RNA compositions targeting claudin-18.2

Abstract

The present disclosure provides RNA technologies for targeting Claudin-18.2 polypeptides. In some embodiments, such RNA technologies can be useful for treatment of diseases associated with positive expression of Claudin-18.2. For example, in some embodiments, such RNA technologies can be useful for treatment of Claudin-18.2 positive cancer, including, e.g., but not limited to biliary cancers, ovarian cancers, gastric cancers, gastro-esophageal cancers, pancreatic cancers. In some embodiments, such RNA technologies can be used in combination therapy (e.g., in combination with a chemotherapeutic agent). The present disclosure further provides RNA backbones containing specific sequences upstream and / or downstream from the coding sequence.

Description

[0001] RNA COMPOSITIONS TARGETING CLAUDIN-18.2

[0002] BACKGROUND

[0003] [1] Cancer is the second leading cause of death globally and is expected to be responsible for an estimated 9.6 million deaths in 2018 (Bray et al. 2018). In general, once a solid tumor has metastasized, with a few exceptions such as germ cell and some carcinoid tumors, 5-year survival rarely exceeds 25%.

[0004] [2] Conventional therapies such as chemotherapy, radiotherapy, surgery, and targeted therapies and recent advances in immunotherapies have improved outcomes in patients with advanced solid tumors. In the last few years, the Food and Drug Administration (FDA) and 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, mainly solid tumors. These approvals have dramatically changed the landscape of cancer treatment. However, certain cancers such as pancreatic adenocarcinoma or metastatic biliary tract cancers still do not yet benefit from existing therapies including immunotherapies.

[0005] SUMMARY

[0006] [3] The poor prognosis of certain cancers such as, e.g. , pancreatic and biliary cancer types, highlights the need for additional treatment approaches.

[0007] [4] The present disclosure, among other things, provides insights and technologies for treating cancer, particularly, cancers that are associated with expression of Claudin-18.2 (CLDN- 18.2). In some embodiments, the present disclosure provides technologies for treating a cancer selected from the group consisting of pancreatic cancers, gastric or gastro-esophageal cancers, biliary cancers, ovarian cancers, etc. In some embodiments, the present disclosure provides technologies for administration of therapy to locally advanced tumors. In some embodiments, the present disclosure provides technologies for treatment of unresectable tumors. In some embodiments, provided technologies provide technologies for treatment of metastatic tumors. Thus, for example, in some embodiments, provided therapy may be administered to a subject or population of subjects suffering from or susceptible to cancer (e.g., to a cancer selected from pancreatic cancers, gastric or gastro-esophageal cancers, biliary cancers, ovarian cancers, and / or otherwise involves one or more pancreatic, gastric, gastroesophageal, biliary, and / or ovarian tumors), which cancer may be or comprise one or more locally advanced tumors, one or more unresectable tumors and / or one or more metastases.

[0008] [5] The present disclosure, among other things, provides an insight that Claudin- 18.2 (CLDN-18.2) represents a particularly useful tumor-associated antigen against which therapies may be targeted. Without wishing to be bound by any particular theory, the present disclosure notes that CLDN-18.2’s tissue expression pattern, including its particularly limited expression in non-cancer tissues, may contribute to its usefulness as a target as described herein. To date, no therapy targeting CLDN-18.2 has been approved for any cancer indication.

[0009] [6] Zolbetuximab (development code IMAB362), which is a monoclonal antibody that targets isoform 2 of Claudin- 18, has been under investigation for the treatment of gastrointestinal adenocarcinomas and pancreatic tumors (Tureci et al. 2019).

[0010] [7] The present disclosure further provides an insight that, in some embodiments, therapy targeting CLDN-18.2, as described herein, may usefully involve administration of RNA (e.g., ssRNA such as mRNA) encoding an antibody agent that targets CLDN-18.2. Still further, the present disclosure provides a particular insight that delivery of RNA via lipid nanoparticles targeting liver cells may be a particularly beneficial strategy for delivering such an antibody agent.

[0011] [8] The present disclosure further provides an insight that a RiboMab format (as illustrated for example, in Figure 13) and, in particular, the RNA sequences and sequence elements described herein may be particularly useful for RNA (e.g., ssRNA such as mRNA) that delivers a CLDN-18.2-targeting agent (e.g., a CLDN-18.2-targeted antibody agent) as described herein.

[0012] [9] The present disclosure, among other things, provides an insight that administration of RNA (e.g., ssRNA such as mRNA) encoding a CLDN-18.2-targeting agent, and in particular a CLDN-18.2-targeting antibody agent, and specifically IMAB362 may represent a particularly desirable strategy for CLDN-18.2-targeted therapy. Without wishing to be bound by any particular theory, the present disclosure proposes that such delivering modality may achieve one or more improvements such as effective administration with reduced incidence (e.g., frequency and / or severity) of TEAEs, and / or with improved relationship between efficacy level and TEAE level (e.g., improved therapeutic window) relative to those observed when a corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements in particular may be achieved by delivering IMAB362 via administration of RNA(s) (e.g., ssRNA(s) such as mRNA(s)) encoding it.

[0013]

[0010] In some embodiments, the present disclosure, among other things, provides insights that mRNA(s) encoding an antibody agent (e.g., IMAB362) or a functional portion thereof that is / or formulated with lipid nanoparticles (LNP) for intravenous (IV) administration can be taken up by target cells (e.g., liver cells) for efficient production of the encoded antibody agent (e.g., IMAB362) at therapeutically relevant plasma concentrations, for example, as illustrated in Figure 14 for the described RiboMab targeting CLDN-18.2.

[0014]

[0011] 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 in lipid nanoparticles (LNPs).

[0015]

[0012] Moreover, the present disclosure, among other things, provides an insight that the capability of a CLDN-18.2-targeted antibody agent as described herein to induce antibodydependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while leveraging immune system of recipient subjects can augment cytotoxic effect(s) of chemotherapy and / or other anti-cancer therapy. In some embodiments, such a combination therapy may prolong progression-free and / or overall survival, e.g., relative to the individual therapies administered alone and / or to another appropriate reference.

[0016]

[0013] Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, for example such as gemcitabine, oxaliplatin, and 5-fluorouracil were shown to upregulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines; moreover, these agents were not observed to increase de novo expression in CLDN-18.2-negative cell lines. See, for example, Tiireci et al. (2019) “Characterization of zolbetuximab in pancreatic cancer models” In Oncoimmimology 8 (1), pp. el523096.

[0017]

[0014] The present disclosure, among other things, provides an insight that CLDN-18.2- targeted therapy as described herein may be particularly useful and / or effective when administered to tumor(s) (e.g., tumor cells, subjects in whom such tumor(s) and / or tumor cell(s) are suspected and / or have been detected, etc.) characterized by (e.g., that have been determined to display and / or that are expected or predicted to display) elevated expression and / or activity of CLDN-18.2 expression in tumor cells (e.g., as may result or have resulted from exposure to one or more chemotherapeutic agents). Indeed, among other things, the present disclosure teaches that provided CLDN-18.2-targeted therapy (e.g., administration of RNA and, more particularly an mRNA encoding a CLDN-18.2-targeting antibody agent) as described herein may provide synergistic therapeutic when administered in combination with (e.g., to a subject who has received and / or is receiving or has otherwise been exposed to) one or more CDLN18.2- enhancing agents (e.g., one or more certain chemotherapeutic agents). Accordingly, in some embodiments, CLDN-18.2-targeted therapy as described herein can be useful in combination with other anti-cancer agents that are expected to and / or have been demonstrated to up-regulate CLDN-18.2 expression in tumor cells.

[0018]

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

[0019]

[0016] 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 as described herein) specifically binds to a 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 ECD 1 that is exposed in cancer cells.

[0017] In some embodiments, at least one RNA (e.g., ssRNA such as mRNA) encodes a variable heavy chain (VH) domain of a CLDN-18.2-targeting antibody agent and a variable light chain (VL) domain of the antibody agent. In some embodiments, such VH domain(s) and VL domain(s) of a CLDN-18.2-targeting antibody agent may be encoded by a single RNA construct; alternatively in some embodiments they may be encoded separately by at least two individual RNA constructs. For example, in some embodiments, an RNA as utilized herein comprises two or more coding regions, which comprises a heavy chain-coding region that encodes at least a VH domain of the antibody agent; and a light chain-coding region that encodes at least a VL domain of the antibody agent. In alternative embodiments, a pharmaceutical composition may comprise: (i) a first RNA comprising a heavy chain-coding region that encodes at least a VH domain of the antibody agent; and (ii) a second RNA comprising a light chain-coding region that encodes at least a VL domain of the antibody agent.

[0020]

[0018] In some embodiments, a heavy chain-coding region can further encode a constant heavy chain (CH) domain; and / or a light chain-coding region can further encode a constant light chain (CL) domain. For example, in some embodiments, a heavy chain-coding region may encode a VH domain, a CHI domain, aCH domain, and a CH domain of an antibody agent in an immunoglobulin G (IgG) form; and / or a light chain-coding region may encode a VL domain and a CL domain of an antibody agent in an IgG form. In some embodiments, an antibody agent in an IgG form is IgGl.

[0021]

[0019] In some embodiments, a heavy chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes a full-length heavy chain of Zolbetuximab or Claudiximab. In some embodiments, a light chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes a full-length light chain of Zolbetuximab or Claudiximab.

[0022]

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

[0023]

[0021] Those skilled in the art will be aware of the burgeoning field of nucleic acid therapeutics, and moreover of RNA (e.g., ssRNA such as mRNA) therapeutics (see, for example, mRNA-encoding proteins and / or cytokines). Various embodiments of technologies 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, an RNA (e.g., ssRNA such as mRNA) may comprise one or more modified nucleotides (e.g., but not limited to pseudouridine), nucleosides, and / or linkages. Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA such as mRNA) may comprise a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and / or translation efficiency. Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA such as mRNA) may comprise a specific combination of at least two 3’UTR sequences (e.g., a combination of a sequence element of an amino terminal enhancer of split RNA and a sequence derived from a mitochondrially encoded 12S RNA). Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA such as mRNA) may comprise a ‘5 UTR sequence that is derived from human a-globin mRNA. Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA such as mRNA) may comprise a 5’ cap analog, e.g., for co-transcriptionally capping.

[0024] Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA such as mRNA) may comprise a secretion signal-coding region with reduced immunogenicity (e.g., a human secretion signal-coding sequence) such that an encoded antibody agent is expressed and secreted. In some embodiments, an RNA may be formulated in or with one or more delivery vehicles (e.g, nanoparticles such as lipid nanoparticles, etc.). Alternatively or additionally, in some embodiments, an RNA may be formulated in or with liver-targeting lipid nanoparticles (e.g., cationic lipid nanoparticles).

[0025]

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

[0023] In some embodiments, RNA(s) that encode a CLDN-18.2-targeting antibody agent may comprise a 5’ cap.

[0026]

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

[0027]

[0025] In some embodiments where a pharmaceutical composition comprises a first RNA encoding a variable heavy chain (VH) domain of a CLDN-18.2-targeting antibody agent, e.g., a heavy chain of a CLDN-18.2-targeting antibody agent, and a second RNA encoding a variable light chain (VL) domain of the antibody agent, e.g., a light chain of a CLDN-18.2-targeting antibody agent, such a first RNA and a second RNA may be present in a molar ratio of about 1.5:1 to about 1:1.5. In some embodiments, such a first RNA and a second RNA may be present in a molar ratio of about 1.30, about 1.29, about 1.28, about 1.27, about 1.26, about 1.25, about 1.24, about 1.23, about 1.22, about 1.21, about 1.20, about 1.19, about 1.18, about 1.17, about 1.16, about 1.15, about 1.14, about 1.13, about 1.12, about 1.11, about 1.10, about 1.09, about 1.08, about 1.07, about 1.06, about 1.05, about 1.04, about 1.03, about 1.02, about 1.01, about

[0028] 1 .00, about 0.99, about 0.98, about 0.97, about 0.96, about 0.95, about 0.94, about 0.93, about 0.92, about 0.91, about 0.90, about 0.89, about 0.88, about 0.87, about 0.86, about 0.85, about 0.84, about 0.83, about 0.82, about 0.81, or about 0.80. In some embodiments, such a first RNA and a second RNA may be present in a weight ratio of 3 : 1 to 1 : 1. In some embodiments, such a first RNA and a second RNA may be present in a weight ratio of about 2:1. In some embodiments, such a first RNA and a second RNA may be present in a weight ratio of about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4: 1 , about 1.3:1, or about 1.2:1.

[0029]

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

[0030]

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

[0031]

[0028] In some embodiments, lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid; a cationic lipid; and a neutral lipid. In some such embodiments, a polymer-conjugated lipid is be present in about 1-2.5 mol% of the total lipids; a cationic lipid is present in 35-65 mol% of the total lipids; and a neutral lipid is present in 35-65 mol% of the total lipids.

[0032]

[0029] Various lipids (including, e.g., polymer-conjugated lipids, cationic lipids, and neutral lipids) are known in the art and can be used herein to form lipid nanoparticles, e.g., lipid nanoparticles targeting a specific cell type (e.g., liver cells). In some embodiments, a polymer- conjugated lipid included in pharmaceutical compositions described herein may be a PEG- conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof). In some embodiments, a cationic lipid included in pharmaceutical compositions described herein may be ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) or a derivative thereof. In some embodiments, neutral lipid included in pharmaceutical compositions described herein may be or comprise a phospholipid or derivative thereof (e.g., l,2-distearoyI-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol.

[0033]

[0030] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant (e.g, sucrose) and / or an aqueous buffered solution, which may in some embodiments include one or more salts (e.g., sodium salts).

[0034]

[0031] In some embodiments, a pharmaceutical composition described herein may further comprises one or more active agents other than RNA (e.g., an ssRNA such as an mRNA) encoding a CLDN-18.2-targeting agent (e.g., antibody agent). For example, in some embodiments, such other active agent may be or comprise a chemotherapeutic agent. An exemplary chemotherapeutic agent may be or comprise a chemotherapeutic agent indicated for treatment of pancreatic cancer.

[0032] In some embodiments, pharmaceutical compositions described herein can be taken up by target cells for production of an 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).

