with another polypeptide). Depending on the host species, a variety of adjuvants can be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunized animal is collected and processed according to known procedures. If the serum containing polyclonal antibodies against the desired epitope also contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antiserum are well known in the art.

[0099] Related to: As used herein, two events or entities are "related" to each other if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, if the presence of a particular biological phenomenon (e.g., expression of CLDN-18.2) is related to the incidence and / or susceptibility or likelihood of response to treatment of a particular disease, disorder, or condition (e.g., cancer) (e.g., in a relevant population), it is considered related to that disease, disorder, and condition.

[0100] Blood-derived sample: As used herein, the term "blood-derived sample" refers to a sample derived from a blood sample (i.e., a whole blood sample) of a subject. Examples of blood-derived samples include, but are not limited to, plasma (which includes, for example, fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood components (including red blood cells (RBCs), platelets, white blood cells, etc.), and cell lysates comprising fractions thereof (e.g., cells such as red blood cells, white blood cells, etc. can be harvested and lysed to obtain a cell lysate). In some embodiments, the blood-derived sample that can be used for the characterization described herein is a plasma sample.

[0101] Cancer: As used herein, the term "cancer" generally refers to a disease or disorder in which the cells of the target tissue exhibit relatively abnormal, uncontrolled, and / or autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. In some embodiments, cancer may comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, cancer may be characterized by solid tumors. In some embodiments, cancer may be characterized by hematological malignancies. Generally, examples of different types of cancers known in the art include, for example, hematopoietic cancers, including leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma, and myeloproliferative disorders; sarcoma; melanoma; adenoma; solid tissue carcinomas; squamous cell carcinomas of the mouth, larynx, pharynx, and lung; liver cancer; genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer; and renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; endocrine system cancers, thyroid cancer; parathyroid cancer; head and neck cancer; ovarian cancer; breast cancer; glioblastoma; colorectal cancer; gastrointestinal cancer, and nervous system cancers; benign lesions such as papilloma, etc.

[0102] Cap: As used herein, the term "cap" refers to a structure that comprises or consists essentially of a nucleoside-5'-triphosphate that is typically linked to the 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'-diphosphate). In some embodiments, the cap is a guanine nucleotide or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, which includes, for example, but is not limited to, a 7-methylguanosine cap having a structure designated as "m7G". In some embodiments, the cap is or comprises a synthetic cap analogue that mimics the RNA cap structure and stabilizes the RNA (If connected thereto), which includes, for example but not limited to, anti-reverse cap analogs (ARCAs) known in the art. Those skilled in the art will understand that methods for attaching a cap to the 5' end of RNA are known in the art. For example, in some embodiments, capped RNA can be obtained by capping RNA with a 5' triphosphate group or RNA with a 5' diphosphate group in vitro using a capping enzyme system, which includes, for example but not limited to, the vaccinia capping enzyme system or the Saccharomyces cerevisiae capping enzyme system. Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a DNA template using methods known in the art, wherein in addition to GTP, the IVT system also contains a dinucleotide cap analog, which includes, for example, the m7GpppG cap analog or the N7-methyl, 2'-O-methyl-GpppG ARCA cap analog or the N7-methyl, 3'-O-methyl-GpppG ARCA cap analog. In some embodiments, the cap is cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. As used herein, the term "cap 0" means the structure "m7GpppN", where N is any nucleoside with an OH moiety at the 2' position. As used herein, the term "cap 1" means the structure "m7GpppNm", where Nm is any nucleoside with an OCH3 moiety at the 2' position. As used herein, the term "cap 2" means the structure "m 7 GpppNmNm", where each Nm is independently any nucleoside with an OCH 3 moiety at the 2' position.

[0103] CLDN-18.2 positive: As used herein, the terms “CLDN-18.2 positive” or “CLDN-18.2+” refer to clinically relevant CLDN-18.2 expression and / or activity, e.g., that may be associated with a particular disease, disorder or condition and / or that may be detected in or on a sample that may be or comprise one or more cell or tissue samples. In some embodiments, CLDN-18.2+ refers to a cancer associated with clinically relevant CLDN-18.2 expression and / or activity. In certain exemplary embodiments, CLDN-18.2 positive expression and / or activity may be de novo CLDN-18.2 overexpression or comprise de novo CLDN-18.2 overexpression, e.g., in cancer cells; alternatively or in addition, in some embodiments, CLDN-18.2 positive expression and / or activity may be associated with or have been associated with exposure to one or more agents or conditions such as one or more chemotherapeutic agents (which include, e.g., gemcitabine and / or cisplatin). In some embodiments, CLDN-18.2 “positive” is evaluated relative to an appropriate reference (e.g., a “negative control” such as the CLDN-18.2 level and / or activity in appropriately comparable non-cancerous cells and / or tissues; a “positive control” such as the CLDN-18.2 level and / or activity as may have been determined for known CLDN-18.2 positive cells and / or tissues; and / or an established threshold of CLDN-18.2 level and / or activity associated with a normal (e.g., healthy, non-cancer) versus an abnormal (e.g., cancer) state). In some embodiments, the term “CLDN-18.2+” is used herein to refer to a tumor sample from a cancer patient when the sample has been determined to show a detectable increase in CLDN-18.2 protein expression relative to an appropriate reference (e.g., the level observed in a sample determined to be or otherwise known to be negative for CLDN-18.2 expression). In some embodiments, a sample is considered to be CLDN-18.2+ when, as evaluated by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) tumor tissue, ≥50% of the tumor cells in the sample have a CLDN-18.2 protein staining intensity of ≥2+; those skilled in the art will recognize that pathologists typically use such a scoring system for interpreting IHC data obtained on tumor samples. See, e.g., Fedchenko and Reifenrath, Diagnostic Pathology (2014) 9:221, which describes different methods for interpreting and reporting the results of IHC analysis (which includes scoring systems). See also Zimmermann et al., Cancer Cytopathology (2014) 48-58. Thus, a pathologist will readily recognize that 2+ refers to a grading score of 2 or higher, indicating that such immunohistochemical assay results are clear.More precisely, 2+ describes moderate or strong staining in a qualitative scale of negative (0), weak (1), moderate (2), strong (3).

[0104] Co - administration: As used herein, the term "co - administration" refers to the use of the pharmaceutical compositions described herein in combination with another treatment (e.g., surgery, radiation, and / or administration of another therapeutic agent, such as a chemotherapeutic agent described herein, and / or an agent that alleviates one or more symptoms or attributes of the related disease, disorder, or condition and / or the treatment being administered [e.g., chemotherapy]) such that the subject receives both. The combined administration of the pharmaceutical compositions described herein and such other treatments can be carried out simultaneously (e.g., by an overlapping regimen) or separately (e.g., sequentially in any order). In some embodiments, the pharmaceutical compositions described herein can comprise two or more active agents combined in a pharmaceutically acceptable carrier (e.g., in a single dosage form). Alternatively, in some embodiments, co - administration involves the administration of two or more physically distinct pharmaceutical compositions, each of which can contain a different combination of active agents or agents; in some such embodiments, one or more (and in some embodiments, all) doses of such different pharmaceutical compositions can be administered substantially simultaneously. In some embodiments, one or more (and in some embodiments, all) doses of such different pharmaceutical compositions can be administered separately, e.g., according to an overlapping regimen or a sequential regimen. Generally, when two or more treatments are delivered or administered close enough in time so that at least some of the biological effects produced by each treatment on the target cells or the subject to which it is administered overlap in time, they are considered to be "co - administered".

[0105] Codon optimization: As used herein, the term "codon optimization" refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism, while preferably not changing the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, the coding region can be codon - optimized for optimal expression in a subject to be treated with the RNA (particularly mRNA) described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies at which tRNAs occur in the cell. Thus, the sequence of the RNA (particularly mRNA) can be modified such that codons for which frequently occurring tRNAs are available are inserted to replace "rare codons".

[0106] Combination Therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of a combination therapy may include administering one or more agents or modalities to a subject who is receiving other agents or modalities in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily at the same time), but in some embodiments, two or more agents or their active moieties may be administered together in a combination composition.

[0107] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, groups of conditions, etc., which may not be identical to one another but are similar enough to permit comparison between them such that one of ordinary skill in the art will understand that conclusions may be reasonably drawn based on the observed differences or similarities. In some embodiments, comparable groups of conditions, environments, individuals, or populations are characterized by a plurality of substantially identical characteristics and one or a small number of varying characteristics. One of ordinary skill in the art will understand that, in context, the degree of identity required for two or more such agents, entities, situations, groups of conditions, etc. to be considered comparable will depend on the circumstances of any given case. For example, one of ordinary skill in the art will understand that groups of environments, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results or phenomena observed under different groups of environments, individuals, or populations or circumstances are caused by or indicative of variations in these varying characteristics.

[0108] Complementary: As used herein, the term "complementary" is used in reference to oligonucleotide hybridization related to base pairing rules. For example, the sequence "C-A-G-T" is complementary to the sequence "G-T-C-A". Complementarity can be partial or complete. Thus, any degree of partial complementarity is intended to be included within the scope of the term "complementary", provided that the partial complementarity permits oligonucleotide hybridization. Partial complementarity is where one or more nucleic acid bases do not match according to the base pairing rules. Full or complete complementarity between nucleic acids is where each and every nucleic acid base matches another base according to the base pairing rules.

[0109] Comprise / include, consist of: The words “comprise / include” and variations thereof will be understood to imply the inclusion of the stated features, elements, members, integers or steps or groups of features, elements, members, integers or steps, but not the exclusion of any other features, elements, members, integers or steps or groups of features, elements, members, integers or steps. The term “consisting essentially of” limits the scope of a claim or the scope of disclosure to specific features, elements, members, integers or steps, and those that do not materially affect the basic and novel features of the claim or disclosure. The term “consisting of” limits the scope of a claim or disclosure to the specified features, elements, members, integers or steps. The term “comprise / include” encompasses the term “consisting essentially of”, and “consisting essentially of” in turn encompasses the term “consisting of”. Thus, each time it appears in this application, the term “comprise / include” may be replaced by the term “consisting essentially of” or “consisting of”. Similarly, each time it appears in this application, the term “consisting essentially of” may be replaced by the term “consisting of”.

[0110] Contact: As used interchangeably herein, the term “deliver” and variations thereof or “contact” means exposing a relevant target (e.g., a cell, tissue, organism, etc.) to an RNA or a composition comprising or delivering it (as described herein) such that the RNA is delivered into the target cell (e.g., the cytosol of the target cell). The target cell can be in in vitro or ex vivo culture or present in a subject (in vivo). Those skilled in the art will understand that different contact methods can be used to achieve such delivery to the target cell in in vitro, ex vivo or in vivo applications. In some embodiments, contacting cells in culture can be or include in vitro transfection. In some embodiments, the contact can utilize one or more delivery carriers (e.g., lipid nanoparticles as described herein). In some embodiments, the contact can be administering to a subject a pharmaceutical composition as described herein or including administering to a subject a pharmaceutical composition as described herein.

[0111] Detection: The term "detection" is used herein broadly to include any suitable means of determining the presence or absence of a target entity in a sample or any form of measurement of the target entity. Thus, "detection" can include determining, measuring, assessing, or assaying the presence or absence, level, quantity, and / or location of the target entity. This includes quantitative and qualitative determinations, measurements, or assessments, which includes semi-quantitative. Such determinations, measurements, or assessments can be relative (e.g., when the target entity is detected relative to a control reference), or can be absolute. Thus, when used in the context of quantifying a target entity, the term "quantify" can refer to absolute or relative quantification. Absolute quantification can be accomplished by correlating the level of the detected target entity to a known control standard (e.g., by generation of a standard curve). Alternatively, relative quantification can be accomplished by comparing the levels or amounts detected between two or more different target entities to provide relative quantification of each of the two or more different target entities (i.e., relative to each other).

[0112] Disease: The term "disease" as used herein refers to a disorder or condition that typically impairs the normal function of a tissue or system in an object (e.g., a human object) and is typically manifested by characteristic signs and / or symptoms. In some embodiments, an exemplary disease is cancer.

[0113] Encode: The term "encode" or variations thereof as used herein refers to the sequence information of a first molecule that directs the production of a second molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process involving a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, if transcription and / or translation of a nucleic acid in a cell or other biological system results in a polypeptide, the nucleic acid encodes the polypeptide.

[0114] Epitope: The term "epitope" as used herein includes any portion specifically recognized by an immunoglobulin (e.g., an antibody or receptor) binding component or an aptamer. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, when the antigen adopts an alternative conformation (e.g., is linearized), at least some of such chemical atoms are physically separated groups.

[0115] Expression: "Expression" of a nucleic acid sequence as used herein refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0116] Fc region: The term "Fc region" as used herein refers to the antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, wherein the Fc sequence comprises at least a hinge region, a CH2 domain, and a CH3 domain.

[0117] 5' untranslated region: The term "5' untranslated region" or "5' UTR" as used herein refers to such a sequence of an mRNA molecule that begins at the transcription start site and ends at a nucleotide (nt) before the start codon (usually AUG) of the RNA coding region.

[0118] Homologous: The term "homologous" or "homologue" as used herein refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to each other if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as being similar to each other as "hydrophobic" or "hydrophilic" amino acids, and / or having "polar" or "nonpolar" side chains. Substituting one amino acid for another of the same type is generally considered to be a "homologous" substitution.

[0119] Identity: As used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. For example, the percent identity between two nucleic acid or polypeptide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. The molecules are identical at a position when the position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence. The percent identity between the two sequences is a function of the number of positions having identical residues, taking into account the number of gaps and the length of each gap (which are introduced for optimal alignment of the two sequences). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989 (which has been incorporated into the ALIGN program (version 2.0)). In some exemplary embodiments, nucleic acid sequence comparison using the ALIGN program uses the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 nucleotides (in some embodiments, contiguous nucleotides). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0120] Immunogenicity: "Immunogenicity" refers to the ability of a foreign substance (such as RNA) to elicit an immune response in a human or other animal. The innate immune system is the relatively non-specific and immediate component of the immune system. Together with the adaptive immune system, it is one of the two main components of the vertebrate immune system.

[0121] Immunoglobulin: As used herein, the term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by structural domains (i.e., immunoglobulin domains) with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also referred to as surface immunoglobulins or membrane immunoglobulins and are typically part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, immunoglobulins generally consist of several chains, usually two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are mainly composed of: immunoglobulin domains or regions such as V L or VL (variable light chain) domains / regions, C L or CL (constant light chain) domains / regions, V H or VH (variable heavy chain) domains / regions and C H or CH (constant heavy chain) domains / regions C H 1 (CH1), C H 2 (CH2), C H 3 (CH3) and C H4(CH4). The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain is typically composed of VL and CL. The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable loops, which can be in the form of sequence-hypervariable and / or structurally defined loops), also known as complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or inconsistent with the context, the CDR sequences herein are identified using DomainGapAlign according to the IMGT rules (Lefranc MP., Nucleic Acids Research 1999; 27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org. However, it should be understood that the present disclosure is not limited to CDR sequences determined only according to the IMGT rules. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which constitute different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. In mammals, there are two types of light chains, namely λ and κ. Immunoglobulin chains contain variable and constant regions. The constant regions are substantially conserved within different isotypes of immunoglobulins, where the variable portions are highly diverse and lead to antigen recognition.

[0122] Isolated: "Isolated" means removed (e.g., purified) from its natural state or from an artificial composition (e.g., a composition from a production process). For example, a nucleic acid or polypeptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or polypeptide can exist in a substantially purified form or can be present in a non-natural environment, such as a host cell.

[0123] Lipid: As used herein, the term "lipid" refers to a molecule that contains one or more hydrophobic moieties or groups and optionally also contains one or more hydrophilic moieties or groups. Molecules that contain both hydrophobic and hydrophilic moieties are also often referred to as amphiphiles. Lipids are generally insoluble or sparingly soluble in water but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. In general, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fat, fat is a subgroup of lipids called triglycerides. Lipids also encompass the following molecules: for example, fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids); and steroids, which are metabolites containing sterols, such as cholesterol or its derivatives. Some examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholestenyl-2'-hydroxyethyl ether, cholestenyl-4'-hydroxybutyl ether, tocopherol, and its derivatives, and mixtures thereof.

