RNA compositions targeting claudin 18.2

Abstract

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

Description

[Background technology]

[0001] Cancer is the second leading cause of death worldwide, and is expected to be responsible for an estimated 9.6 million deaths in 2018 (Bray et al. 2018). In general, once solid tumors metastasize, 5-year survival rates rarely exceed 25%, with a few exceptions, such as germ cell and some carcinoid tumors.

[0002] Recent advances in conventional treatments, such as chemotherapy, radiation therapy, surgery, and targeted therapy, as well as immunotherapy, have improved outcomes for patients with advanced solid tumors. In recent years, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved eight checkpoint inhibitors (one monoclonal antibody targeting the CTLA-4 pathway, ipilimumab, and seven antibodies targeting programmed death receptor / ligand [PD / PD-L1], including atezolizumab, avelumab, durvalumab, nivolumab, cemiplimab, and pembrolizumab) for the treatment of patients with multiple cancer types, primarily solid tumors. These approvals have dramatically changed the landscape of cancer treatment. However, certain cancers, such as pancreatic adenocarcinoma or metastatic biliary tract cancer, have yet to benefit from existing treatments, including immunotherapy. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Bray et al. 2018 Summary of the Invention [Problem to be solved by the invention]

[0004] For example, the poor prognosis of certain cancers, such as pancreatic and cholangiocarcinoma types, highlights the need for additional therapeutic approaches. [Means for solving the problem]

[0005] The present disclosure provides, inter alia, insights and techniques for treating cancer, particularly cancers associated with expression of claudin 18.2 (CLDN-18.2). In some embodiments, the present disclosure provides techniques for treating cancer selected from the group consisting of pancreatic cancer, gastric or gastroesophageal cancer, cholangiocarcinoma, ovarian cancer, etc. In some embodiments, the present disclosure provides techniques for administering therapy to locally advanced tumors. In some embodiments, the present disclosure provides techniques for treating unresectable tumors. In some embodiments, the provided techniques provide techniques for treating metastatic tumors. Thus, for example, in some embodiments, the provided therapies can be administered to a subject or population of subjects suffering from or susceptible to cancer (e.g., a cancer selected from pancreatic cancer, gastric or gastroesophageal cancer, cholangiocarcinoma, ovarian cancer, and / or a cancer comprising one or more pancreatic, gastric, gastroesophageal, bile duct, and / or ovarian tumors), which cancer can be or can include one or more locally advanced tumors, one or more unresectable tumors, and / or one or more metastases.

[0006] This disclosure provides, inter alia, insight that claudin 18.2 (CLDN-18.2) is a particularly useful tumor-associated antigen that can be targeted by therapy. Without wishing to be bound by any particular theory, this disclosure notes that the tissue expression pattern of CLDN-18.2, including its particularly restricted expression in non-cancerous tissues, may contribute to its usefulness as a target as described herein. To date, no therapeutic targeting CLDN-18.2 has been approved for any cancer indication.

[0007] Zolbetuximab (development code IMAB362), a monoclonal antibody targeting isoform 2 of claudin-18, is under investigation for the treatment of gastrointestinal adenocarcinoma and pancreatic tumors (Tureci et al. 2019).

[0008] The present disclosure further provides the insight that, in some embodiments, the CLDN-18.2-targeting treatments described herein may usefully include the administration of RNA (e.g., ssRNA such as mRNA) encoding an antibody agent that targets CLDN-18.2. Furthermore, the present disclosure provides the specific insight that delivery of RNA via lipid nanoparticles targeted to hepatocytes may be a particularly beneficial strategy for delivering such antibody agents.

[0009] The present disclosure further provides insight that the RiboMab format (e.g., as illustrated in Figure 13), and particularly the RNA sequences and sequence elements described herein, may be particularly useful for RNA (e.g., ssRNA such as mRNA) delivering the CLDN-18.2 targeting agents (e.g., CLDN-18.2 targeting antibody agents) described herein.

[0010] The present disclosure provides insight that, among other things, administration of RNA (e.g., ssRNA such as mRNA) encoding a CLDN-18.2-targeting agent, particularly a CLDN-18.2-targeting antibody agent, and in particular IMAB362, may be a particularly desirable strategy for CLDN-18.2-targeting therapy. Without wishing to be bound by any particular theory, the present disclosure proposes that such a delivery mode may achieve one or more improvements, such as a reduced incidence (e.g., frequency and / or severity) of TEAEs and / or effective administration with an improved relationship between efficacy levels and TEAE levels (e.g., an improved therapeutic window), compared to that observed when the corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements may be achieved, inter alia, by delivering IMAB362 via administration of one or more RNAs (e.g., one or more ssRNAs such as one or more mRNAs) encoding IMAB362.

[0011] In some embodiments, the present disclosure provides, inter alia, the insight that one or more mRNAs encoding an antibody agent (e.g., IMAB362) or functional portion thereof that is or is formulated with a lipid nanoparticle (LNP) for intravenous (IV) administration can be taken up by target cells (e.g., hepatocytes) to efficiently produce the encoded antibody agent (e.g., IMAB362) at therapeutically relevant plasma concentrations, as illustrated, for example, in FIG. 14 for the described RiboMab targeting CLDN-18.2.

[0012] In some embodiments, the present disclosure utilizes RiboMabs as CLDN-18.2 targeting agents. In some embodiments, such RiboMabs are antibody agents encoded by mRNA, e.g., engineered for minimal immunogenicity and / or formulated into lipid nanoparticles (LNPs).

[0013] Furthermore, the present disclosure provides, inter alia, the insight that the ability of the CLDN-18.2-targeting antibody agents 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 recipient subject's immune system can enhance the cytotoxic effects of one or more chemotherapy and / or other anticancer therapies. In some embodiments, such combination therapy may, for example, prolong progression-free survival and / or overall survival compared to each individual therapy administered alone and / or another appropriate reference.

[0014] Without wishing to be bound by any particular theory, the present disclosure observes that certain chemotherapeutic agents, such as gemcitabine, oxaliplatin, and 5-fluorouracil, have been shown to upregulate pre-existing CLDN-18.2 expression levels in pancreatic cancer cell lines; furthermore, these agents were not observed to increase de novo expression in CLDN-18.2-negative cell lines. See, for example, Tureci et al. (2019) "Characterization of zolbetuximab in pancreatic cancer models," In Oncoimmunology 8(1), pp. e1523096.

[0015] The present disclosure provides, inter alia, the insight that the CLDN-18.2-targeted therapies described herein may be particularly useful and / or effective when administered to one or more tumors (e.g., tumor cells, subjects in which such one or more tumors and / or one or more tumor cells are suspected and / or detected, etc.) characterized (e.g., determined to exhibit and / or expected or predicted to exhibit elevated expression and / or activity) of CLDN-18.2 expression and / or activity in tumor cells (e.g., which may or may have resulted from exposure to one or more chemotherapeutic agents). Indeed, inter alia, the present disclosure teaches that the provided CLDN-18.2-targeted therapies described herein (e.g., administration of RNA, more specifically, mRNA encoding a CLDN-18.2-targeting antibody agent) may provide synergistic treatment when administered in combination (e.g., to a subject that has received and / or is receiving or has been exposed to) one or more CDLN18.2-potentiating agents (e.g., one or more particular chemotherapeutic agents). Thus, in some embodiments, the CLDN-18.2-targeted therapies described herein may be useful in combination with other anti-cancer agents that are predicted and / or demonstrated to upregulate CLDN-18.2 expression in tumor cells.

[0016] 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) comprising one or more coding regions encoding an antibody agent that binds to a claudin 18.2 (CLDN-18.2) polypeptide, e.g., that selectively binds to a claudin 18.2 (CLDN-18.2) polypeptide compared to a claudin 18.1 (CLDN18.1) polypeptide (a "CLDN-18.2-targeting antibody agent"); and (b) lipid nanoparticles, wherein the at least one RNA is encapsulated within at least one of the lipid nanoparticles. In some embodiments, such pharmaceutical compositions comprise and / or can deliver one or more RNAs encoding an antibody that binds to a CLDN-18.2 polypeptide, e.g., that selectively binds to a CLDN-18.2 polypeptide compared to a CLND18.1 polypeptide. In some embodiments, such pharmaceutical compositions comprise and / or can deliver one or more RNAs encoding an antigen-binding fragment that binds to a CLDN-18.2 polypeptide, e.g., that selectively binds to a CLDN-18.2 polypeptide compared to a CLND18.1 polypeptide.

[0017] In some embodiments, an antibody agent that targets CLDN-18.2 (and may be encoded by an RNA, such as an ssRNA, e.g., an 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 epitope of ECD1 that is exposed in cancer cells.

[0018] In some embodiments, at least one RNA (e.g., ssRNA such as mRNA) encodes the variable heavy chain (V) of a CLDN-18.2-targeting antibody agent. H ) domain and the variable light chain (V L In some embodiments, one or more such V domains of a CLDN-18.2-targeting antibody agent are H A domain and one or more VL The domains can be encoded by a single RNA construct; alternatively, in some embodiments, they can be separately encoded by at least two individual RNA constructs. For example, in some embodiments, the RNA utilized herein comprises at least the V domain of an antibody agent. H a heavy chain coding region encoding a domain and at least a V domain of the antibody agent; L In an alternative embodiment, the pharmaceutical composition comprises two or more coding regions, including (i) a light chain coding region encoding at least the V domain of the antibody agent. H (ii) a first RNA comprising a heavy chain coding region encoding at least a V domain of the antibody agent; L and a second RNA comprising a light chain coding region encoding the domain.

[0019] In some embodiments, the heavy chain coding region comprises a constant heavy chain (C H ) domain, and / or the light chain coding region may further encode a constant light chain (C L For example, in some embodiments, the heavy chain coding region may further encode the V domain of an antibody agent in the form of immunoglobulin G (IgG). H Domain, C H1 Domain, C H2 domain, and C H3 and / or the light chain coding region may encode the V domain of an antibody agent in the IgG form. L Domain and C L In some embodiments, the antibody agent in IgG form is an IgG1.

[0020] 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.

[0021] In some embodiments, the one or more RNAs encoding the CLDN-18.2-targeting antibody agent may include a secretory signal coding region. In some embodiments, such a secretory signal coding region enables the CLDN-18.2-targeting antibody agent encoded by the one or more RNAs to be secreted, for example, upon translation by cells present in the treated subject, thus resulting in plasma concentrations of the biologically active CLDN-18.2-targeting antibody agent.

[0022] Those skilled in the art will recognize the burgeoning field of nucleic acid therapeutics, and even RNA (e.g., ssRNA, such as mRNA) therapeutics (see, e.g., mRNA encoding proteins and / or cytokines). Various embodiments of the technology provided herein may utilize particular features of RNA (e.g., ssRNA, such as mRNA) therapeutic technologies and / or delivery systems. For example, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) may contain one or more modified nucleotides (e.g., but not limited to, pseudouridine), nucleosides, and / or linkages. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) may contain a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and / or translation efficiency. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) may contain a specific combination of at least two 3'UTR sequences (e.g., a combination of a sequence element from an amino-terminal enhancer of a split RNA and a sequence derived from mitochondrially encoded 12S RNA). Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA such as mRNA) may comprise a 5'UTR sequence derived from human alpha globin mRNA. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA such as mRNA) may comprise a 5' cap analog, e.g., for co-transcriptional capping. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA such as mRNA) may comprise a secretory signal coding region with reduced immunogenicity (e.g., a human secretory signal coding sequence) such that the encoded antibody agent is expressed and secreted. In some embodiments, the RNA may be formulated in or with one or more delivery vehicles (e.g., nanoparticles, such as lipid nanoparticles). Alternatively or additionally, in some embodiments, the RNA may be formulated in or with liver-targeting lipid nanoparticles (e.g., cationic lipid nanoparticles).

[0023] In some embodiments, one or more RNAs encoding a CLDN-18.2-targeting antibody agent may contain at least one non-coding sequence element (e.g., to increase RNA stability and / or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, a 3' untranslated region (UTR), a 5' UTR, a cap structure for co-transcriptional capping of mRNA, a polyadenine (polyA) tail, and any combination thereof. For example, in some embodiments, the RNAs (e.g., the first RNA and / or the second RNA) each independently comprise, from 5' to 3', (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 contained in the RNA is or comprises a modified polyA sequence.

[0024] In some embodiments, the one or more RNAs encoding the CLDN-18.2-targeting antibody agent may include a 5' cap.

[0025] In some embodiments, the RNA encoding the CLDN-18.2 targeting antibody agent may contain at least one modified ribonucleotide. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of one or more RNAs may be replaced by a modified ribonucleotide. In some embodiments, such a modified ribonucleotide may be or may include pseudouridine.

[0026] The pharmaceutical composition comprises a variable heavy chain (VH) of a CLDN-18.2-targeting antibody agent. H ) domain, e.g., a first RNA encoding the heavy chain of a CLDN-18.2-targeting antibody agent, and a second RNA encoding the variable light chain (V LIn some embodiments, the 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, the 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 1.09, about 1.10, about 1.11, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.29, about 1.29, about 1.29, about 1.29, about 2 ... The first and second RNAs may be present in a molar ratio of about 7:1, 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, the first and second RNAs may be present in a weight ratio of 3:1 to 1:1. In some embodiments, the first and second RNAs may be present in a weight ratio of about 2:1. In some embodiments, such first and second RNAs 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.

[0027] In some embodiments, the RNA content (e.g., one or more RNAs encoding a CLDN-18.2-targeting antibody agent) of the pharmaceutical compositions described herein is present at a concentration of 0.5 mg / mL to 1.5 mg / mL.

[0028] In some embodiments, the lipid nanoparticles provided in the pharmaceutical compositions described herein are liver-targeted lipid nanoparticles. In some embodiments, the lipid nanoparticles provided in the pharmaceutical compositions described herein are cationic lipid nanoparticles. In some embodiments, the lipid particles provided in the pharmaceutical compositions described herein may have an average size of about 50-150 nm.

[0029] In some embodiments, the lipids forming the lipid nanoparticles include a polymer-bound lipid, a cationic lipid, and a neutral lipid. In some such embodiments, the polymer-bound lipid is present at about 1-2.5 mol% of the total lipid, the cationic lipid is present at 35-65 mol% of the total lipid, and the neutral lipid is present at 35-65 mol% of the total lipid.

[0030] Various lipids (e.g., including 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 targeted to specific cell types (e.g., hepatocytes). In some embodiments, the polymer-conjugated lipid included in the pharmaceutical compositions described herein can be a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof). In some embodiments, the cationic lipid included in the pharmaceutical compositions described herein can be ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) or a derivative thereof. In some embodiments, the neutral lipid included in the pharmaceutical compositions described herein can be or include a phospholipid or a derivative thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol.

[0031] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, which may, in some embodiments, enhance the stability of such compositions under certain conditions. For example, in some embodiments, the pharmaceutical compositions may further comprise a cryoprotectant (e.g., sucrose) and / or an aqueous buffer, which in some embodiments may include one or more salts (e.g., sodium salts).

[0032] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more active agents other than RNA (e.g., ssRNA such as mRNA) encoding a CLDN-18.2 targeting agent (e.g., an antibody agent). For example, in some embodiments, such other active agents may be or may include a chemotherapeutic agent. An exemplary chemotherapeutic agent may be or may include a chemotherapeutic agent indicated for the treatment of pancreatic cancer.

[0033] In some embodiments, the pharmaceutical compositions described herein can be taken up by target cells to produce the encoded CLDN-18.2-targeting antibody agent 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).

[0034] Therefore, another aspect of the present disclosure relates to a method of using the pharmaceutical compositions described herein. For example, one aspect provided herein relates to a method comprising administering a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive solid tumor. Examples of CLDN-18.2-positive solid tumors include, 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 may be characterized by 50% or more of tumor cells exhibiting a CLDN-18.2 protein staining intensity of ≥2+ as assessed by immunohistochemistry in formalin-fixed, paraffin-embedded neoplastic tissue from the subject to which the composition is administered. In some embodiments, a subject suffering from a CLDN-18.2-positive solid tumor may have a locally advanced, unresectable, or metastatic tumor. In some embodiments, a subject suffering from a CLDN-18.2-positive solid tumor may have received sufficient prior treatment to increase CLDN-18.2 levels such that the solid tumor is characterized as a CLDN-18.2-positive solid tumor.

[0035] In some embodiments, the pharmaceutical compositions described herein can be administered as monotherapy. In some embodiments, the pharmaceutical compositions can be administered as part of a combination therapy including such pharmaceutical composition and a chemotherapeutic agent. Thus, in some embodiments, a subject receiving a provided pharmaceutical composition is also receiving a chemotherapeutic agent. In some embodiments, a subject receiving a provided pharmaceutical composition is administered a chemotherapeutic agent such that the subject is receiving 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 4 hours after) administration of the provided pharmaceutical composition.

[0036] In some embodiments, the technology provided herein is useful for treating CLDN-18.2-positive pancreatic tumors. In some embodiments involving administration of a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive pancreatic tumor, the subject may receive the provided pharmaceutical composition as a monotherapy or as part of a combination therapy including the provided pharmaceutical composition and a chemotherapeutic agent indicated for the treatment of pancreatic tumors. In some embodiments, the chemotherapeutic agent may be or include gemcitabine and / or paclitaxel (e.g., nab-paclitaxel). In some embodiments, the chemotherapeutic agent may be or include FOLFIRINOX, a combination of cancer therapeutic agents including folinic acid (FOL), fluorouracil (F), irinotecan (IRIN), and oxaliplatin (OX).

[0037] In some embodiments, the technology provided herein is useful for treating CLDN-18.2-positive biliary tract tumors. In some embodiments involving administration of a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive biliary tract tumor, the subject may receive the provided pharmaceutical composition as a monotherapy or as part of a combination therapy including the provided pharmaceutical composition and a chemotherapeutic agent indicated for the treatment of biliary tract tumors. In some embodiments, the chemotherapeutic agent may be or include gemcitabine and / or cisplatin.

[0038] The pharmaceutical compositions and methods described herein can be applied to subjects of any age who have a CLDN-18.2-positive solid tumor. In some embodiments, the subject who has a CLDN-18.2-positive solid tumor is an adult subject.

[0039] The pharmaceutical compositions described herein can be administered to a subject in need thereof by any suitable method known in the art. For example, in some embodiments, the provided pharmaceutical compositions can be administered to a subject suffering from a CLDN-18.2-positive solid tumor by intravenous injection.

[0040] The dosage of the pharmaceutical compositions described herein can vary depending on many factors, including, but not limited to, the weight of the subject being treated, the type and / or stage of the cancer, and / or the monotherapy or combination therapy. In some embodiments, the pharmaceutical compositions described herein are administered to a subject suffering from a CLDN-18.2-positive solid tumor in at least one or more administration cycles (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more). In some embodiments, each administration cycle can be a three-week administration cycle. In some embodiments, the pharmaceutical compositions described herein are administered in at least one dose per administration cycle. In some embodiments, an administration cycle comprises the administration of a set number and / or pattern of doses; in some embodiments, an administration cycle comprises the administration of a set cumulative dose, e.g., over a specific period of time, and optionally via multiple administrations, which may be administered, e.g., at one or more set intervals and / or according to a set pattern. In some embodiments, each dose or cumulative dose of a pharmaceutical composition described herein may contain one or more RNAs encoding a CLDN-18.2 targeting antibody agent (whether encoded by a single RNA or by two or more RNAs) in an amount in the range of 0.1 mg / kg to 5 mg / kg body weight of the subject to be administered.

[0041] Another aspect of the present disclosure relates to certain improvements in methods for delivering a CLDN-18.2-targeting antibody agent to a subject for cancer treatment, the methods comprising administering a provided pharmaceutical composition to a cancer subject. In some embodiments, the pharmaceutical compositions described herein may achieve one or more improvements, such as effective administration with reduced TEAEs (e.g., frequency and / or severity) and / or an improved relationship between efficacy levels and TEAE levels (e.g., an improved therapeutic window), compared to those observed when the corresponding (e.g., encoded) protein (e.g., antigen) agent itself is administered. In particular, the present disclosure teaches that such improvements may be achieved, inter alia, by delivering IMAB362 via administration of one or more RNAs (e.g., one or more ssRNAs, such as one or more mRNAs) encoding IMAB362.

[0042] Methods for producing a CLDN-18.2-targeting antibody agent are also within the scope of the present disclosure. In some embodiments, the method for producing a CLDN-18.2-targeting antibody agent comprises administering to cells a composition comprising at least one RNA (e.g., as described herein) comprising one or more coding regions encoding a CLDN-18.2-targeting antibody agent, such that the cells express and secrete the CLDN-18.2-targeting antibody agent encoded by such one or more RNAs. In some embodiments, the administered or targeted cells are or comprise hepatocytes.

[0043] In some embodiments, the cells are in cell culture.

[0044] 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 pharmaceutical compositions can be administered to a subject such that the 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 via antibody-dependent cellular cytotoxicity (ADCC). For example, in some embodiments, the therapeutically relevant plasma concentration is 0.3 to 28 μg / mL.

[0045] Among other things, the present disclosure also provides methods for characterizing one or more characteristics of an RNA encoding part or all of an antibody agent, or a composition thereof. In some embodiments, the method includes determining one or more characteristics of the antibody agent expressed from at least one mRNA introduced into a cell, wherein the at least one mRNA includes one or more characteristics of at least one RNA including a coding region encoding an antibody agent that binds to a claudin 18.2 (CLDN-18.2) polypeptide, e.g., that selectively binds to a claudin 18.2 (CLDN-18.2) polypeptide compared to a claudin 18.1 polypeptide, wherein the one or more characteristics include (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent for CLDN-18.2; (iii) the efficacy of the antibody agent in mediating target cell death via ADCC; and (iv) the efficacy of the antibody agent in mediating target cell death via complement-dependent cytotoxicity (CDC).