[0035]

[0033] Accordingly, another aspect of the present disclosure relates to methods of using 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 a CLDN- 18.2-positive solid tumor are but are not limited to a biliary tract tumor, a gastric tumor, a gastro-esophageal tumor, an ovarian tumor, a pancreatic tumor, and a tumor that expresses or exhibits a certain level of a CLDN-18.2 polypeptide. In some embodiments, a CLDN- 18.2-positive tumor may be characterized in that > 50% of tumor cells show > 2+ CLDN-18.2 protein staining intensity as assessed by an immunohistochemistry assay in formalin-fixed, paraffin-embedded neoplastic tissue from a subject to be 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 a pre-treatment sufficient to increase CLDN-18.2 level such that his / her solid tumor is characterized as a CLDN- 18.2-positive solid tumor.

[0036]

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

[0035] In some embodiments, technologies provided herein are useful for treatment of a CLDN-18.2 positive pancreatic tumor. In some embodiments involving administration of a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive pancreatic tumor, such a subject may be receiving such a provided composition as a monotherapy or as part of a combination therapy comprising such a provided pharmaceutical composition and a chemotherapeutic agent indicated for treatment of pancreatic tumor. In some embodiments, such a chemotherapeutic agent may be or comprise gemcitabine and / or paclitaxel (e.g., nab-paclitaxel). In some embodiments, such a chemotherapeutic agent may be or comprise FOLFIRINOX, which is a combination of cancer drugs including: folinic acid (FOL), fluorouracil (F), irinotecan (IRIN), and oxalipatin (OX).

[0037]

[0036] In some embodiments, technologies provided herein are useful for treatment of a CLDN-18.2 positive biliary tract tumor. In some embodiments involving administration of a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive biliary tract tumor, such a subject may be receiving such a provided composition as a monotherapy or as part of a combination therapy comprising such a provided pharmaceutical composition and a chemotherapeutic agent indicated for treatment of biliary tract tumor. In some embodiments, such a chemotherapeutic agent may be or comprise gemcitabine and / or cisplatin.

[0038]

[0037] Pharmaceutical compositions and methods described herein may be applicable to a subject of any age suffering from a CLDN-18.2 positive solid tumor. In some embodiments, a subject suffering from a CLDN-18.2 positive solid tumor is an adult subject.

[0039]

[0038] Pharmaceutical compositions described herein may be administered to a subject in need thereof by appropriate methods known in the art. For example, in some embodiments, a provided pharmaceutical composition may be administered to a subject suffering from a CLDN- 18.2 positive solid tumor by intravenous injection.

[0040]

[0039] Dosage of pharmaceutical compositions described herein may vary with a number of factors including, e.g., but not limited to body weight of a subject to be treated, cancer types and / or cancer stages, and / or monotherapy or combination therapy. In some embodiments, a pharmaceutical composition described herein is administered to a subject suffering from a CLDN-18.2 positive solid tumor in at least one or more (including, 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) dosing cycles. In some embodiments, each dosing cycle may be a three-week dosing cycle. In some embodiments, a pharmaceutical composition described herein is administered is at least one dose per dosing cycle. In some embodiments, a dosing cycle involves administration of a set number and / or pattern of doses; in some embodiments, a dosing cycle involves administration of a set cumulative dose, e.g., over a particular period of time, and optionally via multiple doses, which may be administered, for example, at set interval(s) and / or according to a set pattern. In some embodiments, each dose or a cumulative dose of a pharmaceutical composition described herein may comprise one or more RNAs encoding a CLDN-18.2-targeting antibody agent (whether encoded by a single RNA or two or more RNAs) in an amount within a range of 0.1 mg / kg to 5 mg / kg body weight of a subject to be administered.

[0041]

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

[0042]

[0041] Methods of producing a CLDN-18.2-targeting antibody agent are also within the scope of the present disclosure. In some embodiments, a method of producing a CLDN-18.2- targeting antibody agent comprises administering to cells a composition comprising at least one RNA (e.g., ones as described herein) comprising one or more coding regions that encode a CLDN-18.2-targeting antibody agent so that such cells express and secrete a CLDN-18.2- targeting antibody agent encoded by such RNA(s). In some embodiments, cells to be administered or targeted are or comprise liver cells.

[0043]

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

[0044]

[0043] In some embodiments, cells are present in a subject. In some such embodiments, a pharmaceutical composition described herein may be administered to a subject in need thereof.

[0045] In some embodiments, such a pharmaceutical composition may be administered to a subject such that a CLDN-18.2-targeting antibody agent is produced at a therapeutically relevant plasma concentration. In some embodiments, a therapeutically relevant plasma concentration is sufficient to mediate cancer cell death through antibody-dependent cellular cytotoxicity (ADCC). For example, in some embodiments, a therapeutically relevant plasma concentration is 0.3- 28 pghnL.

[0046]

[0044] Among other things, the present disclosure also provides methods of characterizing one or more features of an RNA or composition thereof, which RNA encodes part or all of an antibody agent. In some embodiments, a method comprising a step of: determining one or more features of an antibody agent expressed from at least one mRNA introduced into cells, wherein such at least one mRNA comprises one or more of features of at least one or more RNA comprising a coding region that encodes an antibody agent that binds to a Claudin-18.2 (CLDN-18.2) polypeptide, e.g., binds preferentially to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 polypeptide, wherein such one or more features comprises: (i) protein expression level of an antibody agent; (ii) binding specificity of an antibody agent to CLDN- 18.2; (iii) efficacy of an antibody agent to mediate target cell death through ADCC; and (iv) efficacy of an antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).

[0047]

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

[0048]

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

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

[0049]

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

[0050]

[0049] In some embodiments, a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise 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 features: (a) protein level of the antibody agent expressed by the cells above a threshold level; (b) preferential binding of the antibody agent to CLDN-18.2 relative to CLDN18.1 ; and (c) killing of at least 50% target cells (e.g., cancer cells) mediated by ADCC and / or CDC.

[0051]

[0050] In some embodiments, a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise characterizing an antibody agent expressed from one or more RNAs described herein as a Zolbetuximab or Claudiximab-equivalent antibody if tested features of the antibody are at least comparable to that of Zolbetuximab or Claudiximab.

[0052]

[0051] In some embodiments involving a step of determining one or more features of an antibody agent expressed from one or more RNAs described herein, such a step may comprise determining one or more of the following features:

[0053] * whether, when assessed 48 hours after contacting or administering, cells express a CLDN-18.2-targeting antibody agent encoded by at least one RNA;

[0054] * whether the antibody agent expressed by the cells binds preferentially to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide; • whether the antibody agent expressed by the cells exhibit comparable target specificity to CLDN-18.2 as observed in a flow cytometric binding assay with a reference CLDN-18.2- targeting monoclonal antibody;

[0055] • whether, when assessed 48 hours after incubating 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, the CLDN-18.2 positive cells, not the control cells, were lysed;

[0056] • whether the antibody agent expressed by the cells exhibit at least comparable ADCC profile of targeted CLDN-18.2 positive cells as observed with a reference CLDN-18.2-targeting monoclonal antibody in the same concentration; and

[0057] • whether, when assessed 2 hours after incubating CLDN-18.2 positive cells or CLDN-18.2 negative control cells with human serum in the presence of the antibody agent, the CLDN- 18.2 positive cells, not the control cells, were lysed.

[0058]

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

[0059]

[0053] Also within the scope of the present disclosure includes a method of manufacture, which comprises steps of:

[0060] (A) determining one or more features of an RNA or composition thereof, which RNA encodes part or all of an antibody agent, which one or more features are selected from the group consisting of:

[0061] (i) length and / or sequence of the RNA;

[0062] (ii) integrity of the RNA;

[0063] (iii) presence and / or location of one or more chemical moieties of the RNA;

[0064] (iv) extent of expression of the antibody agent when the RNA is introduced into a cell; (v) stability of the RNA or composition thereof;

[0065] (vi) level of antibody agent in a biological sample from an organism into which the RNA has been introduced;

[0066] (vii) binding specificity of the antibody agent expressed from the RNA, optionally to CLDN-18.2 and optionally relative to CLDN 18.1 ;

[0067] (viii) efficacy of the antibody agent to mediate target cell death through ADCC;

[0068] (ix) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC);

[0069] (x) lipid identity and amount / concentration within the composition;

[0070] (xi) size of lipid nanoparticles within the composition;

[0071] (xii) polydispersity of lipid nanoparticles within the composition;

[0072] (xiii) amount / concentration of the RNA within the composition;

[0073] (xiv) extent of encapsulation of the RNA within lipid nanoparticles; and

[0074] (xv) combinations thereof;

[0075] (B) comparing such one or more features of the RNA or composition thereof with that of an appropriate reference standard; and

[0076] (C) (i) designating the RNA or composition thereof for one or more further steps of manufacturing and / or distribution if the comparison demonstrates that the RNA or composition thereof meets or exceeds the reference standard; or

[0077] (ii) taking an alternative action if the comparison demonstrates that the RNA or composition thereof does not meet or exceed the reference standard.

[0078]

[0054] In some embodiments of a method of manufacture, when an RNA (e.g., ones described herein) is assessed and one or more features of the RNA meets or exceeds an appropriate reference standard, such an RNA is designated for formulation, e.g., in some embodiments involving formulation with lipid particles described herein.

[0079]

[0055] In some embodiments of a method of manufacture, when a composition comprising an RNA (e.g., ones described herein) is assessed and one or more features of the composition meets or exceeds an appropriate reference standard, such a composition is designated for release and / or distribution of the composition.

[0080]

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

[0081]

[0057] Provided herein is also a method of determining a dosing regimen of a pharmaceutical composition targeting CLDN-18.2. For example, in some embodiments, such a method comprises steps of: (A) administering a pharmaceutical composition (e.g., ones described herein) to a subject suffering from a CLDN-18.2 positive solid tumor under a pre-determined dosing regimen; (B) monitoring or measuring tumor size of the subject periodically over a period of time; (C) evaluating the dosing regimen based on the tumor size measurement(s). For example, a dose and / or dosage frequency can be increased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is not therapeutically relevant; or a dose and / or dosage frequency can be decreased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, but adverse effect (e.g., toxicity effect) is shown in the subject. If reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, and no adverse effect (e.g., toxicity effect) is shown in the subject, no changes is made to a dosage regimen.

[0082]

[0058] In some embodiments, such a method of determining a dosing regimen of a pharmaceutical composition targeting CLDN-18.2 may be performed in a group of animal subjects (e.g., mammalian non-human subjects) each a bearing a human CLDN-18.2 positive xenograft tumor. In some such embodiments, a dose and / or dosage frequency can be increased if less than 30% of the animal subjects exhibit reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) and / or extent of reduction in tumor size exhibited by the animal subjects is not therapeutically relevant; or a dose and / or dosage frequency can be decreased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, but significant adverse effect (e.g., toxicity effect) is shown in at least 30% of the animal subjects. If reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, and no significant adverse effect (e.g., toxicity effect) is shown in the animal subjects, no changes is made to a dosage regimen.

[0059] The present disclosure, among other things, provides, in particular, a composition or medical preparation comprising:

[0083] (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and

[0084] (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17.

[0085] In some embodiments, the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.

[0086] The present disclosure also provides a composition or medical preparation comprising:

[0087] (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and

[0088] (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4. In some embodiments, the RNA, 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.

[0089] 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.

[0090] 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.

[0091] In some embodiments, the RNA, e.g., 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.

[0092] In some embodiments, the RNA, e.g., 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.

[0093] The present disclosure also provides a composition or medical preparation comprising:

[0094] (i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and

[0095] (ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein 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.

[0096] 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.

[0097] 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.

[0098] In some embodiments, the RNA, e.g., 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.

[0099] In some embodiments, the RNA, e.g., 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.

[0100] In some embodiments,

[0101] (a) the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and / or

[0102] (b) the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.

[0103] In some embodiments, the coding region under (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, and the coding region under (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, 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.

[0104] In some embodiments, the RNA under (i) is a first RNA molecule and the RNA under (ii) is a second RNA molecule.

[0105] In some embodiments, at least 90% is at least 95%, 96%, 97%, 98%, 99%.

[0106] In some embodiments, the antibody agent binds preferentially to CLDN-18.2 relative to Claudin-

[0107] 18.1 (CLDN-18.1).

[0108] In some embodiments, the antibody agent binds to a first extracellular domain (ECD1) of CLDN-18.2.

[0109] In some embodiments, the antibody agent binds to an epitope of ECD 1 of CLDN-18.2 that is exposed in cancer cells.

[0110] In some embodiments, the antibody agent that binds to CLDN-18.2 comprises two binding arms wherein each binding arm comprises a heavy chain of an antibody agent that binds to CLDN-

[0111] 18.2 and a light chain of an antibody agent that binds to CLDN-18.2.

[0112] In some embodiments, the antibody agent is IgGl .

[0113] In some embodiments, the IgGl is human IgGl.

[0114] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form a binding domain that binds to CLDN-18.2.

[0115] In some embodiments, the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)).

[0116] In some embodiments, the VH(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH(CLDN-18.2) comprises CDR1 , CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 5, 6, and 7, respectively.

[0117] In some embodiments, the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)).

[0118] In some embodiments, the VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 15.

[0119] In some embodiments, the VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 8, 9, and 10, respectively.

[0120] In some embodiments, the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising CDR1 , CDR2 and CDR3 of the amino acid sequence SEQ ID NO: 14, and the the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising CDR1 , CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 15.

[0121] In some embodiments, the first polypeptide chain comprises a variable domain of a 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 as set forth in SEQ ID NO: 5, 6, and 7, respectively, and the the second polypeptide chain comprises a variable domain of a 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 as set forth in SEQ ID NO: 8, 9, and 10, respectively.

[0122] In some embodiments, the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising the amino acid sequence SEQ ID NO: 14, and the the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising the amino acid sequence of SEQ ID NO: 15.

[0123] In some embodiments, the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and the the second polypeptide chain comprises a variable domain of a light chain (VL) 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). In some embodiments, the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), a constant domain 1 of a heavy chain (CHI) of an antibody agent, a constant domain 2 of a heavy chain (CH2) of an antibody agent, and a constant domain 3 of a heavy chain (CH3) of an antibody agent.

[0124] In some embodiments, the VH(CLDN-18.2), CHI, CH2 and CH3 are present in the first polypeptide chain in an immunoglobulin G (IgG) form.