[0124] Locally advanced tumor: As used herein, the term "locally advanced tumor" or "locally advanced cancer" refers to its generally recognized meaning in the art, which can vary depending on the type of cancer. For example, in some embodiments, a locally advanced tumor refers to a relatively large tumor that has not spread to another body site. In some embodiments, a locally advanced tumor is used to describe a cancer that has grown outside the tissue or organ where it originated but has not spread to distant sites within the subject's body. By way of example only, in some embodiments, locally advanced pancreatic cancer generally refers to stage III disease with tumor extension to adjacent organs (e.g., lymph nodes, liver, duodenum, superior mesenteric artery, and / or celiac trunk) but without signs of metastatic disease; however, complete surgical resection with negative pathologic margins is not possible.

[0125] Mol%: As used herein, "mol%" is defined as the ratio of the number of moles of a component to the total number of moles of all components multiplied by 100. "Mol% of total lipids" as used in the present disclosure is defined as the ratio of the number of moles of a lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0126] Non-immunogenic RNA: As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to an RNA that does not induce a response of the immune system, or induces a weaker response than the same RNA, when administered (e.g., to a mammal), and that differs from the same RNA only in that the same RNA has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., weaker than that induced by standard RNA (stdRNA).

[0127] Nucleic Acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is DNA or contains DNA. In some embodiments, the nucleic acid is RNA or contains RNA. In some embodiments, the nucleic acid is peptide nucleic acid (PNA) or contains peptide nucleic acid (PNA). In some embodiments, the nucleic acid is a single-stranded nucleic acid or contains a single-stranded nucleic acid. In some embodiments, the nucleic acid is a double-stranded nucleic acid or contains a double-stranded nucleic acid. In some embodiments, the nucleic acid contains both single-stranded and double-stranded portions. In some embodiments, the nucleic acid contains a backbone containing one or more phosphodiester bonds. In some embodiments, the nucleic acid contains a backbone containing both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may contain a backbone containing one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, such as in "peptide nucleic acid". In some embodiments, the nucleic acid contains one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid contains one or more or all non-natural residues. In some embodiments, the non-natural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residues contain one or more modified sugars compared to those in natural residues (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence containing one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from natural sources, enzymatic synthesis (e.g., by polymerization based on complementary templates, e.g., in vivo or in vitro, proliferation in recombinant cells or systems), or chemical synthesis.In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0128] Nucleotide: As used herein, the term "nucleotide" has its recognized meaning in the art. When the number of nucleotides is used as an indication of the size (e.g., of a polynucleotide), the specific number of nucleotides refers to the number of nucleotides on a single strand (e.g., of a polynucleotide).

[0129] Optional: As used herein, the term "optional" or "optionally" means that the subsequent described event, circumstance, or condition may or may not occur, and the description includes the case where the event, circumstance, or condition occurs and the case where it does not occur.

[0130] Patient: As used herein, the term "patient" refers to any organism that has a disease, disorder, or condition or is at risk of a disease, disorder, or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient has or is susceptible to one or more diseases, disorders, or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the patient has been diagnosed with one or more diseases, disorders, or conditions. In some embodiments, the disease, disorder, or condition to which the provided technology is applicable is or includes cancer, or there is one or more tumors. In some embodiments, the patient is undergoing or has undergone a specific treatment for diagnosing and / or treating a disease, disorder, or condition. In some embodiments, the patient is a cancer patient.

[0131] Polypeptide: As used herein, the term "polypeptide" generally has its generally recognized meaning in the art - a polymer of at least three or more amino acids. One of ordinary skill in the art will understand that the term "polypeptide" is intended to be broad enough to cover not only polypeptides having the complete sequences described herein, but also polypeptides representing functional, bioactive or characteristic fragments, portions or domains of such complete polypeptides (e.g., fragments, portions or domains that retain at least one activity). In some embodiments, the polypeptide may comprise L-amino acids, D-amino acids or both and / or may comprise any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, synthetic amino acids and combinations thereof (e.g., may be peptidomimetics or contain peptidomimetics).

[0132] Pharmacologically active polypeptide: As used herein, the term "pharmacologically active polypeptide" means a peptide or polypeptide that can be used to treat an individual in whom the expression of the peptide or polypeptide would be beneficial, e.g., in ameliorating the symptoms of a disease. Preferably, the pharmacologically active peptide or polypeptide has curative or palliative properties and can be administered to improve, relieve, alleviate, reverse a disease, delay the onset of a disease or reduce the severity of one or more symptoms of a disease. In some embodiments, when administered to an individual in a therapeutically effective amount, the pharmacologically active peptide or polypeptide has a positive or beneficial effect on the condition or disease state of the individual. The pharmacologically active peptide or polypeptide may have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmacologically active peptide" or "pharmacologically active polypeptide" includes the complete peptide or polypeptide and may also refer to its pharmacologically active fragments. It may also include pharmacologically active variants and / or analogs of the peptide or polypeptide. Specific examples of pharmacologically active peptides and polypeptides include, but are not limited to, immunostimulants such as cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome engineering proteins and blood proteins. In some embodiments, the pharmacologically active peptides and polypeptides include replacement proteins. An "immunostimulant" is any substance that stimulates the immune system by inducing the activation or enhancing the activity of any component of the immune system, particularly immune effector cells. Immunostimulants can be pro-inflammatory (e.g., when treating infections or cancer) or anti-inflammatory (e.g., when treating autoimmune diseases). According to one aspect, the immunostimulant is a cytokine or a variant thereof. Some examples of cytokines include interferons, such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ); interleukins, such as IL2, IL7, IL12, IL15, and IL23; colony-stimulating factors, such as M-CSF and GM-CSF; and tumor necrosis factor. According to a further aspect, the immunostimulant includes adjuvant-type immunostimulants, such as APC Toll-like receptor agonists or co-stimulatory / cell adhesion membrane proteins. Some examples of Toll-like receptor agonists include co-stimulatory / adhesion proteins, such as CD80, CD86, and ICAM-1. The term "cytokine" refers to a protein having a molecular weight of about 5 to 60 kDa and involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, when released, cytokines affect the behavior of cells surrounding their release site. Some examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that: (i) they generally act at more variable concentrations than hormones, and (ii) they are generally produced by a wide range of cells (virtually all nucleated cells can produce cytokines). Specific examples of cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon α (IFNα), interferon β (IFNβ), interferon γ (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as variants and derivatives thereof. According to the present disclosure, the cytokine can be a naturally occurring cytokine or a functional fragment or variant thereof. The cytokine can be a human cytokine and can be derived from any vertebrate, particularly any mammal. A particularly preferred cytokine is interferon-α. Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and tumor cells. In a typical scenario, virus-infected cells will release interferons, causing nearby cells to enhance their antiviral defenses. Interferons are generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that prevent intracellular virus replication by binding to interferon receptors on the surface of the regulated cells and altering and regulating the transcription of intracellular genes. Based on the type of receptor through which interferons signal, interferons are generally classified into three categories: type I interferons (type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω), type II interferons (IFNγ in humans), and type III interferons. According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα. According to the present disclosure, the interferon can be a naturally occurring interferon or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal. Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major categories based on different structural characteristics. However, their amino acid sequence similarity is rather weak (usually 15% to 25% identity). The human genome encodes more than 50 interleukins and related proteins. According to the present disclosure, the interleukin can be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin can be a human interleukin and can be derived from any vertebrate, particularly any mammal. The immunostimulatory polypeptides described herein can be prepared as fusion polypeptides or chimeric polypeptides that comprise an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulatory agent can be fused to a pharmacokinetic (PK) extender moiety to increase the circulating half-life. Some examples of non-limiting PK extender moieties are serum albumin or fragments thereof or variants of serum albumin or fragments thereof (e.g., HAS or fragments or variants thereof), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (such as those disclosed in U.S. Pub. No. 2005 / 0287153 and 2007 / 0003549). Other exemplary PK extender moieties are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, which is incorporated herein by reference in its entirety. In some embodiments, the bioactive peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides methods for treating a subject having a condition (e.g., a protein deficiency) that requires protein replacement, comprising administering to the subject an RNA (particularly an mRNA) encoding the replacement protein as described herein. The term "protein replacement" refers to the introduction of a protein (including functional variants thereof) into a subject that lacks such protein. The term also refers to the introduction of a protein in other ways into a subject that requires or would benefit from the provision of a protein (e.g., a subject having a protein deficit). The term "condition characterized by protein deficiency" refers to any condition that exhibits a pathological condition caused by protein deficiency or an insufficient amount of protein. The term encompasses protein folding disorders that result in biologically inactive protein products, i.e., conformational disorders. Protein deficits can relate to infectious diseases, immunosuppression, organ failure, glandular problems, radiation sickness, nutritional deficiencies, poisoning, or other environmental or external insults. The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction generally involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the receptor to the hormone; (v) transmission and amplification of the signal; and (vi) breakdown of the hormone. Hormones differ from cytokines in that: (1) hormones generally act at relatively constant concentrations, and (2) are typically produced by specific types of cells. In some embodiments, the "hormone" is a peptide or polypeptide hormone such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormones, growth hormone (e.g., human growth hormone or bovine growth hormone), oxytocin, atrial-natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin. The term "adhesion molecule" refers to proteins located on the cell surface and involved in the binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are generally transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Some specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins. Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cellular signaling pathways such as those of transmembrane protein kinases (e.g., receptor tyrosine kinases (RTK)). Such regulation can lead to cell growth, division, survival, or differentiation or apoptosis. Some specific examples of integrins include: α 1 β 1 , α 2 β 1 , α 3 β 1 , α 4 β 1 , α 5 β 1 , α 6 β 1 , α 7 β 1 , α L β 2 , α M β 2 , α IIb β 3 , α V β 1 , α V β3 , α V β 5 , α V β 6 , α V β 8 , and α 6 β 4 。 The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in the processes of cell recognition, binding, and / or adhesion. A common feature of molecules belonging to this superfamily is that they contain regions called immunoglobulin domains or folds. Some members of the immunoglobulin superfamily include antibodies (such as IgG), T cell receptors (TCR), major histocompatibility complex (MHC) molecules, co-receptors (such as CD4, CD8, CD19), antigen receptor accessory molecules (such as CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (such as CD28, CD80, CD86), etc. The term "immunologically active compound" refers to any compound that modifies the immune response, for example, by inducing and / or inhibiting the maturation of immune cells, inducing and / or inhibiting cytokine biosynthesis, and / or by stimulating B cells to produce antibodies to modify humoral immunity. Immunologically active compounds have potent immunostimulatory activities, including but not limited to antiviral and antitumor activities, and can also downregulate other aspects of the immune response, such as shifting the immune response from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds can be used as vaccine adjuvants. Some specific examples of immunologically active compounds include interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, particularly tumor-associated antigens, pathogen-associated antigens (such as antigens of bacteria, parasites, or viruses), allergens, and autoantigens. Immunologically active compounds can be vaccine antigens, i.e., antigens that, when inoculated into a subject, induce an immune response. In some embodiments, the RNA (particularly mRNA) described in the present disclosure contains a nucleic acid sequence encoding a peptide or polypeptide that contains an epitope for inducing an immune response against an antigen in a subject. A "peptide or polypeptide that contains an epitope for inducing an immune response against an antigen in a subject" is also referred to herein as a "vaccine antigen", a "peptide and protein antigen", or simply an "antigen". In some embodiments, the RNA encoding the vaccine antigen is expressed in a cell of a subject (e.g., a muscle cell or an antigen-presenting cell (APC)) to provide the vaccine antigen. In some embodiments, the vaccine antigen is expressed on the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the RNA encoding the vaccine antigen is administered systemically (e.g., intravenously). In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, the RNA encoding the vaccine antigen is expressed in the spleen. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, the RNA encoding the vaccine antigen is expressed in antigen-presenting cells, preferably in professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from dendritic cells, macrophages, and B cells. In some embodiments, the RNA encoding the vaccine antigen is administered intramuscularly. The vaccine antigen comprises an epitope for inducing an immune response against the antigen in a subject. Thus, the vaccine antigen comprises an antigenic sequence for inducing an immune response against the antigen in a subject. Such an antigenic sequence can correspond to a target antigen or a disease-associated antigen, such as a protein of an infective agent (e.g., a viral antigen or a bacterial antigen) or a tumor antigen, or can correspond to an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or its immunogenic variant. Thus, the antigenic sequence can comprise at least an epitope of the target antigen or disease-associated antigen or its immunogenic variant. The antigenic sequence or its processed product (e.g., a fragment thereof) can bind to an antigen receptor (e.g., TCR or CAR) carried by an immune effector cell. In some embodiments, the antigenic sequence is selected from an antigen or a fragment thereof expressed by a target cell targeted by an immune effector cell, or a variant of such an antigenic sequence or fragment. In some embodiments, the RNA encoding the vaccine antigen is expressed in a cell of a subject to provide an antigen or its processed product for binding to an antigen receptor expressed by an immune effector cell, said binding resulting in stimulation, sensitization, and / or expansion of the immune effector cell. "Antigen" according to the present disclosure encompasses any substance that will elicit an immune response and / or any substance that an immune response or immune mechanism (e.g., a cellular response and / or a humoral response) is directed against. This also includes cases where the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more of the antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance that specifically reacts with an antibody or a T lymphocyte (T cell), such as a peptide or polypeptide. The term "antigen" may encompass a molecule containing at least one epitope (e.g., a T cell epitope). In some embodiments, the antigen is a molecule that optionally induces an immune reaction after processing, and the immune reaction may be specific for the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen. The term "autoantigen" or "self-antigen" refers to an antigen that is derived from within an individual (i.e., an autoantigen may also be referred to as a "self-antigen") and that elicits an abnormally vigorous immune response against a normal part of the body. Such a vigorous immune reaction against an autoantigen may be the cause of an "autoimmune disease". According to the present disclosure, any suitable antigen that is a candidate for an immune response may be used, where the immune response may comprise a humoral immune response, a cellular immune response, or both. In the case of some embodiments of the present disclosure, the antigen is presented by a cell, such as an antigen-presenting cell (in the context of MHC molecules), which results in an immune response against the antigen. The antigen may be a product corresponding to or derived from a naturally occurring antigen. Such a naturally occurring antigen may include or be derived from an allergen, a virus, a bacterium, a fungus, a parasite, and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present disclosure, the antigen may correspond to a naturally occurring product, such as a viral protein, or a portion thereof. The term "disease-related antigen" is used in its broadest sense and refers to any antigen that is related to a disease. A disease-related antigen is a molecule that contains epitopes that will stimulate the host's immune system to generate an antigen-specific cellular immune response and / or a humoral antibody response against the disease. Disease-related antigens include pathogen-related antigens, i.e., antigens related to an infection caused by a microorganism, typically microbial antigens (e.g., bacterial or viral antigens), or antigens related to cancer (typically a tumor), such as tumor antigens. In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly those that are predominantly intracellular or predominantly present as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen from a virus, bacterium, single-celled organism, or parasite, such as a viral antigen like viral ribonucleoprotein or envelope protein. In some embodiments, the antigen should be presented by MHC molecules, which leads to the modulation of cells of the immune system (e.g., CD4+ and CD8+ lymphocytes), particularly the activation of cells of the immune system (e.g., CD4+ and CD8+ lymphocytes), particularly through the modulation of the activity of the T cell receptor. The term "epitope" refers to the antigenic determinant in a molecule (e.g., an antigen), i.e., a part or fragment of the molecule that is recognized by the immune system, e.g., recognized by T cells or B cells, particularly when presented in the context of MHC molecules. The epitope of a protein can comprise contiguous or non-contiguous portions of the protein and can be, for example, about 5 to about 100 amino acids, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length. The term "T cell epitope" refers to a part or fragment of a protein that is recognized by T cells when present in the context of MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and refer to a gene complex present in all vertebrates. According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation and / or the amino acid sequence that disrupts immune tolerance is directly fused or fused via a linker to an antigen peptide or polypeptide (antigen sequence). The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional meaning and refer to the overall body response to an antigen and can refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the term "immune response to" or "immune response against" with respect to a substance (e.g., an antigen, cell, or tissue) refers to the immune response against the said substance, e.g., a cellular response. The immune response can include one or more reactions selected from the following: the appearance of antibodies against one or more antigens and the expansion of antigen-specific T lymphocytes (e.g., CD4 + and CD8 + T lymphocytes, such as CD8 + T lymphocytes). The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the following operations: administering to the individual one or more immunogens or antigens as described herein or derivatives thereof (especially in the form of RNA encoding them (especially mRNA)), and stimulating an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens. The term "allergen" refers to a class of antigens that originate outside the body of the subject (i.e., an allergen can also be referred to as a "heterologous antigen") and produce an abnormally intense immune response, where the subject's immune system reacts against a perceived threat that is otherwise harmless to the subject. "Allergy" is a disease caused by such an intense immune response against an allergen. Allergens are typically antigens that can stimulate a type I hypersensitivity reaction in atopic individuals through an immunoglobulin E (IgE) response. Specific examples of allergens include allergens derived from peanut proteins (e.g., Ara h2.02), ovalbumin, grass pollen proteins (e.g., Phlp 5), and dust mite proteins (e.g., Der p 2). The term "growth factor" refers to a molecule that can stimulate cell growth, proliferation, healing, and / or cell differentiation. Generally speaking, growth factors act as signaling molecules between cells. The term "growth factor" includes specific cytokines and hormones that bind to specific receptors on the surface of their target cells. Some examples of growth factors include bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) (e.g., VEGFA), epidermal growth factor (EGF), insulin-like growth factor, ephrin, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulin, neurotrophic factor (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renin (RNLS) (anti-apoptotic survival factor), T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor (transforming growth factor α (TGF-α), transforming growth factor β (TGF-β)), and tumor necrosis factor-α (TNF-α). In some embodiments, the "growth factor" is a peptide or polypeptide growth factor. The term "protease inhibitor" refers to a molecule that inhibits the function of a protease, particularly a peptide or polypeptide. Protease inhibitors can be classified by the protease being inhibited (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serine protease inhibitors). Specific examples of protease inhibitors include serine protease inhibitors, such as α1 - antitrypsin, aprotinin, and bestatin. The term "enzyme" refers to a macromolecular biological catalyst that accelerates chemical reactions. Like any catalyst, enzymes are not consumed in the reactions they catalyze and do not alter the equilibrium of said reactions. Unlike many other catalysts, enzymes are much more specific. In some embodiments, enzymes are essential for the homeostasis of an organism. For example, any dysfunction of an enzyme (particularly a decrease in activity, which can be caused by any of a mutation, deletion, or decreased production) results in a disease. Some examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1 - TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase. The term "receptor" refers to a protein molecule that receives signals (particularly chemical signals called ligands) from outside the cell. The binding of a signal (such as a ligand) to a receptor causes some type of response in the cell, such as the intracellular activation of a kinase. Receptors include transmembrane receptors (e.g., ion - channel - linked (ionotropic) receptors, G - protein - linked (metabotropic) receptors, and enzyme - linked receptors) and intracellular receptors (e.g., cytoplasmic receptors and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors for which the ligand is a peptide), such as P - selectin glycoprotein ligand - 1 (PSGL - 1). The term "growth factor receptor" refers to a receptor that binds to a growth factor. The term "apoptosis regulator" refers to a molecule that regulates apoptosis (i.e., activates or inhibits apoptosis), particularly a peptide or polypeptide. Apoptosis regulators can be divided into two major categories: apoptosis regulators that regulate mitochondrial function and apoptosis regulators that regulate caspases. The first category includes proteins (such as BCL - 2, BCL - xL) that are used to maintain mitochondrial integrity by preventing the loss of mitochondrial membrane potential and / or preventing the release of pro - apoptotic proteins (such as cytochrome C) into the cytosol. Pro - apoptotic proteins that promote the release of cytochrome C (such as BAX, BAK, BIM) also belong to this first category. The second category includes proteins such as the inhibitor of apoptosis proteins (such as XIAP) or FLIP that block caspase activation. The term "transcription factor" refers to a protein that regulates the rate of transcription of genetic information from DNA to messenger RNA, particularly by binding to specific DNA sequences. Transcription factors may regulate cell division, cell growth, and cell death throughout life; regulate cell migration and organization during embryonic development; and / or respond to signals from outside the cell, such as hormones. A transcription factor contains at least one DNA-binding domain that binds to a specific DNA sequence, which is typically adjacent to the gene regulated by the transcription factor. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family. The term "tumor suppressor protein" refers to a molecule, particularly a peptide or polypeptide, that protects cells from a step on the carcinogenic pathway. Tumor suppressor proteins (usually encoded by the corresponding tumor suppressor genes) exhibit a weakening or inhibitory effect in regulating the cell cycle and / or promote apoptosis. Its functions can be one or more of the following: inhibiting genes necessary for continuous cell cycle; coupling the cell cycle to DNA damage (as long as there is damaged DNA in the cell, cell division should not occur); initiating apoptosis if the damaged DNA cannot be repaired; metastasis inhibition (e.g., preventing the spread of tumor cells, preventing the loss of contact inhibition and inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI / SNF (SWItch / Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), ST5, suppression of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3). The term "structural protein" refers to proteins that confer hardness and rigidity to biological components that are otherwise fluid. Structural proteins are mainly fibrous (e.g., collagen and elastin), but can also be spherical (e.g., actin and tubulin). Generally, spherical proteins can dissolve as monomers, but polymerize to form long fibers, such as those that can constitute the cytoskeleton. Some other structural proteins are motor proteins (e.g., myosin, kinesin, and dynein) that can generate mechanical force, as well as surfactant proteins. Specific examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin. The term "reprogramming factor" or "reprogramming transcription factor" refers to such molecules, particularly peptides or polypeptides, which when expressed in somatic cells (optionally together with other substances such as other reprogramming factors) cause the somatic cells to be reprogrammed or de-differentiated into cells with stem cell characteristics, particularly pluripotency. Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG. The term "genome engineering protein" refers to proteins that can insert, delete, or replace DNA in the genome of an object. Specific examples of genome engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9). The term "blood protein" refers to peptides or polypeptides present in the plasma of an object, particularly in the plasma of a healthy object. Blood proteins have various functions, such as transport (e.g., albumin, transferrin), enzyme activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., α1-antitrypsin), etc. Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (G-CSF), modified factor VIII, and anticoagulants. Thus, in some embodiments, the bioactive peptide or polypeptide is (i) a cytokine, preferably selected from erythropoietin (EPO), interleukin 4 (IL-4), and interleukin 10 (IL-10), more preferably EPO; (ii) an adhesion molecule, particularly integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunoreactive compound, particularly an antigen, such as a viral antigen or a bacterial antigen, such as an antigen of SARS-CoV-2, such as the spike (S) protein of SARS-CoV-2 or a variant thereof; (v) a hormone, particularly vasopressin, insulin, or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly α1-antitrypsin; (viii) an enzyme, preferably selected from herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes, and lactase; (ix) a receptor, particularly a growth factor receptor; (x) an apoptosis regulator, particularly BAX; (xi) a transcription factor, particularly FOXP3; (xii) a tumor suppressor protein, particularly p53; (xiii) a structural protein, particularly surfactant protein B; (xiv) a reprogramming factor, e.g., selected from OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) a genome engineering protein, particularly clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9); and (xvi) a blood protein, particularly fibrinogen. In some embodiments, the bioactive peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administering the peptide or polypeptide to a subject elicits an immune response in the subject against one or more antigens or one or more epitopes, and the immune response can be therapeutic or partially or fully protective. In some embodiments, the RNA encodes at least one epitope, such as at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. In some embodiments, the target antigen is a tumor antigen and the antigenic sequence (e.g., epitope) is derived from a tumor antigen. The tumor antigen can be a "standard" antigen, which is generally known to be expressed in multiple cancers. The tumor antigen can also be a "neoantigen", which is specific to an individual's tumor and has not been previously recognized by the immune system. The neoantigen or neoepitope can be caused by one or more cancer-specific mutations in the cancer cell genome, resulting in amino acid changes. If the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen comprising one or more amino acid changes. In some embodiments, the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or an immunogenic variant thereof. Thus, in some embodiments, the RNA (e.g., mRNA) used in the present disclosure encodes an amino acid sequence comprising: a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or an immunogenic variant thereof. In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. Thus, in some embodiments, the RNA (particularly mRNA) described in the present disclosure encodes an amino acid sequence comprising: a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