[0046] In some embodiments, methods for characterizing pharmaceutical compositions targeting CLDN-18.2 are provided herein. Such methods include: (a) contacting cells with at least one composition or pharmaceutical composition described herein (encoding part or all of a CLDN-18.2-targeting antibody agent); and detecting the antibody agent produced by the cells. In some embodiments, the cells may be or may include hepatocytes.

[0047] In some embodiments, such methods may further include determining one or more characteristics of the antibody agent expressed from one or more RNAs described herein, such one or more characteristics including (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 efficacy of the antibody agent in mediating target cell death via ADCC; and (iv) the efficacy of the antibody agent in mediating target cell death via complement-dependent cytotoxicity (CDC). In some embodiments, determining one or more characteristics of the antibody agent expressed from one or more RNAs described herein may include comparing such characteristics of the CLDN-18.2-targeting antibody agent with characteristics of a reference CLDN-18.2-targeting antibody.

[0048] In some embodiments, determining one or more characteristics of an antibody agent expressed from one or more RNAs described herein can include assessing a protein expression level of the antibody agent above a threshold level, e.g., in some embodiments, the threshold level corresponds to a therapeutically relevant plasma concentration.

[0049] In some embodiments, determining one or more characteristics of an antibody agent expressed from one or more RNAs described herein may include assessing binding of the antibody agent to a CLDN-18.2 polypeptide. In some embodiments, such binding assessment may include determining binding of the antibody agent to a CLDN-18.2 polypeptide compared to binding of the antibody agent to a CLDN18.1 polypeptide. In some embodiments, such binding assessment may include determining a binding selectivity profile of the antibody agent that is at least comparable to the binding selectivity 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.

[0050] In some embodiments, the provided methods of characterizing pharmaceutical compositions targeting CLDN-18.2 or a component thereof may further include characterizing an antibody agent expressed from one or more RNAs described herein as a CLDN-18.2-targeting antibody agent if the antibody agent comprises the following characteristics: (a) a protein level of the antibody agent expressed by a cell that exceeds a threshold level; (b) selective binding of the antibody agent to CLDN-18.2 compared to CLDN18.1; and (c) killing of at least 50% of target cells (e.g., cancer cells) mediated by ADCC and / or CDC.

[0051] In some embodiments, provided methods of characterizing pharmaceutical compositions targeting CLDN-18.2 or a component thereof may further include characterizing an antibody agent expressed from one or more RNAs described herein as a zolbetuximab- or claudiximab-equivalent antibody if the tested characteristics of the antibody are at least comparable to those of zolbetuximab or claudiximab.

[0052] In some embodiments that involve determining one or more characteristics of an antibody agent expressed from one or more RNAs described herein, such a step includes determining one or more of the following characteristics: whether the cells express at least one RNA-encoded CLDN-18.2-targeting antibody agent when assessed 48 hours after contact or administration; whether the antibody agent expressed by the cell selectively binds to a CLDN-18.2 polypeptide compared to a CLDN18.1 polypeptide; · whether the antibody agent expressed by the cells exhibits equivalent target specificity for CLDN-18.2 as observed in a flow cytometry binding assay using a reference CLDN-18.2-targeting monoclonal antibody; · Whether CLDN-18.2-positive cells, but not control cells, were lysed when assessed 48 hours after incubation of immune effector cells (e.g., PBMC cells) and CLDN-18.2-positive cells or CLDN-18.2-negative control cells in the presence of the antibody agent; whether the antibody agent expressed by the cells exhibits at least a comparable ADCC profile of the targeted CLDN-18.2-positive cells as observed with a reference CLDN-18.2-targeting monoclonal antibody at the same concentration; and Lysis of CLDN-18.2-positive cells, but not control cells, was assessed after 2 hours of incubation of CLDN-18.2-positive cells or CLDN-18.2-negative control cells with human serum in the presence of the antibody agent. The method may include determining one or more of:

[0053] In some embodiments, the cells used in the provided methods of characterizing a pharmaceutical composition targeting CLDN-18.2 or a component thereof are present in vivo, e.g., in a subject (e.g., a mammalian subject, e.g., a mammalian non-human subject, e.g., a mouse or monkey subject). In some such embodiments, determining one or more characteristics of an antibody agent expressed from one or more RNAs described herein may include determining antibody levels in one or more tissues of such a subject. In some embodiments, such characterization methods, when such a composition or pharmaceutical composition is characterized as a CLDN-18.2-targeting antibody agent, may further include administering a composition or pharmaceutical composition described herein to a group of animal subjects, each bearing a human CLDN-18.2-positive xenograft tumor, to determine anti-tumor activity.

[0054] Also included within the scope of this disclosure is a method of manufacture comprising the steps of: (A) determining one or more characteristics of an RNA encoding part or all of an antibody agent, or a composition thereof, wherein the one or more characteristics are: (i) RNA length and / or sequence; (ii) RNA integrity; (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 cells; (v) stability of the RNA or composition thereof; (vi) the level of the antibody agent in a biological sample from the organism into which the RNA was introduced; (vii) the binding specificity of the antibody agent expressed from the RNA, optionally relative to CLDN-18.2, and optionally relative to CLDN18.1; (viii) the efficacy of antibody agents in mediating target cell death via ADCC; (ix) the efficacy of antibody agents in mediating target cell death via complement-dependent cytotoxicity (CDC); (x) the identity and amount / concentration of lipids in the composition; (xi) the size of the lipid nanoparticles within the composition; (xii) the polydispersity of the lipid nanoparticles within the composition; (xiii) the amount / concentration of RNA in the composition; (xiv) the extent of RNA encapsulation within the lipid nanoparticles; and (xv) combinations thereof a step selected from the group consisting of: (B) comparing such one or more characteristics of the RNA or composition thereof with characteristics of a suitable reference standard; and (C)(i) designating the RNA or composition thereof for one or more further steps of manufacture and / or distribution if the comparison demonstrates that the RNA or composition thereof meets or exceeds the reference standard; or (ii) Taking alternative action if the comparison demonstrates that the RNA or composition thereof does not meet or exceed the reference standard.

[0055] In some embodiments of the manufacturing methods, RNA (e.g., those described herein) is evaluated, and if one or more characteristics of the RNA meet or exceed appropriate reference standards, such RNA is designated for formulation, in some embodiments, including, for example, formulation with lipid particles described herein.

[0056] In some embodiments of the manufacturing methods, compositions comprising RNA (e.g., those described herein) are evaluated, and if one or more characteristics of the composition meet or exceed appropriate reference standards, such compositions are designated for shipping and / or distribution of the composition.

[0057] In some embodiments of the manufacturing methods, when RNA (e.g., as described herein) is designated for formulation and / or a composition comprising RNA (e.g., as described herein) is designated for shipping and / or distribution of the composition, such methods may further include administering the formulation and / or composition to a group of animal subjects each bearing a human CLDN-18.2-positive xenograft tumor to determine anti-tumor activity.

[0058] Also provided herein is a method for determining the dosing regimen of a pharmaceutical composition targeting CLDN-18.2. For example, in some embodiments, such a method includes: (A) administering a pharmaceutical composition (e.g., as described herein) to a subject suffering from a CLDN-18.2-positive solid tumor under a predetermined dosing regimen; (B) periodically monitoring or measuring the subject's tumor size over a period of time; and (C) evaluating the dosing regimen based on one or more tumor size measurements. For example, if the reduction in tumor size after administration of a pharmaceutical composition (e.g., as described herein) is not therapeutically appropriate, the dose and / or dosing frequency can be increased; or, if the reduction in tumor size after administration of a pharmaceutical composition (e.g., as described herein) is therapeutically appropriate but the subject shows adverse effects (e.g., toxic effects), the dose and / or dosing frequency can be decreased. If the reduction in tumor size after administration of a pharmaceutical composition (e.g., as described herein) is therapeutically appropriate and the subject shows no adverse effects (e.g., toxic effects), the dosing regimen is not changed.

[0059] In some embodiments, such a method for determining the administration regimen of a pharmaceutical composition targeting CLDN-18.2 can be carried out on a group of animal subjects (e.g., mammalian non-human subjects), each bearing a human CLDN-18.2-positive xenograft tumor. In some such embodiments, if less than 30% of the animal subjects show a decrease in tumor size after administration of a pharmaceutical composition (e.g., as described herein) and / or the degree of tumor size decrease shown by the animal subjects is not therapeutically relevant, the dose and / or administration frequency can be increased; or, if the decrease in tumor size after administration of a pharmaceutical composition (e.g., as described herein) is therapeutically relevant but at least 30% of the animal subjects show significant adverse effects (e.g., toxic effects), the dose and / or administration frequency can be decreased. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., as described herein) is therapeutically relevant and the animal subjects show no significant adverse effects (e.g., toxic effects), the administration regimen is not changed.

[0060] The present disclosure relates, inter alia, to (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2). 1. A composition or pharmaceutical formulation comprising: the coding region of (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; (ii) The coding region of SEQ ID NO: 17 comprises the nucleotide sequence of nucleotides 79 to 738, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17; A composition or pharmaceutical formulation is provided. 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; 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 (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2). 1. A composition or pharmaceutical formulation comprising: 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; 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; A composition or pharmaceutical formulation is provided. In some embodiments, the RNA, e.g., each RNA, comprises a 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, e.g., each RNA, comprises a 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, eg, each RNA, comprises a 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, eg, each RNA, comprises a 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, eg, each RNA, comprises a 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 (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2). 1. A composition or pharmaceutical formulation comprising: RNAs, e.g., each RNA, comprising a 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 / or a 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; A composition or pharmaceutical formulation is provided. In some embodiments, the RNA, e.g., each RNA, comprises a 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 a 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, e.g., each RNA, comprises a 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 a 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, e.g., each 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 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, eg, each RNA, comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO:18, and a 3' UTR comprising the nucleotide sequence of SEQ ID NO:19. In some embodiments, the RNA, eg, each RNA, comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20, and a 3'UTR comprising the nucleotide sequence of SEQ ID NO:21. In some embodiments, the coding region of (a)(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; (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; and / or (b) the first polypeptide chain comprises an 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; 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 coding region of (i) 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; The coding region of (ii) 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; 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 in (i) is a first RNA molecule and the RNA 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 selectively binds to claudin 18.1 (CLDN-18.2) relative to 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 on cancer cells. In some embodiments, the antibody agent that binds to CLDN-18.2 comprises two binding arms, each binding arm comprising 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 an 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 an antibody agent that binds CLDN-18.2 (VH(CLDN-18.2)). In some embodiments, VH(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:14. In some embodiments, VH(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively. In some embodiments, the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds CLDN-18.2 (VL(CLDN-18.2)). In some embodiments, VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO:15. In some embodiments, VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NOs: 8, 9 and 10, respectively. In some embodiments, the first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), which comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14, and 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)), which comprises CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15. 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)), comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, 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)), comprising CDR1, CDR2, and CDR3 comprising the sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively. In some embodiments, the first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), comprising the amino acid sequence of SEQ ID NO: 14, and 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)), comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and 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)), wherein VH(CLDN-18.2) and VL(CLDN-18.2) interact 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 (VH) of the heavy chain 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, VH(CLDN-18.2), CH1, CH2 and CH3 are present in a first polypeptide chain in an immunoglobulin G (IgG) format. 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 a second polypeptide chain in an IgG format. In some embodiments, CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain. In some embodiments, the first polypeptide chain and the second polypeptide chain each independently comprise a secretion signal, and the secretion signal is preferably located at the N-terminus of the first polypeptide chain and the second polypeptide chain. In some embodiments, the secretory 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 of (i) comprises the nucleotide sequence of SEQ ID NO:16 and the coding region of (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, eg, each RNA, comprises a polyA sequence. In some embodiments, the polyA sequence is an interrupted sequence of A nucleotides. In some embodiments, the polyA sequence comprises at least 100 nucleotides. In some embodiments, the polyA sequence is x -LA y A x is a sequence of at least 20 A nucleotides, and 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 polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:23. In some embodiments, the composition or pharmaceutical formulation comprises: (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) 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. Includes. In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 Includes. In some embodiments, the composition or pharmaceutical formulation comprises: (i) RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 27 Includes. The present disclosure also provides (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27 The present invention provides a composition or pharmaceutical formulation comprising: The present disclosure also provides (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 The present invention provides a composition or pharmaceutical formulation comprising: The present disclosure also provides (i) RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 27 The present invention provides a composition or pharmaceutical formulation comprising: In some embodiments, the RNA, eg, each RNA, comprises a modified nucleoside in place of a uridine. In some embodiments, the RNA, eg, each RNA, comprises a modified nucleoside in place of each 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, eg, each RNA, comprises a 5' cap. In some embodiments, the RNA, e.g., each RNA, is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG. In some embodiments, the RNA, eg, the RNA, is single-stranded RNA. In some embodiments, the RNA, eg, each RNA, is an mRNA. In some embodiments, the RNAs, eg, each RNA, is formulated in a lipid nanoparticle (LNP), eg, each RNA is co-formulated in a lipid nanoparticle (LNP). In some embodiments, the lipids that form the lipid nanoparticles include cationic lipids, polymer-conjugated lipids, and neutral lipids. In some embodiments, a. Cationic lipids are present at 35-65 mol% of the total lipids; b. the polymer-bound lipid is present at approximately 1-2.5 mol% of the total lipid; and c. Neutral lipids are present at 35-65 mol% of total lipids. In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate). In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In some embodiments, the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. In some embodiments, the lipid nanoparticles have an average size of about 50-150 nm. In some embodiments, the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 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, eg, each RNA, and optionally the particle-forming components, are in separate vials. In some embodiments, the pharmaceutical preparation further comprises instructions for using the composition or pharmaceutical preparation to treat or prevent cancer. The present disclosure also provides a composition or pharmaceutical formulation described herein for pharmaceutical use. In some embodiments, the medical use includes the therapeutic or prophylactic treatment of a disease or disorder. In some embodiments, therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing cancer. In some embodiments, the cancer comprises a CLDN-18.2-positive cancer. In some embodiments, the cancer comprises a 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 a CLDN-18.2-positive biliary tract tumor. In some embodiments, the cancer comprises a CLDN-18.2-positive locally advanced, unresectable, or metastatic cancer. In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder further comprises administering an additional therapy. In some embodiments, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy. In some embodiments, the additional therapy comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the compositions or pharmaceutical formulations described herein are for administration to humans. In some embodiments, the compositions or pharmaceutical formulations described herein are for intravenous administration. The present disclosure also provides a method of treating cancer in a subject, comprising administering to the subject a composition described herein. In some embodiments, the cancer comprises a CLDN-18.2-positive cancer. In some embodiments, the cancer comprises a 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 a CLDN-18.2-positive biliary tract tumor. In some embodiments, the cancer comprises a CLDN-18.2-positive locally advanced, unresectable, or metastatic cancer. In one embodiment, the methods described herein further comprise administering an additional therapy. In some embodiments, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy. In some embodiments, the additional therapy comprises 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 compositions described herein for use in the methods described herein. In some embodiments, when the polypeptide chains of an antibody agent that binds to claudin 18.2 (CLDN-18.2) are expressed, the polypeptide chains are secreted into the bloodstream as fully assembled and / or functional antibodies. A fully assembled antibody is a tetramer composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2). A functional antibody is an antibody that has the expected biological activity of an antibody, e.g., binding to its target and / or recruitment and / or stimulation of the immune system, e.g., ADCC, at the same or similar level as the corresponding antibody expressed in vitro. In some embodiments, the compositions or pharmaceutical formulations described herein are for introducing RNA into hepatocytes and expressing the polypeptide chain encoded by the RNA in the hepatocytes. In some embodiments, the compositions or pharmaceutical formulations described herein are for systemic delivery of polypeptide chains. In some embodiments, the compositions or pharmaceutical formulations described herein are for systemic delivery of the polypeptide chains following expression of the polypeptide chains 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 a composition described herein such that the RNA is introduced into hepatocytes; and (b) expressing in hepatocytes the polypeptide chain encoded by the RNA. The present invention provides a method comprising: 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 a composition described herein such that the RNA is introduced into hepatocytes; and (b) expressing in hepatocytes the polypeptide chain encoded by the RNA. wherein, after expression, the polypeptide chain is secreted into the bloodstream; A method is provided. The present disclosure also provides a method for systemic delivery of an antibody agent that binds to claudin 18.2 (CLDN-18.2) in a subject, comprising: (a) administering a composition described herein such that the RNA is introduced into hepatocytes; and (b) expressing in hepatocytes the polypeptide chain encoded by the RNA. wherein, after expression, the polypeptide chain is secreted into the bloodstream; A method is provided. In some embodiments, the administration is parenteral. In some embodiments, the administration is intravenous.