[0125] In some embodiments, the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)), and a constant domain of a light chain (CL) of an antibody agent.

[0126] In some embodiments, the VL(CLDN-18.2) and the CL are present in the second polypeptide chain in an IgG form.

[0127] In some embodiments, the CHI on the first polypeptide chain interacts with the CL on the second polypeptide chain.

[0128] In some embodiments, the first polypeptide chain and the second polypeptide chain each independently comprise a secretion signal, wherein the secretion signal is preferably located at the N-terminus of the first polypeptide chain and the second polypeptide chain.

[0129] In some embodiments, the secretion signal of the first polypeptide chain and / or the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13.

[0130] In some embodiments, the coding region under (i) comprises the nucleotide sequence of SEQ ID NO: 16, and the coding region under (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, e.g., each RNA, comprises a poly-A sequence.

[0131] In some embodiments, the poly-A sequence is an interrupted sequence of A nucleotides.

[0132] In some embodiments, the poly-A sequence comprises at least 100 nucleotides.

[0133] In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence Ax- L- Ay, wherein Axis a sequence of at least 20 A nucleotides, Ayis 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. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 23.

[0134] In some embodiments, the composition or medical preparation 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

[0135] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 25 or 27.

[0136] In some embodiments, the composition or medical preparation comprises:

[0137] (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and

[0138] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25.

[0139] In some embodiments, the composition or medical preparation comprises:

[0140] (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and

[0141] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27.

[0142] The present disclosure also provides a composition or medical preparation comprising:

[0143] (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and

[0144] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27.

[0145] The present disclosure also provides a composition or medical preparation comprising:

[0146] (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and

[0147] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25.

[0148] The present disclosure also provides a composition or medical preparation comprising:

[0149] (i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and

[0150] (ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27.

[0151] In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside in place of uridine.

[0152] In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside in place of each uridine.

[0153] In some embodiments, the modified nucleoside is pseudouridine (y) and / or N1 -methylpseudouridine (mly).

[0154] In some embodiments, the modified nucleoside is N1 -methyl -pseudouridine (mly).

[0155] In some embodiments, the RNA, e.g., each RNA, comprises a 5’ cap.

[0156] In some embodiments, the RNA, e.g., each RNA, comprises the 5’ cap m27 3 _°Gppp( r ’0)ApG. In some embodiments, the RNA, e.g., each RNA, is single- stranded RNA.

[0157] In some embodiments, the RNA, e.g., each RNA, is mRNA. In some embodiments, the RNA, e.g., each RNA, is formulated in lipid nanoparticles (LNP), e.g., each RNA is co-formulated in lipid nanoparticles (LNP).

[0158] In some embodiments, lipids that form the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid.

[0159] In some embodiments, a. the cationic lipid is present in 35-65 mol% of the total lipids; b. the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; and c. the neutral lipid is present in 35-65 mol% of the total lipids.

[0160] In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate).

[0161] In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

[0162] In some embodiments, the neutral lipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

[0163] In some embodiments, the lipid nanoparticles have an average size of about 50-150 nm.

[0164] 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, l,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In some embodiments, the composition is a pharmaceutical composition.

[0165] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0166] In some embodiments, the medical preparation is a kit.

[0167] In some embodiments, the RNA, e.g., each RNA, and optionally the particle forming components are in separate vials.

[0168] In some embodiments, the medical preparation further comprises instructions for use of the composition or medical preparation for treating or preventing cancer.

[0169] The present disclosure also provides the composition or medical preparation described herein for pharmaceutical use.

[0170] In some embodiments, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder. In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing cancer.

[0171] In some embodiments, the cancer comprises a CLDN-18.2-positive cancer.

[0172] In some embodiments, the cancer comprises a CLDN-18.2-positive solid tumor.

[0173] In some embodiments, the cancer comprises a CLDN-18.2-positive pancreatic cancer.

[0174] In some embodiments, the cancer comprises a CLDN-18.2-positive gastric cancer.

[0175] In some embodiments, the cancer comprises a CLDN-18.2-positive biliary tract tumor.

[0176] In some embodiments, the cancer comprises a CLDN-18.2-positive locally advanced, unresectable, or metastatic cancer.

[0177] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder further comprises administering a further therapy.

[0178] In some embodiments, the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) radiotherapy, and (iii) chemotherapy.

[0179] In some embodiments, the further therapy comprises administering a further therapeutic agent. In some embodiments, the further therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the composition or medical preparation described herein is for administration to a human.

[0180] In some embodiments, the composition or medical preparation described herein is for intravenous administration.

[0181] The present disclosure also provides a method of treating cancer in a subject comprising administering to the subject the composition described herein.

[0182] In some embodiments, the cancer comprises a CLDN-18.2-positive cancer.

[0183] In some embodiments, the cancer comprises a CLDN-18.2-positive solid tumor.

[0184] In some embodiments, the cancer comprises a CLDN-18.2-positive pancreatic cancer.

[0185] In some embodiments, the cancer comprises a CLDN-18.2-positive gastric cancer.

[0186] In some embodiments, the cancer comprises a CLDN-18.2-positive biliary tract tumor.

[0187] In some embodiments, the cancer comprises a CLDN-18.2-positive locally advanced, unresectable, or metastatic cancer.

[0188] In some embodiments, the method described herein further comprises administering a further therapy. In some embodiments, the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) radiotherapy, and (iii) chemotherapy.

[0189] In some embodiments, the further therapy comprises administering a further therapeutic agent. In some embodiments, the further therapeutic agent comprises an anti-cancer therapeutic agent. In some embodiments, the subject is a human.

[0190] In some embodiments, the composition is administered intravenously.

[0191] The present disclosure also provides the composition described herein for use in a method described herein.

[0192] In some embodiments, upon expression of the polypeptide chains of the antibody agent that binds to Claudin-18.2 (CLDN-18.2) the polypeptide chains are secreted into the bloodstream as fully assembled antibodies and / or as 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 the antibody, such as binding to its target and / or recruitment and / or stimulation of the immune system, for example ADCC, e.g., to the same or a similar level as a corresponding antibody expressed in vitro.

[0193] In some embodiments, the composition or medical preparation described herein is for introducing the RNA into liver cells and expressing the polypeptide chains encoded by the RNA in liver cells.

[0194] In some embodiments, the composition or medical preparation described herein is for systemic delivery of the polypeptide chains.

[0195] In some embodiments, the composition or medical preparation described herein is for systemic delivery of the polypeptide chains following expression of the polypeptide chains in liver cells. The present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising:

[0196] (a) administering a composition described herein such that the RNA is introduced into liver cells; and

[0197] (b) expressing the polypeptide chains encoded by the RNA in the liver cells.

[0198] The present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising: (a) administering a composition described herein such that the RNA is introduced into liver cells; and

[0199] (b) expressing the polypeptide chains encoded by the RNA in the liver cells, wherein, following expression, the polypeptide chains are secreted into the bloodstream.

[0200] 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, said method comprising:

[0201] (a) administering a composition described herein such that the RNA is introduced into liver cells; and

[0202] (b) expressing the polypeptide chains encoded by the RNA in the liver cells, wherein, following expression, the polypeptide chains are secreted into the bloodstream.

[0203] In some embodiments, administration is parenteral administration.

[0204] In some embodiments, administration is intravenous administration.

[0205] 160] The present disclosure, among other things, also provides the following items:

[0206] 1. A pharmaceutical composition comprising: a. at least one single- stranded RNA comprising one or more coding regions that encode an antibody agent that binds preferentially to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 polypeptide; and b. lipid nanoparticles; wherein the at least one single-stranded RNA is encapsulated within at least one of the lipid nanoparticles.

[0207] 2. The pharmaceutical composition of item 1, wherein the antibody agent specifically binds to a first extracellular domain (ECD1) of a CLDN-18.2 polypeptide.

[0208] 3. The pharmaceutical composition of item 2, wherein the antibody agent specifically binds to an epitope of ECD 1 that is exposed in cancer cells.

[0209] 4. The pharmaceutical composition of any one of items 1-3, wherein the antibody agent is or comprises an antibody or an antigen binding fragment thereof.

[0210] 5. The pharmaceutical composition of any one of items 1-4, wherein the at least one singlestranded RNA encodes both of: a variable heavy chain (VH) domain of the antibody agent; and a variable light chain (VL) domain of the antibody agent. The pharmaceutical composition of item 5, wherein the at least one single-stranded RNA is a first single-stranded RNA comprising a heavy chain-coding region that encodes at least a VH domain of the antibody agent; and a. wherein the first single-stranded RNA further comprises a light chain-coding region that encodes at least a VL domain of the antibody agent; or b. wherein the pharmaceutical composition further comprises a second singlestranded RNA comprising a light chain-coding region that encodes at least a VL domain of the antibody agent. The pharmaceutical composition of item 6, wherein the heavy chain-coding region further encodes a constant heavy chain (CH) domain; and / or the light chain-coding region further encodes a constant light chain (CL) domain. The pharmaceutical composition of item 6, wherein the heavy chain-coding region encodes a VH domain, a CHI domain, aCn2 domain, and a CH3 domain of the antibody agent in an immunoglobulin G (IgG) form; and / or the light chain-coding region encodes a VL domain and a CL domain of the antibody agent in an IgG form. The pharmaceutical composition of item 8, wherein the IgG is IgGl. The pharmaceutical composition of any one of items 6-9, wherein the heavy chain-coding region consists of or comprises a nucleotide sequence that encodes a full-length heavy chain of Zolbetuximab or Claudiximab. The pharmaceutical composition of any one of items 6-9, wherein the light chain-coding region consists of or comprises a nucleotide sequence that encodes a full-length light chain of Zolbetuximab or Claudiximab. The pharmaceutical composition of any one of items 6-11, wherein the first single- stranded and / or the second single- stranded RNA each independently comprise a secretion signalencoding region. The pharmaceutical composition of any one of items 6-12, wherein the first single-stranded and / or the second single- stranded RNA each independently comprise at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). The pharmaceutical composition of 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 poly adenine (polyA) tail. The pharmaceutical composition of any one of items 6-14, wherein the first single-stranded RNA comprises, in a 5’ to 3’ direction: a. a 5’UTR-coding region; b. a secretion signal-coding region; c. the heavy chain-coding region; d. a 3’ UTR-coding region; and e. a polyA tail-coding region. The pharmaceutical composition of any one of items 6-15, wherein the second singlestranded RNA comprises, in a 5’ to 3’ direction: a. a 5’UTR-coding region; b. a secretion signal-coding region; c. the light chain-coding region; d. a 3’ UTR-coding region; and e. a polyA tail-coding region. The pharmaceutical composition of item 14 or 15, wherein the polyA tail is or comprises a modified polyA sequence. The pharmaceutical composition of any one of items 6-16, wherein the first single-stranded and / or the second single-stranded RNA comprises a 5’ cap. The pharmaceutical composition of any one of items 6-18, wherein the first single- stranded and / or the second single-stranded RNA comprises at least one modified ribonucleotide. The pharmaceutical composition of item 19, wherein the modified ribonucleotide comprises pseudouridine. The pharmaceutical composition of any one of items 6-20, wherein the at least one singlestranded RNA comprises the first single-stranded RNA and the second single-stranded RNA. The pharmaceutical composition of any one of items 6-21, wherein the first single-stranded RNA and the second single-stranded RNA are present in a weight ratio of 3 : 1 to 1 :1. The pharmaceutical composition of any one of items 1-22, wherein the lipid nanoparticles are liver-targeting lipid nanoparticles. The pharmaceutical composition of any one of items 1-23, wherein the lipid nanoparticles are cationic lipid nanoparticles. The pharmaceutical composition of item 24, wherein lipids that form the lipid nanoparticles comprise:

[0211] - a polymer-conjugated lipid;

[0212] - a cationic lipid; and

[0213] - a neutral lipid. The pharmaceutical composition of item 25, wherein: a. the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; b. the cationic lipid is present in 35-65 mol% of the total lipids; and c. the neutral lipid is present in 35-65 mol% of the total lipids. The pharmaceutical composition of item 25 or 26, wherein the polymer-conjugated lipid is a PEG-conjugated lipid e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). The pharmaceutical composition of any one of items 25-27, wherein the cationic lipid is ((3- hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2 -butyloctanoate). The pharmaceutical composition of any one of items 25-28, wherein the neutral lipid comprises 1 ,2-Distearoyl-sn-glycero-3 -phosphocholine (DPSC) and / or cholesterol. The pharmaceutical composition of any one of items 1-29, wherein the lipid nanoparticles have an average size of about 50-150 nm. The pharmaceutical composition of any one of items 1-30, further comprising a cryoprotectant (e.g., sucrose). The pharmaceutical composition of any one of items 1-31, comprising an aqueous buffered solution. The pharmaceutical composition of item 32, wherein the aqueous buffered solution includes sodium ions. The pharmaceutical composition of any one of items 1 -33, further comprising a chemotherapeutic agent. The pharmaceutical composition of item 34, wherein the chemotherapeutic agent is a chemotherapeutic agent indicated for treatment of pancreatic cancer. The pharmaceutical composition of any one of items 1-35, wherein the at least one singlestranded RNA is present at a concentration of 0.5 mg / mL to 1.5 mg / mL. A method comprising administering a pharmaceutical composition of any one of item 1-36 to a subject suffering from a CLDN-18.2-positive solid tumor. The method of item 37, wherein the CLDN- 18.2-positive tumor is a pancreatic tumor. The method of item 37, wherein the CLDN- 18.2-positive tumor is a gastric tumor. The method of item 37, wherein the CLDN- 18.2-positive tumor is a biliary tract tumor. The method of any one of items 37-40, wherein the CLDN-18.2-positive solid tumor is locally advanced, unresectable, or metastatic. The method of any one of items 37-41, wherein the subject has received a pre-treatment sufficient to increase CLDN-18.2 level such that a solid tumor from which the subject is suffering is characterized as a CLDN-18.2-positive solid tumor. The method of any one of items 37-42, wherein the CLDN-18.2-positive tumor is characterized in that > 50% of tumor cells show > 2+ CLDN-18.2 protein staining intensity as assessed by an immunohistochemistry assay in formalin-fixed, paraffin-embedded neoplastic tissue from the subject. The method of any one of items 37-43, wherein the pharmaceutical composition is administered as monotherapy. The method of any one of items 37-44, wherein the pharmaceutical composition is administered as part of combination therapy comprising the pharmaceutical composition and a chemotherapeutic agent. The method of any one of items 37-45, wherein the subject has received the chemotherapeutic agent. The method of item 45, further comprising administering to the subject the chemotherapeutic agent such that the subject is receiving the combination therapy. The method of item 47, wherein the chemotherapeutic agent is administered at least four hours after the administration of the pharmaceutical composition. The method of any one of items 45-48, wherein the chemotherapeutic agent is or comprises gemcitabine and / or paclitaxel (e.g., nab-paclitaxel) for a subject suffering from a CLDN-

[0214] 18.2-positive pancreatic tumor. The method of any one of items 45-48, wherein the chemotherapeutic agent is or comprises FOLFIRINOX for a subject suffering from a CLDN- 18.2-positive pancreatic tumor. The method of any one of items 45-48, wherein the chemotherapeutic agent is or comprises gemcitabine and / or cisplatin for a subject suffering from a CLDN-18.2-positive biliary tract cancer. 52. The method of any one of items 37-51, wherein the subject is an adult subject.