[0133] Recombinant: As used herein, the term "recombinant" means "prepared by genetic engineering". In some embodiments, the "recombinant object" in the context of the present disclosure is not naturally occurring.

[0134] Reference / reference standard: As used herein, "reference" describes the standard or control against which a comparison is made. For example, in some embodiments, a test agent, animal, individual, population, sample, sequence, or value is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or assayed substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, the reference or control is or comprises a set of specifications (e.g., relevant acceptance criteria). Generally, as understood by those skilled in the art, the reference or control is assayed or characterized under conditions or circumstances comparable to those under evaluation. Those skilled in the art will understand when there is sufficient similarity to justify reliance on a particular possible reference or control and / or comparison to a particular possible reference or control.

[0135] Ribonucleotides: As used herein, the term "ribonucleotide" includes unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can include one or more modifications, including but not limited to, for example, (a) terminal modifications, such as 5'-end modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse ligation, etc.), 3'-end modifications (e.g., conjugation, reverse ligation, etc.), (b) base modifications, such as replacement with a modified base, a stabilizing base, a destabilizing base, or a base that pairs with an expanded library of ligands or a conjugated base, (c) sugar modifications (e.g., at the 2'-position or 4'-position) or sugar replacement, and (d) internucleoside bond modifications, which include modification or replacement of the phosphodiester bond. The term "ribonucleotide" also encompasses ribonucleoside triphosphates, which include modified and unmodified ribonucleoside triphosphates.

[0136] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA contains both single-stranded and double-stranded portions. In some embodiments, the RNA can contain a backbone structure as described in the definition of "nucleic acid / polynucleotide" above. The RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments in which the RNA is mRNA, the RNA typically contains a poly(A) region at its 3'-end. In some embodiments in which the RNA is mRNA, the RNA typically contains a cap structure recognized in the art at its 5'-end, e.g., for identifying the mRNA and linking it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0137] Secretory signal: As used herein, the term "secretory signal" or "signal peptide" refers to an amino acid sequence present in a polypeptide that targets the polypeptide to the secretory pathway. Generally, the secretory signal is cleaved after translocation to the endoplasmic reticulum following RNA translation. Generally, the secretory signal is a short (e.g., 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids in length) peptide. The secretory signal can be present at the N-terminus of the polypeptide.

[0138] Selective or specific: As understood by those skilled in the art, when used herein in connection with an active agent, the terms “selective” or “specific” mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to “specifically” bind to a target if it preferentially binds to its target in the presence of one or more competing alternative targets. In many embodiments, the specific interaction depends on the presence of specific structural features of the target entity (e.g., epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the target binding portion of one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding portion. In some embodiments, specificity is evaluated relative to the specificity of a reference non-specific binding portion. In some embodiments, a CLDN-18.2 targeting antibody agent encoded by one or more RNAs (e.g., the RNAs described herein) does not detectably bind to a competing alternative target (e.g., a CLDN18.1 polypeptide) under conditions where it binds to a CLDN-18.2 polypeptide. In some embodiments, the CLDN-18.2 targeting antibody agent binds to the CLDN-18.2 polypeptide with a higher binding rate, lower dissociation rate, increased affinity, reduced dissociation, and / or increased stability compared to its competing alternative targets, including, for example, a CLDN18.1 polypeptide.

[0139] Specific binding: As used herein, the term “specific binding” refers to the ability to discriminate between possible binding partners in the context in which binding occurs. An antibody agent that interacts with a particular target in the presence of other potential targets is said to “specifically bind” to the target with which it interacts. In some embodiments, specific binding is evaluated by detecting or determining the degree of association between the CDR of the antibody agent and its partner; in some embodiments, specific binding is evaluated by detecting or determining the degree of dissociation of the antibody agent-partner complex; in some embodiments, specific binding is evaluated by detecting or determining the ability of the antibody agent to compete for the alternative interaction between its partner and another entity. In some embodiments, specific binding is evaluated by performing such detection or determination over a range of concentrations.

[0140] Subject: As used herein, the term "subject" refers to an organism to which the compositions described herein are to be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject is predisposed to a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject is a human having one or more characteristics of susceptibility or risk for a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or treatment has been administered and / or is being administered.

[0141] Predisposed to: An individual "predisposed to" a disease, disorder, or condition is an individual at risk of developing the disease, disorder, or condition. In some embodiments, an individual predisposed to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual predisposed to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual predisposed to a disease, disorder, or condition is an individual who has been exposed to an environment associated with the development of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., a family member of an individual with the disease, disorder, or condition; a carrier of a genetic or other biomarker associated with the disease, disorder, or condition, etc.).

[0142] Having: An individual "having" a disease, disorder, and / or condition has been diagnosed as having and / or exhibiting one or more symptoms of the disease, disorder, or condition.

[0143] Synthetic: As used herein, the term "synthetic" refers to an entity that is artificial, or made by human intervention, or produced by synthesis rather than by natural production. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized (e.g., by solid-phase synthesis in some embodiments). In some embodiments, the term "synthetic" refers to an entity made outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) produced by in vitro transcription using a template.

[0144] Therapeutic agent: As used interchangeably herein, the phrase “therapeutic agent” or “treatment” refers to an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject or patient. In some embodiments, a therapeutic agent or treatment is any substance that can be used to alleviate, improve, relieve, inhibit, prevent one or more symptoms or characteristics of a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce its incidence. In some embodiments, a therapeutic agent or treatment is a medical intervention (e.g., surgery, radiation, light therapy) that can be performed to alleviate, relieve, inhibit, prevent one or more symptoms or characteristics of a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce its incidence.

[0145] 3′ untranslated region: As used herein, the term “3′ untranslated region” or “3′ UTR” refers to the sequence of an mRNA molecule that begins after the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3′ UTR begins immediately after the stop codon of the coding region of the open reading frame sequence. In other embodiments, the 3′ UTR does not begin immediately after the stop codon of the coding region of the open reading frame sequence.

[0146] Threshold level (e.g., acceptance criterion): As used herein, the term “threshold level” refers to a level that is used as a reference to obtain information about a measurement result (e.g., a measurement result obtained in an assay) and / or to classify a measurement result (e.g., a measurement result obtained in an assay). For example, in some embodiments, a threshold level refers to a value measured in an assay that represents the boundary between two subsets of a defined population (e.g., batches that meet quality control criteria versus batches that do not meet quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines another subset of the population. The threshold level can be determined based on one or more control samples or between a population of control samples. The threshold level can be determined before, simultaneously with, or after the measurement of interest is performed. In some embodiments, the threshold level can be a range of values.

[0147] Transfection: As used herein, the term "transfection" refers to the introduction of nucleic acids (particularly RNA) into cells. For the purposes of the present disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject (e.g., a patient) or the cells may be in vitro (e.g., outside the patient). Thus, according to the present disclosure, the cells used to transfect the nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to the present disclosure, transfection can be transient or stable. For some applications of transfection, it may be sufficient to only transiently express the transfected genetic material. RNA can be transfected into cells to transiently express the protein it encodes. Since the nucleic acids introduced during transfection generally do not integrate into the nuclear genome, the foreign nucleic acids will be diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cells and their daughter cells, stable transfection must occur. Such stable transfection can be achieved by using, for example, virus-based systems or transposon-based systems for transfection. RNA can be transfected into cells to transiently express the protein it encodes.

[0148] Treatment: As used herein, the term "treatment" and variations thereof refer to any method for partially or completely alleviating, ameliorating, relieving, inhibiting, preventing one or more symptoms or features of a disease, disorder, and / or condition, delaying its onset, reducing its severity, and / or reducing its incidence. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who only exhibits early signs of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing a pathological condition associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject in the late stage of a disease, disorder, and / or condition.