[0061] The present disclosure also provides, among other things: 1. A pharmaceutical composition comprising: a. at least one single-stranded RNA comprising one or more coding regions encoding an antibody agent that selectively binds to a claudin 18.2 (CLDN-18.2) polypeptide relative to a claudin 18.1 polypeptide; and b. Lipid nanoparticles wherein at least one single-stranded RNA is encapsulated in at least one of the lipid nanoparticles. Pharmaceutical compositions. 2. The pharmaceutical composition according to item 1, wherein the antibody agent specifically binds to the first extracellular domain (ECD1) of the CLDN-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. At least one single-stranded RNA encodes the variable heavy chain (V H ) domain and the variable light chain (V L 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the composition encodes both the nucleotide sequence and the nucleotide sequence of the target gene. 6. At least one single-stranded RNA is present in at least one V of the antibody agent. H a first single-stranded RNA comprising a heavy chain coding region encoding a domain; and a. The first single-stranded RNA comprises at least the V L or further comprising a light chain coding region encoding a domain; b. The pharmaceutical composition comprises at least V of the antibody drug. L a second single-stranded RNA comprising a light chain coding region encoding a domain; Item 6. The pharmaceutical composition according to item 5. 7. The heavy chain coding region is a constant heavy chain (C H ) domain, and / or the light chain coding region further encodes a constant light chain (C L 7. The pharmaceutical composition of item 6, further encoding a .) domain. 8. The heavy chain coding region is V of an antibody agent in the form of immunoglobulin G (IgG). H Domain, C H1 Domain, C H2 domain, and C H3 and / or the light chain coding region encodes the V domain of an IgG form of an antibody agent. L Domain and C L 7. The pharmaceutical composition according to item 6, encoding a domain. 9. The pharmaceutical composition of 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 claudiximab. 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 claudiximab. 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 contain a secretory 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 comprise at least one non-coding sequence element (e.g., to increase 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. A first single-stranded RNA is synthesized in the 5' to 3' direction as follows: a.5'UTR coding region; b. secretion signal coding region; c. heavy chain coding region; d. 3'UTR coding region; and e. Poly A tail coding region 15. The pharmaceutical composition according to any one of items 6 to 14, comprising: 16. A second single-stranded RNA is coupled in the 5' to 3' direction to: a.5'UTR coding region; b. secretion signal coding region; c. light chain coding region; d. 3'UTR coding region; and e. Poly A tail coding region 16. The pharmaceutical composition according to any one of items 6 to 15, comprising: 17. The pharmaceutical composition according to item 14 or 15, wherein the poly A tail is or comprises a modified poly A 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 a first single-stranded RNA and a 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 nanoparticles are liver-targeting lipid nanoparticles. 24. The pharmaceutical composition according to any one of items 1 to 23, wherein the lipid nanoparticles are cationic lipid nanoparticles. 25. The lipids forming the lipid nanoparticles are -polymer-bound lipids; cationic lipids; and -Neutral lipids 25. The pharmaceutical composition according to item 24, comprising: 26.a Polymer-bound lipids are present at approximately 1-2.5 mol% of total lipids; b. cationic lipids are present at 35-65 mol% of the total lipids; and c. Neutral lipids are present at 35-65 mol% of the total lipids; Item 26. The pharmaceutical composition according to item 25. 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-ditetradecylacetamide). 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-butyloctanoate). 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 lipid nanoparticles have an average size of about 50 to 150 nm. 31. The pharmaceutical composition according to any one of items 1 to 30, further comprising a cryoprotectant (e.g., sucrose). 32. The pharmaceutical composition according to any one of items 1 to 31, comprising an aqueous buffer. 33. The pharmaceutical composition according to item 32, wherein the aqueous buffer contains sodium ions. 34. The pharmaceutical composition according to any one of items 1 to 33, further comprising a chemotherapeutic agent. 35. The pharmaceutical composition according to item 34, wherein the chemotherapeutic agent is a chemotherapeutic agent indicated 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 in a concentration of 0.5 mg / mL to 1.5 mg / mL. 37. A method comprising administering to a subject suffering from a CLDN-18.2-positive solid tumor a 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. 40. The method according to item 37, wherein the CLDN-18.2-positive tumor is a biliary tract tumor. 41. 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. 42. The method of any one of items 37 to 41, wherein the subject has undergone a pretreatment sufficient to elevate CLDN-18.2 levels such that the solid tumor from which the subject is afflicted is characterized as a CLDN-18.2-positive solid tumor. 43. The method according to any one of items 37 to 42, wherein the CLDN-18.2-positive tumor is characterized in that 50% or more of the tumor cells exhibit a ≥2+ CLDN-18.2 protein staining intensity when assessed by immunohistochemistry assay in formalin-fixed, paraffin-embedded neoplastic tissue from the subject. 44. The method according to any one of items 37 to 43, wherein the pharmaceutical composition is administered as monotherapy. 45. 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. 46. ​​The method according to any one of items 37 to 45, wherein the subject has received a chemotherapy agent. 47. The method of item 45, further comprising administering a chemotherapeutic agent to the subject such that the subject is receiving combination therapy. 48. The method according to item 47, wherein the chemotherapeutic agent is administered at least 4 hours after administration of the pharmaceutical composition. 49. The method according to any one of items 45 to 48, wherein the chemotherapeutic agent is or comprises gemcitabine and / or paclitaxel (e.g., nab-paclitaxel) for a subject suffering from a CLDN-18.2-positive pancreatic tumor. 50. The method according to any one of items 45 to 48, wherein the chemotherapeutic agent is or comprises FOLFIRINOX for a subject suffering from a CLDN-18.2-positive pancreatic tumor. 51. The method according to any one of items 45 to 48, wherein the chemotherapeutic agent is or comprises gemcitabine and / or cisplatin for a subject suffering from CLDN-18.2-positive biliary tract cancer. 52. 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 carried out 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 administration cycles. 55. The method according to item 54, wherein the pharmaceutical composition is administered as one or more doses per administration cycle. 56. The method of item 55, wherein each administration cycle is a 3-week administration cycle. 57. The method of item 55 or 56, wherein one or more administrations comprise at least one single-stranded RNA in the range of 0.1 mg / kg to 5 mg / kg body weight of the subject. 58. A method for delivering a CLDN-18.2-targeting antibody for the treatment of cancer in a subject, comprising administering to the subject a pharmaceutical composition according to any one of items 1 to 36. 59. A method for producing a CLDN-18.2 targeting antibody, comprising 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 at least one single-stranded RNA of the pharmaceutical composition. 60. The method according to item 59, wherein the cells are hepatocytes. 61. The method of item 59 or 60, wherein the cell is in a subject. 62. The method of 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 via antibody-dependent cellular 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 determining one or more characteristics of an antibody agent expressed from 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 comprising a coding region encoding an antibody agent that selectively binds to a claudin 18.2 (CLDN-18.2) polypeptide relative to a claudin 18.1 polypeptide, wherein the one or more characteristics are determined based on: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent for CLDN-18.2; (iii) the efficacy of the antibody agent in mediating target cell death by ADCC; and (iv) the efficacy of the antibody agent in mediating target cell death via complement-dependent cytotoxicity (CDC). Including, a method. 66. A method for characterizing a pharmaceutical composition targeting CLDN-18.2, comprising: contacting the cells with at least one pharmaceutical composition according to any one of items 1 to 35; and Detecting the antibody agent produced by the cells A method comprising: 67. The method of item 66, further comprising determining one or more characteristics of the antibody agent, the one or more characteristics including (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 efficacy of the antibody agent in mediating target cell death via ADCC; and (iv) the efficacy of the antibody agent in mediating target cell death via complement-dependent cytotoxicity (CDC). 68. The method according to any one of items 65 to 67, wherein the cells are hepatocytes. 69. The method of item 65 or 67, wherein the determining step comprises comparing one or more characteristics of the antibody agent with characteristics of a reference CLDN-18.2-targeting antibody. 70. The method of any one of items 65 and 67-69, wherein the determining step comprises assessing the protein expression level of the antibody agent above a threshold level. 71. The method according to item 70, wherein the threshold level is a level sufficient to induce ADCC. 72. The method of any one of items 65 and 67 to 71, wherein the determining step comprises assessing binding of the antibody agent to the CLDN-18.2 polypeptide. 73. The method of item 72, wherein evaluating comprises determining binding of the antibody agent to a CLDN-18.2 polypeptide compared to binding to a CLDN18.1 polypeptide. 74. The method of item 72 or 73, wherein evaluating comprises determining a binding selectivity profile of the antibody agent that is at least comparable to the binding selectivity profile of a reference CLDN-18.2-targeting antibody. 75. The method of item 69 or 74, wherein the reference CLDN-18.2 targeting antibody is zolbetuximab or claudiximab. 76. An antibody drug has the following characteristics: a. a protein level of the antibody agent expressed by the cells above a threshold level sufficient to induce ADCC; b. selective binding of the antibody agent to CLDN-18.2 compared to CLDN18.1; and c. At least 50% of target cells killed by ADCC and / or CDC 76. The method of any one of items 65 to 75, wherein the antibody agent is further characterized as a CLDN-18.2-targeting antibody agent if it comprises: 77. The method of item 76, wherein the antibody agent is further characterized as a zolbetuximab- or claudiximab-equivalent antibody if the antibody characteristics 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 cells are cancer cells. 79. The method of any one of items 65 and 66-78, wherein the determining step comprises determining whether the cells express the anti-CLDN18-2 antibody agent encoded by the at least one single-stranded RNA when assessed 48 hours after contacting. 80. The process for determining the following characteristics: - whether the antibody agent expressed by the cell selectively binds to a CLDN-18.2 polypeptide compared to a CLDN18.1 polypeptide; - whether the antibody agent expressed by the cells exhibits equivalent target specificity for CLDN-18.2 as observed in a flow cytometry binding assay using a reference CLDN-18.2-targeting monoclonal antibody; - Whether CLDN-18.2-positive cells, but not control cells, were lysed when assessed 48 hours after incubation of immune effector cells (e.g., PBMC cells) and CLDN-18.2-positive cells or CLDN-18.2-negative control cells in the presence of the antibody agent; - whether the antibody agent expressed by the cells exhibits at least a comparable ADCC profile of the targeted CLDN-18.2-positive cells as observed with a reference CLDN-18.2-targeting monoclonal antibody at the same concentration; and - Whether CLDN-18.2-positive cells, but not control cells, were lysed when assessed 2 hours after incubation of CLDN-18.2-positive cells or CLDN-18.2-negative control cells with human serum in the presence of the antibody agent 80. The method of any one of items 65 and 66-79, comprising determining one or more of: 81. The method of any one of items 66 to 80, wherein the cell is present in a subject (e.g., a mouse or monkey subject). 82. The method of item 81, wherein the one or more characteristics include antibody levels in one or more tissues of the subject. 83. If the pharmaceutical composition is characterized as targeting CLDN-18.2, 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. 83. The method according to any one of items 66 to 82, further comprising: 84. The following process: (A) determining one or more characteristics of a single-stranded RNA (ssRNA) encoding part or all of an antibody agent, or a composition thereof, wherein the one or more characteristics are: (i) the length and / or sequence of the ssRNA; (ii) ssRNA integrity; (iii) the presence and / or location of one or more chemical moieties in the ssRNA; (iv) the degree of expression of the antibody drug when ssRNA is introduced into cells; (v) stability of the ssRNA or composition thereof; (vi) the level of the antibody agent in a biological sample derived from an organism into which the ssRNA was introduced; (vii) the binding specificity of the antibody agent expressed from the ssRNA, optionally to CLDN-18.2, and optionally relative to CLDN18.1; (viii) the efficacy of antibody agents in mediating target cell death by ADCC; (ix) the efficacy of antibody agents in mediating target cell death by complement-dependent cytotoxicity (CDC); (x) the identity and amount / concentration of lipids in the composition; (xi) the size of the lipid nanoparticles within the composition; (xii) the polydispersity of the lipid nanoparticles within the composition; (xiii) the amount / concentration of ssRNA in the composition; (xiv) the extent of encapsulation of ssRNA within lipid nanoparticles; and (xv) combinations thereof a step selected from the group consisting of: (B) comparing one or more characteristics of the ssRNA or composition thereof with characteristics of a suitable reference standard; and (C)(i) designating the ssRNA or composition thereof for one or more further steps of manufacture and / or distribution if the comparison demonstrates that the ssRNA or composition thereof meets or exceeds the reference standard; or (ii) taking alternative action if the comparison demonstrates that the ssRNA or composition thereof does not meet or exceed the reference standard. A manufacturing method comprising: 85. The method according to item 84, wherein ssRNA is evaluated and the one or more further steps of step (C)(i) are or include at least formulation of ssRNA. 86. The method according to item 84 or 85, wherein a composition is evaluated, the composition comprises lipid nanoparticles, and the one or more further steps of step (C)(i) is or include shipping and distribution of 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 the administration regimen of a pharmaceutical composition targeting CLDN-18.2, comprising the steps of: (A) administering the pharmaceutical composition according to any one of items 1 to 35 to a group of animal subjects, each of which bears a human CLDN-18.2-positive xenograft tumor, under a predetermined dosing regimen; (B) periodically measuring the tumor size of the animal subject; (C)(i) increasing the dose and / or frequency of administration if the reduction in tumor size following administration of the pharmaceutical composition is not therapeutically adequate; or (ii) reducing the dose and / or frequency of administration if the reduction in tumor size following administration of the pharmaceutical composition is therapeutically relevant and toxic effects are exhibited in at least 30% of the animal subjects; or (iii) not altering the dosing regimen if the reduction in tumor size following administration of the pharmaceutical composition is therapeutically relevant and no toxic effects are noted in the animal subject. A method comprising: This disclosure further provides insight that the 3'-terminal region of mRNA is a highly sensitive and exceptional region with respect to mRNA translation ability and functionality. Both in vitro and in vivo results suggest that single nucleotide substitutions upstream of the poly(A) tail affect mRNA translation ability and functionality. Thus, the present disclosure provides, inter alia, a composition or pharmaceutical formulation comprising RNA, RNA, (i) a coding sequence encoding a polypeptide; (ii) 3'UTR sequence; (iii) a polyA sequence, and (iv) a nucleotide sequence linking the 3'UTR sequence and the polyA sequence, comprising the sequence CUXGAGCUAGC (where X is C, A, or U); Also provided is a composition or pharmaceutical formulation comprising: In some embodiments, the nucleotide sequence linking the 3'UTR sequence and the polyA sequence comprises the sequence CUCGAGCUAGC. In some embodiments, the RNA comprises, in the 5' to 3' direction, a coding sequence encoding a polypeptide, a 3' UTR sequence, a nucleotide sequence linking the 3' UTR sequence and the polyA sequence, and a polyA 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 to the nucleotide sequence of SEQ ID NO:22. In some embodiments, the RNA comprises a 3'UTR 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. In some embodiments, the RNA comprises a 3'UTR comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37. In some embodiments, the polyA sequence is an interrupted sequence of A nucleotides. In some embodiments, the polyA sequence comprises at least 100 nucleotides. In some embodiments, the polyA sequence is x -LA y A x is a sequence of at least 20 A nucleotides, and 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 polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:23, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:23. In some embodiments, the RNA comprises a 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 comprises a 5'UTR comprising the nucleotide sequence of nucleotides 14-53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14-53 of SEQ ID NO:20, preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4AACUAGU, where 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. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of nucleotides 14-53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14-53 of SEQ ID NO:20, preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, where 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 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, preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU. In some embodiments, the RNA comprises a 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, preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of nucleotides 7-53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7-53 of SEQ ID NO:20. In some embodiments, 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. In some embodiments, 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. In some embodiments, the RNA comprises a 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, and, downstream of the coding sequence encoding the polypeptide, a 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 polyA sequence. In some embodiments, 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, downstream of the coding sequence encoding the polypeptide, a 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 polyA sequence. In some embodiments, 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 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 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, downstream of the coding sequence encoding the polypeptide, a 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 polyA sequence. In some embodiments, 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 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 a 5'UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20, and downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 20, and downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of SEQ ID NO:36. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 38, and downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 38, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the RNA comprises a 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, and, downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, and a polyA sequence. In some embodiments, 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, downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, and a polyA sequence. In some embodiments, 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 downstream of the coding sequence encoding the polypeptide 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. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20, and downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a polyA sequence. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 20, and downstream of the coding sequence encoding the polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a polyA sequence. In some embodiments, the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20, and a sequence downstream of the coding sequence encoding the polypeptide comprising 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 that encode two or more polypeptides. In some embodiments, the polypeptide encoded by the coding sequence is an antibody or polypeptide chain thereof, such as an antibody or polypeptide chain thereof that binds to CLDN-18.2. In some embodiments, the antibody or polypeptide chain thereof that binds to CLDN-18.2 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 a binding 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 a binding 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 a binding agent that binds to claudin-18.2 (CLDN-18.2). In some embodiments, the RNA encodes an antibody or antibody-like molecule. In some embodiments, the RNA comprises at least two, e.g., two, RNA molecules, and at least one, e.g., all, of the RNA molecules comprise a 5'UTR, a 3'UTR, a 3'UTR sequence, a polyA sequence, and / or a nucleotide sequence linking the 3'UTR sequence and the polyA sequence as defined. In some embodiments, the RNA is (i) an RNA comprising a coding sequence encoding a first polypeptide chain comprising a heavy chain of an antibody agent; and (ii) RNA comprising a coding sequence encoding a second polypeptide chain comprising a light chain of the antibody agent. Includes. 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 comprising the heavy chain of the antibody agent that binds to CLDN-18.2 and the coding sequence encoding the second polypeptide chain comprising the light chain of the antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the first polypeptide chain comprising the heavy chain of the antibody agent that binds to CLDN-18.2 and the second polypeptide chain comprising the light chain of the antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the RNA, eg, each RNA, comprises a modified nucleoside in place of a uridine. In some embodiments, the RNA, eg, each RNA, comprises a modified nucleoside in place of each 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, eg, each RNA, comprises a 5' cap. In some embodiments, the RNA, e.g., each RNA, is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG. In some embodiments, the RNA, eg, the RNA, is single-stranded RNA. In some embodiments, the RNA, eg, each RNA, is an mRNA. In some embodiments, the RNAs, eg, each RNA, is formulated in a lipid nanoparticle (LNP), eg, each RNA is co-formulated in a lipid nanoparticle (LNP). In some embodiments, the lipids that form the lipid nanoparticles include cationic lipids, polymer-conjugated lipids, and neutral lipids. In some embodiments, a. Cationic lipids are present at 35-65 mol% of the total lipids; a. the polymer-bound lipid is present at approximately 1-2.5 mol% of the total lipid; and c. Neutral lipids are present at 35-65 mol% of total lipids. In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate). In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In some embodiments, the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol. In some embodiments, the lipid nanoparticles have an average size of about 50-150 nm. In some embodiments, the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 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, eg, each RNA, and optionally the particle-forming components, are in separate vials. In some embodiments, the composition or pharmaceutical preparation is for administration to a human. In some embodiments, the composition or pharmaceutical preparation is for introducing 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, hi some embodiments, the composition or pharmaceutical formulation is for systemic delivery of the polypeptide following expression of the polypeptide in hepatocytes. The present disclosure also provides, inter alia, a method for expressing a polypeptide in a subject, comprising: (a) administering the composition so that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in hepatocytes The present invention provides a method comprising: The present disclosure also provides, inter alia, a method for expressing a polypeptide in a subject, comprising: (a) administering the composition so that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in hepatocytes wherein, after expression, the polypeptide is secreted into the bloodstream. A method is provided. The present disclosure also provides, inter alia, a method for systemic delivery of a polypeptide in a subject, comprising: (a) administering the composition so that RNA encoding the polypeptide is introduced into hepatocytes; and (b) expressing the polypeptide in hepatocytes wherein, after expression, the polypeptide is secreted into the bloodstream. A method is provided. In some embodiments, the administration is parenteral, hi some embodiments, the administration is intravenous. [Brief explanation of the drawings]

[0062] [Figure 1] This figure shows that a CLDN-18.2-targeting antibody (RiboMab01), encoded by two RNAs encoding the heavy and light chains of the CLDN-18.2-targeting antibody, was expressed in primary human hepatocytes and CHO-K1 cells. (Figure 1A) Primary human hepatocytes were lipofected with 0.22–55.50 μg / mL of a composition (RB_RMAB01) containing two or more RNAs encoding the heavy and light chains of the CLDN-18.2-targeting antibody. (Left) ELISA analysis of RiboMab01 concentration 48 hours after transfection. (Right) Western blot analysis of cell culture supernatants from the indicated lipofections. Recombinant purified IMAB362 was used as a standard for Western blot analysis. Analysis was performed under non-reducing conditions using 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). Supernatant from untransfected primary human hepatocytes was used as a mock control. (Figure 1B) CHO-K1 cells were lipofected with 2.00–182.00 ng / mL of RB_RMAB01. RiboMab01 concentrations measured by ELISA 48 h after transfection are shown. Error bars represent the standard error of the mean (n = 3). [Figure 2]RiboMab01 binds to a target specific for CLDN-18.2. Target binding of RiboMab01 to CLDN-18.2 was determined by a flow cytometry binding assay visualized using a fluorescently labeled antibody against the F(ab')2 fragment of human IgG(H+L). (Figure 2A) Dilution series of RiboMab01-containing CHO-K1 cell culture supernatant (left) or IMAB362 reference protein (right) were incubated with 5 x 105 CLDN-18.2 + HEK293 transfectants. (Figure 2B) Dilution series of RiboMab01-containing CHO-K1 cell culture supernatant (left) or IMAB362 reference protein (right) were incubated with 5 x 105 CLDN-18.1 + HEK293 transfectants. [Figure 3] High target-specific cytotoxicity mediated by in vitro-expressed RiboMab01 is shown. (Figure 3A) RiboMab01-containing cell culture supernatant from CHO-K1 cells lipofected with RB_RMAB01 was subjected to ADCC assay. 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 with the indicated RiboMab01 and IMAB362 reference protein concentrations for 48 hours. Specific cell lysis measured by a luciferase-based assay is shown. (Figure 3B) RiboMab01-containing cell culture supernatant from CHO-K1 cells lipofected with RB_RMAB01 was subjected to CDC assay. For the CDC assay, CLDN-18.2+ CHO-K1 transfectants (solid line) were used as target cells, and CLDN-18.2-negative CHO-K1 (dotted line) were used as control cells. Target and control cells were incubated with the indicated concentrations of human serum and RiboMab01 for 2 hours. CDC measured with a luciferase-based assay is shown. Error bars represent the standard error of the mean (n = 3). [Figure 4]Figure 4A shows specific tumor cell lysis mediated by RiboMab01 generated in mice. Plasma from mice administered five repeated injections of either 1 μg (approximately 0.04 mg / kg), 3 μg (approximately 0.10 mg / kg), 10 μg (approximately 0.40 mg / kg), and 30 μg (approximately 1.20 mg / kg) of RB_RMAB01 or 80 μg (approximately 3.20 mg / kg) of IMAB362 was collected 24 h after the fifth injection and subjected to a luciferase-based ex vivo ADCC assay. Plasma from untreated mice supplemented with IMAB362 was used as the assay reference. CLDN-18.2+NUG-C4 transfectants were used as targets, and human PBMCs were used as effector cells. (Figure 4A) RiboMab01-mediated ADCC of NUG-C4 cells after 48 h of incubation with 1% plasma is shown. (Figure 4B) No nonspecific lysis of target-negative MDA-MB-231 cells. Error bars are standard error of the mean (n=3). [Figure 5]We demonstrate that RiboMab01 expressed in non-human primates mediates dose-dependent ADCC. Non-human primates (NHPs) were administered three repeated doses of RB_RMAB01 at 0.1, 0.4, or 1.6 mg / kg once weekly. RiboMab01-containing serum from all monkeys collected 24 hours (black bars) and 168 hours (white bars) after the first injection was subjected to a luciferase-based ex vivo ADCC assay. 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 5A) RiboMab01-mediated ADCC of NUG-C4 cells after 48 hours of incubation is shown. (Figure 5B) Nonspecific lysis of target-negative MDA-MB-231 cells is shown. Error bars represent the standard error of the mean (n = 3). (Figure 5C) Serum from NHP No. 14 (1.6 mg / kg RB_RMAB01, RiboMab01 serum concentration 232 μg / mL) collected 48 h after the third dose was used in a luciferase-based ex vivo ADCC assay. CLDN-18.2+NUG-C4 transfectants (solid line) were used as target cells, and CLDN-18.2-negative MDA-MB-231 cells (dotted line) were used as control cells. Human PBMCs from a healthy donor were used as effector cells. ADCC of NUG-C4 cells mediated by RiboMab01-containing serum (red solid line) or recombinant IMAB362 reference protein (black solid line) with EC50s of 66 pM and 151 pM, respectively, is shown. The red and black dotted lines represent weak nonspecific lysis of MDA-MB-231 control cells. Incubation time was 48 hours. Error bars are standard error of the mean (n=3). [Figure 6]This figure shows that systemic availability of RiboMab01 mediates tumor growth inhibition in vivo. Mice bearing subcutaneous CLDN-18.2 + NCI-N87 xenograft tumors were injected IV with 1 μg (approximately 0.04 mg / kg), 3 μg (approximately 0.10 mg / kg), 10 μg (approximately 0.40 mg / kg), and 30 μg (approximately 1.20 mg / kg) of RB_RMAB01, 800 μg (approximately 32 mg / kg) of IMAB362 reference protein, 30 μg (approximately 1.20 mg / kg) of luciferase mRNA, or saline alone on test days 15, 22, 29, 36, 43, and 50 after tumor cell inoculation. Median tumor growth in treatment and control groups is shown. Dotted lines indicate injections. Significance was calculated by two-way ANOVA. ns indicates not significant. [Figure 7] The concentration-time profile of RiboMab01 in mouse serum after a single dose is shown. Balb / cJRj mice received a single IV injection of 1 μg (approximately 0.040 mg / kg), 3 μg (approximately 0.10 mg / kg), 10 μg (approximately 0.40 mg / kg), or 30 μg (approximately 1.20 mg / kg) of the RB_RMAB01 drug formulation and 40 μg (approximately 1.60 mg / kg) of the IMAB362 reference protein. Plasma was collected 6, 24, 96, 168, 264, 336, and 504 hours after administration. RiboMab01 concentrations in plasma, measured by ELISA, are shown. Error bars represent standard error of the mean (n=3). [Figure 8] Concentration-time profiles of RiboMab01 in rat serum after a single dose are shown. RjHan:Wister rats received a single IV injection of 0.04, 0.10, 0.40, or 1.20 mg / kg RB_RMAB01 and 3.60 mg / kg IMAB362 reference protein. Plasma was collected 2, 6, 8, 10, 22, 24, 27, 30, 48, 72, 96, 168, 216, 264, and 336 hours after administration. RiboMab01 concentrations in plasma are shown as measured by ELISA. Error bars are standard error of the mean (n=3). [Figure 9]Figure 1 shows the kinetics of RB_RMAB01 expression in mice after weekly injections. Balb / cJRj mice received IV injections of 1 μg (approximately 0.04 mg / kg), 3 μg (approximately 0.10 mg / kg), 10 μg (approximately 0.40 mg / kg), or 30 μg (approximately 1.20 mg / kg) of RB_RMAB01 and 80 μg (approximately 3.20 mg / kg) of IMAB362 reference protein on test days 1, 8, 15, 21, and 29. Plasma was collected 24 hours before and 24 hours after administration. RiboMab01 concentrations in plasma, measured by ELISA, are shown. Dotted lines indicate injections. Error bars are standard error of the mean (n=3). [Figure 10]