[0215] 53. The method of any one of items 37-52, wherein the administering is performed by intravenous injection.

[0216] 54. The method of any one of items 37-53, wherein the pharmaceutical composition is administered in at least one, at least two, at least three or more dosing cycles.

[0217] 55. The method of item 54, wherein the pharmaceutical composition is administered as one or more doses per dosing cycle.

[0218] 56. The method of item 55, wherein each dosing cycle is a three-week dosing cycle.

[0219] 57. The method of item 55 or 56, wherein the one or more doses comprise the at least one singlestranded RNA within a range of 0.1 mg / kg to 5 mg / kg body weight of the subject.

[0220] 58. In a method of delivering a CLDN-18.2-targeting antibody for cancer treatment in a subject, the improvement comprising administering to the subject the pharmaceutical composition of any one of items 1-36.

[0221] 59. A method of producing a CLDN-18.2-targeting antibody comprising administering to cells the pharmaceutical composition of any one of items 1-35 so that the cells express and secrete the CLDN-18.2-targeting antibody encoded by the at least one single-stranded RNA of the pharmaceutical composition.

[0222] 60. The method of item 59, wherein the cells are liver cells.

[0223] 61. The method of item 59 or 60, wherein the cells are in a subject.

[0224] 62. The method of item 61, wherein the CLDN-18.2-targeting antibody is produced at a therapeutically relevant plasma concentration.

[0225] 63. The method of item 62, wherein the therapeutically relevant plasma concentration is sufficient to mediate cancer cell death through antibody-dependent cellular cytotoxicity (ADCC).

[0226] 64. The method of item 63, wherein the therapeutically relevant plasma concentration is 0.3-

[0227] 28 μg / mL.

[0228] 65. A method comprising a step of: determining one or more features of an antibody agent expressed from at least one mRNA introduced into cells, wherein the at least one mRNA comprises one or more of features of at least one or more single- stranded RNA comprising a coding region that encodes an antibody agent that binds preferentially to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin- 18.1 polypeptide, wherein the one or more features comprises: (i) protein expression level of the antibody agent; (ii) binding specificity of the antibody agent to CLDN-18.2; (iii) efficacy of the antibody agent to mediate target cell death through ADCC; and (iv) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).

[0229] 66. A method of characterizing a pharmaceutical composition targeting CLDN-18.2, the method comprising steps of: contacting cells with at least one pharmaceutical composition as set forth in any one of items 1-35; and detecting the antibody agent produced by the cells.

[0230] 67. The method of item 66, further comprising determining one or more features of the antibody agent, wherein the one or more features comprises: (i) protein expression level of the antibody agent; (ii) binding specificity of the antibody agent to a CLDN-18.2 polypeptide;

[0231] (iii) efficacy of the antibody agent to mediate target cell death through ADCC; and (iv) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).

[0232] 68. The method of any one of items 65-67, wherein the cells are liver cells.

[0233] 69. The method of item 65 or 67, wherein the step of determining comprises comparing the one or more features of the antibody agent with that of a reference CLDN-18.2-targeting antibody. 0. The method of any one of items 65 and 67-69, wherein the step of determining comprises assessing the protein expression level of the antibody agent above a threshold level.

[0234] 71. The method of item 70, wherein the threshold level is a level that is sufficient to induce ADCC. 2. The method of any one of items 65 and 67-71 , wherein the step of determining comprises assessing binding of the antibody agent to a CLDN-18.2 polypeptide. 3. The method of item 72, wherein the assessing comprises determining binding of the antibody agent to a CLDN-18.2 polypeptide relative to its binding to a CLDN18.1 polypeptide. 4. The method of item 72 or 73, wherein the assessing comprises determining a binding preference profile of the antibody agent at least comparable to that of a reference CLDN- 18.2-targeting antibody. The method of item 69 or 74, wherein the reference CLDN-18.2-targeting antibody is Zolbetuximab or Claudiximab. The method of any one of items 65-75, further characterizing the antibody agent as a CLDN- 18.2-targeting antibody agent if the antibody agent comprises the following features: a. protein level of the antibody agent expressed by the cells above a threshold level that is sufficient to induce ADCC; b. preferential binding of the antibody agent to CLDN- 18.2 relative to CLDN 18.1; and c. killing of at least 50% target cells mediated by ADCC and / or CDC. The method of item 76, further characterizing the antibody agent as a Zolbetuximab or Claudiximab-equivalent antibody if the features of the antibody are at least comparable to that of Zolbetuximab or Claudiximab. The method of any one of items 65-77, wherein the target cells are cancer cells. The method of any one of items 65 and 66-78, wherein the step of determining comprises determining whether, when assessed 48 hours after the contacting, the cells express an anti- CLDN18-2 antibody agent encoded by the at least one single-stranded RNA. The method of any one of items 65 and 66-79, wherein the step of determining comprises determining one or more of the following features:

[0235] - whether the antibody agent expressed by the cells binds preferentially to a CLDN- 18.2 polypeptide relative to a CLDN18.1 polypeptide;

[0236] - whether the antibody agent expressed by the cells exhibit comparable target specificity to CLDN- 18.2 as observed in a flow cytometric binding assay with a reference CLDN- 18.2- targeting monoclonal antibody;

[0237] - whether, when assessed 48 hours after incubating 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, the CLDN-18.2 positive cells, not the control cells, were lysed;

[0238] - whether the antibody agent expressed by the cells exhibit at least comparable ADCC profile of targeted CLDN-18.2 positive cells as observed with a reference CLDN-18.2- targeting monoclonal antibody in the same concentration; and - whether, when assessed 2 hours after incubating CLDN-18.2 positive cells or CLDN-18.2 negative control cells with human serum in the presence of the antibody agent, the CLDN- 18.2 positive cells, not the control cells, were lysed. The method of any one of items 66-80, wherein the cells are present in a subject (e.g., a mouse or monkey subject). The method of item 81, wherein the one or more features include antibody level in one or more tissues in the subject. The method of any one of items 66-82, further comprising: administering the pharmaceutical composition to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor to determine anti-tumor activity if the pharmaceutical composition is characterized as CLDN-18.2-targeting. A method of manufacture, the method comprising steps of:

[0239] (A) determining one or more features of a single stranded RNA (ssRNA) or composition thereof, which ssRNA encodes part or all of an antibody agent, which one or more features are selected from the group consisting of:

[0240] (i) length and / or sequence of the ssRNA;

[0241] (ii) integrity of the ssRNA;

[0242] (iii) presence and / or location of one or more chemical moieties of the ssRNA;

[0243] (iv) extent of expression of the antibody agent when the ssRNA is introduced into a cell;

[0244] (v) stability of the ssRNA or composition thereof;

[0245] (vi) level of antibody agent in a biological sample from an organism into which the ssRNA has been introduced;

[0246] (vii) binding specificity of the antibody agent expressed from the ssRNA, optionally to CLDN-18.2 and optionally relative to CLDN18.1;

[0247] (viii) efficacy of the antibody agent to mediate target cell death through ADCC;

[0248] (ix) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC);

[0249] (x) lipid identity and amount / concentration within the composition;

[0250] (xi) size of lipid nanoparticles within the composition;

[0251] (xii) polydispersity of lipid nanoparticles within the composition; (xiii) amount / concentration of the ssRNA within the composition;

[0252] (xiv) extent of encapsulation of the ssRNA within lipid nanoparticles; and

[0253] (xv) combinations thereof;

[0254] (B) comparing the one or more features of the ssRNA or composition thereof with that of an appropriate reference standard; and

[0255] (C) (i) designating the ssRNA or composition thereof for one or more further steps of manufacturing and / or distribution if the comparison demonstrates that the ssRNA or composition thereof meets or exceeds the reference standard; or

[0256] (ii) taking an alternative action if the comparison demonstrates that the ssRNA or composition thereof does not meet or exceed the reference standard. The method of item 84, wherein the ssRNA is assessed and the one or more further steps of step (C)(i) are or comprise at least formulation of the ssRNA. The method of item 84 or 85, wherein the composition is assessed and the composition comprises lipid nanoparticles and the one or more further steps of step (C)(i) are or comprise include release and distribution of the composition. The method of item 85, further comprising administering the formulation to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor to determine antitumor activity. A method of determining a dosing regimen of a pharmaceutical composition targeting

[0257] CLDN-18.2-targeting, the method comprising steps of:

[0258] (A) administering a pharmaceutical composition set forth in any one of items 1-35 to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor under a pre-determined dosing regimen;

[0259] (B) measuring tumor size of the animal subjects periodically;

[0260] (C) (i) increasing the dose and / or dosage frequency if reduction in tumor size after the administration of the pharmaceutical composition is not therapeutically relevant; or

[0261] (ii) decreasing the dose and / or dosage frequency if reduction in tumor size after the administration of the pharmaceutical composition is therapeutically relevant, and toxicity effect is shown in at least 30% of the animal subjects; or (iii) making no changes to the dosage regimen if reduction in tumor size after the administration of the pharmaceutical composition is therapeutically relevant, and no toxicity effect is shown in the animal subjects.

[0262] The present disclosure further provides an insight that the 3 ’end region of mRNA is a very sensitive and exceptional area in terms of translational capacity as well as functionality of mRNA. Both in vitro and in vivo results suggest that a single nucleotide substitution upstream of the poly(A) tail has an impact on translational capacity and functionality of mRNA.

[0263] Accordingly, the present disclosure, among other things, also provides a composition or medical preparation comprising RNA, wherein the RNA comprises:

[0264] (i) a coding sequence that encodes a polypeptide,

[0265] (ii) a 3’ UTR sequence,

[0266] (iii) a poly-A sequence, and

[0267] (iv) a nucleotide sequence linking the 3’ UTR sequence and the poly-A sequence comprising the sequence CUXGAGCUAGC, wherein X is C, A, or U.

[0268] In some embodiments, the nucleotide sequence linking the 3’ UTR sequence and the poly-A sequence comprises the sequence CUCGAGCUAGC.

[0269] In some embodiments, the RNA comprises in the 5' — > 3' direction the coding sequence that encodes a polypeptide, the 3’ UTR sequence, the nucleotide sequence linking the 3’ UTR sequence and the poly-A sequence, and the poly-A sequence.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] In some embodiments, the poly-A sequence is an interrupted sequence of A nucleotides.

[0274] In some embodiments, the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence Ax- L-Ay, wherein Axis a sequence of at least 20 A nucleotides, Ayis 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. In some embodiments, the poly-A 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.

[0275] 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.

[0276] 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 which is preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4AACUAGU, wherein XI is any nucleotide, preferably A or C, X2 is any nucleotide, preferably A or C, X3 is any nucleotide, preferably C, U or G, and X4 is A or is missing.

[0277] 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 which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, wherein XI is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.

[0278] 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 which is preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU.

[0279] 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 which is preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU.

[0280] 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. 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.

[0281] 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.

[0282] 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 that encodes 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 poly-A sequence.

[0283] 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 that encodes 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 poly-A sequence.

[0284] 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.

[0285] 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 that encodes 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 poly-A sequence.

[0286] 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.

[0287] 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

[0288] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

[0289] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36.

[0290] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 38 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

[0291] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 38 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36.

[0292] 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 that encodes 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 poly-A 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 that encodes 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 poly-A sequence.

[0293] 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 37.

[0294] 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a poly-A sequence.

[0295] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a poly-A sequence.

[0296] In some embodiments, the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 37.

[0297] In some embodiments, at least 90% is at least 95%, 96%, 97%, 98%, or 99%.

[0298] In some embodiments, the RNA comprises two or more coding sequences encoding two or more polypeptides.

[0299] In some embodiments, the polypeptide encoded by the coding sequence is an antibody or a polypeptide chain thereof, for example, an antibody binding to CLDN-18.2 or a polypeptide chain thereof. In some embodiments, an antibody binding to CLDN-18.2 or a polypeptide chain thereof is as described herein. The polypeptide encoded by the coding sequence may, however, be any polypeptide, including, but not limited to, pharmaceutically active polypeptides and peptides, in particular those described herein.

[0300] In some embodiments, the RNA does not encode a polypeptide which binds to Claudin-6 (CLDN-6) and / or CD3.

[0301] In some embodiments, the RNA does not encode one or more polypeptide chains of a binding agent which binds to Claudin-6 (CLDN-6) and / or CD3. In some embodiments, the RNA does not encode a cytokine.

[0302] In some embodiments, the RNA does not encode IL2 and / or IL7.

[0303] In some embodiments, the RNA does not encode a polypeptide which binds to HIV.

[0304] In some embodiments, the RNA does not encode one or more polypeptide chains of a binding agent which binds to HIV.

[0305] In some embodiments, the RNA does not encode a polypeptide which binds to Claudin-18.2 (CLDN-18.2).

[0306] In some embodiments, the RNA does not encode one or more polypeptide chains of a binding agent which binds to Claudin-18.2 (CLDN-18.2).

[0307] In some embodiments, the RNA encodes an antibody or an antibody-like molecule.

[0308] 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 poly-A sequence, and / or a nucleotide sequence linking a 3’ UTR sequence and a poly-A sequence as defined.