[0149] Unresectable tumor: As used herein, the term "unresectable tumor" generally refers to a tumor that is characterized by one or more features that, in the reasonable medical judgment, are considered to indicate that the tumor cannot be safely (e.g., without undue harm to the subject) removed by surgery; and / or for which a competent medical professional has determined that the risks of tumor resection to the subject outweigh the benefits associated with such resection. In some embodiments, an unresectable tumor refers to a tumor that involves and / or has grown into essential organs or tissues (including blood vessels that may not be reconstructable), and / or is located in a position that cannot be easily accessed surgically without posing an unreasonable risk of damage to one or more other critical or essential organs and / or tissues (including blood vessels). In some embodiments, the "unresectability" of a tumor refers to the likelihood of achieving a negative surgical margin (R0) resection. In the case of pancreatic cancer, encasement of the tumor around major blood vessels such as the superior mesenteric artery (SMA) or celiac axis, portal vein obstruction, and the presence of celiac or para-aortic lymphadenopathy are generally considered findings that preclude R0 surgery. Those skilled in the art will appreciate the parameters for determining whether a tumor is unresectable.

[0150] Those skilled in the art reading this specification will understand that, in many embodiments, standard techniques are available and can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and / or transformation (e.g., electroporation, liposome transfection, transfection). Enzymatic reactions and / or purification techniques can generally be carried out according to the manufacturer's instructions or as commonly practiced in the art or as described herein. In many embodiments, the foregoing techniques and operations can generally be carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Detailed Description

[0151] For many cancer patients, and particularly for patients with recurrent or refractory advanced solid tumors, the outcomes of Standard of Care (SOC) treatment remain poor. Treatment options typically also include palliative chemotherapy (which may be less tolerable after prior repeated exposure to cytotoxic compounds) or best supportive care, as well as investigational treatments of unproven benefit. Treatment in this population is not curative and overall survival is expected to be in the order of months. Immunotherapy has emerged as an effective treatment option for some cancers with high unmet medical needs. Specifically, immune checkpoint inhibitors have been approved for the treatment of multiple cancer indications and act by activating pre-existing anti-tumor specific T cells. The medical need for multiple cancer types remains high. The present disclosure particularly provides insights and techniques for treating cancer (e.g., pancreatic cancer and / or cholangiocarcinoma) with treatments targeting Claudin-18.2 (CLDN-18.2).

[0152] In some embodiments, the present disclosure particularly provides an RNA technology for delivering a monoclonal antibody targeting CLDN-18.2 (which combines both effective anti-tumor characteristics and excellent safety characteristics), bypassing the hurdles of the slow and cumbersome antibody manufacturing process. Without wishing to be bound by any particular theory, the present disclosure proposes that such an RNA delivery modality can achieve one or more improvements, such as effective administration with a reduced incidence of treatment-emergent adverse events (“TEAEs”) (e.g., frequency and / or severity) and / or an improved relationship between the potency level and the TEAE level (e.g., improved therapeutic window), as compared to those observed when administering the corresponding (e.g., encoded) protein (e.g., antibody) agent itself. In particular, the present disclosure teaches that such improvements can be achieved, in particular, by delivering IMAB362 via administration of RNA encoding IMAB362 (e.g., ssRNA, e.g., mRNA).

[0153] In some embodiments, the present disclosure particularly provides the insight that mRNA encoding an antibody agent (e.g., IMAB362) or a functional portion thereof (optionally formulated with a lipid nanoparticle (LNP) for intravenous (IV) administration to a subject (e.g., a human patient, a model organism, etc.)) can be taken up by target cells (e.g., hepatocytes) to effectively produce a therapeutically relevant plasma concentration of the encoded antibody agent (e.g., IMAB362), e.g., as Figure 14As shown herein for CLDN-18.2-targeting antibody agents expressed by RNA (e.g., the RNAs described herein). In some embodiments, the antibody agent is expressed by mRNA, e.g., engineered for minimal immunogenicity and / or formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA encoding the antibody agent can comprise modified nucleotides (e.g., but not limited to pseudouridine and / or 1-methyl-pseudouridine).

[0154] Furthermore, the present disclosure particularly provides the insight that the ability of a CLDN-18.2-targeting antibody agent delivered as described herein to induce antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while engaging the recipient's immune system can enhance the cytotoxic effects of chemotherapy and / or other anti-cancer therapies. In some embodiments, such combination therapies can prolong progression-free and / or overall survival, e.g., relative to individual therapies administered alone and / or relative to another suitable reference.

[0155] Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, such as gemcitabine, oxaliplatin, and 5-fluorouracil, show an upregulation of existing CLDN-18.2 expression levels in pancreatic cancer cell lines; furthermore, no de novo expression was observed for these agents in CLDN-18.2-negative cell lines. See, e.g., Türeci et al., (2019) “Characterization of Zolbetuximab in pancreatic cancer models.” In Oncoimmunology 8(1), pp.e1523096.

[0156] The present disclosure particularly provides the insight that a treatment targeting CLDN-18.2 as described herein can be particularly useful and / or effective when administered to a tumor (e.g., tumor cells, a subject suspected and / or detected with such a tumor and / or tumor cells, etc.) characterized by (e.g., determined to exhibit and / or expected or predicted to exhibit) elevated expression and / or activity of CLDN-18.2 in tumor cells (e.g., possibly or already caused by exposure to one or more chemotherapeutic agents). Indeed, the present disclosure particularly teaches that the provided treatment targeting CLDN-18.2 as described herein (e.g., administering RNA, and more particularly, mRNA encoding a CLDN-18.2 targeting antibody reagent) can provide a synergistic treatment when administered in combination with one or more CDLN-18.2 enhancers (e.g., one or more specific chemotherapeutic agents) (e.g., administered to a subject who has received and / or is receiving or otherwise exposed to the one or more CDLN18.2 enhancers (e.g., one or more specific chemotherapeutic agents)). Thus, in some embodiments, a targeted CLDN-18.2 treatment as described herein in combination with other anti-cancer agents expected and / or demonstrated to upregulate CLDN-18.2 expression and / or activity in tumor cells can be useful.

[0157] Accordingly, the present disclosure particularly provides insights and techniques for treating cancer, particularly cancer associated with the expression of CLDN-18.2. In some embodiments, the provided techniques are effective for treating pancreatic cancer. In some embodiments, the provided techniques are effective for treating gastric cancer or gastroesophageal cancer. In some embodiments, the provided techniques are effective for treating cholangiocarcinoma. In some embodiments, the provided techniques are effective for treating ovarian cancer. In some embodiments, the provided techniques are effective when applied to locally advanced tumors. In some embodiments, the provided techniques are effective when applied to unresectable tumors. In some embodiments, the provided techniques are effective when applied to metastatic tumors. I. Claudin-18.2 Polypeptide

[0158] Claudin-18.2 (CLDN-18.2) is a cancer-associated splice variant of claudin-18. CLDN-18.2 is a member of the claudin family of more than 20 structurally related proteins that are involved in the formation of tight junctions in epithelia and endothelia.

[0159] CLDN18 Expression in Healthy Tissues. Claudin 18.2 is a 27.8 kDa protein with four transmembrane domains and two small extracellular loops (Niimi et al. 2001). CLDN-18.2 is a tight junction molecule of gastric epithelial cells. Gastric tight junctions are highly specialized to exclude gastric acid, which can damage the gastric lining.

[0160] CLDN-18.2 is a highly selective gastric lineage antigen (Sahin et al. 2008). Typically, its expression is restricted to short-lived differentiated gastric epithelial cells in the pits and basal regions of gastric glands. The stem cell compartment that continuously replenishes the differentiated epithelial cells of gastric glands is CLDN-18.2 negative. Without wishing to be bound by theory, it is generally believed that no other normal human cell type expresses CLDN-18.2 at the transcriptional or protein level.

[0161] CLDN18 Expression in Cancer. CLDN-18.2 is expressed in a variety of human cancers including gastric cancer, gastroesophageal cancer (GE), and pancreatic cancer (PC) (Karanjawala et al. 2008; Coati et al. 2019), as well as in precancerous lesions ( etal. 2014; Tanaka et al. 2011). Tumor-associated CLDN-18.2 expression has also been detected in ovarian cancer (Sahin et al. 2008), biliary tract cancer (Shinozakiet al. 2011), and lung cancer (Micke et al. 2014).

[0162] Approximately 77% of primary gastric adenocarcinomas (GAC) are CLDN-18.2+. 56% of GAC show strong CLDN-18.2 expression in at least 60% of tumor cells (defined by immunohistochemical analysis as staining intensity ≥2+). CLDN-18.2 expression is more frequent in diffuse gastric cancer than in intestinal-type gastric cancer. CLDN-18.2 protein is also frequently detected in lymph node metastases and distant metastases to the ovary (so-called Krukenberg tumor) of gastric cancer. In addition, 50% of esophageal adenocarcinomas show significant CLDN-18.2 expression.

[0163] In pancreatic cancer, CLDN-18.2 is expressed in pancreatic ductal adenocarcinoma (PDAC) at an incidence of 60% to 90% (Karanjawala et al. 2008; et al. 2014). PDAC accounts for more than 80% of all pancreatic tumors, is the seventh most common cancer in Europe, and is the fourth leading cause of cancer-related death in the European Union (Ferlay et al. 2010; Jemal et al. 2011; Seufferlein et al. 2012). Almost 60% of patients with PDAC express membrane-bound CLDN-18.2, and CLDN-18.2 is ectopically activated in 20% of patients with pancreatic neuroendocrine tumors. CLDN-18.2 is expressed in primary and metastatic PDAC lesions ( etal. 2014).

[0164] Downregulation of CLDN-18.2 by siRNA technology has been shown to result in inhibition of gastric cancer cell proliferation (Niimi et al. 2001), indicating involvement in the proliferation of CLDN-18.2+ tumor cells.

[0165] Exemplary sequences of CLDN-18.2 (SEQ ID NO: 32) and the splice variant CLDN18.1 (SEQ ID ID: 33) are shown below: II. Exemplary antibody agents targeting Claudin-18.2 polypeptides

[0166] In some embodiments, the antibody agent targeting CLDN-18.2 specifically binds to the CLDN-18.2 polypeptide. In some embodiments, the antibody agent targeting CLDN-18.2 specifically binds to the first extracellular domain (ECD1) of the CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to the ECD1 epitope exposed in cancer cells. In some embodiments, such an antibody agent may have at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9M, or lower binding affinity (e.g., as measured by the dissociation constant). Those skilled in the art will understand that in some cases, the binding affinity (e.g., as measured by the dissociation constant) can be affected by non-covalent intermolecular interactions (such as hydrogen bonds, electrostatic interactions, hydrophobic forces, and van der Waals forces) between two molecules. As an alternative or in addition, the binding affinity between a ligand and its target molecule can be affected by the presence of other molecules. Those skilled in the art will be familiar with a variety of techniques for measuring binding affinity and / or dissociation constant according to the present disclosure, including, for example, but not limited to ELISA, gel shift assay, pull-down assay, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, grating-coupled interferometry, and spectroscopy.

[0167] In some embodiments, an antibody targeting CLDN-18.2 can specifically bind to the CLDN-18.2 polypeptide relative to the CLDN18.1 polypeptide. In some embodiments, an antibody targeting CLDN-18.2 does not bind to any other claudin family members, including the closely related claudin-18 splice variant 1 (CLDN18.1) that is mainly expressed in tissues such as the lung.

[0168] In some embodiments, the antibody agent targeting CLDN-18.2 can be any one of the CLDN-18.2 targeting antibodies described in WO 2007 / 059997, WO2008 / 145338, and WO2013 / 174510 (the contents of which are incorporated herein by reference in their entireties for the purposes described herein).

[0169] In some embodiments, the antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR selected from the following (including, for example, 1 CDR, 2 CDRs, and 3 CDRs): (i) CDR1 represented by the amino acid residues (GYTFTSYW); (ii) CDR2 represented by the amino acid residues (IYPSDSYT); and (iii) CDR3 represented by the amino acid residues (TRSWRGNSFDY); and / or (b) a variable light chain domain having at least one CDR selected from the following (including, for example, 1 CDR, 2 CDRs, and 3 CDRs): (i) CDR1 represented by the amino acid residues (QSLLNSGNQKNY); (ii) CDR2 represented by the amino acid residues (WAS); and (iii) CDR3 represented by the amino acid residues (QNDYSYPFT).

[0170] In some embodiments, as described in U.S. 9,751,934, an antibody agent targeting CLDN-18.2 has a heavy chain amino acid sequence and a light chain amino acid sequence that are or include related sequences (e.g., variable region sequences, such as CDR and / or framework (FR) sequences). For example, in some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence represented by amino acid residues 20 to 467 of SEQ ID NO:1 as shown below (wherein SEQ ID NO:1 herein corresponds to SEQ ID NO:118 of U.S. 9,751,934, and the underlined amino acid sequence of SEQ ID NO:1 corresponds to the secretion signal sequence), and a light chain consisting of or comprising the amino acids represented by amino acid residues 21 to 240 of SEQ ID NO:2 as shown below (wherein SEQ ID NO:2 herein corresponds to SEQ ID NO:125 of U.S. 9,751,934, and the underlined amino acid sequence of SEQ ID NO:2 corresponds to the secretion signal sequence).

[0171] In some embodiments, an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR selected from the following (including, for example, 1 CDR, 2 CDRs, and 3 CDRs): (i) CDR1 represented by amino acid residues 45 to 52 of SEQ ID NO:1; (ii) CDR2 represented by amino acid residues 70 to 77 of SEQ ID NO:1; and (iii) CDR3 represented by amino acid residues 116 to 126 of SEQ ID NO:1; and / or (b) a variable light chain domain having at least one CDR selected from the following (including, for example, 1 CDR, 2 CDRs, and 3 CDRs): (i) CDR1 represented by amino acid residues 47 to 58 of SEQ ID NO:2; (ii) CDR2 represented by amino acid residues 76 to 78 of SEQ ID NO:2; and (iii) CDR3 represented by amino acid residues 115 to 123 of SEQ ID NO:2.

[0172] In some embodiments, an antibody agent targeting CLDN-18.2 comprises a variable heavy chain domain containing the amino acid sequence of SEQ ID NO:14 and a variable light chain domain containing the amino acid sequence of SEQ ID NO:15.

[0173] In some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence of SEQ ID NO:1 and a light chain consisting of or comprising the amino acid sequence of SEQ ID NO:2.

[0174] In some embodiments, an antibody agent targeting CLDN-18.2 can be engineered to reduce potential immunogenicity and / or improve secretion. For example, in some embodiments, the murine secretion signal sequence of an antibody agent targeting CLDN-18.2 can be replaced with a human secretion signal sequence.

[0175] In some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID NO:3 as shown below (where the underlined amino acid sequence corresponds to the secretion signal sequence); and a light chain consisting of or comprising the amino acids represented by amino acid residues 27 to 246 of SEQ ID NO:4 as shown below (where the underlined amino acid sequence corresponds to the secretion signal sequence).

[0176] In some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence of SEQ ID NO:3 and a light chain consisting of or comprising the amino acid sequence of SEQ ID NO:4.

[0177] In some embodiments, an antibody agent targeting CLDN-18.2 comprises one or more Fc regions having a lysine at its C-terminus. The source of this lysine is the naturally occurring sequence present in humans, and these Fc regions are derived from said sequence. During the production of recombinant antibodies in cell culture, this terminal lysine can be proteolytically cleaved off by endogenous carboxypeptidases, resulting in a constant region having the same sequence but lacking the C-terminal lysine. Antibodies produced from nucleic acid sequences encoding or not encoding the terminal lysine are substantially identical in sequence and function because, for example, when using antibodies produced in a CHO-based production system, the processing degree of the terminal lysine is usually high (Dick, L.W. et al. Biotechnol. Bioeng. 2008; 100:1132–1143). Thus, it should be understood that the proteins (such as antibodies) according to the present invention can be produced with or without encoding or having a terminal lysine. It should also be understood according to the present invention that a sequence having a terminal lysine (such as a constant region sequence having a terminal lysine) can be understood as the corresponding sequence without a terminal lysine, and a sequence without a terminal lysine can also be understood as the corresponding sequence having a terminal lysine.