[0023] Figure 1 shows the kinetics of RB_RMAB01 expression after repeated dosing in NHPs. NHPs received IV injections of 0.1, 0.4, or 1.6 mg / kg RB_RMAB01 on study days 1, 8, and 15. Plasma was collected 6, 24, 48, 72, 96, and 168 hours after the first and third doses, 48, 72, and 168 hours after the second dose, and 264, 336, and 504 hours after the third dose. Plasma RiboMab01 concentrations measured by ELISA are shown. Error bars are standard error of the mean (n=3). [Figure 11] Liver targeting of LNP-formulated mRNA in vivo is shown. Mice received a single IV injection of LNP-formulated firefly luciferase mRNA. Bioluminescence was monitored 6, 24, 48, 72, and 144 hours after administration. (Figure 11A) Bioluminescence images 6 hours after administration are shown for individual mice in the ventral position (left) and for single organs of mice No. 1 and 2 (right). (Figure 11B) Quantification of luciferase signal (photons / second) is shown for all analyzed time points (n=5 or 3, average). LN indicates lymph node. [Figure 12]Exemplary embodiments of RNA technology useful for encoding various antibody drug formats ("RiboMabs") and their formulations and applications are shown. (Figure 12A) The RiboMabs® platform is applicable to provide RNA constructs encoding various antibody formats, including, but not limited to, monospecific IgG antibodies, bispecific bi-(scFv)2 antibodies, and bispecific Fab-(scFv)2 antibodies. (Figure 12B) In some embodiments, therapeutic antibodies such as IgG can be encoded by purified mRNA containing modified ribonucleotides (e.g., uridines replaced with pseudouridines) and encapsulated in lipid nanoparticles (mRNA / LNPs). Such mRNA constructs can further include one or more non-coding sequence elements (e.g., to increase RNA stability and / or translation efficiency). In some embodiments, exemplary non-coding sequence elements include, but are not limited to, a cap structure, a 5' UTR, a 3' UTR, a polyadenylation tail, and any combination thereof. In some embodiments, the lipid nanoparticles may contain a conjugated lipid (e.g., a PEG-conjugated lipid), a cationic lipid, and a neutral helper lipid. Such mRNA / LNP drug formulations can be administered to a subject in vivo so that the mRNA is translated in vivo to express the antibody. (Figure 12C) The patient's own somatic cells administered the mRNA / LNP drug formulation described herein can produce the active agent (e.g., an IgG RiboMab) encoded by the mRNA. For example, in some embodiments, upon IV injection, the antibody-encoding mRNA / LNP is internalized by hepatocytes and translated, resulting in systemic plasma concentrations of biologically active RiboMab. Abbreviations: A30L70, poly(A) tail measured at 100 adenosines neutralized by the linker at position 30; bi, bispecific; C, C-terminus; CDS, coding sequence; CH, constant heavy domain; CL, constant light domain; Fab, antigen-binding fragment; IgG, immunoglobulin G; LNP, lipid nanoparticle; mΨ, 1-methylpseudouridine; N, N-terminus; scFv, single-chain variable fragment; TAA, tumor-associated antigen; UTR, untranslated region; VH, variable heavy domain; VL, variable light domain. [Figure 13] 13 is a schematic diagram of an exemplary RNA construct encoding the heavy chain (HC) and light chain (LC), respectively, of an antibody drug. As shown in FIG. 13, such an RNA construct encoding the HC and LC forms an RNA composition (RB_RMAB01), which, in some embodiments, can be formulated into lipid nanoparticles to form an RNA / LNP drug formulation. Abbreviations: polyA, polyadenine tail; CH, constant heavy domain; CL, constant light domain; Sec, secretion signal; UTR, untranslated region; VH, variable heavy domain; VL, variable light domain. [Figure 14] 1 is a graph showing the dose-exposure relationship of RB_RMAB01 at tmax in cynomolgus monkeys. Cynomolgus monkeys (n=3) were given an IV injection of 0.1, 0.4, or 1.6 mg / kg of RB_RMAB01. Dose-dependent RiboMab01 concentrations in plasma at Cmax (mean, n=3) measured by ELISA are shown. The green line indicates the dose that can be administered to human subjects and its corresponding expected serum concentration. [Figure 15] 1 is an exemplary electropherogram of an exemplary RNA mixture containing a first RNA encoding an antibody heavy chain (HC) and a second RNA encoding an antibody light chain (LC). The electropherogram shows two peaks, one for the LC and one for the HC, respectively. A: area under the peak, h: peak height. [Figure 16] Figure 1 shows anti-CLDN18.2 RiboMabs expression in vitro. HEK293T / 17 cells were electroporated with mRNAs encoding anti-CLDN18.2 RiboMabs, all with the same backbone but different coding sequences. Anti-CLDN18.2 RiboMabs concentrations were measured by ELISA 48 hours after transfection. Error bars are standard error of the mean (n=2). [Figure 17]Anti-CLDN18.2 RiboMab exposure in mice after repeated RNA-LNP administration is shown. Balb / cJRj mice were intravenously injected twice weekly with 3 μg or 30 μg of RNA-LNPs containing mRNAs separately encoding the HC and LC of anti-CLDN18.2 RiboMab, along with either scaffold A or B. Serum was collected at the indicated time points after the first administration. The arithmetic mean (n = 3) and standard error of the anti-CLDN18.2 RiboMab concentrations in serum measured by ELISA are shown. The detection limit was 0.074 ng / mL. Downward arrows correspond to the first and second RNA-LNP injections. Luc-RNA-LNPs were used as a negative control. ELISA = enzyme-linked immunosorbent assay; Luc = luciferase. [Figure 18] Figure 1 shows the cytotoxic activity of anti-CLDN18.2 RiboMabs encoded by RNAs utilizing scaffolds A and B. Ex vivo ADCC mediated by anti-CLDN18.2 RiboMabs is shown in mouse serum collected 24 hours after administration of RNA-LNPs at the indicated doses. CLDN18.2-transduced NUGC-4 transfectants were used as target cells, and human PBMCs from healthy donors were used as effector cells at an E:T ratio of 20:1 (top panel). CLDN18.2-negative MDA-MB-231 cells were used as a negative control (bottom panel). Target and effector cells were incubated for 24 hours. Results for the control antibody are shown in the right panel. Data are the mean ± SD of triplicate determinations per mouse. Ab = antibody; ADCC = antibody-dependent cellular cytotoxicity; E:T ratio = effector cell-to-target cell ratio; M1 = mouse No. 1; PBMC = peripheral blood mononuclear cells. [Figure 19A] We show that EPO mRNA transcribed from Scaffold C is superior to Scaffold A in vivo, but inferior to that derived from the Scaffold B / Scaffold D cassette. [Figure 19B] We show that EPO mRNA transcribed from Scaffold C is superior to Scaffold A in vivo, but inferior to that derived from the Scaffold B / Scaffold D cassette. [Figure 20A]A comparison of mRNA translation from scaffolds B, C, and D, which have different coding sequences, is shown, demonstrating that the difference in performance between scaffold C and scaffold B / D is independent of the coding sequence. (Figure 20A) Firefly luciferase mRNA in scaffolds B (●), C (■), and D (▲) was electroporated twice into hiDCs (solid and dashed lines). Bright-Glo assays were performed at the times indicated. [Figure 20B] (FIG. 20B) hiDCs were electroporated twice (solid and dashed lines) with eGFP mRNA in scaffolds C (■) and D (▲). Cells were harvested and assayed by FACS for eGFP expression at the indicated times. [Figure 20C] (Figure 20C) Primary human hepatocytes were lipofected with hIL-18 mRNA in scaffolds B (●), C (■), and D (▲). Supernatants from transfected cells were harvested at the indicated times and assayed by ELISA for the presence of hIL-18. [Figure 21A] Figure 21A shows the translation of firefly luciferase mRNA derived from scaffolds B and C containing different nucleotides at position -9 upstream of the polyA in hiDCs, demonstrating that the 3'UTR end sequence affects long-term translation in vitro. (Figure 21A) Firefly luciferase mRNA derived from scaffold B with A (◆), G (■), T (▲), or C (●) at position -9 upstream of the polyA was electroporated into hiDCs in separate experiments, and luciferase expression was assayed at the indicated time points. [Figure 21B] (Figure 21B) Firefly luciferase mRNA from backbone C with A (◆), G (■), T (▲), or C (●) at position −9 upstream of the polyA was electroporated into hiDCs in separate experiments, and luciferase expression was assayed at the indicated time points. [Figure 22A] Scaffold B is used to demonstrate that the 3'UTR end sequence significantly influences long-term translation in vivo. [Figure 22B] Scaffold B is used to demonstrate that the 3'UTR end sequence significantly influences long-term translation in vivo. [Figure 23A]Using Scaffold D to demonstrate that 3'UTR end sequences significantly affect long-term translation in vivo [Figure 23B] Using Scaffold D to demonstrate that 3'UTR end sequences significantly affect long-term translation in vivo [Figure 24] We show that sequences 5' upstream of the coding sequence also affect long-term translation in vivo. [Figure 25] 1 shows that the combination of 5′ and 3′ sequence elements affects long-term translation in vivo. [Figure 26] Scaffold B performed significantly better than the earlier scaffold versions, scaffolds A and G, for long-term translation in vivo.

[0063] Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, which may vary. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0064] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0065] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0066] 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, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0067] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0068] Specific Definitions a, an, the: As used herein, the terms "a," "an," and "the" are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0069] About or Approximately: As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. Generally, a person skilled in the art familiar with the context will understand the associated degree of dispersion encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" can encompass a range of values ​​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 referenced value.

[0070] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of the composition or agent contained therein to a target site or site to be treated. Those skilled in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), oral, cutaneous (e.g., may be or may include one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may include only a single dose. In some embodiments, administration may include the application of a fixed number of doses. In some embodiments, administration may include administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) for at least a selected period of time.

[0071] And / or: As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0072] 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 contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may contain one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may contain one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, antibody agents utilized in accordance with the present disclosure include intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (such as, for example, Zybody®); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probody®); small modular immunopharmaceuticals (Small Modular ImmunoPharmaceuticals ("SMIP™"); single chain or tandem diabodies (TandAb®); VHH; Anticalin®; Nanobody® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimer®; DART; TCR-like antibodies; Adnectin®; Affilin®; Trans-body®; Affibody®; TrimerX®; microproteins; Fynomer®, Centyrin®; and KALBITOR®.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 consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). In some embodiments, each light chain consists of 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 and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of VL are referred to as 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 antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxy terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins 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. The term "full length" when used in reference to an antibody indicates that the antibody is not a fragment, but includes all of the domains of a particular isotype that are normally 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 one heavy-light chain pair and is used interchangeably herein with "half molecule."In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, an antibody may include covalent modifications (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, an antibody agent is or includes a polypeptide that includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are sequence-identical or contain 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are at least 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 1 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR has been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR have been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has an amino acid sequence that is identical to the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been substituted relative to the reference CDR, but the included CDRs are otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0073] Antibody agents can be produced 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 and phage display technologies. Additional 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)).

[0074] Antibodies can be produced by standard techniques, such as by immunization with an appropriate polypeptide or one or more portions thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing one or more desired epitopes, optionally haptenized to another polypeptide. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunized animals is collected and processed according to known procedures. If the serum containing polyclonal antibodies against the desired epitope 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 antisera are well known in the art.

[0075] Associated: As that term is used herein, two events or entities are "associated" with one another if the presence, level, and / or form of one correlates with that of the other. For example, a particular biological phenomenon (e.g., expression of CLDN-18.2) is considered to be associated with a particular disease, disorder, or condition (e.g., cancer) if its presence correlates (e.g., across a relevant population) with the incidence and / or susceptibility of the disease, disorder, or condition, or correlates with the likelihood of responsiveness to treatment.

[0076] Blood-derived sample: As used herein, the term "blood-derived sample" refers to a sample derived from a subject's blood sample (i.e., a whole blood sample). Examples of blood-derived samples include, but are not limited to, plasma (including, for example, fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood fractions including red blood cells (RBCs), platelets, white blood cells, etc., and cell lysates containing fractions thereof (e.g., cells such as red blood cells, white blood cells, etc. can be harvested and lysed to obtain cell lysates). In some embodiments, the blood-derived sample used in the characterization described herein is a plasma sample.

[0077] Cancer: The term "cancer" is used herein generally to refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, cancer may be characterized by a solid tumor. In some embodiments, cancer may be characterized by a hematological tumor. In general, examples of various types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the oral cavity, throat, larynx and lung, genitourinary cancers such as liver cancer, 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, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, ovarian cancer, breast cancer, glioblastoma, colorectal cancer, gastrointestinal cancer, and cancers of the nervous system, benign lesions such as papilloma, etc.

[0078] Cap: As used herein, the term "cap" refers to a structure comprising or consisting essentially of a nucleoside-5'-triphosphate that is typically attached to the 5' end of an uncapped RNA (e.g., an uncapped RNA having a 5'-diphosphate). In some embodiments, the cap is or includes a guanine nucleotide. In some embodiments, the cap is or includes a naturally occurring RNA 5' cap, including, but not limited to, a 7-methylguanosine cap having the structure referred to as "m7G." In some embodiments, the cap is or includes a synthetic cap analog that resembles the RNA cap structure and has the ability to stabilize RNA when attached thereto, including, 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 an RNA are known in the art. For example, in some embodiments, capped RNA can be obtained by in vitro capping of RNA with a 5' triphosphate group or RNA with a 5' diphosphate group using a capping enzyme system (e.g., 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, where, in addition to GTP, the IVT system also contains a dinucleotide cap analog (e.g., m7GpppG cap analog or N7-methyl,2'-O-methyl-GpppG ARCA cap analog or N7-methyl,3'-O-methyl-GpppG ARCA cap analog). In some embodiments, the cap is Cap0, Cap1, or Cap2, preferably Cap1 or Cap2. According to the present application, the term "Cap 0" refers to the structure "m7GpppN", where N is any nucleoside with an OH moiety at the 2' position.As used herein, the term "cap 1" refers to the structure "m7GpppNm," where Nm is any nucleoside having an OCH3 moiety at the 2' position. As used herein, the term "cap 2" refers to the structure "m7GpppNmNm," where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0079] CLDN-18.2 positive: As used herein, the term "CLDN-18.2 positive" or "CLDN-18.2+" refers to clinically relevant CLDN-18.2 expression and / or activity that may be associated with, for example, a particular disease, disorder, or condition, and / or that may be detected in or on a sample that may be or contain 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 or include, for example, de novo CLDN-18.2 overexpression in cancer cells; alternatively or additionally, in some embodiments, CLDN-18.2 positive expression and / or activity may be associated with or potentially related to exposure to one or more agents or conditions, such as one or more chemotherapeutic agents (e.g., including gemcitabine and / or cisplatin). In some embodiments, CLDN-18.2 "positive" is assessed against an appropriate reference (e.g., a "negative control" such as CLDN-18.2 levels and / or activity in one or more appropriately comparable non-cancerous cells and / or one or more tissues; a "positive control" such as CLDN-18.2 levels and / or activity that may have been determined for one or more known CLDN-18.2-positive cells and / or one or more tissues; and / or an established threshold for CLDN-18.2 levels and / or activity associated with normal (e.g., healthy, non-cancerous) versus non-normal (e.g., cancerous) conditions. In some embodiments, the term "CLDN-18.2+" is used herein to refer to a tumor sample from a cancer patient when determined to exhibit detectable elevated CLDN-18.2 protein expression compared to an appropriate standard (e.g., the level observed in a sample determined to be negative or otherwise known to be negative for CLDN-18.2 expression).In some embodiments, a sample is considered to be CLDN-18.2+ if 50% or more of the tumor cells in the sample are determined to have a CLDN-18.2 protein staining intensity of ≥ 2+ as assessed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) neoplastic tissue; those skilled in the art will recognize that pathologists generally use such scoring systems to interpret IHC data obtained for one or more tumor samples. For example, see Fedchenko and Reifenrath, Diagnostic Pathology (2014) 9:221, which describes various approaches for interpreting and reporting IHC analysis results, including scoring systems. See also Zimmermann et al., Cancer Cytopathology (2014) 48-58. Thus, pathologists will readily recognize that 2+ refers to a malignancy score of 2 or greater, indicating that the results of such immunohistochemistry assays are unfavorable. More precisely, 2+ represents moderate or strong staining on a qualitative scale from "negative" (0), "weak" (1), "moderate" (2), and "strong" (3).

[0080] Concurrent administration: As used herein, the term "concurrent administration" refers to the use of a pharmaceutical composition described herein in combination with another treatment (e.g., administration of another therapeutic agent, such as surgery, radiation, and / or a chemotherapeutic agent described herein, and / or an agent and / or therapy administered that alleviates one or more symptoms or attributes of an associated disease, disorder, or condition [e.g., chemotherapy]) such that the subject receives both. The combined administration of a pharmaceutical composition described herein with such other therapy can be performed simultaneously (e.g., via an overlapping protocol) or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition described herein can contain two or more active agents combined in a single pharmaceutically acceptable carrier (e.g., in a single dosage form). Alternatively, in some embodiments, concurrent administration can involve the administration of two or more physically distinct pharmaceutical compositions, each of which can contain a different active agent or combination of agents; in some such embodiments, one or more (in some embodiments, all) doses of such separate pharmaceutical compositions can be administered substantially simultaneously. In some embodiments, one or more (in some embodiments, all) doses of such separate pharmaceutical compositions can be administered separately, for example, according to an overlapping or sequential regimen. In general, two or more therapies may be considered to be "co-administered" if they are delivered or administered sufficiently close in time that there is at least some temporal overlap in one or more biological effects produced by each on the target cell or subject to which they are administered.

[0081] Codon optimization: As used herein, the term "codon optimization" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, the coding region can be codon-optimized for optimal expression in a subject treated using the RNA (especially mRNA) described herein. Codon optimization is based on the observation that translation efficiency is also determined by the different frequencies of occurrence of tRNAs in cells. Therefore, the sequence of RNA (especially mRNA) can be modified so that codons for which frequently occurring tRNAs are available are inserted instead of "rare codons."

[0082] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the 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 overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administering one or more agents or modalities to a subject receiving one or more other agents or one or more modalities in combination. For clarity, combination therapy does not require that the individual agents be administered together (or necessarily simultaneously) in a single composition, although in some embodiments, two or more agents, or active portions thereof, may be administered together in a combination composition.

[0083] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but are sufficiently similar to permit comparison between them, so that one of skill in the art would understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few varying characteristics. One of skill in the art would understand what degree of identity is required in any given situation for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered equivalent in context. For example, one of skill in the art would understand that sets of circumstances, individuals, or populations are equivalent 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 obtained or phenomena observed under or with respect to different sets of circumstances, individuals, or populations are caused by or indicate changes in these varying characteristics.

[0084] Complementary: As used herein, the term "complementary" is used in reference to oligonucleotide hybridization related by base pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be partial or total. Therefore, any degree of partial complementarity is intended to be included within the scope of the term "complementary", as long as the partial complementarity allows oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to the base pairing rules. Total complementarity or complete complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base pairing rules.

[0085] Comprises, consists: The words "comprises" and variations such as "comprising" are understood to imply the inclusion of a stated feature, element, member, integer, or step or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and to those that do not materially affect one or more of the basic and novel characteristics 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 "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0086] Contacting: As used interchangeably herein, the terms "delivery," "delivering," or "contacting" refer to exposing a relevant target (e.g., a cell, tissue, organism, etc.) to one or more RNAs described herein or a composition comprising or delivering the same, such that the RNA is delivered to the target cell (e.g., the cytosol of the target cell). The target cell can be cultured in vitro or ex vivo, or can be present in a subject (in vivo). Those skilled in the art will understand that various contacting methods can be utilized to achieve such delivery to target cells in in vitro, ex vivo, or in vivo applications. In some embodiments, contacting of cells in culture can be or can include in vitro transfection. In some embodiments, contacting can utilize one or more delivery vehicles (e.g., lipid nanoparticles described herein). In some embodiments, contacting can be or can include administering a pharmaceutical composition described herein to a subject.