[0309] In some embodiments, the RNA comprises:

[0310] (i) an RNA comprising a coding sequence that encodes a first polypeptide chain comprising a heavy chain of an antibody agent, and

[0311] (ii) an RNA comprising a coding sequence that encodes a second polypeptide chain comprising a light chain of an antibody agent.

[0312] In some embodiments, the RNA under (i) is a first RNA molecule and the RNA under (ii) is a second RNA molecule.

[0313] In some embodiments, the antibody agent binds to Claudin-18.2 (CLDN-18.2).

[0314] In some embodiments, the antibody agent that binds to CLDN-18.2 is as described herein. In some embodiments, the coding sequence that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to CLDN-18.2, and the coding sequence that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the first polypeptide chain comprising a heavy chain of an antibody agent that binds to CLDN-18.2, and the second polypeptide chain comprising a light chain of an antibody agent that binds to CLDN-18.2 are as described herein. In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside in place of uridine.

[0315] In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside in place of each uridine.

[0316] In some embodiments, the modified nucleoside is pseudouridine (ψ ) and / or N1 -methyl - pseudouridine (m 1 ψ ).

[0317] In some embodiments, the modified nucleoside is Nl-methyl-pseudouridine (m1 ).

[0318] In some embodiments, the RNA, e.g., each RNA, comprises a 5’ cap.

[0319] In some embodiments, the RNA, e.g., each RNA, comprises the 5’ cap m27’3‘()Gppp(m12 0,)ApG.

[0320] In some embodiments, the RNA, e.g., each RNA, is single-stranded RNA.

[0321] In some embodiments, the RNA, e.g., each RNA, is mRNA.

[0322] In some embodiments, the RNA, e.g., each RNA, is formulated in lipid nanoparticles (LNP), e.g., each RNA is co-formulated in lipid nanoparticles (LNP).

[0323] In some embodiments, lipids that form the lipid nanoparticles comprise a cationic lipid, apolymer- conjugated lipid; and a neutral lipid.

[0324] In some embodiments: a. the cationic lipid is present in 35-65 mol% of the total lipids; a. the polymer-conjugated lipid is present in about 1 -2.5 mol% of the total lipids; and c. the neutral lipid is present in 35-65 mol% of the total lipids.

[0325] In some embodiments, the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bi s(2-butyloctano ate) .

[0326] In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

[0327] In some embodiments, the neutral lipid comprises l ,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

[0328] In some embodiments, the lipid nanoparticles have an average size of about 50-150 nm.

[0329] In some embodiments, the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-

[0330] 9, l-diyl)bis(2 -butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2- distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0331] 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.

[0332] In some embodiments, the medical preparation is a kit.

[0333] In some embodiments, the RNA, e.g., each RNA, and optionally the particle forming components are in separate vials.

[0334] In some embodiments, the composition or medical preparation is for intravenous administration.

[0335] In some embodiments, the composition or medical preparation is for introducing the RNA into liver cells and expressing the polypeptide encoded by the RNA in liver cells.

[0336] In some embodiments, the composition or medical preparation is for systemic delivery of the polypeptide. In some embodiments, the composition or medical preparation is for systemic delivery of the polypeptide following expression of the polypeptide in liver cells.

[0337] The present disclosure, among other things, also provides a method for expressing a polypeptide in a subject, said method comprising:

[0338] (a) administering a composition described above such that RNA encoding the polypeptide is introduced into liver cells; and

[0339] (b) expressing the polypeptide in the liver cells.

[0340] The present disclosure, among other things, also provides a method for expressing a polypeptide in a subject, said method comprising:

[0341] (a) administering a composition described above such that RNA encoding the polypeptide is introduced into liver cells; and

[0342] (b) expressing the polypeptide in the liver cells, wherein, following expression, the polypeptide is secreted into the bloodstream.

[0343] The present disclosure, among other things, also provides a method for systemic delivery of a polypeptide in a subject, said method comprising:

[0344] (a) administering a composition described above such that RNA encoding the polypeptide is introduced into liver cells; and

[0345] (b) expressing the polypeptide in the liver cells, wherein, following expression, the polypeptide is secreted into the bloodstream.

[0346] In some embodiments, administration is parenteral administration. In some embodiments, administration is intravenous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0347]

[0061] Figure 1 shows that a CLDN-18.2-targeting antibody (RiboMabOl) encoded by two RNAs encoding a heavy chain and a light chain, respectively, of a CLDN-18.2-targeting antibody is expressed in primary human hepatocytes and CHO-K1 cells. (Panel A) Primary human hepatocytes were lipofected with 0.22-55.50 μg / mL a composition comprising two or more RNAs encoding heavy chain and light chain, respectively, of a CLDN-18.2-targeting antibody (RB RMAB01). (Left) ELISA analyses of RiboMabOl concentrations 48 hours post transfection. (Right) Western Blot analysis of cell culture supernatant from indicated lipofections. Recombinant purified IMAB362 served as reference for Western Blot analysis. Analysis was performed under non-reducing conditions and with HRP-conjugated anti-human antibodies. A mixture of Fcy-fragment specific and anti-kappa light chain specific antibodies was used for detection of full length IgG, free heavy (HC) and free light chains (LC). Supernatant of untransfected primary human hepatocytes served as mock control. (Panel B) CHO-K1 cells were lipofected with 2.00-182.00 ng / mL RB RMABOl . RiboMabOl concentration determined via ELISA 48 hours post transfection are shown. Error bars are standard errors of the mean (n=3).

[0348]

[0062] Figure 2 shows that RiboMabOl binds target specific to CLDN-18.2. Targeted binding of RiboMabOl to CLDN-18.2 was determined by flow cytometric binding assays visualized using a fluorescently labeled antibody directed against the F(ab')2 fragment of human IgG (H+L). A dilution row of RiboMabOl -containing CHO-K1 cell culture supernatant (Panels A and B, left) or IMAB362 reference protein (Panels A and B, right) was incubated with 5 x 105(Panel A) CLDN-18.2+ or (Panel B) CLDN18.1+ HEK293 transfectants.

[0349]

[0063] Figure 3 shows high target specific cell cytotoxicity mediated by in vitro expressed RiboMabOl. RiboMabOl -containing cell culture supernatant from CHO-K1 cells lipofected with RB RMABOl was subjected to (Panel A) ADCC and (Panel B) CDC assays. (Panel A) For ADCC assays, CLDN-18.2+ NUG-C4 transfectants served as target cells and CLDN-18.2-negative MDA-MB-231 cells as control cells. Human PBMCs of three different healthy donors were utilized as effector cells (E:T ratio 30:1). Target or control and effector cells were incubated for 48 hours with the indicated RiboMabOl and IMAB362 reference protein concentrations. Specific cell lysis as determined in a luciferase-based assay is shown. (Panel B) For CDC assays, CLDN- 18.2+ CH0-K1 transfectants (solid lines) served as target cells and CLDN- 18.2-negative CHO-K1 (dotted lines) as control cells. Target and control cells were incubated with human serum and RiboMabOl concentrations as indicated for 2 hours. CDC determined in a luciferase-based assay is shown. Error bars are standard errors of the mean (n=3).

[0350]

[0064] Figure 4 shows specific tumor cell lysis mediated by RiboMabOl generated in mice. Plasma of mice dosed with five repetitive injections of either 1 pg (-0.04 mg / kg), 3 pg (-0.10 mg / kg), 10 pg (-0.40 mg / kg) and 30 pg (-1.20 mg / kg) RB_RMAB01 or 80 pg (-3.20 mg / kg) of IMAB362 was sampled 24 hours post 5th injection and directed to luciferasebased ex vivo ADCC assays. Plasma of untreated mice spiked with IMAB362 served as assay reference. CLDN- 18.2+ NUG-C4 transfectants served as target and human PBMCs as effector cells. (Panel A) RiboMabOl mediated ADCC of NUG-C4 cells after 48 hours incubation with 1% of plasma is shown. (Panel B) No unspecific lysis on target-negative MDA-MB-231 cells. Error bars are standard errors of the mean (n=3).

[0351]

[0065] Figure 5 shows that RiboMabOl expressed by non-human primates mediates dose-dependent ADCC. Non-Human Primates (NHP) received three repetitive doses of 0.1, 0.4 or 1.6 mg / kg RB_RMAB01 once weekly. RiboMabOl -containing serum of all monkeys sampled 24 hours (black bars) and 168 hours (white bars) post 1st injection was directed to luciferasebased ex vivo ADCC assays. CLDN- 18.2+ NUG-C4 transfectants served as target cells. Human PBMCs from two different healthy donors (24 h, donor 1, 168 h, donor 2) served as effector cells. (Panel A) RiboMabOl -mediated ADCC of NUG-C4 cells after 48 hours incubation is shown. (Panel B) Unspecific lysis on target-negative MDA-MB-231 cells is shown. Error bars are standard error of the mean (n=3). (Panel C) Serum of NHP No. 14 (1.6 mg / kg

[0352] RB RMAB01, RiboMabOl serum concentration 232 μg / mL) collected 48 hours after the 3rd dosing was used for a luciferase-based ex vivo ADCC assays. CLDN-18.2+ NUG-C4 transfectants (solid lines) served as target cells, CLDN- 18.2-negative MDA-MB-231 cells (dotted lines) as control cells. Human PBMCs of a healthy donor served as effector cells. ADCC of NUG-C4 cells mediated by RiboMabOl -containing serum (solid red line) or by the recombinant - IMAB362 reference protein (solid black line) - with an EC50 of 66 pM and 151 pM respectively - is shown. Dotted red and black lines represent weak unspecific lysis on MDA- MB-231 control cells. Incubation time was 48 hours. Error bars are standard errors of the mean (n=3).

[0353]

[0066] Figure 6 shows that systemic availability of RiboMabOl mediates tumor growth inhibition in vivo. Mice bearing subcutaneous CLDN-18.2+ NCI-N87 xenograft tumors received IV injections of 1 gg (-0.04 mg / kg), 3 gg (-0.10 mg / kg), 10 gg (-0.40 mg / kg) and 30 μg (-1.20 mg / kg) RB RMABOl, 800 gg (-32 mg / kg) 1MAB362 reference protein, 30 μg

[0354] (-1.20 mg / kg) luciferase mRNA or saline only on test days 15, 22, 29, 36, 43 and 50 post tumor cell inoculation. Median tumor growth of treatment and control groups is shown. Dotted lines indicate injections. Significance was calculated by Two-way ANOVA. Ns indicates not significant.

[0355]

[0067] Figure 7 shows concentration-time profile of RiboMabOl in mouse serum after single dosing. Balb / cJRj mice received a single IV injection of 1 gg (-0.040 mg / kg), 3 gg (-0.10 mg / kg), 10 gg (-0.40 mg / kg) or 30 gg (-1.20 mg / kg) RB_RMAB01 drug product and 40 gg (-1.60 mg / kg) IMAB362 reference protein. Plasma was sampled 6, 24, 96, 168, 264, 336 and 504 hours post administration. RiboMabOl concentrations in plasma measured via ELISA are shown. Error bars are standard errors of the mean (n=3).

[0356]

[0068] Figure 8 shows concentration-time profile of RiboMabOl in rat serum after single dosing. RjHan: Wister rats received a single IV injection of 0.04, 0.10, 0.40 or 1 .20 mg / kg of RB RMABOl and 3.60 mg / kg of IMAB362 reference protein. Plasma was sampled 2, 6, 8, 10, 22, 24, 27, 30, 48, 72, 96, 168, 216, 264 and 336 hours post administration. RiboMabOl concentrations in plasma measured via ELISA are shown. Error bars are standard errors of the mean (n=3).

[0357]

[0069] Figure 9 shows kinetics of RB RMABOl expression in mice after weekly injection. Balb / cJRj mice received IV injections of 1 gg (-0.04 mg / kg), 3 gg (-0.10 mg / kg), 10 gg (-0.40 mg / kg) or 30 gg (-1.20 mg / kg) RB RMABOl and 80 gg (-3.20 mg / kg) IMAB362 reference protein at test days 1, 8, 15, 21 and 29. Plasma was sampled 24 hours pre- and 24 hours post-dosing. RiboMabOl concentrations in plasma measured via ELISA are shown. Dotted lines indicate injections. Error bars are standard errors of the mean (n=3).

[0358]

[0070] Figure 10 shows kinetics of RB RMABOl expression after repetitive dosing in NHP. NHP received IV injections of 0.1, 0.4 or 1.6 mg / kg RB RMABOl at test days 1, 8 and 15. Plasma was sampled 6, 24, 48, 72, 96 and 168 hours post 1st and 3rd dosing and 48, 72 and 168 hours post 2nd dosing as well as 264, 336 and 504 hours post 3rd dosing. RiboMabOl concentrations in plasma measured via ELISA are shown. Error bars are standard errors of the mean (n=3).

[0359]

[0071] Figure 11 shows liver targeting of LNP formulated mRNA in vivo. Mice received a single IV injection of LNP formulated firefly luciferase mRNA. Bioluminescence was monitored 6, 24, 48, 72 and 144 hours after administration. (Panel A) Bioluminescent images 6 hours post administration are shown for (left) individual mice in ventral position (n=5) and (right) single organs of mice #1 and 2. (Panel B) Quantification of luciferase signals (photons / second) is shown for all time points of analysis (n=5 or 3, mean). LN indicates lymph nodes.

[0360]

[0072] Figure 12 illustrates exemplary embodiments of RNA technology useful for encoding various antibody agent formats (“RiboMab”) and formulations thereof as well as its applications. (Panel A) The RiboMab® platform is applicable to provide RNA constructs encoding various antibody formats, including, e.g. , but not limited to monospecific antibody IgG, bispecific antibody bi-(scFv)2, and bispecific antibody Fab-(svFv)2. (Panel B) In some embodiments, therapeutic antibodies such as IgG can be encoded by purified mRNA comprising modified ribonucleotides (e.g., uridines replaced by pseudouridines) mRNA and encapsulated in lipid nanoparticles (mRNA / LNP). Such an mRNA construct may further comprise one or more non-coding sequence elements (e.g., to enhance RNA stability and / or translation efficiency). In some embodiments, exemplary non-coding sequence elements include but are not limited to a cap structure, 5’ UTR, 3’ UTR, a polyadenyl tail, and any combinations thereof. In some embodiments, lipid nanoparticles may comprises a conjugated lipid (e.g., PEG -conjugated lipid), a cationic lipid, and a neutral helper lipid. Such mRNA / LNP drug product formulation can be administered to a subject in vivo such that the mRNA is translated in vivo to express an antibody. (Panel C) The patient’s own body cells administered with mRNA / LNP drug product formulations described herein are capable to produce active drug encoded by mRNA (e.g., IgG RiboMab). For example, in some embodiments, upon IV injection, antibody-encoding mRNA / LNP are internalized and translated by liver cells, yielding systemic plasma concentrations of the biologically active RiboMab. Abbreviations: A30L70, Poly(A) tail, measuring 100 adenosines abrogated by a 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; ml'P, 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.