[0178] In some embodiments, the antibody targeting CLDN-18.2 is IMAB362 (also known as zolbetuximab, claudiximab). IMAB362 (the antibody targeting CLDN-18.2) is in late-stage clinical development (NCT01630083, NCT03816163, NCT03653507, NCT03505320, NCT03504397) and is known in the art (see, e.g., Sahin et al. 2018; Sahin et al. 2017; Al-Batran et al. 2017a; Al-Batran et al. 2017b; Türeci et al. 2019; Trarbach et al. 2014; Morlock et al. 2018a; Schuler et al. 2016; Lordick et al. 2016; Morlock et al. 2018b). Its target, CLDN-18.2, is a highly selective tumor-associated surface marker.

[0179] IMAB362, developed by Ganymed Pharmaceuticals GmbH and acquired by Astellas Pharma Inc., is a full-length IgG1 antibody targeting the tight junction protein CLDN-18.2 and mediates cell death through antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). IMAB362 recognizes the first extracellular domain (ECD1) of CLDN-18.2 with high affinity and specificity (Sahin et al. 2008; Türeci et al. 2011). This epitope is inaccessible in the normal epithelial barrier of the antibody. Disruption of tight junctions and loss of cell polarization are early markers of cancer. During this process, the epitope of IMAB362 is exposed. IMAB362 does not bind to any other claudin family members, including the closely related claudin 18 splice variant 1 (CLDN18.1) that is mainly expressed in tissues such as the lung.

[0180] In the phase 2 FAST trial of EOX (NCT01630083) in first-line patients with gastric cancer and gastroesophageal cancer, IMAB362 was tested in combination with epirubicin, oxaliplatin, and capecitabine (EOX) (Morlock et al. 2018a; Schuler et al. 2016; Al-Batran et al. 2016; Lordick et al. 2016; Morlock et al. 2018b). The FAST patient population included patients whose tumors had ≥40% tumor cells expressing CLDN-18.2 with moderate to strong (≥2+) staining intensity. A patient subset whose tumors had ≥70% tumor cells with ≥2+ CLDN-18.2 staining intensity derived the greatest benefit from IMAB362 treatment at the 800 / 600 mg / kg 2 dose, with their median overall survival (OS) almost doubling (Al-Batran et al. 2016; Lordick et al. 2016). The OS benefit of IMAB362 at ≥70% CLDN-18.2 expression (+33.1 weeks; p<0.0005) was accompanied by a significant delay in centrally independent reviewed progression (+14.5 weeks; p<0.0005) and a higher objective response rate (ORR) (35.1% versus 27.1%). Adding IMAB362 to EOX did not have a negative impact on patient-related outcomes. Throughout the study, no significant differences between treatment groups were observed in the mixed effect model repeat measurement of global health status or total ST022 score, but IMAB362 plus EOX significantly delayed the deterioration of the global health score by 2.6 months compared to EOX alone (p = 0.008).

[0181] IMAB362 was also tested in phase 2 and 3 trials of Astellas Pharma Inc.'s global development program in patients with CLDN-18.2+ gastric cancer / gastroesophageal cancer and pancreatic cancer.

[0182] IMAB362 has been tested in a variety of clinical trials, as shown in Table 1 below. Table 1: Summary of certain clinical trials involving the administration of IMAB362

[0183] The safety profile of IMAB362 has been well characterized in patients and has been tolerated at repeated doses up to 1000 mg / m 2 q3w (up to a c of 603 μg / mL max ) without dose-limiting toxicity (Sahin et al. 2018; Türeci et al. 2019).

[0184] Without wishing to be bound by a particular theory, the main pharmacological mode of action of IMAB362 for effecting tumor cell killing involves antibody-dependent cell cytotoxicity (ADCC). Based on the dose-response curve obtained from in vitro ADCC assays, the drug concentration producing a 95% response was observed at IMAB362 concentrations in serum of 0.3 to 28 μg / mL (Sahin et al. 2018). For example, effective lysis of CLDN-18.2+ cells by ADCC has been reported, with an EC 95 of 0.3 to 28 μg / mL (Sahin et al. 2018).

[0185] In multiple trials, IMAB362 has been well tolerated, with nausea and vomiting being the main adverse events (AEs), and no dose-limiting toxicity (DLT) and clinical activity as a single agent and in combination with chemotherapy were observed.

[0186] The present disclosure particularly provides the insight that IMAB362 or variants thereof (e.g., variants that share one or more characteristics of IMAB362, including, for example, one or more (and in many embodiments all) CDR sequences, one or more (and in many embodiments all) FR sequences, and / or variable heavy and / or light chain sequences, etc.), and / or variants that are a class of variants such as IgG1, IgM, IgA, etc.) may represent particularly desirable antibodies for delivery by ribonucleic acid as described herein. Without wishing to be bound by any particular theory, the present disclosure proposes that such a delivery mode may enable effective administration relative to those observed when administering the IMAB362 antibody itself, as well as a reduction in the incidence (e.g., frequency and / or severity) of IMAB362 treatment-related adverse events (TEAEs). In the phase 2a MONO trial of IMAB362 (NCT01197885), TEAEs occurred in 82% (n = 44 / 54) of patients; nausea (61%), vomiting (50%), and fatigue (22%) were the most common TEAEs. Grade 3 vomiting was reported in 12 patients (22%), and grade 3 nausea was reported in 8 patients (15%). These patients received 600 mg / m 2The dose. Nausea and vomiting observed in this study were controlled by pausing or slowing the infusion of IMAB362, indicating that the AE is related to C max (Türeci et al. 2019).

[0187] In particular, the present disclosure especially shows that the pharmacokinetic (PK) profile of IMAB362 delivered as ribonucleic acid (“RiboMab01”) as described herein shows a gradual increase in antibody concentration and C max is significantly lower compared to IMAB362 at 48 to 72 hours after administration. The altered PK profile of RiboMab01 may reduce the C max -related AEs seen in patients after treatment with IMAB362. The present disclosure also provides non-human primate study data, which shows that no systemic side effects such as diarrhea were observed.

[0188] The present disclosure particularly understands the favorable risk / benefit profile observed for the administered IMB362 antibody, especially in certain indications with high medical need, and proposes that the delivery as described herein can be effective and / or particularly desirable. III. RNA Technology for Delivering Antibody-Based Therapeutics

[0189] Recombinant protein antibodies are widely used biologic agents for treating diseases or disorders (e.g., cancer) but show many limitations, including, for example, a lengthy manufacturing process development and a short serum half-life for antibody derivatives. The present disclosure particularly provides a technology to address certain limitations of recombinant antibody technology (including, for example, a lengthy manufacturing process development and a short serum half-life for antibody derivatives) by utilizing RNA technology as a mode to directly express antibody agents (referred to as RiboMab) in the cells of a patient, as a new class of antibody-based therapeutics. In some embodiments, the present disclosure particularly provides the insight that RiboMab formulated with lipid nanoparticles (LNPs) for intravenous (IV) administration can be taken up by cells (e.g., hepatocytes) to effectively generate therapeutically relevant plasma concentrations of the encoded RiboMab antibody ( Figure 14 ). In some embodiments, RiboMab is an antibody agent encoded by, for example, mRNA engineered for minimal immunogenicity and / or formulated in lipid nanoparticles (LNPs). In some embodiments, the mRNA encoding the antibody agent may contain modified nucleotides (e.g., but not limited to pseudouridine and / or 1-methyl-pseudouridine).

[0190] The RiboMab technology can be used to deliver various antibody forms. For example, in some embodiments, the RiboMab technology can be used to express intact immunoglobulins (Igs), which include, for example but not limited to, IgG. In some embodiments, an intact immunoglobulin (Ig) can be encoded by a single RNA comprising a first coding region encoding an antibody heavy chain and a second coding region encoding an antibody light chain variable domain, wherein the single RNA comprises or encodes an internal ribosome entry side (IRES) or another internal promoter or peptide sequence, such as a "self-cleaving" 2A or 2A-like sequence (see, e.g., Szymczak et al. Nat Biotechnol 22:589, May 2004; ePub April 4 2004) to produce the corresponding heavy and light chains, which can then be processed to form an intact IgG. In some embodiments, an intact Ig can be encoded by two separate RNAs: a first RNA comprising a coding region encoding an antibody heavy chain; and a second RNA comprising a coding region encoding an antibody light chain. Such first and second RNAs are then translated in a target cell into the corresponding antibody chains and an intact Ig antibody is formed.

[0191] In some embodiments, the RiboMab technology can be used to express bispecific antibody variants, such as, for example, as Figure 12 (shown in Figure A) or as described in Stadler et al. (2016) Oncoimmunology 5(3):e1091555 and / or in Stadler et al. (2017) Nature Medicine 23(7):815-817. For example, in some embodiments, a bivalent antibody agent can be encoded by a single RNA comprising a first coding region encoding a single-chain variable fragment (scFv) of a first target and a second coding region encoding an scFv of a second target. In some embodiments, a bivalent antibody agent can be encoded by two separate RNAs: a first RNA comprising a coding region encoding an scFv of a first target and a coding region encoding a heavy chain antigen-binding fragment (Fab) of a second target; and a second RNA comprising a coding region encoding an scFv of the same first target and a coding region encoding a light chain Fab of the same second target. Such first and second RNAs are then translated in a target cell into the subunits of the antibody and a bispecific antibody is formed.

[0192] In some embodiments, an RNA agent (e.g., the ssRNA described herein) can be delivered together with a vector. In some embodiments, the RNA / LNP is administered intravenously (IV) and taken up by target cells (e.g., hepatocytes) to effectively produce a therapeutically relevant plasma concentration of the encoded RiboMab antibody. A. RNA and compositions thereof that encode an antibody agent against Claudin-18.2 polypeptide

[0193] In some embodiments, at least one RNA comprises one or more coding regions encoding an antibody agent as described in the section entitled "Exemplary Antibody Agents Targeting Claudin-18.2 Polypeptide" above. In some embodiments, at least one RNA comprises one or more coding regions encoding the antibody agent IMAB362 as described above or exemplified herein.

[0194] Without being bound by any particular theory, the present disclosure particularly provides the insight that in some embodiments, the antibody agent IMAB362 can be particularly useful and / or effective (at least in part) because it specifically binds to CLDN-18.2, and furthermore, preferentially binds to CLDN-18.2 relative to CLDN18.1. In some embodiments, the teachings provided herein can be applicable to other antibody agents specific for CLDN-18.2, and particularly applicable to such antibodies that even preferentially bind to CLDN-18.2 relative to CLDN18.1. For example, in some embodiments, at least one RNA comprises one or more coding regions encoding an antibody agent that preferentially binds to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide. In some embodiments, the binding affinity of such an antibody agent for a CLDN-18.2 polypeptide is at least 50% or more higher than the binding affinity for a CLDN18.1 polypeptide, including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher. In some embodiments, the binding affinity of such an antibody agent for a CLDN-18.2 polypeptide is higher than the binding affinity for a CLDN18.1 polypeptide by the following multiples: at least 1.1-fold or more, including, for example, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10,000-fold or higher. In some embodiments, such an antibody agent does not detectably bind to any other claudin family member (including CLDN18.1). In some embodiments, the antibody agent can be an antibody or comprise an antibody. In some embodiments, the antibody agent can be an antigen-binding fragment or comprise an antigen-binding fragment.

[0195] In some embodiments, an antibody agent targeting CLDN-18.2 (and which can be encoded by an RNA such as ssRNA, such as the mRNA described herein) specifically binds to the first extracellular domain (ECD1) of the CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an ECD1 epitope exposed in cancer cells.

[0196] In some embodiments, at least one RNA encodes the variable heavy chain (V H ) domain of a CLDN-18.2-targeting antibody agent and the variable light chain (V L ) domain of the antibody agent. In some embodiments, such V H domains and V L domains of the CLDN-18.2-targeting antibody agent can be encoded by a single RNA construct; alternatively, in some embodiments, they can be encoded separately by at least two separate RNA constructs. For example, in some embodiments, the RNA as used herein comprises two or more coding regions, which comprise a heavy chain coding region encoding at least the V H domain of the CLDN-18.2-targeting antibody agent; and a light chain coding region encoding at least the V L domain of the CLDN-18.2-targeting antibody agent. In some alternative embodiments, the composition comprises (i) a first RNA comprising a heavy chain coding region encoding at least the V H domain of the CLDN-18.2-targeting antibody agent; and (ii) a second RNA comprising a light chain coding region encoding at least the V L domain of the CLDN-18.2-targeting antibody agent.

[0197] In some embodiments, the heavy chain coding region may further encode a constant heavy chain (C H ) domain; and / or the light chain coding region may further encode a constant light chain (C L ) domain. For example, in some embodiments, the heavy chain coding region may encode the V H domain, C H1 domain, C H2 domain, and C H3 domain of a CLDN-18.2-targeting antibody agent in immunoglobulin form (e.g., IgG); and / or the light chain coding region may encode the V L domain and C LDomains. For example, in some embodiments, a full-length immunoglobulin (Ig) can be encoded by a single RNA that contains a first coding region encoding the heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG) and a second coding region encoding the light chain variable domain of a CLDN-18.2 Ig antibody (e.g., IgG), where the single RNA requires protein translation to produce a fusion protein containing the heavy and light chains of the antibody, and post-translational cleavage of the fusion protein by a suitable protease into the corresponding heavy and light chains, which can then be processed to form a full-length Ig (e.g., IgG). In some embodiments, the full-length Ig can be encoded by two separate RNAs: a first RNA that contains the coding region encoding the heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG); and a second RNA that contains the coding region encoding the light chain of a CLDN-18.2 Ig antibody (e.g., IgG). Such first and second RNAs are then translated in a target cell into the corresponding antibody chains and a full-length Ig antibody (e.g., IgG) is formed. In some embodiments, the antibody agent in the form of IgG encoded by one or more RNAs is IgG1.

[0198] In some embodiments, the heavy chain coding region of the RNA consists of or contains a nucleotide sequence encoding at least one CDR selected from the group consisting of, for example, 1 CDR, 2 CDRs, and 3 CDRs: (i) CDR1 represented by the amino acid residues (GYTFTSYW); (ii) CDR2 represented by the amino acid residues (IYPSDSYT); and (iii) CDR3 represented by the amino acid residues (TRSWRGNSFDY). In some embodiments, the light chain coding region of the RNA consists of or contains a nucleotide sequence encoding at least one CDR selected from the group consisting of, for example, 1 CDR, 2 CDRs, and 3 CDRs: (i) CDR1 represented by the amino acid residues (QSLLNSGNQKNY); (ii) CDR2 represented by the amino acid residues (WAS); and (iii) CDR3 represented by the amino acid residues (QNDYSYPFT).

[0199] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 20 to 467 of SEQ ID NO:1. In some embodiments, one or more amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:1 (e.g., to reduce immunogenicity and / or stability). For example, in some embodiments, SEQ ID NO:1 may comprise at least one or more (including, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more) amino acid modifications for one or more non-CDR regions (which include, for example, amino acid insertions, deletions, and / or substitutions). In some embodiments, no more than 50 (including, for example, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 or fewer) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:1. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 21 to 240 of SEQ ID NO:2. In some embodiments, one or more amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:2 (e.g., to reduce immunogenicity and / or stability). For example, in some embodiments, SEQ ID NO:2 may comprise at least one or more (including, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more) amino acid modifications for one or more non-CDR regions (which include, for example, amino acid insertions, deletions, and / or substitutions). In some embodiments, no more than 50 (including, for example, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 or fewer) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:2.

[0200] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2.

[0201] In some embodiments, the heavy chain coding region of the RNA consists of or comprises: a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID NO:3. In some embodiments, one or more amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:3 (e.g., to reduce immunogenicity and / or stability). For example, in some embodiments, SEQ ID NO:3 may comprise at least one or more (including, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more) amino acid modifications for one or more non-CDR regions (which include, for example, amino acid insertions, deletions, and / or substitutions). In some embodiments, no more than 50 (which include, for example, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 or fewer) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:3. In some embodiments, the light chain coding region of the RNA consists of or comprises: a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 27 to 246 of SEQ ID NO:4. In some embodiments, one or more amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:4 (e.g., to reduce immunogenicity and / or stability). For example, in some embodiments, SEQ ID NO:4 may comprise at least one or more (including, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more) amino acid modifications for one or more non-CDR regions (which include, for example, amino acid insertions, deletions, and / or substitutions). In some embodiments, no more than 50 (which include, for example, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 or fewer) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:4.