[0087] Detection: The term "detecting" is used broadly herein to include any suitable means for determining the presence or absence of an entity of interest in a sample or any form of measurement of the entity of interest. Thus, "detecting" can include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of the entity of interest. Quantitative and qualitative determinations, measurements, or assessments, including semi-quantitative, are included. Such determinations, measurements, or assessments may be relative, for example, when the entity of interest is detected compared to a control standard, or may be absolute. Thus, the term "quantifying," when used in the context of quantifying an entity of interest, can refer to absolute or relative quantification. Absolute quantification can be achieved by correlating the detected level of the entity of interest with a known control standard (e.g., via the creation of a standard curve). Alternatively, relative quantification can be achieved by comparing the detected levels or amounts of two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0088] Disease: As used herein, the term "disease" refers to a disorder or condition that typically impairs the normal function of a tissue or system of a subject (e.g., a human subject) and is typically manifested as characteristic signs and / or symptoms. In some embodiments, an exemplary disease is cancer.

[0089] Encode: As used herein, the term "encode" or "encoding" refers to the sequence information of a first molecule that leads to the production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. 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, a nucleic acid encodes a polypeptide when transcription and / or translation of the nucleic acid produces a polypeptide in a cell or other biological system.

[0090] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component or 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, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0091] Expression: As used herein, "expression" of a nucleic acid sequence 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.

[0092] Fc region: As used herein, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least a hinge region, a CH2 domain, and a CH3 domain.

[0093] 5 prime untranslated region: As used herein, the term "5 prime untranslated region" or "5'UTR" refers to the sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region of the RNA.

[0094] Homology: As used herein, the term "homology" or "homologue" 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 one another 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 one another 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., contain residues with related chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains. Substitution of one amino acid for another amino acid of the same type can often be considered a "homologous" substitution.

[0095] 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 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 one another 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. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence to be 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.If a 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 molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.Sequence comparison and determination of percent identity between two sequences can be achieved 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 is incorporated into the ALIGN program (version 2.0).In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program use a 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 provided over 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 entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is provided over 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 provided over the entire length of the reference sequence.

[0096] Immunogenicity: "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is a relatively nonspecific, immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0097] Immunoglobulin: As used herein, the term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antibodies or antigen receptors such as 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 and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2002).nd ed. Raven Press, NY (1989). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily V L or VL (variable light chain) domain / region, C L or CL (constant light chain) domain / region, V H or a VH (variable heavy chain) domain / region, and C H or CH (constant heavy chain) domain / region C H 1(CH1), C H 2(CH2), C H 3(CH3) and C HThe heavy chain constant region is typically composed of immunoglobulin domains or regions, such as CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds within the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically consists of a VL and a CL. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions in which the sequence and / or the configuration of structurally defined loops may be hypervariable), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradicted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc MP Nucleic Acids Research 1999;27:209-212). 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 solely to the CDR sequences determined 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 carboxy termini. In mammals, there are two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region.The constant regions are essentially conserved among the different isotypes of immunoglobulins, while the variable parts are highly diverse and are responsible for antigen recognition.

[0098] Isolated: "Isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid or polypeptide naturally occurring in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0099] Lipid: As used herein, the term "lipid" refers to a molecule containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are also often referred to as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, the amphiphilic nature allows the molecules to self-assemble into organized structures and various phases. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0100] Locally Advanced Tumor: As used herein, the terms "locally advanced tumor" or "locally advanced cancer" refer to their art-recognized meanings, which may vary for different types of cancer. For example, in some embodiments, a locally advanced tumor refers to a tumor that is large but has not yet spread to another body part. In some embodiments, a locally advanced tumor is used to describe a cancer that has grown outside the tissue or organ in which it began, but has not yet spread to distant sites within the subject's body. By way of example only, in some embodiments, locally advanced pancreatic cancer typically refers to stage III disease, which involves tumor spread to adjacent organs (e.g., lymph nodes, liver, duodenum, superior mesenteric artery, and / or celiac artery) but no signs of metastatic disease; however, complete surgical resection with negative pathological margins is not possible.

[0101] Mol %: As used herein, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100. As used in this disclosure, "mol % of total lipids" is defined as the ratio of the number of moles of one 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.

[0102] Non-immunogenic RNA: As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that does not induce a response by the immune system when administered to, for example, a mammal, or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and processing that render the non-immunogenic RNA non-immunogenic, i.e., a standard RNA (stdRNA).

[0103] Nucleic Acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid can comprise one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or a backbone comprising one or more peptide bonds, e.g., as in "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, 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 residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the modified sugars in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by in vivo or in vitro polymerization based on a complementary template, renaturation in a recombinant cell or system, 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, 400 0, 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.

[0104] Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used, for example, as an indicator of the size of a polynucleotide, the specific number of nucleotides refers, for example, to the number of nucleotides on one strand of the polynucleotide.

[0105] Optional: As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0106] Patient: As used herein, the term "patient" refers to any organism suffering from or at risk of a disease or 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 human. In some embodiments, the patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition amenable to the provided techniques is or includes cancer or the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat the disease, disorder, or condition. In some embodiments, the patient is a cancer patient.

[0107] Polypeptide: As used herein, the term "polypeptide" typically has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides that are functional, biologically active, 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, polypeptides may contain L-amino acids, D-amino acids, or both, and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, polypeptides may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or may include peptidomimetics).

[0108] Pharmaceutically active polypeptide: As used herein, the term "pharmaceutically active polypeptide" refers to a peptide or polypeptide that can be used to treat an individual in whom expression of the peptide or polypeptide would be beneficial, for example, to ameliorate symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or pathology. A pharmaceutically active peptide or polypeptide can have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide" or "pharmaceutically active polypeptide" includes the entire peptide or polypeptide and can also refer to a pharmaceutically active fragment thereof. The term can also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0109] Specific examples of pharmaceutically 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, pharmaceutically active peptides and polypeptides comprise substituted proteins.

[0110] An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components 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).

[0111] In one embodiment, the immunostimulatory agent is a cytokine or a variant thereof. 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. In another embodiment, the immunostimulatory agent comprises an adjuvant-type immunostimulatory agent, such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins, such as CD80, CD86, and ICAM-1.

[0112] The term "cytokine" refers to a protein having a molecular weight of approximately 5-60 kDa and involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, upon release, cytokines affect the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones, and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (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), and variants and derivatives thereof.

[0113] According to the present disclosure, cytokine can be naturally occurring cytokine or its functional fragment or variant.Cytokine can be human cytokine and can be derived from any vertebrate, particularly any mammal.One particularly preferred cytokine is interferon alpha.

[0114] 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 release interferons to enhance the antiviral defenses of nearby cells. Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to interferon receptors on the surface of the cells, thereby preventing viral replication within the cells.

[0115] Based on the type of receptor through which the interferons signal, they are typically divided into three classes: 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.

[0116] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0117] 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.

[0118] Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on characteristic structural features. However, their amino acid sequence similarity is fairly weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

[0119] According to the present disclosure, the interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be a human interleukin and may be derived from any vertebrate, particularly any mammal.

[0120] The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulatory agent can be fused to an extended pharmacokinetic (PK) group that increases its circulatory half-life. Non-limiting examples of extended PK groups include serum albumin or a fragment thereof or a variant of serum albumin or a fragment thereof (e.g., HSA or a fragment or variant thereof), immunoglobulin Fc or an Fc fragment and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, the entire contents of which are incorporated herein by reference.

[0121] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides a method for treating a subject having a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject an RNA (particularly mRNA) described herein that encodes the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) into a subject having a deficiency of such a protein. The term also refers to the introduction of a protein into a subject who requires or would benefit from the provision of a protein, e.g., a subject suffering from a protein deficiency. The term "disorder characterized by a protein deficiency" refers to any disorder exhibiting a pathology caused by the absence or insufficient amount of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein deficiency may be involved in infection, immunosuppression, organ failure, glandular disorders, radiation damage, nutritional deficiency, poisoning, or other environmental or external insults.

[0122] The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction typically 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 hormone by a receptor; (v) relaying and amplification of the signal; and (vi) degradation of the hormone. Hormones differ from cytokines in that (1) they typically act at more consistent concentrations and (2) they are generally produced by specific cell types. In some embodiments, a "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0123] The term "adhesion molecule" refers to a protein located on the surface of a cell and involved in binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0124] Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cell signaling pathways, for example, transmembrane protein kinase pathways such as receptor tyrosine kinases (RTKs). Such regulation can result in cell growth, division, survival or differentiation, or apoptosis. Specific examples of integrins include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α L β2, α M β2, α IIb β3, α V β1, α V β3, α V β5, α V β6, α V β8, and α6β4.

[0125] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in cell recognition, binding, and / or adhesion processes. Molecules belonging to this superfamily share the characteristic of containing regions known as immunoglobulin domains or immunoglobulin folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD3-γ, CD3-δ, CD-3ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and the like.

[0126] The term "immunologically active compound" refers to any compound that alters the immune response, for example, by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and can also downregulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants. Specific examples of immunologically active compound include interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferon, integrin, addressin, selectin, homing receptor, and antigen, particularly tumor-associated antigen, pathogen-associated antigen (such as bacterial antigen, parasite antigen or virus antigen), allergen and autoantigen.Immunologically active compound can be vaccine antigen, that is, the antigen that inoculates to subject induces immune response.

[0127] In some embodiments, the RNA (particularly mRNA) described in this disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject. "Peptides or polypeptides comprising an epitope for inducing an immune response against an antigen in a subject" are also referred to herein as "vaccine antigens," "peptide antigens and protein antigens," or simply "antigens."

[0128] In some embodiments, RNA encoding the vaccine antigen is expressed in cells of a subject, such as muscle cells or antigen-presenting cells (APCs), to provide the vaccine antigen. In some embodiments, expression of the antigen is on the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, RNA encoding the vaccine antigen is administered systemically, for example, intravenously. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, RNA encoding the vaccine antigen is administered intramuscularly.

[0129] Vaccine antigens comprise epitopes for inducing immune responses against antigens in subjects.Therefore, vaccine antigens comprise antigen sequences for inducing immune responses against antigens in subjects.Such antigen sequences can correspond to target antigens or disease-related antigens, such as proteins or tumor antigens of infectious agents (e.g., viral antigens or bacterial antigens), or their immunogenic variants, or immunogenic fragments or immunogenic variants of target antigens or disease-related antigens.Therefore, antigen sequences can comprise at least one epitope of target antigens or disease-related antigens or their immunogenic variants.

[0130] The antigen sequence or its processing product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR or CAR, carried by an immune effector cell. In some embodiments, the antigen sequence is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigen sequence or fragment.

[0131] In some embodiments, RNA encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, said binding resulting in stimulation, priming and / or expansion of the immune effector cells.

[0132] According to the present disclosure, an "antigen" encompasses any substance that elicits an immune response and / or any substance against which an immune response or mechanism, such as a cellular and / or humoral response, is directed. This also includes situations in which an immune response or mechanism is directed against one or more antigenic peptides, particularly when the antigen is processed into antigenic peptides and presented in the context of MHC molecules. In particular, "antigen" relates to any substance, such as a peptide or polypeptide, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen" can include molecules that contain at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0133] The term "autoantigen" (or "self-antigen") refers to an antigen that originates within a subject's body (i.e., an autoantigen may also be called a "self-antigen") and that produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen may be the cause of an "autoimmune disease."

[0134] According to the present disclosure, any suitable antigen that is a candidate for an immune response may be used, and the immune response may include a humoral or cellular immune response, or both. In some embodiments of the present disclosure, the antigen is presented by cells, such as antigen-presenting cells, in association with MHC molecules, resulting in an immune response to the antigen. The antigen may correspond to a naturally occurring antigen or a product derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may 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.

[0135] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0136] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is present primarily intracellularly or as a surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen derived from a virus, bacterium, unicellular organism, or parasite, e.g., a viral antigen such as a viral ribonucleoprotein or coat protein. In some embodiments, the antigen should be presented by an MHC molecule that leads to modulation, particularly activation of cells of the immune system, such as CD4+ and CD8+ lymphocytes, particularly through modulation of the activity of T cell receptors.

[0137] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by an antibody, T cell, or B cell, particularly when presented in the context of an MHC molecule. An epitope of a protein can include continuous or discontinuous portions of the protein and can be, for example, about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length.

[0138] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to the complex of genes that comprises MHC class I and MHC class II molecules and is present in all vertebrates.

[0139] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation and / or the amino acid sequence that breaks immune tolerance is fused to the antigenic peptide or polypeptide (antigen sequence) directly or via a linker.

[0140] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the integrated body's response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, to the agent. An immune response is characterized by the development of antibodies against one or more antigens, as well as antigen-specific T lymphocytes, e.g., CD4, which can be detected in various in vitro proliferation or cytokine production tests. + and CD8 + T lymphocytes, e.g., CD8 + The reaction may include one or more responses selected from the group consisting of: T lymphocyte expansion.

[0141] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons and, as described herein, relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding same, to stimulate an immune response against said one or more immunogens or antigens or against cells characterized by the presentation of said one or more immunogens or antigens.

[0142] The term "allergen" refers to a type of antigen that originates outside the subject's body (i.e., allergens are sometimes called "xenoantigens") and causes the subject's immune system to mount an unusually vigorous immune response to repel a perceived threat that would otherwise be harmless to the subject. "Allergy" is a disease caused by such a vigorous immune response to allergens. Allergens are typically antigens that can stimulate type I hypersensitivity reactions in atopic individuals via an immunoglobulin E (IgE) response. Specific examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and house dust mite proteins (e.g., Der p 2).

[0143] The term "growth factor" refers to a molecule that can stimulate cell growth, proliferation, healing, and / or cell differentiation. Typically, growth factors function 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. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs) such as VEGFA, epidermal growth factor (EGF), insulin-like growth factors, ephrins, macrophage colony-stimulating factors, granulocyte colony-stimulating factors, granulocyte-macrophage colony-stimulating factors, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (an anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β)), and tumor necrosis factor alpha (TNF-α). In some embodiments, a "growth factor" is a peptide or polypeptide growth factor.

[0144] The term "protease inhibitor" refers to a molecule, particularly a peptide or polypeptide, that inhibits the function of a protease. Protease inhibitors can be classified by the protease they inhibit (e.g., aspartic acid protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serpins). Specific examples of protease inhibitors include serpins, such as alpha-1-antitrypsin, aprotinin, and bestatin.

[0145] The term "enzyme" refers to a macromolecular biological catalyst that promotes chemical reactions. Like any catalyst, enzymes are not consumed by 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 a subject; for example, enzyme dysfunction (particularly reduced activity, which can be caused by either mutation, deletion, or reduced production) results in disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0146] The term "receptor" refers to a protein molecule that receives a signal (e.g., a chemical signal called a ligand) from outside the cell. Binding of a signal (e.g., a ligand) to a receptor triggers some kind of cellular response, such as the intracellular activation of a kinase. Receptors include transmembrane receptors (such as ion channel-linked (ionotropic) receptors, G protein-coupled (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (such as cytoplasmic and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to a growth factor.

[0147] The term "apoptosis regulator" refers to molecules, particularly peptides or polypeptides, that regulate apoptosis, i.e., activate or inhibit apoptosis. Apoptosis regulators can be divided into two broad classes: those that regulate mitochondrial function and those that regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) that maintain mitochondrial integrity by preventing the loss of mitochondrial membrane potential and / or the release of proapoptotic proteins such as cytochrome C into the cytosol. Proapoptotic proteins that promote the release of cytochrome C (e.g., BAX, BAK, BIM) also belong to this first class. The second class includes inhibitors of apoptosis proteins (e.g., XIAP) or proteins such as FLIP that block caspase activation.

[0148] 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 can regulate cell division, proliferation, and death throughout life, cell migration and organization during embryonic development, and / or in response to signals from outside the cell, such as hormones. Transcription factors typically contain at least one DNA-binding domain that binds to a specific DNA sequence 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.

[0149] The term "tumor suppressor protein" refers to a molecule, particularly a peptide or polypeptide, that protects cells from a step on the path to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exert a dampening or suppressive effect on cell cycle regulation and / or promote apoptosis. Their functions may be one or more of the following: suppression of genes essential for cell cycle continuation; coupling of the cell cycle to DNA damage (cell division should not occur as long as damaged DNA is present in the cell); initiation of apoptosis when damaged DNA cannot be repaired; metastasis suppression (e.g., preventing tumor cell dispersal, blocking 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, suppressor of tumorigenicity 5 (ST5), suppressor of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).

[0150] The term "structural protein" refers to a protein that imparts rigidity and stiffness to otherwise fluid biological components. Structural proteins are mostly fibrous (such as collagen and elastin), but can also be globular (such as actin and tubulin). Globular proteins are usually soluble as monomers but polymerize to form long fibers that can, for example, constitute the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin, and dynein) that can generate mechanical force, and 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.

[0151] The term "reprogramming factor" or "reprogramming transcription factor" relates to a molecule, particularly a peptide or polypeptide, which, when expressed in a somatic cell, optionally together with further agents such as further reprogramming factors, results in the reprogramming or dedifferentiation of said somatic cell into a cell with stem cell properties, particularly pluripotency. Particular examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0152] The term " genome engineering protein " refers to the protein that can insert DNA, delete DNA or replace DNA in the genome of interest.Specific examples of genome engineering protein include meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9).

[0153] The term "blood protein" refers to peptides or polypeptides present in a subject's plasma, particularly the plasma of a healthy subject. Blood proteins have diverse functions, such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), and protease inhibitors (e.g., α1-antitrypsin). 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.

[0154] Thus, in some embodiments, the pharmaceutically active peptide or polypeptide is selected from the group consisting of: (i) a cytokine, more preferably EPO, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2) and interleukin 10 (IL-11); (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunologically active compound, particularly an antigen such as a viral or bacterial antigen, for example 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 alpha 1-antitrypsin; (viii) preferably herpes simplex virus type 1 thymidine kinase. (xii) tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, for example, selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome engineering proteins, particularly clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

[0155] In some embodiments, the pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response in the subject against the one or more antigens or one or more epitopes, which may be therapeutic or partially or fully protective.

[0156] In some embodiments, the RNA encodes at least one epitope, e.g., 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.

[0157] In some embodiments, the target antigen is a tumor antigen, and the antigen sequence (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen that contains one or more amino acid changes.

[0158] 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 its immunogenic variant. 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 its immunogenic variant.

[0159] In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. Thus, in some embodiments, the RNA (particularly mRNA) described in this disclosure encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. 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.

[0160] Recombinant: As used herein, the term "recombinant" means "produced by genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0161] Reference / Reference Standard: As used herein, "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest 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 determined 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 includes established specifications (e.g., relevant acceptance criteria). Typically, as will be understood by one of skill in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. One of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0162] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses 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 may include one or more modifications, including, but not limited to, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (e.g., conjugation, reverse linkage, etc.), (b) base modifications, such as substitution with a modified base, a stabilized base, a destabilized base, or a base that forms a base pair with an expanded repertoire of partners, or a conjugated base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) internucleoside bond modifications, including phosphodiester bond modifications or substitutions. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0163] 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 includes both single-stranded and double-stranded portions. In some embodiments, the RNA may include a backbone structure as described in the definition of "nucleic acid / polynucleotide" above. The RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, the RNA is an mRNA. In some embodiments, the RNA is an mRNA, the RNA typically includes a poly(A) region at its 3' end. In some embodiments, the RNA is an mRNA, the RNA typically includes an art-recognized cap structure at its 5' end, e.g., for recognition of the mRNA and binding to ribosomes to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic and / or chemical synthesis).

[0164] Secretory signal: As used herein, the term "secretory signal" or "signal peptide" refers to an amino acid sequence present in a polypeptide that can target the polypeptide to the secretory pathway. Typically, secretory signals are cleaved after translocation of the RNA into the endoplasmic reticulum following translation. Secretory signals are typically short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids in length) peptides. Secretory signals can be present at the N-terminus of a polypeptide.

[0165] Selective or specific: The terms "selective" or "specific," as used herein with respect to an active agent, are understood by those skilled in the art to mean that the agent discriminates between potential target entities, conditions, or cells. For example, in some embodiments, an agent is said to "specifically" bind to a target if it selectively binds to that target in the presence of one or more competing surrogate targets. In many embodiments, the specific interaction depends on the presence of a particular structural feature (e.g., an epitope, cleft, binding site) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity can be assessed relative to the specificity of the target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding moiety. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding moiety. In some embodiments, a CLDN-18.2-targeting antibody agent encoded by one or more RNAs (e.g., those described herein) does not detectably bind to a competing surrogate target (e.g., a CLDN18.1 polypeptide) under conditions that bind to a CLDN-18.2 polypeptide. In some embodiments, a CLDN-18.2-targeting antibody agent binds to a CLDN-18.2 polypeptide with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to one or more of its competing surrogate targets, including, for example, a CLDN18.1 polypeptide.

[0166] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in an environment in which binding occurs. An antibody agent that interacts with one specific 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 assessed by detecting or determining the degree of association between the CDRs of the antibody agent and their partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the antibody agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the antibody agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0167] Subject: As used herein, the term "subject" refers to an organism to which a composition described herein is administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, household pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject is susceptible to a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject exhibits one or more symptoms or characteristics 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 characteristics of a disease, disorder, or condition (e.g., cancer). In some embodiments, the subject is a person having one or more characteristics characteristic of susceptibility to, 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 and / or who has been diagnosed and / or treated.

[0168] Susceptible: An individual who is "susceptible" to a disease, disorder, or condition is at risk of developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions 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., family members of an individual suffering from the disease, disorder, or condition; carriers of genetic markers or other biomarkers associated with the disease, disorder, or condition, etc.).