[0361]

[0073] Figure 13 is a schematic representation of exemplary RNA constructs encoding a heavy chain (HC) and a light chain (LC), respectively, of an antibody agent. As presented in Figure 13, such HC- and LC-encoding RNA constructs form an RNA composition (RB_RMAB01), which in some embodiments may be formulated into lipid nanoparticles to form a RNA / LNP drug product formulation. Abbreviations: Poly A, poly adenine tail; CH, constant heavy domain; CL, constant light domain; Sec, secretion signal; UTR, untranslated region; VH, variable heavy domain; VL, variable light domain

[0362]

[0074] Figure 14 is a graph showing dose-exposure correlation of RBJRMAB01 in cynomolgus monkey at tmax. Cynomolgus monkeys (n-3) received IV injections of 0.1, 0.4 or 1 .6 mg / kg RB RMAB01 . Dose-dependent RiboMabOl concentrations (mean, n=3) in plasma measured via ELISA at Cmax are depicted. A green line indicates a dose that can be administered to a human subject and its corresponding anticipated serum concentration.

[0363]

[0075] Figure 15 is an example electropherogram of an exemplary RNA mixture comprising a first RNA encoding a heavy chain (HC) of an antibody and a second RNA encoding a light chain (LC) of the antibody. The electropherogram depicts two peaks for LC and HC, respectively. A: area under the peak, h: height of the peak.

[0364]

[0076] Figure 16 shows anti-CLDN18.2 RiboMab expression in vitro. HEK293T / 17 cells were electroporated with mRNAs all encoding the anti-CLDN18.2 RiboMab with the same backbone but with different coding sequences. Anti-CLD 18.2 RiboMab concentrations were measured 48 hours post transfection by ELISA. Error bars are standard errors of the mean (n=2).

[0365]

[0077] Figure 17 shows anti-CLDN18.2 RiboMab exposure in mice after repeated RNA- LNP dosing. Balb / c.TRj mice received two weekly IV injections of 3 or 30 pg RNA-LNP each containing mRNAs separately encoding the HC and LC of the anti-CLDN18.2 RiboMab, either in with Backbone A or Backbone B. Serum was sampled at the indicated timepoints post first administration. Arithmetic means (n = 3) and standard error of anti-CLDNl 8.2 RiboMab concentrations in serum measured via ELISA are shown. Limit of detection was 0.074 ng / mL. Downward-facing arrows correspond to first and second RNA-LNP injection. Luc-RNA-LNP served as negative control. ELISA = enzyme-linked immunosorbent assay; Luc = luciferase.

[0366]

[0078] Figure 18 shows cytotoxic activity of anti-CLDN18.2 RiboMab encoded by RNAs utilizing Backbone A and B. Shown is ex vivo ADCC mediated by the anti-CLDN18.2 RiboMab in mouse serum collected 24 hours after RNA-LNP administration at the indicated doses. CLDN18.2-transduced NUGC-4 transfectants served as target cells and human PBMCs from a healthy donor as effector cells in an E:T ratio of 20:1 (upper panel). CLDN 18.2-negative MDA-MB-231 served as negative control (lower panel). Target and effector cells were incubated for 24 hours. Results of the control antibody are shown in the right panel. Data are means ± SD of three measurements per mouse. Ab = antibody; ADCC = Antibody-dependent cellular cytotoxicity; E:T-ratio = effector to target cell ratio;Ml= mouse number one;

[0367] PBMCs = Peripheral blood mononuclear cells.

[0368]

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

[0369]

[0080] Figure 20 shows a comparison of mRNA translation derived from Backbones B, C and D with different coding sequences and demonstrates that the differences in performance between Backbone C and Backbone B / D is independent of the coding sequence. A, Firefly Luciferase mRNAs in Backbones B (•), C(«) and D ( Δ) were electroporated two times (solid and dashed lines) in hiDCs. Bright-Glo assay was performed at the times indicated. B, eGFP mRNAs in Backbones C(«) and D ( ) were electroporated in hiDCs twice (solid and dashed lines). Cells were harvested and assayed with FACS for eGFP expression at the times indicated. C, primary human hepatocytes were lipofected with hIL-18 mRNAs in Backbones B (•), C(H) and D ( Δ). Supernatants from transfected cells were collected at the indicated times and assayed for the presence of hIL-18 with ELISA.

[0370]

[0081] Figure 21 shows the translation of Firefly Luciferase mRNAs derived from Backbones B and C containing different nucleotides at position -9 upstream of polyA in hiDCs and demonstrates that the 3‘ UTR end sequence impacts long-term translation in vitro. Firefly Luciferase mRNAs from Backbones B (panel A) and C (Panel B) having A( ), G(H), T( Δ) or C(®) at the position -9 upstream of polyA sequence were electroporated in hiDCs in two separate experiments and Luciferase expression was assayed at the indicated time points.

[0082] Figure 22 shows that the 3‘ UTR end sequence significantly impacts long-term translation in vivo using Backbone B.

[0371]

[0083] Figure 23 shows that the 3‘ UTR end sequence significantly impacts long-term translation in vivo using Backbone D

[0372]

[0084] Figure 24 shows that the 5’ sequence upstream of the coding sequence also impacts long-term translation in vivo.

[0373]

[0085] Figure 25 shows that the combination of the 5’ sequence elements and the 3’ sequence elements impacts long-term translation in vivo.

[0374]

[0086] Figure 26 shows that Backbone B performed significantly better that earlier backbone versions, Backbones A and G, in terms of long-term translation in vivo.

[0375]

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

[0376]

[0088] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0377]

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

[0378]

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

[0379]

[0091] 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, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0380] CERTAIN DEFINITIONS

[0381]

[0092] A, an, the: As used herein, the terms "a", "an" and "the" are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0382]

[0093] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are 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 referred value.

[0383]

[0094] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0384]

[0095] And / or: As used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0385]

[0096] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements which are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody agent utilized in accordance with the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.) 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 Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. 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 inter-connected by disulfide bonds. In some embodiments, each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). In some embodiments, each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is 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. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3), the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI , a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxyterminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as Clq. The term "full-length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g. the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody. As used herein, the term "Fab-arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably with "half molecule" herein. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g, attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1 -5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it shows at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an 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, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0386]

[0097] Antibody agents can be made by the skilled person using methods and commercially available services and kits known in the art. For example, methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further 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)).

[0387]

[0098] Antibodies may be produced by standard techniques, for example by immunization with the appropriate polypeptide or portion(s) 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 a desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to 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.

[0099] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated 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 with incidence of and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population), or likelihood of responsiveness to a treatment.

[0388]

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

[0389]

[0101] Cancer: The term “cancer” is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer may be characterized by a solid tumor. In some embodiments, cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, ovarian cancer, breast cancer, glioblastomas, colorectal cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0102] Cap: As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally- occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as "m7G." In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g. , but not limited to anti-reverse cap analogs (ARCAs) known in the art. Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, wherein, in addition to the GTP, an IVT system also contains a dinucleotide cap analog (including, e.g., a m7GpppG cap analog or an N7-methyl, 2’-O- methyl -GpppG ARCA cap analog or an N7-methyl, 3'-O-methyl-GpppG ARCA cap analog) using methods known in the art. In some embodiments, a cap is a cap0, cap1, or cap2, preferably cap1 or cap2. As used herein, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. As used herein, the term "cap1" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. As used herein, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.

[0390]

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

[0391]

[0104] Co-administration: As used herein, the term “co-administration” refers to use of a pharmaceutical composition described herein in combination with another therapy (e.g., surgery, radiation, and / or administration of an another therapeutic agent such as a chemotherapeutic agent described herein, and / or an agent that relieves one or more symptoms or attributes of the relevant disease, disorder or condition and / or of administered therapy [e.g., chemotherapy]), so that a subject receives both. The combined administration of a pharmaceutical composition described herein and such other therapy may be performed concurrently (e.g., via overlapping protocols) or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition described herein may include two or more active agents combined in one pharmaceutically- acceptable carrier (e.g., in a single dosage form). Alternatively, in some embodiments, coadministration involves administration of two or more physically distinct pharmaceutical compositions, each of which may contain a different active agent or combination of agents; in some such embodiments, one or more (and, in some embodiments, all) doses of such distinct pharmaceutical compositions may be administered substantially simultaneously. In some embodiments, one or more (and, in some embodiments, all) doses of such distinct pharmaceutical compositions may be administered separately, e.g., according to overlapping regimens or sequential regimens. In general, two or more therapies may be considered to be “coadministered” when delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject to which they are administered.

[0392]

[0105] Codon optimization-. As used herein, the term "codon-optimization" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".

[0393]

[0106] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0394]

[0107] Comparable-. As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0395]

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

[0396]

[0109] Comprise, consist: The word "comprise", and variations such as "comprises" and "comprising", will be 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 those that do not materially affect the basic and novel characteristic(s) 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, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of or "consisting of. Likewise, at each occurrence in the present application, the term "consisting essentially of may be replaced with the term "consisting of.

[0397]

[0110] Contacting: As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to exposing a relevant target (e.g., cell, tissue, organism, etc.) to RNA(s) or a composition that comprises or delivers the same as described herein, so that the RNA is delivered into a target cell (e.g., cytosol of a target cell). A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Those skilled in the art will appreciate that different methods of contacting may be utilized to achieve such delivery to a target cell in in vitro, ex vivo, or in vivo applications. In some embodiments, contacting cells in culture may be or comprise in vitro transfection. In some embodiments, contacting may utilize one or more delivery vehicles (e.g., lipid nanoparticles described herein). In some embodiments, contacting may be or comprise administering a pharmaceutical composition described herein to a subject.

[0398]

[0111] Detecting: The term “detecting” is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between 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.

[0399]

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

[0400]

[0113] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides 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 that includes 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 if transcription and / or translation of the nucleic acid produces the polypeptide in a cell or other biological system.

[0401]

[0114] 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 an aptamer. In some embodiments, an epitope is comprised of a plurality of 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 near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

[0402]

[0115] 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 an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0403]

[0116] Fc region As used herein, the term “Fc region” refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.

[0117] Five prime untranslated region: As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a 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 an RNA.

[0404]

[0118] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “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., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill 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 “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0405]

[0119] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between 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, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence 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 a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (c.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the 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, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. In some embodiments, the degree of identity is given for a region which 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 given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0406]

[0120] Immunogenicity: "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

[0407]

[0121] Immunoglobulin: As used herein, the term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains or regions, such as the VL or VL (variable light chain) domain / region, CL or CL (constant light chain) domain / region, VH or VH (variable heavy chain) domain / region, and the CH or CH (constant heavy chain) domains / regions CHI (CHI), CH2 (CH2), CH3 (CH3), and CH4 (CH4). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between the CHI and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a VL and a CL. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed 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 stated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also internet http address www.imgt.org. However, it should be understood that the present disclosure is not limited to CDR sequences only determined according to the IMGT rules. There are five types of mammalian immunoglobulin heavy chains, i.e., a, 6, c. y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

[0408]

[0122] Isolated: " Isolated" means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid or polypeptide naturally present 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 substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0409]

[0123] Lipid: As used herein, the term "lipid" relates to molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl -2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0410]

[0124] Locally advanced tumor: As used herein, the term “locally advanced tumor” or “locally advanced cancer” refers to its art-recognized meaning, which may vary with 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 cancer that has grown outside the tissue or organ it started but has not yet spread to distant sites in the body of a subject. By way of example only, in some embodiments, locally advanced pancreatic cancer typically refers to stage III disease with tumor extension to adjacent organs (e.g., lymph nodes, liver, duodenum, superior mesenteric artery, and / or celiac trunk) but no signs of metastatic disease; yet complete surgical excision with negative pathologic margins is not possible.

[0411]

[0125] 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 the present disclosure, "mol % of the total lipid" 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 lipid" includes lipids and lipid-like material.

[0412]

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

[0413]

[0127] Nucleic acid / Polynucleotide'. As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. 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 a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis). In some embodiments, a 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, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0414]

[0128] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0415]

[0129] Optional: The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

[0130] Patient: As used herein, the term “patient” refers to any organism who is suffering 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, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes cancer, or presence of one or more tumors. In some embodiments, a patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition. In some embodiments, a patient is a cancer patient.

[0416]

[0131] Polypeptide'. The term “polypeptide”, as used herein, typically has its art- recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. 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, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

[0417]

[0132] Pharmaceutically active polypeptide'. The term “pharmaceutically active polypeptide”, as used herein, means a peptide or polypeptide that can be used in the treatment of an individual where the expression of the peptide or polypeptide would be of benefit, e.g., in ameliorating the symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount. A pharmaceutically active peptide or polypeptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease. The term "pharmaceutically active peptide" or "pharmaceutically active polypeptide" includes entire peptides or polypeptides, and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active variants and / or analogs of a peptide or polypeptide.

[0418] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, immunostimulants, e.g., cytokines, honnones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins. In some embodiments, the pharmaceutically active peptide and polypeptide includes a replacement protein.

[0419] An “immunostimulant” is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells. The immunostimulant may be pro-inflammatory (e.g., when treating infections or cancer), or anti-inflammatory (e.g., when treating autoimmune diseases).

[0420] According to one aspect, the immunostimulant is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-alpha (IFN-a) or interferon- amma (IFN-Υ), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. According to another aspect, the immunostimulant includes an adjuvant-type immunostimulatory agent such as APC Toll-like Receptor agonists or costimulatory / cell adhesion membrane proteins. Examples of Toll-like Receptor agonists include costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0421] The term "cytokines" relates to proteins which have a molecular weight of about 5 to 60 kDa and which participate in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include honnones or growth factors. Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than honnones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines). Particular examples of cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFNa), interferon beta (IFNP), 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), as well as variants and derivatives thereof.