[0202] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:4.

[0203] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full-length heavy chain of zolbetuximab or claudiximab (e.g., as described and / or exemplified herein). In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full-length light chain of zolbetuximab or claudiximab.

[0204] In some embodiments, one or more RNAs can be used to encode a bispecific or multispecific antibody agent that binds to two or more target molecules, e.g., one of which is a CLDN-18.2 polypeptide. For example, Figure 12 A shows an exemplary bispecific antibody encoded by one or more RNAs. See also, e.g., Stadler et al. (2016) Oncoimmunology 5(3):e1091555; and / or in Stadler et al. (2017) Nature Medicine 23(7):815-817. In some embodiments, a bivalent antibody agent can be encoded by a single RNA comprising a first coding region encoding a single-chain variable fragment (scFv) that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a second coding region encoding an scFv of a second target (e.g., in some embodiments, it can be a T cell receptor). In some embodiments, a bivalent antibody agent can be encoded by two separate RNAs: a first RNA that comprises a coding region encoding an scFv that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a coding region encoding a heavy chain antigen-binding fragment (Fab) of a second target (e.g., in some embodiments, it can be a T cell receptor); and a second RNA that comprises a coding region encoding an scFv targeting a CLDN-18.2 polypeptide and a coding region encoding a light chain Fab of the same second target. In some embodiments, a bivalent antibody agent can be encoded by two separate RNAs: a first RNA that comprises a coding region encoding an scFv of a first target (e.g., in some embodiments, it can be a T cell receptor) and a coding region encoding a heavy chain antigen-binding fragment (Fab) that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide); and a second RNA that comprises a coding region encoding an scFv of the same first target and a coding region encoding a light chain Fab targeting a CLDN-18.2 polypeptide. Such first and second RNAs are then translated in a target cell into subunits of the antibody and a bispecific antibody is formed.

[0205] Secretory signal coding region: In some embodiments, the RNA encoding a CLDN-18.2 targeting antibody agent may comprise a secretory signal coding region. In some embodiments, such a secretory signal coding region allows the CLDN-18.2 targeting antibody agent encoded by one or more RNAs to be secreted by cells such as those present in a subject to be treated after translation, thereby generating a certain plasma concentration of the bioactive CLDN-18.2 targeting antibody agent. In some embodiments, the secretory signal coding region contained in the RNA consists of or comprises a nucleotide sequence encoding a non-human secretory signal. For example, in some embodiments, such a non-human secretory signal may be a murine secretory signal, which in some embodiments may be the amino acid sequence of MGWSCIILFLVATATGVHS or MESQTQVLMSLLFWVSGTCG or comprise the amino acid sequence of MGWSCIILFLVATATGVHS or MESQTQVLMSLLFWVSGTCG. In some embodiments, the secretory signal coding region contained in the RNA consists of or comprises a nucleotide sequence encoding a human secretory signal, which in some embodiments may be the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the secretory signal coding region contained in the RNA encoding the heavy chain domain of the CLDN-18.2 targeting antibody agent may comprise: (i) a nucleotide sequence encoding a murine secretory signal amino acid sequence, which in some embodiments may be the amino acid sequence of MGWSCIILFLVATATGVHS or comprise the amino acid sequence of MGWSCIILFLVATATGVHS; or (ii) a nucleotide sequence encoding a human secretory signal amino acid sequence, which in some embodiments may be the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS.In some embodiments, the secretion signal coding region contained in the RNA encoding the light chain domain of a CLDN-18.2 targeting antibody agent may comprise: (i) a nucleotide sequence encoding a murine secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MESQTQVLMSLLFWVSGTCG or an amino acid sequence comprising MESQTQVLMSLLFWVSGTCG; or (ii) a nucleotide sequence encoding a human secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or an amino acid sequence comprising MRVMAPRTLILLLSGALALTETWAGS.

[0206] In some embodiments, the RNA encoding a CLDN-18.2 targeting antibody agent may comprise at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). Some examples of non-coding sequence elements include but are not limited to 3' untranslated region (UTR), 5' UTR, a cap structure for co-transcriptional capping of mRNA, a polyadenine (poly A) tail, and any combination thereof.

[0207] UTR (5' UTR and / or 3' UTR): In some embodiments, the provided RNA may comprise a nucleotide sequence encoding a desired 5' UTR and / or a desired 3' UTR. Those skilled in the art will understand that the untranslated regions of an mRNA sequence (e.g., 3' UTR and / or 5' UTR) can contribute to mRNA stability, mRNA localization, and / or translation efficiency.

[0208] In some embodiments, the provided RNA may comprise a 5' UTR nucleotide sequence and / or a 3' UTR nucleotide sequence. In some embodiments, such a 5' UTR sequence may be operably linked 3' to a coding sequence (e.g., which comprises one or more coding regions). As a supplement or alternative, in some embodiments, a 3' UTR sequence may be operably linked 5' to a coding sequence (e.g., which comprises one or more coding regions).

[0209] In some embodiments of any aspect described herein, the 5' and 3' UTR sequences contained in the RNA may consist of or contain 5' and 3' UTR sequences that are native or endogenous to the open reading frame of the gene of interest. Alternatively, in some embodiments, the 5' and / or 3' UTR sequences contained in the RNA are not endogenous to the coding sequence (e.g., which contains one or more coding regions); in some such embodiments, such 5' and / or 3' UTR sequences can be used to modify the stability and / or translation efficiency of the transcribed RNA sequence. For example, those skilled in the art will understand that AU-rich elements in the 3' UTR sequence can reduce the stability of mRNA. Thus, as those skilled in the art will understand, 3' and / or 5' UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs known in the art.

[0210] For example, those skilled in the art will understand that in some embodiments, a nucleotide sequence consisting of or containing a Kozak sequence of the open reading frame sequence of the gene of interest or nucleotide sequence can be selected and used as the nucleotide sequence encoding the 5' UTR. As those skilled in the art will understand, Kozak sequences are known to increase the translation efficiency of some RNA transcripts, but not all RNAs necessarily require a Kozak sequence for efficient translation. In some embodiments, the provided RNA polynucleotide may contain a nucleotide sequence encoding a 5' UTR derived from an RNA virus, the RNA genome of which is stable in cells. In some embodiments, various modified ribonucleotides (e.g., as described herein) can be used for the 3' and / or 5' UTRs, for example to prevent exonucleolytic degradation of the transcribed RNA sequence.

[0211] In some embodiments, the 5' UTR contained in the RNA can be derived from human α-globin mRNA in combination with the Kozak region.

[0212] In some embodiments, the RNA may comprise one or more 3’UTRs. For example, in some embodiments, the RNA may comprise two copies of the 3’-UTR derived from globin mRNA (e.g., such as α2-globin, α1-globin, β-globin (e.g., human β-globin) mRNA). In some embodiments, two copies of the 3’UTR derived from human β-globin mRNA may be used. For example, in some embodiments, it may be placed between the coding sequence of the RNA and the poly(A) tail to increase the protein expression level and / or extend the persistence of the RNA. In some embodiments, the 3’UTR comprised in the RNA may be or comprise one or more (e.g., 1, 2, 3 or more) of the 3’UTR sequences disclosed in WO 2017 / 060314 (the entire content of which is incorporated herein by reference for the purposes described herein). In some embodiments, the 3’-UTR may be a combination (FI element) of at least two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (designated F) and mitochondrially encoded 12S ribosomal RNA (designated I). These were identified by an in vitro selection process of sequences that confer RNA stability and enhanced total protein expression (see WO 2017 / 060314, incorporated herein by reference).

[0213] In some embodiments, the 5’-UTR comprises the nucleotide sequence of SEQ ID NO:18 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95% or 90% identity to the nucleotide sequence of SEQ ID NO:18 or 20.

[0214] In some embodiments, the 3’-UTR comprises the nucleotide sequence of SEQ ID NO:19 or 21, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95% or 90% identity to the nucleotide sequence of SEQ ID NO:19 or 21.

[0215] PolyA Tail: In some embodiments, the provided RNA may comprise a nucleotide sequence encoding a polyA tail. A polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, the length of which can vary (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenosine nucleotides. In some embodiments, the polyA tail is a nucleotide sequence comprising at least 30 adenosine nucleotides or more adenosine nucleotides, including, for example, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120 or more adenosine nucleotides. In some embodiments, the polyA tail is a polyA homopolymer tail or comprises a polyA homopolymer tail. In some embodiments, the polyA tail may comprise one or more modified adenosine nucleosides, including but not limited to cordiocipin and 8-azidoadenosine. In some embodiments, the polyA tail may comprise one or more non-adenosine nucleotides. In some embodiments, the polyA tail may be or comprise the following: a disrupted or modified polyA tail as described in WO 2016 / 005324 (the entire content of which is incorporated herein by reference for the purposes described herein). For example, in some embodiments, the polyA tail comprised in the RNA described herein may be a modified polyA sequence or comprise a modified polyA sequence, the modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 consecutive A nucleotides, which is 5' to the linker sequence; and a second sequence of at least 20 consecutive A nucleotides, which is 3' to the linker sequence. In some embodiments, the modified polyA sequence may comprise: a linker sequence that is not a polyA sequence and that comprises at least ten nucleotides (e.g., T, G, and / or C nucleotides); a first sequence of at least 30 consecutive A nucleotides, which is 5' to the linker sequence; and a second sequence of at least 70 consecutive A nucleotides, which is 3' to the linker sequence.

[0216] In some embodiments, no nucleotide other than an A nucleotide flanks the 3' end of the polyA tail, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0217] 5’ Cap: In some embodiments, the RNA described herein may comprise a 5’ cap, which may be incorporated into such RNA during transcription or ligated to such RNA after transcription. In some embodiments, the RNA may comprise a 5’ cap structure for co-transcriptional capping of the RNA. Some examples of cap structures for co-transcriptional capping are known in the art and include, for example, as described in WO 2017 / 053297, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, the 5’ cap contained in the RNA described herein is m7G(5′)ppp(5′)(2′OMeA)pG or comprises m7G(5′)ppp(5′)(2′OMeA)pG. In some embodiments, the 5’ cap contained in the RNA described herein is a cap 1 structure [e.g., m 2 7,3'-O Gppp(m 1 2'-O )ApG] or comprises a cap 1 structure [e.g., m 2 7,3'-O Gppp(m 1 2'-O )ApG]. When the RNA sequence described herein has a 5’ end with the nucleotide 5’-AG and it is described that the RNA comprises a 5’ cap containing the second and third nucleotides A and G, respectively [e.g., m 2 7,3'-O Gppp(m 1 2'-O )ApG], it should be understood that in some embodiments, the second and third nucleotides of the cap correspond to the nucleotides 5’-AG of the RNA sequence.

[0218] Chemical Modification: In some embodiments, the RNA encoding the CLDN-18.2 targeting antibody agent may comprise at least one modified ribonucleotide. For example, in some embodiments, to improve the stability of such RNA and / or reduce the immunogenicity of such RNA and / or reduce the cytotoxicity of such RNA. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of the RNA may be replaced with a modified ribonucleotide. For example, in some embodiments, some or all of the cytidine residues present in the RNA may be replaced with a modified cytidine, which in some embodiments may be, for example, 5-methylcytidine. As an alternative or in addition, in some embodiments, some or all of the uridine residues present in the RNA may be replaced with a modified uridine, which in some embodiments may be: 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halogenated-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-hydroxyacetate (cmo5U), methyl uridine 5-hydroxyacetate (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxymethylhydroxymethyl-uridine (chm5U), methyl 5-carboxymethylhydroxymethyl-uridine (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine (τm5U), 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine (τm5s2U), 1-tauromethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylviny)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, some or all of the uridine residues present in the RNA can be replaced with a modified uridine selected from: pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof. In some embodiments, some or all of the uridine residues present in the RNA can be replaced with pseudouridine or a derivative thereof (e.g., 1-methylpseudouridine). In some embodiments, some or all of the uridine residues present in the RNA can be replaced with pseudouridine. In some embodiments, some or all of the uridine residues present in the RNA can be replaced with 1-methylpseudouridine. In some embodiments, all of the uridine residues present in the RNA are replaced with pseudouridine. In some embodiments, all of the uridine residues present in the RNA are replaced with 1-methylpseudouridine.,

[0219] Codon Optimization and GC Enrichment: The codons of the RNA (especially mRNA) described in the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or replace rare codons in the cell (or subject) in which the target peptide or polypeptide is to be expressed with synonymous common codons in the cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (especially mRNA) described in the present disclosure is encoded by such a coding sequence that is codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In some embodiments, codon optimization and / or the increase in G / C content preferably do not change the sequence of the encoded amino acid sequence. In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (especially mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of the wild-type RNA, wherein the amino acid sequence encoded by this RNA is preferably not modified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of the RNA. A sequence with an increased G (guanosine) / C (cytosine) content is more stable than a sequence with an increased A (adenosine) / U (uracil) content. Regarding the fact that several codons encode one and the same amino acid (so-called genetic code degeneracy), the codons most favorable for stability (so-called alternative codon usage) can be determined. Depending on the amino acid encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U or contain a lower content of A and / or U nucleotides. In several embodiments, the G / C content of the coding region of the RNA (especially mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% or even more compared to the G / C content of the coding region of the wild-type RNA.

[0220] Non-Immunogenic RNA: In certain embodiments, by incorporating modified nucleosides that inhibit the activation of RNA-mediated innate immune receptors into the RNA and / or restricting double-stranded RNA The amount of (dsRNA) (e.g., by limiting the formation of double-stranded RNA (dsRNA) during in vitro transcription and / or by removing double-stranded RNA (dsRNA) after in vitro transcription, for example) renders the RNA described herein non-immunogenic. In certain embodiments, the non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides that inhibit the activation of RNA-mediated innate immune receptors and / or by removing double-stranded RNA (dsRNA) after in vitro transcription, for example.

[0221] To render non-immunogenic non-immunogenic RNA (especially mRNA) by incorporating modified nucleosides, any modified nucleoside can be used as long as it reduces or inhibits the immunogenicity of the RNA. Particularly preferred are modified nucleosides that inhibit the activation of RNA-mediated innate immune receptors. In some embodiments, the modified nucleoside comprises the replacement of one or more uridines with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside containing the modified nucleobase is selected from: 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-hydroxyacetate (cmo 5 U), methyl uridine 5-hydroxyacetate (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), methyl 5-carboxyhydroxymethyl-uridine (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine (τm 5 U), 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine (τm5s2U), 1-tauromethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0222] In some embodiments, replacing one or more uridines with a nucleoside comprising a modified nucleobase includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridines.