[0169] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0170] Synthetic: As used herein, the term "synthetic" refers to an entity that is artificial, or created with human intervention, or that originates synthetically rather than naturally. For example, in some embodiments, a synthetic nucleic acid or synthetic polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments, by solid-phase synthesis. In some embodiments, the term "synthetic" refers to an entity that is created outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or synthetic polynucleotide refers to a nucleic acid molecule (e.g., RNA) that is produced by in vitro transcription using a template.

[0171] Therapeutic Agent: As used interchangeably herein, the phrases "therapeutic agent" or "therapy" refer to an agent or intervention that, when administered to a subject or patient, has a therapeutic effect and / or induces a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, alleviate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0172] 3 prime untranslated region: As used herein, the term "3 prime 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 an open reading frame sequence. In other embodiments, the 3'UTR does not begin immediately after the stop codon of the coding region of an open reading frame sequence.

[0173] Threshold level (e.g., acceptance criteria): As used herein, the term "threshold level" refers to a level used as a reference for obtaining information about and / or classifying measurement results, e.g., measurement results obtained in an assay. For example, in some embodiments, the threshold level refers to a value measured in an assay that defines a dividing line between two subsets of a population (e.g., batches that meet quality control standards versus batches that do not meet quality control standards). Thus, values ​​above the threshold level define one subset of the population, and values ​​below the threshold level define the other subset of the population. The threshold level can be determined based on one or more control samples or across a population of control samples. The threshold level can be determined before, simultaneously with, or after the measurement of interest. In some embodiments, the threshold level can be a range of values.

[0174] Transfection: As used herein, the term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this 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 present in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can 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 this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, resulting in the exogenous nucleic acid being diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved, for example, by using a virus-based or transposon-based system for transfection. RNA can be transfected into cells to transiently express its encoded protein.

[0175] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later stage of the disease, disorder, and / or condition.

[0176] Unresectable tumor: As used herein, the term "unresectable tumor" typically refers to a tumor characterized by one or more features that, according to sound medical judgment, are considered to indicate that the tumor cannot be safely removed by surgery (e.g., without undue harm to the subject), and / or a tumor for which a competent medical professional has determined that the risks to the subject of tumor removal outweigh the benefits associated with such removal. In some embodiments, an unresectable tumor refers to a tumor that involves and / or has grown into an essential organ or tissue (including blood vessels that may not be reconstructable) and / or a tumor in a location that cannot be easily accessed surgically without undue risk of damage to one or more other vital or essential organs and / or tissues (including blood vessels). In some embodiments, the "unresectableness" of a tumor refers to the possibility of achieving a margin-negative (R0) resection. In the context of pancreatic cancer, irregular stenosis of major vessels by tumor, such as the superior mesenteric artery (SMA) or celiac artery, portal vein obstruction, and the presence of celiac or para-aortic lymphadenopathy, are generally recognized findings that preclude R0 surgery. Those skilled in the art will understand the parameters that determine whether a tumor is unresectable.

[0177] Those skilled in the art reading this specification will understand that, in many embodiments, standard techniques are available and may be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and / or transformation (e.g., electroporation, lipofection, transfection). Enzymatic reactions and / or purification techniques may typically be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. In many embodiments, the foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF THE INVENTION

[0178] Outcomes of standard-of-care (SOC) therapy remain poor for many cancer patients, particularly those with relapsed or refractory advanced solid tumors. Treatment options typically include further palliative chemotherapy, which may be poorly tolerated after prior repeated exposure to cytotoxic compounds, or best supportive care, and investigational therapies without demonstrated benefit. Treatment in this population is not curative, and expected overall survival is several months. Immunotherapy has emerged as an effective treatment option for some cancers with high unmet medical need. Specifically, immune checkpoint inhibitors have been approved for treatment across a variety of cancer indications and act by activating pre-existing anti-tumor-specific T cells. Medical need remains high for various cancer types. The present disclosure provides insights and techniques for treating cancer (e.g., pancreatic cancer and / or cholangiocarcinoma) with therapies targeting claudin 18.2 (CLDN-18.2), among others.

[0179] In some embodiments, the present disclosure provides RNA technologies for delivering monoclonal antibodies targeting CLDN-18.2 that, among other things, combine both potent antitumor properties and an excellent safety profile, eliminating the hurdles of the time-consuming and laborious antibody manufacturing process. Without wishing to be bound by any particular theory, the present disclosure proposes that such RNA delivery modes may achieve one or more improvements, such as a reduced incidence (e.g., frequency and / or severity) of treatment-emergent adverse events ("TEAEs") and / or effective administration with an improved relationship between efficacy levels and TEAE levels (e.g., an improved therapeutic window), compared to that observed when the corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements may be achieved, inter alia, by delivering IMAB362 via administration of one or more RNAs (e.g., one or more ssRNAs, such as one or more mRNAs) encoding IMAB362.

[0180] In some embodiments, the present disclosure provides, inter alia, the insight that one or more mRNAs encoding an antibody agent (e.g., IMAB362) or a functional portion thereof, optionally formulated with lipid nanoparticles (LNPs) 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 efficiently produce the encoded antibody agent (e.g., IMAB362) at therapeutically relevant plasma concentrations, e.g., as shown in FIG. 14 for a CLDN-18.2-targeting antibody agent expressed from RNA (e.g., one described herein). In some embodiments, the antibody agent is expressed from mRNA, e.g., engineered for minimal immunogenicity, and / or formulated into lipid nanoparticles (LNPs). In some embodiments, the mRNA encoding the antibody agent may include modified nucleotides (e.g., but not limited to, pseudouridine and / or 1-methyl-pseudouridine).

[0181] Furthermore, the present disclosure provides insight into, among other things, the ability of CLDN-18.2-targeting antibody agents delivered as described herein to induce antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells), while potentiating the cytotoxic effects of one or more chemotherapy and / or other anticancer therapies by harnessing the recipient subject's immune system. In some embodiments, such combination therapy may, for example, prolong progression-free survival and / or overall survival compared to each individual therapy administered alone and / or another appropriate reference.

[0182] Without wishing to be bound by any particular theory, the present disclosure observes that certain chemotherapeutic agents, such as gemcitabine, oxaliplatin, and 5-fluorouracil, have been shown to upregulate pre-existing CLDN-18.2 expression levels in pancreatic cancer cell lines; furthermore, these agents were not observed to increase de novo expression in CLDN-18.2-negative cell lines. See, for example, Tureci et al. (2019) "Characterization of Zolbetuximab in pancreatic cancer models." In Oncoimmunology 8(1), pp. e1523096.

[0183] The present disclosure provides, inter alia, the insight that the CLDN-18.2-targeted therapies described herein may be particularly useful and / or effective when administered to one or more tumors (e.g., tumor cells, subjects in which such one or more tumors and / or one or more tumor cells are suspected and / or detected, etc.) characterized (e.g., determined to exhibit and / or expected or predicted to exhibit elevated expression and / or activity) of CLDN-18.2 expression and / or activity in tumor cells (e.g., which may or may have resulted from exposure to one or more chemotherapeutic agents). Indeed, inter alia, the present disclosure teaches that the provided CLDN-18.2-targeted therapies described herein (e.g., administration of RNA, more specifically, mRNA encoding a CLDN-18.2-targeting antibody agent) may provide synergistic treatment when administered in combination (e.g., to a subject that has received and / or is receiving or has been exposed to) one or more CDLN-18.2-potentiating agents (e.g., one or more particular chemotherapeutic agents). Thus, in some embodiments, the CLDN-18.2-targeted therapies described herein may be useful in combination with other anti-cancer agents that are expected and / or have been demonstrated to upregulate CLDN-18.2 expression and / or activity in tumor cells.

[0184] Thus, the present disclosure provides, among other things, insights and techniques for treating cancer, particularly cancers associated with CLDN-18.2 expression. In some embodiments, the provided techniques are effective in treating pancreatic cancer. In some embodiments, the provided techniques are effective in treating gastric or gastroesophageal cancer. In some embodiments, the provided techniques are effective in treating cholangiocarcinoma. In some embodiments, the provided techniques are effective in 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.

[0185] I. Claudin 18.2 Polypeptide 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, a family of over 20 structurally related proteins involved in the formation of tight junctions in epithelia and endothelia.

[0186] 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 in the gastric epithelium. Gastric tight junctions are highly specialized to repel gastric acid, which can damage the stomach lining.

[0187] CLDN-18.2 is a highly selective gastric lineage antigen (Sahin et al. 2008). Typically, its expression is restricted to short-lived differentiated cells of the gastric epithelium in the pits and basal regions of the gastric glands. The stem cell zone, where differentiated epithelial cells of the gastric glands are continuously replenished, is CLDN-18.2 negative. Without wishing to be bound by theory, it is generally believed that other normal cell types in the human body do not express CLDN-18.2 at the transcriptional or protein level.

[0188] CLDN18 expression in cancer. CLDN-18.2 is expressed in a variety of human cancers, including gastric, gastroesophageal (GE), and pancreatic cancer (PC) (Karanjawala et al. 2008; Coati et al. 2019), as well as precancerous lesions (Woll et al. 2014; Tanaka et al. 2011). Tumor-associated expression of CLDN-18.2 has also been detected in ovarian cancer (Sahin et al. 2008), cholangiocarcinoma (Shinozaki et al. 2011), and lung cancer (Micke et al. 2014).

[0189] Approximately 77% of primary gastric adenocarcinomas (GACs) are CLDN-18.2+. 56% of GACs show strong CLDN-18.2 expression, defined as staining intensity ≥ 2+ by immunohistochemical analysis, in at least 60% of tumor cells. CLDN-18.2 expression is more frequent in diffuse gastric cancer than in intestinal gastric cancer. CLDN-18.2 protein is also frequently detected in lymph node metastases of gastric cancer and distant metastases to the ovaries (so-called Krukenberg tumors). Furthermore, 50% of esophageal adenocarcinomas show significant CLDN-18.2 expression.

[0190] In pancreatic cancer, CLDN-18.2 is expressed in pancreatic ductal adenocarcinoma (PDAC) with an incidence of 60–90% (Karanjawala et al. 2008; Woll et al. 2014). PDAC, which accounts for over 80% of all pancreatic neoplasms, is the seventh most common cancer in Europe and the fourth leading cause of cancer-related death in the European Union (Ferlay et al. 2010; Jemal et al. 2011; Seufferlein et al. 2012). Nearly 60% of patients with PDAC express membrane-bound CLDN-18.2, and 20% of patients with pancreatic neuroendocrine neoplasms have ectopically activated CLDN-18.2. CLDN-18.2 is expressed in primary and metastatic PDAC lesions (Woll et al. 2014).

[0191] 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 its involvement in the proliferation of CLDN-18.2+ tumor cells.

[0192] Exemplary sequences for CLDN-18.2 (SEQ ID NO: 32) and the splice variant CLDN18.1 (SEQ ID NO: 33) are shown below: [Table 1]

[0193] II. Exemplary Antibody Agents Targeting Claudin 18.2 Polypeptides In some embodiments, an antibody agent targeting CLDN-18.2 specifically binds to a CLDN-18.2 polypeptide. In some embodiments, an antibody agent targeting CLDN-18.2 specifically binds to the first extracellular domain (ECD1) of a 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. In some embodiments, such an antibody agent specifically binds to a CLDN-18.2 polypeptide, e.g., an epitope of ECD1 of a CLDN-18.2 polypeptide, with an affinity of 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 -9 The ligand may have a binding affinity (e.g., as measured by a dissociation constant) of M or less. Those skilled in the art will understand that in some cases, binding affinity (e.g., as measured by a dissociation constant) may be influenced by non-covalent intermolecular interactions, such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. Alternatively, or in addition, the binding affinity between a ligand and its target molecule may be influenced by the presence of other molecules. Those skilled in the art are familiar with various techniques for measuring binding affinity and / or dissociation constants in accordance with the present disclosure, including, but not limited to, ELISA, gel shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, grating-binding interferometry, and spectroscopic assays.

[0194] In some embodiments, an antibody targeting CLDN-18.2 may specifically bind to a CLDN-18.2 polypeptide relative to a 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 splice variant 1 of claudin 18 (CLDN18.1), which is expressed primarily in tissues, such as the lung.

[0195] In some embodiments, the antibody agent targeting CLDN-18.2 may be any one of the CLDN-18.2-targeting antibodies described in WO 2007 / 059997, WO 2008 / 145338, and WO 2013 / 174510, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.

[0196] In some embodiments, an antibody agent targeting CLDN-18.2 comprises: (a) a variable heavy chain domain having at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) a CDR1 represented by amino acid residues (GYTFTSYW); (ii) a CDR2 represented by amino acid residues (IYPSDSYT); and (iii) a CDR3 represented by amino acid residues (TRSWRGNSFDY); and / or (b) a variable light chain domain having at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) a CDR1 represented by amino acid residues (QSLLNSGNQKNY); (ii) a CDR2 represented by amino acid residues (WAS); and (iii) a CDR3 represented by amino acid residues (QNDYSYPFT).

[0197] In some embodiments, an antibody agent targeting CLDN-18.2 has heavy and light chain amino acid sequences that are or include related sequences (e.g., variable region sequences, e.g., CDR and / or framework (FR) sequences) described in U.S. Patent No. 9,751,934. For example, in some embodiments, an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or including the amino acid sequence represented by amino acid residues 20 to 467 of SEQ ID NO:1 shown below (where SEQ ID NO:1 corresponds to SEQ ID NO:118 in U.S. Patent No. 9,751,934, and the underlined amino acid sequence in SEQ ID NO:1 corresponds to the secretory signal sequence), and a light chain consisting of or including amino acid residues 21 to 240 of SEQ ID NO:2 shown below (where SEQ ID NO:2 corresponds to SEQ ID NO:125 in U.S. Patent No. 9,751,934, and the underlined amino acid sequence in SEQ ID NO:2 corresponds to the secretory signal sequence).

[0198] [Table 2]

[0199] In some embodiments, an antibody agent targeting CLDN-18.2 comprises: (a) a variable heavy chain domain having at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues 45-52 of SEQ ID NO: 1; (ii) CDR2 represented by amino acid residues 70-77 of SEQ ID NO: 1; and (iii) CDR3 represented by amino acid residues 116-126 of SEQ ID NO: 1; and / or (b) a variable light chain domain having at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues 47-58 of SEQ ID NO: 2; (ii) CDR2 represented by amino acid residues 76-78 of SEQ ID NO: 2; and (iii) CDR3 represented by amino acid residues 115-123 of SEQ ID NO: 2.

[0200] In some embodiments, an antibody agent targeting CLDN-18.2 comprises a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 14 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 15.

[0201] [Table 3]

[0202] [Table 4]

[0203] 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.

[0204] In some embodiments, antibody agents targeting CLDN-18.2 can be engineered to reduce potential immunogenicity and / or improve secretion. For example, in some embodiments, the mouse secretory signal sequence of an antibody agent targeting CLDN-18.2 can be replaced with a human one.

[0205] 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 shown below (the underlined amino acid sequence corresponds to the secretory signal sequence), and a light chain consisting of or comprising amino acid residues 27 to 246 of SEQ ID NO: 4 shown below (the underlined amino acid sequence corresponds to the secretory signal sequence).

[0206] [Table 5]

[0207] 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.

[0208] In some embodiments, antibody agents targeting CLDN-18.2 contain one or more Fc regions with a C-terminal lysine. The origin of this lysine is the naturally occurring sequence found in the human from which these Fc regions are derived. During cell culture production of recombinant antibodies, this terminal lysine can be proteolytically cleaved by one or more endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. Antibodies produced from nucleic acid sequences that do or do not encode a terminal lysine are substantially identical in sequence and function, since the degree of terminal lysine processing is typically high, for example, when using antibodies produced in CHO-based production systems (Dick, L. Wet et al. Biotechnol. Bioeng. 2008;100:1132-1143). Therefore, it is understood that proteins according to the present invention, such as antibodies, can be produced regardless of whether they encode or have a terminal lysine. It is also understood in accordance with the present invention that a sequence with a terminal lysine, e.g., a constant region sequence with a terminal lysine, can be understood as the corresponding sequence without the terminal lysine, and vice versa.

[0209] In some embodiments, the antibody targeting CLDN-18.2 is IMAB362 (also known as zolbetuximab, claudiximab). IMAB362, an antibody targeting CLDN-18.2, is in advanced 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; Tureci 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.

[0210] IMAB362, developed by Ganymed Pharmaceuticals GmbH and acquired by Astellas Pharma Inc., is a full IgG1 antibody targeting the tight junction protein CLDN-18.2, mediating cell death via antibody-dependent cellular 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; Tureci et al. 2011). The epitope is inaccessible to antibodies through normal epithelial barriers. Disruption of tight junctions and loss of cell polarization are early hallmarks of cancer. During this process, the IMAB362 epitope is exposed. IMAB362 does not bind to any other claudin family members, including the closely related splice variant 1 of claudin 18 (CLDN18.1), which is primarily expressed in tissues such as the lung.

[0211] IMAB362 plus epirubicin, oxaliplatin, and capecitabine (EOX) was tested in the phase 2 FAST trial (NCT01630083) against EOX in first-line patients with gastric and gastroesophageal cancer (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% or more tumor cells expressing CLDN-18.2 at moderate to strong (≥2+) staining intensity. The subset of patients whose tumors had 70% or more tumor cells with ≥2+ CLDN-18.2 staining intensity received 800 / 600 mg / kg IMAB362 plus epirubicin, oxaliplatin, and capecitabine (EOX). 2 Patients with ≥70% CLDN-18.2 expression achieved the greatest benefit from IMAB362 treatment at a dose of 100 mg / kg / day, nearly doubling median overall survival (OS) (Al-Batran et al. 2016; Lordick et al. 2016). The benefit of IMAB362 in OS in patients with ≥70% CLDN-18.2 expression (+33.1 weeks; p<0.0005) was accompanied by a significant delay in disease progression by central independent review (+14.5 weeks; p<0.0005) and a higher objective response rate (ORR) (35.1% vs. 27.1%). The addition of IMAB362 to EOX did not adversely affect patient-related outcomes. No significant differences were observed between treatment groups in mixed-effects, repeated-measures models for general health status or total STO22 scores throughout the study, but IMAB362 plus EOX significantly delayed the deterioration of general health scores by 2.6 months compared with EOX alone (p=0.008).

[0212] IMAB362 is also being studied by Astellas Pharma Inc. in a global development program of Phase 2 and 3 trials in patients with CLDN-18.2+ gastric / gastroesophageal and pancreatic cancer.

[0213] IMAB362 is being tested in various clinical trials as shown in Table 1 below. [Table 6]

[0214] The safety profile of IMAB362 in patients has been well characterized and is administered at doses up to 1000 mg / m 2 q3w (maximum C of 603 μg / mL) max ) has been tolerated without dose-limiting toxicity (Sahin et al. 2018; Tureci et al. 2019).

[0215] Without wishing to be bound by any particular theory, the primary pharmacological mechanism of action of IMAB362 to kill tumor cells involves antibody-dependent cellular cytotoxicity (ADCC). Based on the dose-response curve obtained by in vitro ADCC testing, the drug concentration resulting in a 95% response is observed at IMAB362 concentrations of 0.3 μg / mL to 28 μg / mL in serum (Sahin et al. 2018). For example, the EC of 0.3 to 28 μg / mL 95 Efficient lysis of CLDN-18.2+ cells via ADCC with IL-18 has been reported (Sahin et al. 2018).

[0216] Across various studies, IMAB362 was well tolerated, with nausea and vomiting being the main adverse events (AEs), and no dose-limiting toxicities (DLTs) and no clinical activity was observed as a single agent or in combination with chemotherapy.

[0217] Among other things, the present disclosure provides the insight that IMAB362 or variants thereof (e.g., variants sharing one or more characteristics of IMAB362, such as one or more (in many embodiments, all) CDR sequences, one or more (in many embodiments, all) FR sequences, and / or heavy and / or light chain variable sequences, and / or variants that are class variants of IMAB362, such as IgG1, IgM, IgA, etc.) may be particularly desirable antibodies for delivery by administration of the ribonucleic acids described herein. Without wishing to be bound by any particular theory, the present disclosure proposes that such modes of delivery may achieve effective administration with a reduced incidence (e.g., frequency and / or severity) of IMAB362 treatment-related adverse events (TEAEs) compared to those observed when the IMAB362 antibody itself is administered. In the phase 2a MONO trial with 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 2 Nausea and vomiting observed in this study were managed by pausing or slowing the infusion of IMAB362, and AEs were reported at C max It was shown to be related (Tureci et al. 2019).

[0218] In particular, the present disclosure provides, inter alia, the pharmacokinetic (PK) profile of IMAB362 delivered as a ribonucleic acid ("RiboMab01") described herein, which demonstrates a gradual increase in antibody concentration 48-72 hours after administration and a significantly lower C than IMAB362. max The changes in the PK profile of RiboMab01 were consistent with the C observed in patients after treatment with IMAB362. max The present disclosure also provides non-human primate study data showing that no systemic side effects, such as diarrhea, were observed.

[0219] Among other things, the present disclosure recognizes the favorable risk / benefit profile observed for administered IMB362 antibodies, particularly in certain indications where there is a high medical need, and proposes that delivery as described herein may be effective and / or particularly desirable.