[0422] According to the disclosure, a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. A cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal. One particularly preferred cytokine is interferon-a.

[0423] Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. Interferons are usually characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells.

[0424] Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon (the type I interferons present in humans are IFNα, IFNβ , IFNε, IFNK and IFNco), type II interferon (IFNγ in humans), and type III interferon.

[0425] According to the disclosure, a type I interferon is preferably IFNa or IFN , more preferably IFNα. According to the disclosure, an interferon may be a naturally occurring interferon or a functional fragment or variant thereof. An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.

[0426] Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.

[0427] According to the disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal. Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant). The immunostimulant may be fused to an extended- pharmacokinetic (PK) group, which increases circulation half-life. Non-limiting examples of extended-PK groups are serum albumin or fragments thereof or variants of the serum albumin or fragments thereof (e.g., HSA or fragments or variants thereof), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. 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 which is herein incorporated by reference in its entirety.

[0428] In some embodiments, a pharmaceutically active peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides a method for treatment of a subject having a disorder requiring protein replacement (e.g., protein deficiency disorders) comprising administering to the subject RNA (in particular, mRNA) as described herein encoding a replacement protein. The term "protein replacement" refers to the introduction of a protein (including functional variants thereof) into a subject having a deficiency in such protein. The term also refers to the introduction of a protein into a subject otherwise requiring or benefiting from providing a protein, e.g., suffering from protein insufficiency. The term "disorder characterized by a protein deficiency" refers to any disorder that presents with a pathology caused by absent or insufficient amounts of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein insufficiency can be involved in infectious diseases, immunosuppression, organ failure, glandular problems, radiation illness, nutritional deficiency, poisoning, or other environmental or external insults.

[0429] The term "hormones" relates to a class of signaling molecules produced by glands, wherein signaling usually includes the following steps: (i) synthesis of a hormone in a particular tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) relay and amplification of the signal; and (vi) breakdown of the hormone. Hormones differ from cytokines in that (1) hormones usually act in less variable concentrations and (2) generally are made by specific kinds of cells. In some embodiments, a "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human grown hormone or bovine somatotropin), oxytocin, atrial-natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptins.

[0430] The term "adhesion molecules" relates to proteins which are located on the surface of a cell and which are involved in binding of the cell with other cells or with 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). Particular examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0431] Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins modulate cell signaling pathways, e.g., pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTK). Such regulation can lead to cellular growth, division, survival, or differentiation or to apoptosis. Particular examples of integrins include: o β1, α2β1 , α3β1, α4β1 , α5β1, α6β1 , α7β1 , αLβ2, αLβ2, αIIbβ3, αvβ1, αvβ3, αvβ5 , αvβ6 , αvβ8, and α6β4.

[0432] The term "immunoglobulins" or "immunoglobulin superfamily" refers to molecules which are involved in the recognition, binding, and / or adhesion processes of cells. Molecules belonging to this superfamily share the feature that they contain a region known as immunoglobulin domain or fold. 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., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co- stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and other.

[0433] The term "immunologically active compound" relates to any compound altering an immune response, e.g., by inducing and / or suppressing maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate 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. Particular examples of immunologically active compounds include interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, in particular tumor-associated antigens, pathogen- associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens. An immunologically active compound may be a vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response.

[0434] In some embodiments, RNA (in particular, mRNA) described in the present 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. The "peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject" is also designated herein as "vaccine antigen", "peptide and protein antigen" or simply "antigen".

[0435] In some embodiments, the RNA encoding the vaccine antigen is expressed in cells , e.g., muscle cells or antigen-presenting cells (APCs), of the subject to provide the vaccine antigen. In some embodiments, expression of the vaccine antigen is at the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, the RNA encoding the vaccine antigen is administered systemically, e.g., intravenously. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in spleen occurs. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in antigen presenting cells, preferably professional antigen presenting cells occurs. In some embodiments, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In some embodiments, the RNA encoding the vaccine antigen is administered intramuscularly.

[0436] The vaccine antigen comprises an epitope for inducing an immune response against an antigen in a subject. Accordingly, the vaccine antigen comprises an antigenic sequence for inducing an immune response against an antigen in a subject. Such antigenic sequence may correspond to a target antigen or disease-associated antigen, e.g., a protein of an infectious agent (e.g., viral or bacterial antigen) or tumor antigen, or may correspond to an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or the immunogenic variant thereof. Thus, the antigenic sequence may comprise at least an epitope of a target antigen or disease-associated antigen or an immunogenic variant thereof. The antigenic sequence or a procession product thereof, e.g., a fragment thereof, may bind to the antigen receptor such as TCR or CAR carried by immune effector cells. In some embodiments, the antigenic sequence is selected from the group consisting of the antigen expressed by a target cell to which the immune effector cells are targeted or a fragment thereof, or a variant of the antigenic sequence or the fragment.

[0437] In some embodiments, the RNA encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or a procession product thereof for binding by the antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and / or expansion of the immune effector cells.

[0438] An "antigen" according to the present disclosure covers any substance that will elicit an immune response and / or any substance against which an immune response or an immune mechanism such as a cellular response and / or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, such as a peptide or polypeptide, that reacts specifically with antibodies or T-lymphocytes (T-cells). The term "antigen" may comprise a molecule that comprises at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen). In some embodiments, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.

[0439] The term "autoantigen" or "self-antigen" refers to an antigen which originates from within the body of a subject (z.e., the autoantigen can also be called "autologous antigen") and which produces an abnormally vigorous immune response against this normal part of the body. Such vigorous immune reactions against autoantigens maybe the cause of "autoimmune diseases".

[0440] According to the present disclosure, any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may comprise a humoral or cellular immune response, or both. In the context of some embodiments of the present disclosure, the antigen is presented by a cell, such as by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.

[0441] 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 which contains epitopes that will stimulate a host's immune system to make 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 which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0442] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein. In some embodiments, the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, such as CD4+ and CD8+ lymphocytes, in particular via the modulation of the activity of a T-cell receptor.

[0443] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies, T cells or B cells, in particular when presented in the context of MHC molecules. An epitope of a protein may comprises a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 30, or about 10 and about 25 amino acids in length.

[0444] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class 1 and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. According to some embodiments, an amino acid sequence enhancing antigen processing and / or presentation and / or an amino acid sequence which breaks immunological tolerance is fused, either directly or through a linker, to an antigenic peptide or polypeptide (antigenic sequence).

[0445] The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4+and CD8+T-lymphocytes, e.g. CD8+T-lymphocytes, which maybe detected in various proliferation or cytokine production tests in vitro.

[0446] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s).

[0447] The term "allergen" refers to a kind of antigen which originates from outside the body of a subject (i.e., the allergen can also be called "heterologous antigen") and which produces an abnormally vigorous immune response in which the immune system of the subject fights off a perceived threat that would otherwise be harmless to the subject. "Allergies" are the diseases caused by such vigorous immune reactions against allergens. An allergen usually is an antigen which is able to stimulate a type-I hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE) responses. Particular examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phi p 5), and proteins of dust mites (e.g., Der p 2).

[0448] The term "growth factors" refers to molecules which are able to stimulate cellular growth, proliferation, healing, and / or cellular differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factors" include particular cytokines and hormones which 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 factor, ephrins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain- derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P)), and tumor necrosis factor-alpha (TNF-a). In some embodiments, a "growth factor" is a peptide or polypeptide growth factor.

[0449] The term "protease inhibitors" refers to molecules, in particular peptides or polypeptides, which inhibit the function of proteases. Protease inhibitors can be classified by the protease which is inhibited (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serpins). Particular examples of protease inhibitors include serpins, such as alpha 1 -antitrypsin, aprotinin, and bestatin.

[0450] The term "enzymes" refers to macromolecular biological catalysts which accelerate chemical reactions. Like any catalyst, enzymes are not consumed in the reaction they catalyze and do not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are much more specific. In some embodiments, an enzyme is essential for homeostasis of a subject, e.g., any malfunction (in particular, decreased activity which may be caused by any of mutation, deletion or decreased production) of the enzyme results in a disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0451] The term "receptors" refers to protein molecules which receive signals (in particular chemical signals called ligands) from outside a cell. The binding of a signal (e.g., ligand) to a receptor causes some kind of response of the cell, e.g., the intracellular activation of a kinase. Receptors include transmembrane receptors (such as ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors). Particular 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 receptors" refers to receptors which bind to growth factors. The term "apoptosis regulators" refers to molecules, in particular peptides or polypeptides, which modulate apoptosis, i.e., which either activate or inhibit apoptosis. Apoptosis regulators can be grouped into two broad classes: those which modulate mitochondrial function and those which regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) which act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and / or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Also to this first class belong proapoptotic proteins (e.g., BAX, BAK, BIM) which promote release of cytochrome C. The second class includes proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP which block the activation of caspases.

[0452] The term "transcription factors" relates to proteins which regulate the rate of transcription of genetic information from DNA to messenger RNA, in particular by binding to a specific DNA sequence. Transcription factors may regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and / or in response to signals from outside the cell, such as a hormone. Transcription factors contain at least one DNA-binding domain which binds to a specific DNA sequence, usually adjacent to the genes which are regulated by the transcription factors. Particular examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0453] The term "tumor suppressor proteins" relates to molecules, in particular peptides or polypeptides, which protect a cell from one step on the path to cancer. Tumor-suppressor proteins (usually encoded by corresponding tumor-suppressor genes) exhibit a weakening or repressive effect on the regulation of the cell cycle and / or promote apoptosis. Their functions may be one or more of the following: repression of genes essential for the continuing of the cell cycle; coupling the cell cycle to DNA damage (as long as damaged DNA is present in a cell, no cell division should take place); initiation of apoptosis, if the damaged DNA cannot be repaired; metastasis suppression (e.g., preventing tumor cells from dispersing, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair. Particular examples of tumor-suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI / SNF (SWItch / Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (STI 4), and Yippee-like 3 (YPEL3). The term "structural proteins" refers to proteins which confer stiffness and rigidity to otherwisefluid biological components. Structural proteins are mostly fibrous (such as collagen and elastin) but may also be globular (such as actin and tubulin). Usually, globular proteins are soluble as monomers, but polymerize to form long, fibers which, for example, may make up the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin, and dynein) which are capable of generating mechanical forces, and surfactant proteins. Particular examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

[0454] The term "reprogramming factors" or "reprogramming transcription factors" relates to molecules, in particular peptides or polypeptides, which, when expressed in somatic cells optionally together with further agents such as further reprogramming factors, lead to reprogramming or dedifferentiation of said somatic cells to cells having stem cell characteristics, in particular pluripotency. Particular examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0455] The term "genomic engineering proteins" relates to proteins which are able to insert, delete or replace DNA in the genome of a subject. Particular examples of genomic engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9).

[0456] The term "blood proteins" relates to peptides or polypeptides which are present in blood plasma of a subject, in particular blood 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), protease inhibitors (e.g., alpha 1 -antitrypsin), etc. Particular 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.

[0457] Thus, in some embodiments, the pharmaceutically active peptide or polypeptide is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10 (IL-11), more preferably EPO; (ii) an adhesion molecule, in particular an integrin; (iii) an immunoglobulin, in particular an antibody; (iv) an immunologically active compound, in particular an antigen, such as a viral or bacterial antigen, e.g., an antigen of SARS-CoV-2, e.g., a spike (S) protein of SARS-CoV-2 or a variant thereof; (v) a hormone, in particular vasopressin, insulin or growth hormone; (vi) a growth factor, in particular VEGFA; (vii) a protease inhibitor, in particular alpha 1 -antitrypsin; (viii) an enzyme, preferably selected from the group consisting of herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes, and lactase; (ix) a receptor, in particular growth factor receptors; (x) an apoptosis regulator, in particular BAX; (xi) a transcription factor, in particular FOXP3; (xii) a tumor suppressor protein, in particular p53; (xiii) a structural protein, in particular surfactant protein B; (xiv) a reprogramming factor, e.g., selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28 and NANOG; (xv) a genomic engineering protein, in particular clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) a blood protein, in particular fibrinogen.

[0458] In some embodiments, a 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 against the one or more antigens or one or more epitopes in a subject which may be therapeutic or partially or fully protective.

[0459] 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.

[0460] In some embodiments, the target antigen is a tumor antigen and the antigenic sequence (e.g., an epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen, which is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen", which is specific to an individual’s tumor and has not been previously recognized by the immune system. A neo-antigen or neo-epitope may result from one or more cancer-specific mutations in the genome of cancer cells resulting in amino acid changes. If the tumor antigen is a neo-antigen, the vaccine antigen preferably comprises an epitope or a fragment of said neo-antigen comprising one or more amino acid changes.

[0461] In some embodiments, the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof. Thus, in some embodiments, the RNA, e.g., mRNA, used in the present disclosure encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.

[0462] 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 the immunogenic variant thereof. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or the immunogenic variant thereof. In some embodiments, the coronavirus is MERS- CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

[0463]

[0133] Recombinant: The term "recombinant", as used herein, means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.

[0464]

[0134] Reference / Reference standard: As used herein, “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference or control is or comprises a set specification (e.g., relevant acceptance criteria). Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0465]

[0135] 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, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) intemucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0466]

[0136] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “ Nucleic acid / Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0467]

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

[0468]

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

[0469]

[0139] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. An antibody agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between CDRs of an antibody agent and their partners; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of an antibody agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of an antibody agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0470]

[0140] Subject'. As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0471]

[0141] Susceptible to: An individual who is “susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display 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 development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of individuals suffering from the disease, disorder, or condition; carrier of a genetic marker or other biomarker associated with the disease, disorder or condition, etc.).

[0472]

[0142] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0473]

[0143] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solidphase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0474] |144] Therapeutic agent: As used interchangeably herein, the phrase “therapeutic agent” or “therapy” refers to an agent or intervention that, when administered to a subject or a patient, has a therapeutic effect and / or elicits 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, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features 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, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0475]

[0145] Three prime untranslated region '. As used herein, the terms "three prime untranslated region" or "3' UTR" refer to the sequence of an mRNA molecule that begins following 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 stop codon of the coding region of an open reading frame sequence

[0476]

[0146] Threshold level (e.g., acceptance criteria). ' As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and / or classify the results of a measurement, for example, the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0147] Transfection: As used herein, the term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or the body of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example. RNA can be transfected into cells to transiently express its coded protein.