[0223] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, due to the unconventional activity of the enzyme, a large number of abnormal products are produced, including double-stranded RNA (dsRNA). DsRNA induces inflammatory cytokines and activates effector enzymes, resulting in the inhibition of protein synthesis. During the synthesis of mRNA by in vitro transcription (IVT), the formation of dsRNA can be restricted, for example, by restricting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP can be added one or more times during mRNA synthesis. Similarly, dsRNA can be removed from RNA (such as IVT RNA) by, for example, ion-pair reverse-phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using RNase III from Escherichia coli (E. coli) can be used, which specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from the IVT RNA preparation. In addition, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA preparation is contacted with the cellulose material, and the ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material but do not allow ssRNA to bind to the cellulose material. For example, suitable methods for providing ssRNA are disclosed in WO 2017 / 182524. In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, for example, removing dsRNA (especially dsmRNA) from non-immunogenic RNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA is dsRNA. In some embodiments, non-immunogenic RNA (especially mRNA) does not contain or is substantially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside-modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside-modified RNA (especially mRNA) and is substantially free of double-stranded RNA (dsRNA).In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997% or at least 99.998% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.). A variety of methods can be used to determine the amount of dsRNA. For example, a sample can be contacted with a dsRNA-specific antibody, and the amount of antibody bound to the RNA can be used as a measure of the amount of dsRNA in the sample. A sample containing a known amount of dsRNA can be used as a reference. For example, RNA can be spotted onto a membrane, such as a nylon blotting membrane. The membrane can be blocked, for example, in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v / v) Tween-20) containing 5% (w / v) non-fat dry milk powder. To detect dsRNA, the membrane can be incubated with a dsRNA-specific antibody such as a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirák, Hungary). After washing, for example, with TBS-T, the membrane can be incubated with a secondary antibody such as HRP-conjugated donkey anti-mouse IgG ((Jackson ImmunoResearch, catalog number 715-035-150), and the signal provided by the secondary antibody can be detected. In some embodiments, the non-immunogenic RNA (especially mRNA) is more efficient in translation in cells than a standard RNA with the same sequence. In some embodiments, the factor by which translation is enhanced relative to its unmodified counterpart is 2-fold. In some embodiments, translation is enhanced to a 3-fold factor. In some embodiments, translation is enhanced to a 4-fold factor. In some embodiments, translation is enhanced to a 5-fold factor. In some embodiments, translation is enhanced to a 6-fold factor. In some embodiments, translation is enhanced to a 7-fold factor. In some embodiments, translation is enhanced to an 8-fold factor. In some embodiments, translation is enhanced to a 9-fold factor. In some embodiments, translation is enhanced to a 10-fold factor. In some embodiments, translation is enhanced to a 15-fold factor. In some embodiments, translation is enhanced to a 20-fold factor. In some embodiments, translation is enhanced to a 50-fold factor. In some embodiments, translation is enhanced to a 100-fold factor. In some embodiments, translation is enhanced to a 200-fold factor. In some embodiments, translation is enhanced to a 500-fold factor. In some embodiments, translation is enhanced to a 1000-fold factor. In some embodiments, translation is enhanced to a 2000-fold factor.In some embodiments, the factor is from 10 to 1000 fold. In some embodiments, the factor is from 10 to 100 fold. In some embodiments, the factor is from 10 to 200 fold. In some embodiments, the factor is from 10 to 300 fold. In some embodiments, the factor is from 10 to 500 fold. In some embodiments, the factor is from 20 to 1000 fold. In some embodiments, the factor is from 30 to 1000 fold. In some embodiments, the factor is from 50 to 1000 fold. In some embodiments, the factor is from 100 to 1000 fold. In some embodiments, the factor is from 200 to 1000 fold. In some embodiments, the translation is enhanced to any other significant amount or range of amounts. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower innate immunogenicity than standard RNA with the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is one-half lower than its unmodified counterpart. In some embodiments, the innate immunogenicity is reduced to one-third. In some embodiments, the innate immunogenicity is reduced to one-fourth. In some embodiments, the innate immunogenicity is reduced to one-fifth. In some embodiments, the innate immunogenicity is reduced to one-sixth. In some embodiments, the innate immunogenicity is reduced to one-seventh. In some embodiments, the innate immunogenicity is reduced to one-eighth. In some embodiments, the innate immunogenicity is reduced to one-ninth. In some embodiments, the innate immunogenicity is reduced to one-tenth. In some embodiments, the innate immunogenicity is reduced to one-fifteenth. In some embodiments, the innate immunogenicity is reduced to one-twentieth. In some embodiments, the innate immunogenicity is reduced to one-fiftieth. In some embodiments, the innate immunogenicity is reduced to one-hundredth. In some embodiments, the innate immunogenicity is reduced to one-two-hundredth. In some embodiments, the innate immunogenicity is reduced to one-five-hundredth. In some embodiments, the innate immunogenicity is reduced to one-one-thousandth. In some embodiments, the innate immunogenicity is reduced to one-two-thousandth. The term "exhibits significantly lower innate immunogenicity" means a detectable reduction in innate immunogenicity. In some embodiments, the term means a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term means a reduction such that non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA.

[0224] In some embodiments, the RNA encoding the heavy chain of the CLDN-18.2 targeting antibody agent comprises, in the 5' to 3' direction: (a) a 5' UTR; (b) a secretion signal coding region; (c) a heavy chain coding region; (d) a 3' UTR; and (e) a polyA tail. See, for example Figure 13 . In some embodiments, the 5' UTR is a sequence derived from human α-globin mRNA in combination with a Kozak region or comprises a sequence derived from human α-globin mRNA in combination with a Kozak region. In some embodiments, the secretion signal coding region is a nucleotide sequence encoding the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS or comprises a nucleotide sequence encoding the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the heavy chain coding region encodes the V H domain, C H1 domain, C H2 domain, and C H3 domain of an IgG-form CLDN-18.2 targeting antibody agent (e.g., those as described herein, such as IMAB262), or the amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID NO:3. In some embodiments, the 3' UTR is the following or comprises the following: a combination of at least two sequence elements from the "split aminoterminal enhancer" (AES) mRNA (designated F) and mitochondrially encoded 12S ribosomal RNA (designated I) (FI element). In some embodiments, the polyA tail is the following or comprises the following: a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately after 30 consecutive adenosines). In some embodiments, such RNA comprises a 5' cap structure, which comprises a CAP1 structure, or m 2 7，3’...

Claims

1. A composition or pharmaceutical preparation, comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein (i) the coding region comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO:16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO:16, and (ii) the coding region comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:

17.

2. The composition or pharmaceutical preparation according to claim 1, wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:

4.

3. A composition or pharmaceutical preparation, comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:

4.

4. The composition or pharmaceutical preparation according to any one of claims 1 to 3, wherein the RNA, such as each RNA, comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:

20.

5. The composition or pharmaceutical preparation according to any one of claims 1 to 4, wherein the RNA, such as each RNA, comprises a 5' UTR, and the 5' UTR comprises the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:

20.

6. The composition or pharmaceutical preparation according to any one of claims 1 to 5, wherein the RNA, such as each RNA, comprises a 5' UTR, and the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 18 or 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18 or 20.

7. The composition or pharmaceutical preparation according to any one of claims 1 to 6, wherein the RNA, such as each RNA, comprises a 3' UTR, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

22.

8. The composition or pharmaceutical preparation according to any one of claims 1 to 7, wherein the RNA, such as each RNA, comprises a 3' UTR, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 or 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19 or 21.

9. A composition or pharmaceutical preparation comprising: (i) an RNA comprising a coding region encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and (ii) an RNA comprising a coding region encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the RNA, such as each RNA, comprises a 5' UTR and / or a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 18 or 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18 or 20, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 or 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19 or 21.

10. The composition or pharmaceutical preparation according to any one of claims 1 to 9, wherein the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 18 or 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18 or 20, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 or 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19 or 21.

11. The composition or pharmaceutical preparation according to any one of claims 1 to 10, wherein the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

19.

12. The composition or pharmaceutical preparation according to any one of claims 1 to 11, wherein the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 20, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

21.

13. The composition or pharmaceutical preparation according to any one of claims 1 to 12, wherein the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 18, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO:

19.

14. The composition or pharmaceutical preparation according to any one of claims 1 to 12, wherein the RNA, such as each RNA, comprises a 5' UTR and a 3' UTR, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 20, and the 3' UTR comprises the nucleotide sequence of SEQ ID NO:

21.

15. The composition or pharmaceutical preparation according to any one of claims 9 to 14, wherein: (a) (i) the coding region described therein comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and (ii) the coding region described therein comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and / or (b) the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity with the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and The second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO:

4.

16. The composition or pharmaceutical preparation according to any one of claims 1 to 15, wherein (i) the coding region described therein comprises the nucleotide sequence of SEQ ID NO:16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:16, and (ii) the coding region described therein comprises the nucleotide sequence of SEQ ID NO:17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

17.

17. The composition or pharmaceutical preparation according to any one of claims 1 to 16, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

4.

18. The composition or pharmaceutical preparation according to any one of claims 1 to 17, wherein the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule.

19. The composition or pharmaceutical preparation according to any one of claims 1 to 18, wherein at least 90% is at least 95%, 96%, 97%, 98%, 99%.

20. The composition or pharmaceutical preparation according to any one of claims 1 to 19, wherein the antibody agent preferentially binds to CLDN-18.2 relative to claudin-18.1 (CLDN-18.1).

21. The composition or pharmaceutical preparation according to any one of claims 1 to 20, wherein the antibody agent binds to the first extracellular domain (ECD1) of CLDN-18.

2.

22. The composition or pharmaceutical preparation according to any one of claims 1 to 21, wherein the antibody agent binds to an epitope of ECD1 of CLDN-18.2 that is exposed in cancer cells.

23. The composition or pharmaceutical preparation according to any one of claims 1 to 22, wherein the antibody agent that binds to CLDN-18.2 comprises two binding arms, wherein each binding arm comprises a heavy chain of the antibody agent that binds to CLDN-18.2 and a light chain of the antibody agent that binds to CLDN-18.

2.

24. The composition or pharmaceutical preparation according to any one of claims 1 to 23, wherein the antibody agent is IgG1.

25. The composition or pharmaceutical preparation according to claim 24, wherein the IgG1 is human IgG1.

26. The composition or pharmaceutical preparation according to any one of claims 1 to 25, wherein the first polypeptide chain interacts with the second polypeptide chain to form a binding domain that binds to CLDN-18.

2.

27. The composition or pharmaceutical preparation according to any one of claims 1 to 26, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)).

28. The composition or pharmaceutical preparation according to claim 27, wherein the VH(CLDN-18.2) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:

14.

29. The composition or pharmaceutical preparation according to claim 27 or 28, wherein the VH(CLDN-18.2) comprises CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively.

30. The composition or pharmaceutical preparation according to any one of claims 1 to 29, wherein the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)).

31. The composition or pharmaceutical preparation according to claim 30, wherein the VL(CLDN-18.2) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:

15.

32. The composition or pharmaceutical preparation according to claim 30 or 31, wherein the VL(CLDN-18.2) comprises CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively.

33. The composition or pharmaceutical preparation according to any one of claims 1 to 32, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) and has CDR1, CDR2, and CDR3 containing the amino acid sequence of SEQ ID NO:14, and the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) and has CDR1, CDR2, and CDR3 containing the amino acid sequence of SEQ ID NO:

15.

34. The composition or pharmaceutical preparation according to any one of claims 1 to 33, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) and has CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) and has CDR1, CDR2, and CDR3 containing the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively.

35. The composition or pharmaceutical preparation according to any one of claims 1 to 34, wherein the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 and contains the amino acid sequence SEQ ID NO: 14 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 and contains the amino acid sequence of SEQ ID NO: 15 (VL(CLDN-18.2)).

36. The composition or pharmaceutical preparation according to any one of claims 1 to 35, wherein the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)), wherein the VH(CLDN-18.2) interacts with the VL(CLDN-18.2) to form a binding domain that binds to Claudin-18.2 (CLDN-18.2).

37. The composition or pharmaceutical preparation according to any one of claims 1 to 36, wherein the first polypeptide chain comprises a variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), a constant domain 1 (CH1) of the heavy chain of the antibody agent, a constant domain 2 (CH2) of the heavy chain of the antibody agent, and a constant domain 3 (CH3) of the heavy chain of the antibody agent.

38. The composition or pharmaceutical preparation according to claim 37, wherein the VH(CLDN-18.2), CH1, CH2, and CH3 exist in the first polypeptide chain in the form of immunoglobulin G (IgG).

39. The composition or pharmaceutical preparation according to any one of claims 1 to 38, wherein the second polypeptide chain comprises a variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) and a constant domain of the light chain of the antibody agent (CL).

40. The composition or pharmaceutical preparation according to claim 39, wherein the VL(CLDN-18.2) and CL exist in the second polypeptide chain in the form of IgG.

41. The composition or pharmaceutical preparation according to claim 39 or 40, wherein CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain.

42. The composition or pharmaceutical preparation according to any one of claims 1 to 41, wherein the first polypeptide chain and the second polypeptide chain each independently comprise a secretion signal, and preferably the secretion signal is located at the N-terminus of the first polypeptide chain and the second polypeptide chain.

43. The composition or pharmaceutical preparation according to claim 42, wherein the secretion signal of the first polypeptide chain and / or the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

13.

44. The composition or pharmaceutical preparation according to any one of claims 1 to 43, wherein (i) the coding region described therein comprises the nucleotide sequence of SEQ ID NO: 16, and (ii) the coding region described therein comprises the nucleotide sequence of SEQ ID NO:

17.

45. The composition or pharmaceutical preparation according to any one of claims 1 to 44, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

4.

46. The composition or pharmaceutical preparation according to any one of claims 1 to 45, wherein the RNA, such as each RNA, comprises a poly-A sequence.

47. The composition or pharmaceutical preparation according to claim 46, wherein the poly-A sequence is a discontinuous sequence of A nucleotides.

48. The composition or pharmaceutical preparation according to claim 46 or 47, wherein the poly-A sequence comprises at least 100 nucleotides.

49. A composition or pharmaceutical preparation according to any one of claims 46 to 48, wherein said poly-A sequence comprises the nucleotide sequence A x -L-A y or consists of the nucleotide sequence A x -L-A y wherein A x is a sequence of at least 20 A nucleotides, A y is a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides which may comprise nucleotides other than A.

50. The composition or pharmaceutical preparation according to any one of claims 46 to 49, wherein the poly-A sequence comprises the nucleotide sequence of SEQ ID NO: 23 or consists of the nucleotide sequence of SEQ ID NO:

23.

51. The composition or pharmaceutical preparation according to any one of claims 1 to 50, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 25 or 27.

52. The composition or pharmaceutical preparation according to claim 51, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO:

25.

53. The composition or pharmaceutical preparation according to claim 51, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO:

27.

54. A composition or pharmaceutical preparation, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27.

55. A composition or pharmaceutical preparation, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO:

25.

56. A composition or pharmaceutical preparation, which contains: (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) an RNA comprising the nucleotide sequence of SEQ ID NO:

27.

57. The composition or pharmaceutical preparation according to any one of claims 1 to 56, wherein the RNA, such as each RNA, comprises a modified nucleoside that replaces uridine.

58. The composition or pharmaceutical preparation according to any one of claims 1 to 57, wherein the RNA, such as each RNA, comprises a modified nucleoside that replaces every uridine.

59. The composition or pharmaceutical preparation according to claim 57 or 58, wherein the modified nucleoside is pseudouridine (ψ) and / or N1-methyl-pseudouridine (m1ψ).

60. The composition or pharmaceutical preparation according to any one of claims 57 to 59, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

61. The composition or pharmaceutical preparation according to any one of claims 1 to 60, wherein the RNA, such as each RNA, comprises a 5' cap.

62. The composition or pharmaceutical preparation according to any one of claims 1 to 61, wherein the RNA, such as each RNA, comprises a 5' cap m 2 7，3’-O Gpp(m 1 2’-O )ApG.

63. The composition or pharmaceutical preparation according to any one of claims 1 to 62, wherein the RNA, such as each RNA, is single-stranded RNA.

64. The composition or pharmaceutical preparation according to any one of claims 1 to 63, wherein the RNA, such as each RNA, is mRNA.

65. The composition or pharmaceutical preparation according to any one of claims 1 to 64, wherein the RNA, such as each RNA, is formulated in a lipid nanoparticle (LNP), such as each RNA is co-formulated in a lipid nanoparticle (LNP).

66. The composition or pharmaceutical preparation according to claim 65, wherein the lipids forming the lipid nanoparticle comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid.

67. The composition or pharmaceutical preparation according to claim 66 wherein: a. the cationic lipid is present at 35 mol% to 65 mol% of the total lipids; b. the polymer-conjugated lipid is present at about 1 mol% to 2.5 mol% of the total lipids; and c. the neutral lipid is present at 35 mol% to 65 mol% of the total lipids.

68. The composition or pharmaceutical preparation according to claim 66 or 67, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate).

69. The composition or pharmaceutical preparation according to any one of claims 66 to 68, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (such as 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide).