[0220] III. RNA Technologies for Delivery of Antibody-Based Therapeutics Recombinant protein antibodies are widely used biologics for the treatment of diseases or disorders (e.g., cancer) but exhibit several limitations, including, for example, lengthy manufacturing process development and, in the case of antibody derivatives, short serum half-lives. The present disclosure provides techniques that address certain limitations of recombinant antibody technology, including, for example, lengthy manufacturing process development and, in the case of antibody derivatives, short serum half-lives, by utilizing RNA technology as a modality to directly express antibody agents, termed RiboMabs, in patient cells as a novel class of antibody-based therapeutics. In some embodiments, the present disclosure provides, among other things, the insight that RiboMabs formulated using lipid nanoparticles (LNPs) for intravenous (IV) administration can be taken up by cells (e.g., hepatocytes) to efficiently produce the encoded RiboMabs at therapeutically relevant plasma concentrations (Figure 14). In some embodiments, RiboMabs are antibody agents encoded by mRNA, e.g., engineered for minimal immunogenicity and / or formulated in lipid nanoparticles (LNPs). In some embodiments, an mRNA encoding an antibody agent may include modified nucleotides (such as, but not limited to, pseudouridine and / or 1-methyl-pseudouridine).

[0221] RiboMab technology can be utilized to deliver a variety of antibody formats. For example, in some embodiments, RiboMab technology can be used to express complete immunoglobulins (Igs), including, but not limited to, IgG. In some embodiments, a complete immunoglobulin (Ig) can be encoded by a single RNA containing a first coding region encoding the antibody heavy chain and a second coding region encoding the antibody light chain variable domain, where the single RNA contains or encodes either an internal ribosome entry site (IRES) or another internal promoter or a 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 generate the respective heavy and light chains, which can then be processed to form a complete IgG. In some embodiments, a complete Ig can be encoded by two separate RNAs: a first RNA containing a coding region encoding the antibody heavy chain and a second RNA containing a coding region encoding the antibody light chain. Such first and second RNAs are then translated into the respective chains of an antibody to form a complete Ig antibody in the target cell.

[0222] In some embodiments, RiboMab technology can be used to express bispecific antibody variants, such as those illustrated in Figure 12 (Panel A) or described in Stadler et al. (2016) Oncoimmunology 5(3):e1091555; and / or 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) against a first target and a second coding region encoding an scFv against 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 against a first target and a coding region encoding a heavy chain antigen-binding fragment (Fab) against a second target; and a second RNA comprising a coding region encoding an scFv against the same first target and a coding region encoding a light chain Fab against the same second target. Such first and second RNAs are then translated into antibody subunits to form the bispecific antibody in the target cell.

[0223] In some embodiments, the RNA agent (e.g., ssRNA described herein) may be delivered with a carrier. In some embodiments, the RNA / LNP is administered intravenously (IV) and taken up by target cells (e.g., hepatocytes) to efficiently produce the encoded RiboMab antibody at therapeutically relevant plasma concentrations.

[0224] A. Provided RNA encoding antibody agents against claudin 18.2 polypeptides and compositions thereof In some embodiments, at least one RNA comprises one or more coding regions encoding an antibody agent described above in the section entitled "Exemplary Antibody Agents Targeting Claudin 18.2 Polypeptides." In some embodiments, at least one RNA comprises one or more coding regions encoding the antibody agent IMAB362 exemplified above or herein.

[0225] Without wishing to be bound by any particular theory, the present disclosure provides, inter alia, the insight that in some embodiments, the antibody agent IMAB362 may be particularly useful and / or effective, at least in part, because it specifically binds to CLDN-18.2 and further selectively binds to CLDN-18.2 relative to CLDN18.1. In some embodiments, the teachings provided herein may be applicable to other antibody agents specific for CLDN-18.2, particularly such antibodies that selectively 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 selectively binds to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide. In some embodiments, such an antibody agent has a binding affinity for a CLDN-18.2 polypeptide that is at least 50% greater than its 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 greater. In some embodiments, such antibody agents have a binding affinity for a CLDN-18.2 polypeptide that is at least 1.1 times higher than that for a CLDN18.1 polypeptide, including, for example, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 500 times, at least 1000 times, at least 5000 times, or at least 10,000 times higher. In some embodiments, such antibody agents do not detectably bind to any other claudin family members, including CLDN18.1. In some embodiments, the antibody agent may be or comprise an antibody. In some embodiments, the antibody agent may be or comprise an antigen-binding fragment.

[0226] In some embodiments, an antibody agent that targets CLDN-18.2 (and may be encoded by an RNA, such as an ssRNA, e.g., an 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 epitope of ECD1 that is exposed in cancer cells.

[0227] In some embodiments, at least one RNA is selected from the group consisting of the variable heavy chain (V H ) domain and the variable light chain (V L In some embodiments, one or more such V domains of a CLDN-18.2-targeting antibody agent are H A domain and one or more V L The domains can be encoded by a single RNA construct; alternatively, in some embodiments, they can be separately encoded by at least two individual RNA constructs. For example, in some embodiments, the RNA utilized herein comprises at least the V domain of a CLDN-18.2 targeting antibody agent. H a heavy chain coding region encoding a domain, and at least a V domain of a CLDN-18.2-targeting antibody agent; L In an alternative embodiment, the composition comprises two or more coding regions, including a light chain coding region encoding at least the V domain of the CLDN-18.2 targeting antibody agent. H and (ii) a first RNA comprising a heavy chain coding region encoding a domain; and (iii) at least a V domain of a CLDN-18.2-targeting antibody agent. L and a second RNA comprising a light chain coding region encoding the domain.

[0228] In some embodiments, the heavy chain coding region comprises a constant heavy chain (C H ) domain, and / or the light chain coding region may further encode a constant light chain (C LFor example, in some embodiments, the heavy chain coding region can further encode the V domain of a CLDN-18.2 targeting antibody agent in immunoglobulin form (e.g., IgG). H Domain, C H1 Domain, C H2 domain, and C H3 and / or the light chain coding region may encode the V domain of a CLDN-18.2 targeting antibody agent in Ig form (e.g., IgG). L Domain and C L For example, in some embodiments, a complete immunoglobulin (Ig) can be encoded by a single RNA comprising 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); this single RNA requires protein translation to obtain a fusion protein comprising the heavy and light chains of the antibody, and post-translational cleavage of the fusion protein by an appropriate protease into the respective heavy and light chains, which can then be processed to form a complete Ig (e.g., IgG). In some embodiments, a complete Ig can be encoded by two separate RNAs: a first RNA comprising a coding region encoding the heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG) and a second RNA comprising a coding region encoding the light chain of a CLDN-18.2 Ig antibody (e.g., IgG). Such first and second RNAs are then translated into the respective chains of an antibody to form a complete Ig antibody (e.g., IgG) in the target cell. In some embodiments, the antibody agent encoded by one or more RNAs in the IgG form is an IgG1.

[0229] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues (GYTFTSYW); (ii) CDR2 represented by amino acid residues (IYPSDSYT); and (iii) CDR3 represented by amino acid residues (TRSWRGNSFDY). In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding at least one CDR (e.g., including one CDR, two CDRs, and three CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues (QSLLNSGNQKNY); (ii) CDR2 represented by amino acid residues (WAS); and (iii) CDR3 represented by amino acid residues (QNDYSYPFT).

[0230] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence represented by amino acid residues 20-467 of SEQ ID NO: 1. In some embodiments, one or more amino acid modifications (e.g., to reduce immunogenicity and / or stability) may be present in one or more non-CDR regions of SEQ ID NO: 1. For example, in some embodiments, SEQ ID NO: 1 may include at least one or more (e.g., including 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 (e.g., including amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, 50 or fewer (e.g., including 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 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 the amino acid sequence represented by amino acid residues 21-240 of SEQ ID NO:2. In some embodiments, one or more amino acid modifications (e.g., to reduce immunogenicity and / or stability) can be present in one or more non-CDR regions of SEQ ID NO:2. For example, in some embodiments, SEQ ID NO:2 can include at least one or more (e.g., including 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 (e.g., including amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, 50 or fewer (e.g., including 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer) amino acid modifications can be present in one or more non-CDR regions of SEQ ID NO:2.

[0231] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence that encodes 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 that encodes the amino acid sequence of SEQ ID NO:2.

[0232] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO:3. In some embodiments, one or more amino acid modifications (e.g., to reduce immunogenicity and / or stability) may be present in one or more non-CDR regions of SEQ ID NO:3. For example, in some embodiments, SEQ ID NO:3 may include at least one or more (e.g., including 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 (e.g., including amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, 50 or fewer (e.g., including 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 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 the amino acid sequence represented by amino acid residues 27-246 of SEQ ID NO:4. In some embodiments, one or more amino acid modifications (e.g., to reduce immunogenicity and / or stability) may be present in one or more non-CDR regions of SEQ ID NO:4. For example, in some embodiments, SEQ ID NO:4 may include at least one or more (e.g., including 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 (e.g., including amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, 50 or fewer (e.g., including 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO:4.

[0233] In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence that encodes 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 that encodes the amino acid sequence of SEQ ID NO: 4.

[0234] 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), hi 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.

[0235] 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, one of which is, for example, a CLDN-18.2 polypeptide. For example, Figure 12A 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 Stadler et al. (2017) Nature Medicine 23(7):815-817. In some embodiments, a bivalent antibody agent can be encoded by a single RNA that includes a first coding region encoding a single-chain variable fragment (scFv) that selectively binds to a CLDN-18.2 polypeptide (compared to a CLDN18.1 polypeptide) and a second coding region encoding an scFv against a second target (e.g., which in some embodiments may be a T cell receptor). In some embodiments, a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region encoding an scFv that selectively binds to a CLDN-18.2 polypeptide (compared to a CLDN18.1 polypeptide) and a coding region encoding a heavy chain antigen-binding fragment (Fab) to a second target (which may be, for example, a T cell receptor in some embodiments); and a second RNA comprising a coding region encoding an scFv that targets a CLDN-18.2 polypeptide and a coding region encoding a light chain Fab to the same second target. In some embodiments, a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region encoding an scFv that targets a first target (which may be, for example, a T cell receptor in some embodiments) and a coding region encoding a heavy chain antigen-binding fragment (Fab) that selectively binds to a CLDN-18.2 polypeptide (compared to a CLDN18.1 polypeptide); and a second RNA comprising a coding region encoding an scFv that is directed to the same first target and a coding region encoding a light chain Fab that targets a CLDN-18.2 polypeptide. Such first and second RNAs are then translated into antibody subunits to form the bispecific antibody in the target cell.

[0236] Secretory signal coding region: In some embodiments, one or more RNAs encoding a CLDN-18.2-targeting antibody agent may include a secretory signal coding region. In some embodiments, such a secretory signal coding region enables the CLDN-18.2-targeting antibody agent encoded by the one or more RNAs to be secreted, for example, upon translation by cells present in a treated subject, thereby resulting in plasma concentrations of a biologically active CLDN-18.2-targeting antibody agent. In some embodiments, the secretory signal coding region included in the RNA consists of or includes a nucleotide sequence encoding a non-human secretory signal. For example, in some embodiments, such a non-human secretory signal may be a mouse secretory signal, which in some embodiments may be or include the amino acid sequence MGWSCIILFLVATATGVHS or MESQTQVLMSLLFWVSGTCG. In some embodiments, the secretory signal coding region included in the RNA consists of or includes a nucleotide sequence encoding a human secretory signal, which in some embodiments may be or include the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the secretory signal coding region included in the RNA encoding the heavy chain domain of a CLDN-18.2-targeting antibody agent may comprise: (i) a nucleotide sequence encoding a mouse secretory signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence MGWSCIILFLVATATGVHS; or (ii) a nucleotide sequence encoding a human secretory signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS.In some embodiments, the secretory signal coding region included in the RNA encoding the light chain domain of a CLDN-18.2-targeting antibody agent may comprise: (i) a nucleotide sequence encoding a mouse secretory signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence MESQTQVLMSLLFWVSGTCG; or (ii) a nucleotide sequence encoding a human secretory signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS.

[0237] In some embodiments, one or more RNAs encoding CLDN-18.2-targeting antibody agents may contain at least one non-coding sequence element (e.g., to increase RNA stability and / or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, a 3' untranslated region (UTR), a 5' UTR, a cap structure for co-transcriptional capping of mRNA, a polyadenine (polyA) tail, and any combination thereof.

[0238] UTR (5'UTR and / or 3'UTR): In some embodiments, the provided RNA may include a nucleotide sequence encoding a 5'UTR of interest and / or a 3'UTR of interest. Those skilled in the art will understand that the untranslated region of an mRNA sequence (e.g., 3'UTR and / or 5'UTR) may contribute to mRNA stability, mRNA localization, and / or translation efficiency.

[0239] In some embodiments, the provided RNA may include a 5'UTR nucleotide sequence and / or a 3'UTR nucleotide sequence. In some embodiments, such a 5'UTR sequence may be operably linked to the 3' end of a coding sequence (e.g., including one or more coding regions). Additionally or alternatively, in some embodiments, a 3'UTR sequence may be operably linked to the 5' end of a coding sequence (e.g., including one or more coding regions).

[0240] In some embodiments of any aspect described herein, the 5' and 3' UTR sequences contained in the RNA may consist of or include naturally occurring or endogenous 5' and 3' UTR sequences for 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., encompassing one or more coding regions); in some such embodiments, such 5' and / or 3' UTR sequences may be useful for modifying 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 may reduce the stability of mRNA. Therefore, as will be understood by those skilled in the art, the 3' and / or 5' UTR can be selected or designed to enhance the stability of the transcribed RNA based on the characteristics of UTRs known in the art.

[0241] For example, those skilled in the art will understand that in some embodiments, a nucleotide sequence consisting of or including a Kozak sequence of the open reading frame sequence of a gene or nucleotide sequence of interest can be selected and used as a nucleotide sequence encoding a 5' UTR. As will be understood by those skilled in the art, Kozak sequences are known to increase the translation efficiency of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation. In some embodiments, the provided RNA polynucleotide can include a nucleotide sequence encoding a 5' UTR derived from an RNA virus whose RNA genome is stable in cells. In some embodiments, various modified ribonucleotides (e.g., as described herein) can be used in the 3' and / or 5' UTR, for example, to prevent exonuclease degradation of the transcribed RNA sequence.

[0242] In some embodiments, the 5'UTR included in the RNA may be derived from human alpha globin mRNA combined with a Kozak region.

[0243] In some embodiments, the RNA may include one or more 3'UTRs. For example, in some embodiments, the RNA may include two copies of a 3'UTR derived from a globin mRNA, such as α2 globin, α1 globin, or β globin (e.g., human β globin) mRNA. In some embodiments, two copies of a 3'UTR derived from human β globin mRNA may be used, e.g., placed between the coding sequence and the poly(A) tail of the RNA to improve protein expression levels and / or long-term persistence of the RNA. In some embodiments, the 3'UTR included in the RNA may be or include one or more (e.g., one, two, three, or more) of the 3'UTR sequences disclosed in International Publication No. WO 2017 / 060314, the entire contents of which are incorporated herein by reference for purposes set forth herein. In some embodiments, the 3'-UTR can be a combination of at least two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (designated F) and the mitochondrially encoded 12S ribosomal RNA (designated I), which were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see WO 2017 / 060314, incorporated herein by reference).

[0244] 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.

[0245] 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.

[0246] Poly-A tail: In some embodiments, the provided RNA may comprise a nucleotide sequence encoding a poly-A tail. A poly-A tail is a nucleotide sequence comprising a stretch of adenosine nucleotides, which may vary in length (e.g., at least five adenine nucleotides) and may comprise up to several hundred adenosine nucleotides. In some embodiments, the poly-A tail is a nucleotide sequence comprising at least 30 or more adenosine nucleotides, e.g., 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 poly-A tail is or comprises a poly-A homopolymer tail. In some embodiments, the polyA tail may contain one or more modified adenosine nucleosides, including, but not limited to, cardiolipin and 8-azaadenosine. In some embodiments, the polyA tail may contain one or more non-adenosine nucleotides. In some embodiments, the polyA tail may be or include a disrupted or modified polyA tail, such as those described in International Publication No. 2016 / 005324, the entire contents of which are incorporated herein by reference for the purposes described herein. For example, in some embodiments, the polyA tail included in the RNA described herein may be or include a modified polyA sequence comprising a linker sequence; a first sequence of at least 20 consecutive A nucleotides 5' of the linker sequence; and a second sequence of at least 20 consecutive A nucleotides 3' of the linker sequence. In some embodiments, the modified polyA sequence may include a linker sequence that is not a polyA sequence and that includes at least 10 nucleotides (e.g., T, G, and / or C nucleotides); a first sequence of at least 30 consecutive A nucleotides that is 5' of the linker sequence; and a second sequence of at least 70 consecutive A nucleotides that is 3' of the linker sequence.

[0247] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0248] 5' Cap: In some embodiments, the RNAs described herein may comprise a 5' cap, which may be incorporated into such RNA during transcription or attached to such RNA after transcription. In some embodiments, the RNA may comprise a 5' cap structure for co-transcriptional capping of the RNA. Examples of cap structures for co-transcriptional capping are known in the art, for example, as described in WO 2017 / 053297, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the 5' cap comprised in the RNAs described herein is or comprises m7G(5')ppp(5')(2'OMeA)pG. In some embodiments, the 5' cap comprised in the RNAs described herein is a cap 1 structure [e.g., m2 7,3’-O Gppp(m1 2’-O ) ApG]. When an RNA sequence described herein is described as having a 5' end with nucleotides 5'-AG, and the RNA includes a 5' cap containing A and G as the second and third nucleotides, respectively [e.g., m2 7,3’-O Gppp(m1 2’-O ) ApG], it should be understood that in some embodiments the second and third nucleotides of the cap correspond to nucleotides 5'-AG of the RNA sequence.

[0249] Chemical modification: In some embodiments, one or more RNAs encoding CLDN-18.2-targeting antibody agents may contain at least one modified ribonucleotide, for example, to increase the stability of such one or more RNAs, and / or reduce the immunogenicity of such one or more RNAs, and / or reduce the cytotoxicity of such RNAs. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of one or more RNAs 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 modified cytidine, and the modified cytidine may be, for example, 5-methylcytidine in some embodiments. Alternatively, or in addition, in some embodiments, some or all of the uridine residues present in the RNA may be replaced with modified uridines, which in some embodiments include 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), and the like. ), 5-carboxymethyluridine (cm5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U),5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm5U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m5s2U), 1-methyl-4-thiopseudouridine (m1s4Ψ), 4-thio-1-methylpseudouridine , 3-methylpseudouridine (m3Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3- (3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2' -O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)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 may be substituted with a modified uridine selected from the group consisting of 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 may be substituted with pseudouridine or a derivative thereof, such as 1-methylpseudouridine. In some embodiments, some or all of the uridine residues present in the RNA may be substituted with pseudouridine. In some embodiments, some or all of the uridine residues present in the RNA may be substituted with 1-methylpseudouridine. In some embodiments, all of the uridine residues present in the RNA are substituted with pseudouridine. In some embodiments, all of the uridine residues present in the RNA are substituted with 1-methylpseudouridine.

[0250] Codon optimization and GC enrichment: The codons of the RNA (particularly mRNA) described in the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or to replace rare codons in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed with codons that are synonymous with high frequency codons in the cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (particularly mRNA) described in the present disclosure is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased 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 region of the wild-type coding sequence. In some embodiments, codon optimization and / or increased 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 an RNA (especially an mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unaltered 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 efficient translation of that RNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode identical amino acids (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acids 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 encoding the same amino acids but containing no or a lower content of A and / or U nucleotides.In various embodiments, the G / C content of the coding region of the RNA (particularly 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.

[0251] Non-immunogenic RNA: In certain embodiments, the RNA described herein is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors, and / or by limiting the amount of double-stranded RNA (dsRNA), for example, by limiting the formation of double-stranded RNA (dsRNA) during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription. In certain embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors, and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription.

[0252] To render non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating modified nucleosides, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Modified nucleosides that suppress RNA-mediated activation of innate immune receptors are particularly preferred. In some embodiments, the modified nucleoside comprises the substitution of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is 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 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (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-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-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 3Ψ), 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-thiouridine (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 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5In certain embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ) or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0253] In some embodiments, the substitution of one or more uridines with nucleosides comprising modified nucleobases comprises substitution of 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.