70. The composition or pharmaceutical preparation according to any one of claims 66 to 69, wherein the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

71. The composition or pharmaceutical preparation according to any one of claims 65 to 70, wherein the average size of the lipid nanoparticle is about 50 to 150 nm. The composition or pharmaceutical preparation according to any one of claims 65 to 71, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

73. The composition according to any one of claims 1 to 72, which is a pharmaceutical composition.

74. The composition according to claim 73, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

75. The pharmaceutical preparation according to any one of claims 1 to 72, which is a kit.

76. The pharmaceutical preparation according to claim 75, wherein the RNA, such as each RNA, and optionally the particle-forming components are in separate vials.

77. The pharmaceutical preparation according to claim 75 or 76, which further comprises instructions for using the composition or pharmaceutical preparation for the treatment or prevention of cancer.

78. The composition or pharmaceutical preparation according to any one of claims 1 to 77, which is for pharmaceutical use.

79. The composition or pharmaceutical preparation according to claim 78, wherein the pharmaceutical use comprises therapeutic or prophylactic treatment of a disease or disorder.

80. The composition or pharmaceutical preparation according to claim 79, wherein the therapeutic or prophylactic treatment of the disease or disorder comprises treating or preventing cancer.

81. The composition or pharmaceutical preparation according to claim 80, wherein the cancer comprises CLDN-18.2 positive cancer.

82. The composition or pharmaceutical preparation according to claim 80 or 81, wherein the cancer comprises CLDN-18.2 positive solid tumors.

83. The composition or pharmaceutical preparation according to any one of claims 80 to 82, wherein the cancer includes CLDN-18.2 positive pancreatic cancer.

84. The composition or pharmaceutical preparation according to any one of claims 80 to 83, wherein the cancer includes CLDN-18.2 positive gastric cancer.

85. The composition or pharmaceutical preparation according to any one of claims 80 to 84, wherein the cancer includes CLDN-18.2 positive biliary tract tumors.

86. The composition or pharmaceutical preparation according to any one of claims 80 to 85, wherein the cancer includes locally advanced, unresectable, or metastatic cancer that is CLDN-18.2 positive.

87. The composition or pharmaceutical preparation according to any one of claims 79 to 86, wherein the therapeutic or prophylactic treatment of the disease or disorder further comprises administering an additional treatment.

88. The composition or pharmaceutical preparation according to claim 87, wherein the additional treatment includes one or more selected from the following: (i) surgery to excise, resect, or debulk the tumor, (ii) radiotherapy, and (iii) chemotherapy.

89. The composition or pharmaceutical preparation according to claim 87 or 88, wherein the additional treatment includes administering an additional therapeutic agent.

90. The composition or pharmaceutical preparation according to claim 89, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.

91. The composition or pharmaceutical preparation according to any one of claims 1 to 90, which is for administration to a human.

92. The composition or pharmaceutical preparation according to any one of claims 1 to 91, which is for intravenous administration.

93. A method for treating cancer in a subject, which comprises administering to the subject a composition according to any one of claims 1 to 74.

94. The method according to claim 93, wherein the cancer comprises CLDN-18.2 positive cancer.

95. The method according to claim 93 or 94, wherein the cancer comprises CLDN-18.2 positive solid tumor.

96. The method according to any one of claims 93 to 95, wherein the cancer comprises CLDN-18.2 positive pancreatic cancer.

97. The method according to any one of claims 93 to 96, wherein the cancer comprises CLDN-18.2 positive gastric cancer.

98. The method according to any one of claims 93 to 97, wherein the cancer comprises CLDN-18.2 positive biliary tract tumor.

99. The method according to any one of claims 93 to 98, wherein the cancer comprises locally advanced, unresectable or metastatic cancer that is CLDN-18.2 positive.

100. The method according to any one of claims 93 to 99, which further comprises administering an additional treatment.

101. The method according to claim 100, wherein the additional treatment comprises one or more selected from the following: (i) surgery to excise, remove the tumor or reduce its volume, (ii) radiotherapy, and (iii) chemotherapy.

102. The method according to claim 100 or 101, wherein the additional treatment comprises administering an additional therapeutic agent.

103. The method according to claim 102, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.

104. The method according to any one of claims 93 to 103, wherein the subject is a human.

105. The method according to any one of claims 93 to 104, wherein the composition is administered intravenously.

106. The composition according to any one of claims 1 to 74, which is for use in the method according to any one of claims 93 to 105.

107. The composition or pharmaceutical preparation according to any one of claims 1 to 92, which is for introducing the RNA into hepatocytes and expressing a polypeptide chain encoded by the RNA in the hepatocytes.

108. The composition or pharmaceutical preparation according to any one of claims 1 to 92, which is for systemic delivery of the polypeptide chain.

109. The composition or pharmaceutical preparation according to any one of claims 1 to 92, which is for systemic delivery of the polypeptide chain after the polypeptide chain is expressed in hepatocytes.

110. A method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprises: (a) administering a composition according to any one of claims 1 to 74 such that the RNA is introduced into hepatocytes; and (b) Express the polypeptide chain encoded by the RNA in the hepatocytes.

111. A method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprising: (a) Administering the composition according to any one of claims 1 to 74 such that the RNA is introduced into hepatocytes; and (b) Expressing the polypeptide chain encoded by the RNA in the hepatocytes, wherein, after expression, the polypeptide chain is secreted into the bloodstream.

112. A method for systemically delivering an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, the method comprising: (a) Administering the composition according to any one of claims 1 to 74 such that the RNA is introduced into hepatocytes; and (b) Expressing the polypeptide chain encoded by the RNA in the hepatocytes, wherein, after expression, the polypeptide chain is secreted into the bloodstream.

113. The method according to any one of claims 110 to 112, wherein the administration is parenteral administration.

114. The method according to any one of claims 110 to 113, wherein the administration is intravenous administration.

115. A composition or pharmaceutical preparation comprising RNA, wherein the RNA comprises: (i) A coding sequence encoding a polypeptide, (ii) A 3'UTR sequence, (iii) A poly-A sequence, and (iv) A nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, which comprises the sequence CUXGAGCUAGC, where X is C, A or U.

116. The composition or pharmaceutical preparation according to claim 115, wherein the nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence comprises the sequence CUXGAGCUAGC.

117. The composition or pharmaceutical preparation according to claim 115 or 116, wherein the RNA comprises, in the 5' to 3' direction, the coding sequence encoding the polypeptide, the 3'UTR sequence, the nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, and the poly-A sequence.

118. The composition or pharmaceutical preparation according to any one of claims 115 to 117, wherein the 3'UTR sequence comprises the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

22.

119. The composition or pharmaceutical preparation according to any one of claims 115 to 118, wherein the RNA comprises a 3'UTR, and the 3'UTR comprises the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:

36.

120. The composition or pharmaceutical preparation according to any one of claims 115 to 118, wherein the RNA comprises a 3'UTR, and the 3'UTR comprises the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:

37.

121. The composition or pharmaceutical preparation according to any one of claims 115 to 120, wherein the poly-A sequence is a discontinuous sequence of A nucleotides.

122. The composition or pharmaceutical preparation according to any one of claims 115 to 121, wherein the poly-A sequence comprises at least 100 nucleotides. The composition or pharmaceutical preparation according to any one of claims 115 to 122, wherein the poly-A sequence comprises the nucleotide sequence A x -L-A y or consists of the nucleotide sequence A x -L-A y wherein A x is a sequence of at least 20 A nucleotides, A y is a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides which may comprise nucleotides other than A.

124. The composition or pharmaceutical preparation according to any one of claims 115 to 123, wherein the poly-A sequence comprises the nucleotide sequence of SEQ ID NO:23 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:23 or consists of the nucleotide sequence of SEQ ID NO:23 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:

23.

125. The composition or pharmaceutical preparation according to any one of claims 115 to 124, wherein the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:

20.

126. The composition or pharmaceutical preparation according to any one of claims 115 to 125, wherein the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4XAACUAGU, wherein X1 is any nucleotide, preferably A or C, X2 is any nucleotide, preferably A or C, X3 is any nucleotide, preferably C, U or G, and X4 is A or absent.

127. The composition or pharmaceutical preparation according to any one of claims 115 to 126, wherein the RNA comprises a 5'UTR, and the 5'UTR comprises the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, wherein X1 is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.

128. The composition or pharmaceutical preparation according to any one of claims 115 to 127, wherein the RNA comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU.

129. The composition or pharmaceutical preparation according to any one of claims 115 to 127, wherein the RNA comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU.

130. The composition or pharmaceutical preparation according to any one of claims 115 to 129, wherein the RNA comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:

20.

131. The composition or pharmaceutical preparation according to any one of claims 115 to 128 and 130, wherein the RNA comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

20.

132. The composition or pharmaceutical preparation according to any one of claims 115 to 127, 129 and 130, wherein the RNA comprises a 5'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

38.

133. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR, which comprises the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.

134. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36; and a poly-A sequence.

135. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

36.

136. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36; and a poly-A sequence.

137. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

36.

138. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of positions 7 to 53 of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36; and a poly-A sequence. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36; and a poly-A sequence. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:

36. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 298 of SEQ ID NO: 36; and a poly-A sequence. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:

36. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of positions 7 to 53 of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of positions 7 to 53 of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 295 of SEQ ID NO: 37 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of positions 1 to 295 of SEQ ID NO: 37; and a poly-A sequence. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of positions 1 to 295 of SEQ ID NO: 37 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of positions 1 to 295 of SEQ ID NO: 37; and a poly-A sequence.

145. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:

37.

146. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37; and a poly-A sequence.

147. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37; and a poly-A sequence.

148. The composition or pharmaceutical preparation according to any one of claims 115 to 132, wherein the RNA comprises: a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:

37.

149. The composition or pharmaceutical preparation according to any one of claims 118 to 142, wherein at least 90% is at least 95%, 96%, 97%, 98% or 99%.

150. The composition or pharmaceutical preparation according to any one of claims 115 to 149, wherein the RNA comprises two or more coding sequences encoding two or more polypeptides.

151. The composition or pharmaceutical preparation according to any one of claims 115 to 150, wherein the RNA does not encode a polypeptide that binds to claudin-6 (CLDN-6) and / or CD3.

152. The composition or pharmaceutical preparation according to any one of claims 115 to 151, wherein the RNA does not encode one or more polypeptide chains of an agent that binds to claudin-6 (CLDN-6) and / or CD3.

153. The composition or pharmaceutical preparation according to any one of claims 115 to 152, wherein the RNA does not encode a cytokine.

154. The composition or pharmaceutical preparation according to any one of claims 115 to 153, wherein the RNA does not encode IL2 and / or IL7.

155. The composition or pharmaceutical preparation according to any one of claims 115 to 154, wherein the RNA does not encode a polypeptide that binds to HIV.

156. The composition or pharmaceutical preparation according to any one of claims 115 to 155, wherein the RNA does not encode one or more polypeptide chains of a binder that binds to HIV.

157. The composition or pharmaceutical preparation according to any one of claims 115 to 156, wherein the RNA does not encode a polypeptide that binds to Claudin-18.2 (CLDN-18.2).

158. The composition or pharmaceutical preparation according to any one of claims 115 to 157, wherein the RNA does not encode one or more polypeptide chains of a binder that binds to Claudin-18.2 (CLDN-18.2).

159. The composition or pharmaceutical preparation according to any one of claims 115 to 158, wherein the RNA encodes an antibody or antibody-like molecule.

160. The composition or pharmaceutical preparation according to any one of claims 115 to 159, wherein the RNA comprises at least two, such as two RNA molecules, and at least one of the RNA molecules, such as all of the RNA molecules, comprises a 5’ UTR, 3’ UTR, 3’ UTR sequence, poly-A sequence and / or a nucleotide sequence connecting the 3’ UTR sequence and the poly-A sequence as defined.

161. The composition or pharmaceutical preparation according to any one of claims 115 to 160, wherein the RNA contains: (i) RNA comprising a coding sequence encoding a first polypeptide chain, the first polypeptide chain comprising a heavy chain of an antibody agent, and (ii) RNA comprising a coding sequence encoding a second polypeptide chain, the second polypeptide chain comprising a light chain of an antibody agent.

162. The composition or pharmaceutical preparation according to claim 161, wherein the RNA in (i) is a first RNA molecule and the RNA in (ii) is a second RNA molecule.

163. The composition or pharmaceutical preparation according to claim 161 or 162, wherein the antibody agent binds to Claudin-18.2 (CLDN-18.2).

164. The composition or pharmaceutical preparation according to any one of claims 115 to 163, wherein the RNA, such as each RNA, comprises a modified nucleoside that replaces uridine.

165. The composition or pharmaceutical preparation according to any one of claims 115 to 164, wherein the RNA, such as each RNA, comprises a modified nucleoside that replaces every uridine.

166. The composition or pharmaceutical preparation according to claim 164 or 165, wherein the modified nucleoside is pseudouridine (ψ) and / or N1-methyl-pseudouridine (m1ψ).

167. The composition or pharmaceutical preparation according to any one of claims 164 to 166, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

168. The composition or pharmaceutical preparation according to any one of claims 115 to 167, wherein the RNA, such as each RNA, comprises a 5’ cap. The composition or pharmaceutical preparation according to any one of claims 115 to 168, wherein the RNA, such as each RNA, comprises a 5'-cap m 2 7，3’ -OGppp(m 1 2’-O )ApG.

170. The composition or pharmaceutical preparation according to any one of claims 115 to 169, wherein the RNA, such as each RNA, is single-stranded RNA. The composition or pharmaceutical preparation according to any one of claims 115 to 170, wherein the RNA, such as each RNA, is mRNA. The composition or pharmaceutical preparation according to any one of claims 115 to 171, wherein the RNA, such as each RNA, is formulated in a lipid nanoparticle (LNP), such as each RNA is co-formulated in a lipid nanoparticle (LNP). The composition or pharmaceutical preparation according to claim 172, wherein the lipids forming the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid. The composition or pharmaceutical preparation according to claim 173, wherein: a. the cationic lipid is present at 35 mol% to 65 mol% of the total lipids; b. the polymer-conjugated lipid is present at about 1 mol% to 2.5 mol% of the total lipids; and c. the neutral lipid is present at 35 mol% to 65 mol% of the total lipids. The composition or pharmaceutical preparation according to claim 173 or 174, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate). The composition or pharmaceutical preparation according to any one of claims 173 to 175, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (such as 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide). The composition or pharmaceutical preparation according to any one of claims 173 to 176, wherein the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. The composition or pharmaceutical preparation according to any one of claims 172 to 177, wherein the average size of the lipid nanoparticles is about 50 to 150 nm. The composition or pharmaceutical preparation according to any one of claims 172 to 178, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyl octanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol. The composition according to any one of claims 115 to 179, which is a pharmaceutical composition. The composition according to claim 180, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients. The pharmaceutical preparation according to any one of claims 115 to 179, which is a kit. The pharmaceutical preparation according to claim 182, wherein the RNA, such as each RNA, and optionally the particle-forming components are in separate vials. The composition or pharmaceutical preparation according to any one of claims 115 to 183, which is for intravenous administration.

185. The composition or pharmaceutical preparation according to any one of claims 115 to 184, for introducing the RNA into hepatocytes and expressing a polypeptide encoded by the RNA in the hepatocytes.

186. The composition or pharmaceutical preparation according to any one of claims 115 to 185, for systemic delivery of the polypeptide.

187. The composition or pharmaceutical preparation according to any one of claims 115 to 186, for systemic delivery of the polypeptide after expression of the polypeptide in hepatocytes.

188. A method for expressing a polypeptide in a subject, the method comprising: (a) administering the composition according to any one of claims 115 to 181 such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes.

189. A method for expressing a polypeptide in a subject, the method comprising: (a) administering the composition according to any one of claims 115 to 181 such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes, wherein, after expression, the polypeptide is secreted into the bloodstream.

190. A method for systemic delivery of a polypeptide in a subject, the method comprising: (a) administering the composition according to any one of claims 115 to 181 such that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in the hepatocytes, wherein, after expression, the polypeptide is secreted into the bloodstream.

191. The method according to any one of claims 188 to 190, wherein the administration is parenteral administration.

192. The method according to any one of claims 188 to 191, wherein the administration is intravenous administration.

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