[0254] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are produced due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to the inhibition of protein synthesis. The formation of dsRNA can be limited during the synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP can be added once or several times during mRNA synthesis. Additionally, dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, using Escherichia coli (E. coli) RNase III, which specifically hydrolyzes dsRNA but not ssRNA, thereby removing dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulose material.In some embodiments, RNA preparation is contacted with cellulose material, and under the condition that dsRNA can be bound to cellulose material, and ssRNA is separated from cellulose material under the condition that ssRNA cannot be bound to cellulose material.Suitable method for providing ssRNA is disclosed in, for example, WO2017 / 182524. In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, and dsRNA (particularly dsmRNA) is removed from non-immunogenic RNA, for example, 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 in non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) does not contain or essentially does not contain dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA.In some embodiments, non-immunogenic RNA (particularly mRNA) composition comprises single-stranded nucleoside modified RNA (particularly mRNA) and does not substantially contain double-stranded RNA (dsRNA).In some embodiments, non-immunogenic RNA (particularly 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 compared to all other nucleic acid molecules (such as DNA, dsRNA).Various methods can be used to determine the amount of dsRNA. For example, the sample can be contacted with a dsRNA-specific antibody, and the amount of antibody bound to RNA can be considered 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 on a membrane, such as a nylon blotting membrane. The membrane can be blocked in, for example, TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For dsRNA detection, the membrane can be incubated with a dsRNA-specific antibody, such as a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing with, for example, 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, non-immunogenic RNA (especially mRNA) is translated more efficiently in cells than standard RNA with the same sequence. In some embodiments, translation is enhanced by 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced by 3-fold.In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the factor is between 10 and 1000-fold. In some embodiments, the factor is between 10 and 100-fold. In some embodiments, the factor is between 10 and 200-fold. In some embodiments, the factor is between 10 and 300-fold. In some embodiments, the factor is between 10 and 500-fold. In some embodiments, the factor is 20-1000 fold. In some embodiments, the factor is 30-1000 fold. In some embodiments, the factor is 50-1000 fold. In some embodiments, the factor is 100-1000 fold. In some embodiments, the factor is 200-1000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts. In some embodiments, the non-immunogenic RNA (particularly mRNA) exhibits significantly less natural immunogenicity than standard RNA having the same sequence. In some embodiments, the non-immunogenic RNA (particularly mRNA) exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced 3-fold. In some embodiments, the natural immunogenicity is reduced 4-fold. In some embodiments, the natural immunogenicity is reduced 5-fold. In some embodiments, the natural immunogenicity is reduced 6-fold. In some embodiments, the natural immunogenicity is reduced 7-fold. In some embodiments, the natural immunogenicity is reduced 8-fold. In some embodiments, the natural immunogenicity is reduced 9-fold. In some embodiments, the natural immunogenicity is reduced by 10-fold.In some embodiments, the natural immunogenicity is reduced by 15-fold. In some embodiments, the natural immunogenicity is reduced by 20-fold. In some embodiments, the natural immunogenicity is reduced by 50-fold. In some embodiments, the natural immunogenicity is reduced by 100-fold. In some embodiments, the natural immunogenicity is reduced by 200-fold. In some embodiments, the natural immunogenicity is reduced by 500-fold. In some embodiments, the natural immunogenicity is reduced by 1000-fold. In some embodiments, the natural immunogenicity is reduced by 2000-fold. The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of non-immunogenic RNA (particularly mRNA) can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction such that non-immunogenic RNA (particularly mRNA) can be repeatedly administered without eliciting a sufficient natural immune response to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA (particularly mRNA) can be administered repeatedly without eliciting a natural immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0255] In some embodiments, the RNA encoding the heavy chain of a CLDN-18.2-targeting antibody agent comprises, from 5' to 3', (a) a 5' UTR; (b) a secretory signal coding region; (c) a heavy chain coding region; (d) a 3' UTR; and (e) a poly-A tail. See, e.g., Figure 13. In some embodiments, the 5' UTR is or comprises a sequence derived from human alpha globin mRNA combined with a Kozak region. In some embodiments, the secretory signal coding region is or comprises a nucleotide sequence encoding the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the heavy chain coding region is the V-terminal end of ...H Domain, C H1 Domain, C H2 domain, and C H3 In some embodiments, the 3'UTR is or includes a combination of at least two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (designated F) and the mitochondrially encoded 12S ribosomal RNA (designated I). In some embodiments, the polyA tail is or includes 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 RNAs have a 5' cap structure, including a cap 1 structure, or an m2 7,3’-O Gppp(m1 2’-O ) ApG. In some embodiments, such RNAs contain all uridines substituted with N1-methylpseudouridine.

[0256] In some embodiments, the RNA encoding the light chain of a CLDN-18.2-targeting antibody agent comprises, from 5' to 3', (a) a 5' UTR; (b) a secretory signal coding region; (c) a light chain coding region; (d) a 3' UTR; and (e) a poly-A tail. See, e.g., Figure 13. In some embodiments, the 5' UTR is or comprises a sequence derived from human alpha globin mRNA combined with a Kozak region. In some embodiments, the secretory signal coding region is or comprises a nucleotide sequence encoding the amino acid sequence MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the light chain coding region is the V-terminal region of an IgG form of a CLDN-18.2-targeting antibody agent (e.g., those described herein, e.g., IMAB262, or the amino acid sequence represented by amino acid residues 27-246 of SEQ ID NO: 4). L Domain and C LIn some embodiments, the 3'UTR is or includes a combination of at least two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (designated F) and the mitochondrially encoded 12S ribosomal RNA (designated I). In some embodiments, the polyA tail is or includes 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 RNAs have a 5' cap structure, including a cap 1 structure, or an m2 7,3’-O Gppp(m1 2’-O ) ApG. In some embodiments, such RNAs contain all uridines substituted with N1-methylpseudouridine.

[0257] In some embodiments, the RNA is or comprises one or more single-stranded RNAs, such as single-stranded mRNAs.

[0258] In some embodiments, the composition comprises a single-stranded mRNA encoding the heavy chain (e.g., open reading frame, ORF) of an antibody agent (e.g., one described herein) targeting CLDN-18.2 and a single-stranded mRNA encoding the light chain (e.g., open reading frame, ORF) of an antibody agent (e.g., one described herein) targeting CLDN-18.2, which, when introduced into a target cell, are translated into their respective subunits to form a complete IgG antibody in...

Claims

1. (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2).

1. A composition or pharmaceutical formulation comprising: (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; Composition or pharmaceutical preparation.

2. the first polypeptide chain comprises the amino acid sequence of amino acids 27-474 of SEQ ID NO:3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27-474 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; 10. The composition or pharmaceutical formulation of claim 1.

3. (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2).

1. A composition or pharmaceutical formulation comprising: the first polypeptide chain comprises the amino acid sequence of amino acids 27-474 of SEQ ID NO:3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27-474 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; Composition or pharmaceutical preparation.

4. 4. The composition or pharmaceutical formulation of any one of claims 1 to 3, wherein the RNA, such as each RNA, comprises a 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. 5. The composition or pharmaceutical formulation of any one of claims 1 to 4, wherein the RNA, such as each RNA, comprises a 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.

6. 6. The composition or pharmaceutical formulation of any one of claims 1 to 5, wherein the RNA, such as each RNA, comprises a 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.

7. 7. The composition or pharmaceutical formulation of any one of claims 1 to 6, wherein the RNA, such as each RNA, comprises a 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.

8. 8. The composition or pharmaceutical formulation of any one of claims 1 to 7, wherein the RNA, such as each RNA, comprises a 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.

9. (i) RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2); and (ii) RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin 18.2 (CLDN-18.2).

1. A composition or pharmaceutical formulation comprising: the RNA, e.g., each RNA, comprises a 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 / or a 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; Composition or pharmaceutical preparation.

10. 10. The composition or pharmaceutical formulation of any one of claims 1 to 9, wherein the RNA, such as each RNA, comprises a 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 a 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.

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

19.

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

21.

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

19.

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

21.

15. (a) the coding region of (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; (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; and / or (b) the first polypeptide chain comprises an amino acid sequence of amino acids 27-474 of SEQ ID NO:3, or an amino acid sequence having at least 90% identity to an amino acid sequence of amino acids 27-474 of SEQ ID NO:3; 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; A composition or pharmaceutical formulation according to any one of claims 9 to 14.

16. (i) the coding region 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 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; A composition or pharmaceutical formulation according to any one of claims 1 to 15.

17. 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; A composition or pharmaceutical formulation according to any one of claims 1 to 16.

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

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

20. 20. The composition or pharmaceutical preparation of any one of claims 1 to 19, wherein the antibody agent selectively binds to claudin 18.1 (CLDN-18.2) compared to CLDN-18.

1.

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

2.

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

23. The composition or pharmaceutical formulation of any one of claims 1 to 22, wherein the antibody agent that binds to CLDN-18.2 comprises two binding arms, and 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. 24. The composition or pharmaceutical preparation of any one of claims 1 to 23, wherein the antibody agent is IgG1.

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

26. 26. The composition or pharmaceutical preparation of 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. 27. The composition or pharmaceutical formulation of 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 of 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 of claim 27 or 28, wherein the VH (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively.

30. The composition or pharmaceutical formulation of 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 of 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 of claim 30 or 31, wherein the VL (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NOs: 8, 9 and 10, respectively.

33. The first polypeptide chain comprises the variable domain of the heavy chain (VH) of an antibody agent that binds to CLDN-18.2, comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 14 (VH(CLDN-18.2)), and the second polypeptide chain comprises the variable domain of the light chain (VL) of an antibody agent that binds to CLDN-18.2, comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO: 15 (VL(CLDN-18.2)). The composition or pharmaceutical formulation of any one of claims 1 to 32.

34. The first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), comprising CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively; and 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)), comprising CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively. The composition or pharmaceutical formulation of any one of claims 1 to 33.

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

36. The first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and 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)), wherein the VH(CLDN-18.2) and the VL(CLDN-18.2) interact to form a binding domain that binds to claudin 18.2 (CLDN-18.2). The composition or pharmaceutical formulation of any one of claims 1 to 35.

37. The composition or pharmaceutical formulation of any one of claims 1 to 36, wherein the first polypeptide chain comprises a variable domain (VH) of the heavy chain 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. 38. The composition or pharmaceutical preparation of claim 37, wherein the VH (CLDN-18.2), CH1, CH2 and CH3 are present in the first polypeptide chain in an immunoglobulin G (IgG) form.

39. 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. The composition or pharmaceutical formulation of any one of claims 1 to 38.

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

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

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

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

13.

44. (i) the coding region comprises the nucleotide sequence of SEQ ID NO: 16; and (ii) the coding region comprises the nucleotide sequence of SEQ ID NO: 17; A composition or pharmaceutical formulation according to any one of claims 1 to 43.

45. 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; A composition or pharmaceutical formulation according to any one of claims 1 to 44.

46. 46. ​​A composition or pharmaceutical formulation according to any one of claims 1 to 45, wherein said RNA, such as each RNA, comprises a polyA sequence.

47. 47. The composition or pharmaceutical preparation of claim 46, wherein the polyA sequence is an interrupted sequence of A nucleotides.

48. 48. The composition or pharmaceutical preparation of claim 46 or 47, wherein the polyA sequence comprises at least 100 nucleotides.

49. The poly A sequence is a nucleotide sequence A ｘ -L-A ｙ wherein A comprises or consists of ｘ is a sequence of at least 20 A nucleotides, 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.

50. 50. The composition or pharmaceutical preparation of any one of claims 46 to 49, wherein said polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:

23.

51. (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) 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.

51. The composition or pharmaceutical formulation of any one of claims 1 to 50, comprising:

52. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 52. The composition or pharmaceutical formulation of claim 51, comprising:

53. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 27 52. The composition or pharmaceutical formulation of claim 51, comprising:

54. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27 10. A composition or pharmaceutical formulation comprising:

55. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 24, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 10. A composition or pharmaceutical formulation comprising:

56. (i) RNA comprising the nucleotide sequence of SEQ ID NO: 26, and (ii) RNA comprising the nucleotide sequence of SEQ ID NO: 27 10. A composition or pharmaceutical formulation comprising:

57. 57. A composition or pharmaceutical formulation according to any one of claims 1 to 56, wherein the RNA, such as each RNA, comprises modified nucleosides in place of uridine.

58. 58. A composition or pharmaceutical formulation according to any one of claims 1 to 57, wherein the RNA, such as each RNA, comprises a modified nucleoside in place of each uridine.

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

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

61. 61. A composition or pharmaceutical formulation according to any one of claims 1 to 60, wherein the RNAs, such as each RNA, comprise a 5' cap.

62. The RNA, e.g., each RNA, ２ ７，３’－Ｏ Gppp (m １ ２’－Ｏ 62. The composition or pharmaceutical preparation of any one of claims 1 to 61, comprising ApG.

63. 63. A composition or pharmaceutical formulation according to any preceding claim, wherein the RNA, such as each RNA, is single-stranded RNA.

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

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

66. 66. The composition or pharmaceutical formulation of claim 65, wherein the lipids forming the lipid nanoparticles comprise cationic lipids, polymer-conjugated lipids, and neutral lipids.

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

68. 68. The composition or pharmaceutical formulation of claim 66 or 67, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate).

69. 69. The composition or pharmaceutical formulation of any one of claims 66 to 68, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

70. 70. A composition or pharmaceutical formulation 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. 71. A composition or pharmaceutical formulation according to any one of claims 65 to 70, wherein the lipid nanoparticles have an average size of about 50 to 150 nm.

72. 72. The composition or pharmaceutical formulation of any one of claims 65 to 71, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

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

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

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

76. 76. The pharmaceutical formulation of claim 75, wherein the RNA, e.g., each RNA, and optionally the particle-forming component, are in separate vials.

77. 77. The pharmaceutical preparation of claim 75 or 76, further comprising instructions for using said composition or pharmaceutical preparation to treat or prevent cancer.

78. A composition or pharmaceutical formulation according to any one of claims 1 to 77 for pharmaceutical use.

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

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

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

82. 82. The composition or pharmaceutical preparation of claim 80 or 81, wherein the cancer comprises a CLDN-18.2-positive solid tumor.

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

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

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

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

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

88. 88. The composition or pharmaceutical preparation of claim 87, wherein the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk a tumor; (ii) radiation therapy; and (iii) chemotherapy.

89. 89. The composition or pharmaceutical preparation of claim 87 or 88, wherein the additional therapy comprises administering an additional therapeutic agent.

90. 90. The composition or pharmaceutical formulation of claim 89, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.

91. 91. A composition or pharmaceutical formulation according to any one of claims 1 to 90, for administration to a human.

92. 92. A composition or pharmaceutical formulation according to any one of claims 1 to 91 for intravenous administration.

93. 75. A method of treating cancer in a subject, comprising administering to said subject a composition according to any one of claims 1 to 74.

94. 94. The method of claim 93, wherein the cancer comprises a CLDN-18.2 positive cancer.

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

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

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

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

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

100. 100. The method of any one of claims 93 to 99, further comprising administering an additional therapy.

101. 101. The method of claim 100, wherein the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy.

102. 102. The method of claim 100 or 101, wherein the additional therapy comprises administering an additional therapeutic agent.

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

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

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

106. A composition according to any one of claims 1 to 74 for use in a method according to any one of claims 93 to 105.

107. 93. The composition or pharmaceutical preparation of any one of claims 1 to 92, wherein the RNA is introduced into a hepatocyte and the polypeptide chain encoded by the RNA is expressed in the hepatocyte.

108. 93. The composition or pharmaceutical formulation of any one of claims 1 to 92, for systemic delivery of the polypeptide chain.

109. 93. The composition or pharmaceutical formulation of any one of claims 1 to 92, for systemic delivery of said polypeptide chain following expression of said polypeptide chain in hepatocytes.

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

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

112. 1. A method for systemic delivery of an antibody agent that binds to claudin 18.2 (CLDN-18.2) in a subject, comprising: (a) administering the composition of any one of claims 1 to 74 so that the RNA is introduced into hepatocytes; and (b) expressing the polypeptide chain encoded by the RNA in the hepatocyte. wherein, after expression, the polypeptide chain is secreted into the bloodstream.

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

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

115. A composition or pharmaceutical formulation comprising RNA, The RNA is (i) a coding sequence encoding a polypeptide; (ii) a 3′ UTR sequence; (iii) a polyA sequence, and (iv) a nucleotide sequence linking the 3′ UTR sequence and the polyA sequence, the nucleotide sequence comprising the sequence CUXGAGCUAGC (wherein X is C, A, or U); 10. A composition or pharmaceutical formulation comprising:

116. 116. The composition or pharmaceutical preparation of claim 115, wherein the nucleotide sequence linking the 3'UTR sequence and the polyA sequence comprises the sequence CUCGAGCUAGC.

117. 117. The composition or pharmaceutical preparation of claim 115 or 116, wherein the RNA comprises, in a 5' to 3' direction, the coding sequence encoding a polypeptide, the 3' UTR sequence, the nucleotide sequence linking the 3' UTR sequence and the polyA sequence, and the polyA sequence.

118. 118. The composition or pharmaceutical preparation of 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. 119. The composition or pharmaceutical preparation of any one of claims 115 to 118, wherein the RNA comprises a 3'UTR 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.

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

37.

121. 121. The composition or pharmaceutical preparation of any one of claims 115 to 120, wherein the polyA sequence is an interrupted sequence of A nucleotides.

122. 122. The composition or pharmaceutical preparation of any one of claims 115 to 121, wherein said polyA sequence comprises at least 100 nucleotides.

123. The poly A sequence is a nucleotide sequence A ｘ -L-A ｙ wherein A comprises or consists of ｘ is a sequence of at least 20 A nucleotides, ｙ is a sequence of at least 60 A nucleotides, and L is a linker of 1 to 20 nucleotides which may include nucleotides other than A.

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

23.

125. 125. The composition or pharmaceutical preparation of any one of claims 115 to 124, wherein the RNA comprises a 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.

126. 126. The composition or pharmaceutical formulation of any one of claims 115 to 125, wherein the RNA comprises a 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, 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 absent.

127. 127. The composition or pharmaceutical formulation of any one of claims 115 to 126, wherein the RNA comprises a 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, wherein X1 is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.

128. 128. The composition or pharmaceutical preparation of any one of claims 115 to 127, wherein the RNA comprises a 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, wherein the nucleotide sequence is preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU.

129. 128. The composition or pharmaceutical preparation of any one of claims 115 to 127, wherein the RNA comprises a 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, wherein the nucleotide sequence is preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU.

130. 130. The composition or pharmaceutical preparation of any one of claims 115 to 129, wherein the RNA comprises a 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. 131. The composition or pharmaceutical preparation of any one of claims 115 to 128 and 130, 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.

132. 131. The composition or pharmaceutical preparation of any one of claims 115 to 127, 129 and 130, 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.

133. The composition or pharmaceutical preparation of 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, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20, and, downstream of the coding sequence encoding a polypeptide, a 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 polyA sequence.

134. The composition or pharmaceutical preparation of 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, downstream of the coding sequence encoding a polypeptide, a 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 polyA sequence.

135. 133. The composition or pharmaceutical preparation of 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 downstream of the coding sequence encoding a 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.

136. The composition or pharmaceutical preparation of 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, downstream of the coding sequence encoding a polypeptide, a 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 polyA sequence.

137. 133. The composition or pharmaceutical preparation of 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 downstream of the coding sequence encoding a 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.

138. 133. The composition or pharmaceutical preparation of 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, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence.

139. 133. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 20, and, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence.

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

36.

141. 133. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 38, and, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a polyA sequence.

142. 133. The composition or pharmaceutical preparation of 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 downstream of the coding sequence encoding a polypeptide comprising the nucleotide sequence of SEQ ID NO:

36.

143. The composition or pharmaceutical preparation of 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, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20, and, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37, and a polyA sequence.

144. The composition or pharmaceutical preparation of 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, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a polyA sequence.

145. 133. The composition or pharmaceutical preparation of 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 downstream of the coding sequence encoding a polypeptide 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. 133. The composition or pharmaceutical preparation of 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, downstream of the coding sequence encoding a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a polyA sequence.

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

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

37.

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

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

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

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

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

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

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

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

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

160. 160. A composition or pharmaceutical formulation 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, such as all, of the RNA molecules comprises a 5'UTR, a 3'UTR, a 3'UTR sequence, a polyA sequence, and / or a nucleotide sequence linking the 3'UTR sequence and the polyA sequence as defined.

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

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

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

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

165. 165. A composition or pharmaceutical preparation according to any one of claims 115 to 164, wherein the RNA, such as each RNA, comprises a modified nucleoside in place of each uridine.

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

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

168. 168. A composition or pharmaceutical preparation according to any one of claims 115 to 167, wherein the RNAs, such as each RNA, comprise a 5' cap.

169. The RNA, e.g., each RNA, ２ ７，３’－Ｏ Gppp (m １ ２’－Ｏ 169. A composition or pharmaceutical preparation according to any one of claims 115 to 168, comprising ApG.

170. 170. A composition or pharmaceutical preparation according to any one of claims 115 to 169, wherein the RNA, such as each RNA, is single-stranded RNA.

171. 171. A composition or pharmaceutical preparation according to any one of claims 115 to 170, wherein the RNA, such as each RNA, is mRNA.

172. 172. The composition or pharmaceutical formulation of any one of claims 115 to 171, wherein the RNA, e.g. each RNA, is formulated in a lipid nanoparticle (LNP), e.g. each RNA is co-formulated in a lipid nanoparticle (LNP).

173. 173. The composition or pharmaceutical formulation of claim 172, wherein the lipids forming the lipid nanoparticles comprise cationic lipids, polymer-bound lipids, and neutral lipids.

174. a. the cationic lipid is present at 35-65 mol% of the total lipid; b. the polymer-bound lipid is present at about 1-2.5 mol % of the total lipid; and c. The neutral lipids are present at 35-65 mol% of the total lipids; 174. The composition or pharmaceutical formulation of claim 173.

175. 175. The composition or pharmaceutical preparation of claim 173 or 174, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate).

176. 176. The composition or pharmaceutical preparation of any one of claims 173 to 175, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

177. 177. A 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.

178. 178. A composition or pharmaceutical formulation according to any one of claims 172 to 177, wherein the lipid nanoparticles have an average size of about 50 to 150 nm.

179. 179. The composition or pharmaceutical formulation of any one of claims 172 to 178, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

180. 180. The composition of any one of claims 115 to 179, which is a pharmaceutical composition.

181. 181. The composition of claim 180, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

182. The pharmaceutical preparation of any one of claims 115 to 179, which is a kit.

183. 183. The pharmaceutical formulation of claim 182, wherein the RNA, e.g., each RNA, and optionally the particle-forming component, are in separate vials.

184. 184. A composition or pharmaceutical preparation according to any one of claims 115 to 183, for intravenous administration.

185. The composition or pharmaceutical preparation of any one of claims 115 to 184, wherein the RNA is introduced into a hepatocyte and the polypeptide encoded by the RNA is expressed in the hepatocyte.

186. 186. A composition or pharmaceutical formulation according to any one of claims 115 to 185, which is for systemic delivery of said polypeptide.

187. 187. The composition or pharmaceutical formulation of any one of claims 115 to 186, for systemic delivery of said polypeptide following expression of said polypeptide in hepatocytes.

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

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

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

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

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

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