[0477]

[0148] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition.

[0478]

[0149] Unresectable tumor. As used herein, the term “unresectable tumor” typically refers to a tumor characterized by one or more features that, in accordance with sound medical judgement, are considered to indicate that the tumor cannot safely (e.g., without undue harm to the subject) be removed by surgery, and / or with respect to which a competent medical profession has determined that risk to the subject of tumor removal outweighs 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 that is otherwise in a location that cannot readily be surgically accessed without unreasonable risk of damage to one or more other critical or essential organs and / or tissues (including blood vessels).. In some embodiments, “unresectability” of a tumor refers to the likelihood of achieving a margin-negative (RO) resection. In the context of pancreatic cancer, encasement of major vessels by a tumor such as superior mesenteric artery (SMA) or celiac axis, portal vein occlusion, and the presence of celiac or para-aortic lymphadenopathy are generally acknowledged as findings that preclude RO surgery. Those skilled in the art will understand parameters that determine whether a tumor is unresectable or not.

[0479]

[0150] Those skilled in the art, reading the present specification, will appreciate 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, foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0480] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0481]

[0151] Outcomes of Standard of Care (SOC) therapy remain poor for many cancer patients, and particularly for those with relapsed or refractory advanced solid tumors. Treatment options typically include further palliative chemotherapy, which might be less tolerated after previous repeated exposure to cytotoxic compounds, or best supportive care, and investigational treatments without proven benefit. Therapy in this population is not curative, with an expected overall survival of a few months. Immunotherapy has emerged as an effective treatment option in some cancers with high unmet medical need. Specifically, immune checkpoint inhibitors are approved for treatment across various cancer indications and act by invigorating pre-existent anti-tumor-specific T cells. The medical need is still high for various cancer types. The present disclosure, among other things, provides insights and technologies for treating cancer (e.g., pancreatic cancer and / or biliary cancer) with a therapy targeting Claudin-18.2 (CLDN-18.2).

[0482]

[0152] In some embodiments, the present disclosure, among other things, provides RNA technologies to deliver a monoclonal antibody targeting CLDN-18.2 that combines both potent anti-tumoral features and an excellent safety profile, skipping the hurdle of slow and cumbersome antibody manufacturing process. Without wishing to be bound by any particular theory, the present disclosure proposes that such RNA delivering modality may achieve one or more improvements such as effective administration with reduced incidence e.g. , frequency and / or severity) of treatment emergent adverse events (“TEAEs”), and / or with improved relationship between efficacy level and TEAE level (e.g., improved therapeutic window) relative to those observed when a corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements in particular may be achieved by delivering IMAB362 via administration of RNA(s) (e.g., ssRNA(s) such as mRNA(s))) encoding it.

[0483]

[0153] In some embodiments, the present disclosure, among other things, provides insights that mRNA(s) encoding an antibody agent (e.g., IMAB362) or a functional portion thereof, optionally formulated with lipid nanoparticles (LNP) for intravenous (IV) administration to a subject (e.g., a human patient, a model organism, etc.), can be taken up by target cells (e.g., liver cells) for efficient production of the encoded antibody agent (e.g., IMAB362) at therapeutically relevant plasma concentrations, for example, as illustrated in Figure 14 for a CLDN-18.2-targeting antibody agent expressed from RNAs (e.g., ones described herein). In some embodiments, antibody agents are expressed from mRNA, e.g., engineered for minimal immunogenicity, and / or formulated in lipid nanoparticles (LNPs). In some embodiments, mRNA that encodes an antibody agent may comprise modified nucleotides (e.g., but not limited to pseudouridine and / or 1-methyl-pseudouridine).

[0484]

[0154] Moreover, the present disclosure, among other things, provides an insight that the capability of a CLDN-18.2-targeting antibody agent delivered as described herein can induce antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while leveraging immune system of recipient subjects can augment cytotoxic effect(s) of chemotherapy and / or other anti-cancer therapy. In some embodiments, such a combination therapy may prolong progression- free and / or overall survival, e.g., relative to the individual therapies administered alone and / or to another appropriate reference.

[0485]

[0155] Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, for example such as gemcitabine, oxaliplatin, and 5-fluorouracil were shown to upregulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines; moreover, 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. el 523096.

[0486]

[0156] The present disclosure, among other things, provides an insight that CLDN-18.2- targeted therapy as described herein may be particularly useful and / or effective when administered to tumor(s) (e.g., tumor cells, subjects in whom such tumor(s) and / or tumor cell(s) are suspected and / or have been detected, etc.) characterized by (e.g., that have been determined to display and / or that are expected or predicted to display) elevated expression and / or activity of CLDN-18.2 expression in tumor cells (e.g., as may result or have resulted from exposure to one or more chemotherapeutic agents). Indeed, among other things, the present disclosure teaches that provided CLDN-18.2-targeted therapy (e.g., administration of RNA and, more particularly an mRNA encoding a CLDN-18.2-targeting antibody agent) as described herein may provide synergistic therapeutic when administered in combination with (e.g., to a subject who has received and / or is receiving or has otherwise been exposed to) one or more CDLN-18.2- enhancing agents (e.g., one or more certain chemotherapeutic agents). Accordingly, in some embodiments, CLDN-18.2-targeted therapy as described herein can be useful in combination with other anti-cancer agents that are expected to and / or have been demonstrated to up-regulate CLDN-18.2 expression and / or activity in tumor cells.

[0487]

[0157] Accordingly, the present disclosure, among other things, provides insights and technologies for treating cancer, particularly, cancers that are associated with expression of CLDN-18.2. In some embodiments, provided technologies are effective for treatment of pancreatic cancers. In some embodiments, provided technologies are effective for treatment of gastric or gastro-esophageal cancers. In some embodiments, provided technologies are effective for treatment of biliary cancers. In some embodiments, provided technologies are effective for treatment of ovarian cancers. In some embodiments, provided technologies are effective when applied to locally advanced tumors. In some embodiments, provided technologies are effective when applied to unresectable tumors. In some embodiments, provided technologies are effective when applied to metastatic tumors.

[0488] I. CIaudin-18.2 polypeptide

[0489]

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

[0490]

[0159] CLDN18 expression in healthy tissues. Claudinl 8.2 is a 27.8 kDa protein with four membrane-spanning domains and two small extracellular loops (Niimi et al. 2001). CLDN- 18.2 is a tight junction molecule of the gastric epithelia. Gastric tight junctions are highly specialized on repelling gastric acid, which may injure the gastric lining.

[0491]

[0160] 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 gastric epithelia in the pit and base regions of gastric glands. The stem cell zone, from which differentiated epithelial cells of the gastric glands are continuously replenished, is CLDN-18.2-negative. Without wishing to be bound by theory, it is commonly believed that no other normal cell type of the human body expresses CLDN-18.2 at transcript level or at protein level.

[0492]

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

[0493]

[0162] About 77% of primary gastric adenocarcinomas (GAC) are CLDN-18.2+ . 56% of GAC display 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 than in intestinal gastric cancers. The CLDN-18.2 protein is also frequently detected in lymph node metastases of gastric cancer and in distant metastases into the ovaries (so-called Krukenberg tumors). Moreover, 50% of esophageal adenocarcinomas display significant expression of CLDN-18.2.

[0494]

[0163] In pancreatic cancer, CLDN-18.2 is expressed with a prevalence of 60-90% in pancreatic ductal adenocarcinoma (PDAC) (Karanjawala et al. 2008; Woll et al. 2014). PDAC, accounting for over 80% of all pancreatic neoplasms, is the seventh most frequent cancer in Europe and fourth of cancer-related causes of death in the European Union (Ferlay et al. 2010; Jemal et al. 2011; Seufferlein et al. 2012). Almost 60% of patients with PDAC express membrane-bound CLDN-18.2 and in 20% of patients with pancreatic neuroendocrine neoplasms CLDN-18.2 is ectopically activated. CLDN-18.2 is expressed in primary and metastatic PDAC lesions (Wbll et al. 2014).

[0495]

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

[0496]

[0165] Exemplary sequences of CLDN-18.2 (SEQ ID NO: 32) and the splice variant CLDN18.1 (SEQ ID NO: 33) are shown below: MAVTACQGLGFWSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGF TECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLT SGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWV AGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKI YDGGARTEDEVQSYPSKHDYV (SEQ ID NO : 32 )

[0497] MSTTTCQWAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGF TECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLT SGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWV AGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKI YDGGARTEDEVQSYPSKHDYV ( SEQ ID NO : 33 )

[0498] II. Exemplary antibody agents targeting Claudin-18.2 polypeptides

[0499]

[0166] 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 a 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 ECD 1 that is exposed in cancer cells. In some embodiments, such an antibody agent may have a binding affinity (e.g., as measured by a dissociation constant) for a CLDN-18.2 polypeptide, e.g., an epitope of ECD 1 of a CLDN-18.2 polypeptide) of at least about 10 -M, at least about 10-5M, at least about 10-6M, at least about 10-7M, at least about 10-8M, at least about 10-9M, or lower. Those skilled in the art will appreciate 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 the two molecules. Alternatively or additionally, binding affinity between a ligand and its target molecule may be affected by the presence of other molecules. Those skilled in the art will be familiar with a variety of technologies for measuring binding affinity and / or dissociation constants in accordance with the present disclosure, including, e.g., but not limited to ELISAs, gel-shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), bio-layer interferometry, grating-coupled interferometry, and spectroscopic assays.

[0500]

[0167] In some embodiments, an antibody targeting CLDN-18.2 may bind specifically 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 member including the closely related splice variant 1 of Claudin-18 (CLDN18.1) that is predominantly expressed in tissues, e.g., lung.

[0501]

[0168] In some embodiments, an antibody agent targeting CLDN-18.2 may be any one of CLDN-18.2-targeting antibodies described in WO 2007 / 059997, WO2008 / 145338, and W02013 / 174510, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.

[0502]

[0169] In some embodiments, an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 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); and / or (b) a variable light chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 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).

[0503]

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

[0504] MGWS C 11 L FL VATATGVHS QVQLQQPGAELVRPGAS VKL S CKASGYTFTS YW INWVKQRP GQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWR GNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO : 1 )

[0505] MESQTQVLMSLLFWVSGTCGDIVMTQSPSS LTVTAGEKVTMS CKS SQSLLNSGNQKNYLT WYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSY PFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 2 )

[0506] 1171] In some embodiments, an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 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 (including, e.g., 1 CDR, 2 CDRs, and 3 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.

[0507]

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

[0508] QVQLQQPGAELVRPGAS VKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATL TVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS ( SEQ ID NO : 14 ) DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTG

[0509] SGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK (SEQ ID NO : 15 )

[0510] [173J 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.

[0511]

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

[0512]

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

[0513] MRVMAPRTLILLLSGALALTETWAGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWI NWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVY YCTRSWRGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO : 3 )

[0514] MRVMAPRTLILLLSGALALTETWAGSDI VMTQS PS S LTVTAGE KVTMS CKS SQSLLNSGN QKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC ( SEQ ID NO : 4 )

[0515]

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

[0516]

[0177] In some embodiments, an antibody agent targeting CLDN-18.2 comprises one or more Fc regions which have at their C-terminus a lysine. The origin of this lysine is a naturally occurring sequence found in humans from which these Fc regions are derived. During cell culture production of recombinant antibodies, this terminal lysine can be cleaved off by proteolysis by endogenous carboxypeptidase(s), resulting in a constant region having the same sequence but lacking the C-terminal lysine. Antibodies produced from nucleic acid sequences that either do, or do not encode a terminal lysine are substantially identical in sequence and in function since the degree of processing of the terminal lysine is typically high when e.g. using antibodies produced in CHO-based production systems (Dick, L.W. et al. Biotechnol. Bioeng. 2008; 100: 1132-1143). Hence, it is understood that proteins in accordance with the invention, such as antibodies, can be generated with or without encoding or having a terminal lysine. It is also understood in accordance with the invention that, sequences with a tenninal lysine, such as a constant region sequence having a terminal lysine, can be understood as the corresponding sequences without a terminal lysine, and that sequences without a tenninal lysine can also be understood as the corresponding sequences with a terminal lysine.

[0517]

[0178] In some embodiments, an 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 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; Tiireci 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.

[0518]

[0179] IMAB362, developed by Ganymed Pharmaceuticals GmbH and acquired by Astellas Pharma Inc., is a full IgGl antibody targeting the tight junction protein CLDN-18.2 and mediates cell death through antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). IMAB362 recognizes the first extracellular domain (ECD1) of CLDN-18.2 with high affinity and specificity (Sahin et al. 2008; Tiireci et al. 2011). The epitope is not accessible in normal epithelial barriers to the antibody. Disruption of tight junctions and loss of cell polarization are early hallmarks of cancer. In this process, the epitope of IMAB362 is exposed. IMAB362 does not bind to any other claudin family member including the closely related splice variant 1 of Claudin 18 (CLDN18.1) that is predominantly expressed in tissues, e.g., lung.

[0519]

[0180] IMAB362 plus epirubicin, oxaliplatin, and capecitabine (EOX) were tested in phase 2 FAST trial (NCT01630083) against EOX in first-line patients with gastric and gastro- esophageal 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% of tumor cells expressing CLDN-18.2 with a moderate-to-strong (> 2+) staining intensity. The subset of patients whose tumors had > 70% of tumor cells with > 2+ CLDN-18.2 staining intensity derived the greatest benefit from IMAB362 treatment at the 800 / 600 mg / kg2dose with near-doubling of their median overall survival (OS) (Al-Batran et al. 2016; Lordick et al. 2016). The benefit of IMAB362 in OS in the > 70% CLDN-18.2 expression (+33.1 weeks; p < 0.0005) was accompanied by a significant delay in central independent reviewed progression (+14.5 weeks; p < 0.0005) and a higher objective response rate (ORR) (35.1% vs 27.1%). Addition of IMAB362 to EOX did not negatively impact patient-related outcome. No significant differences between the treatment arms were observed in the Mixed effect Model Repeat Measurement for global health state or total STO22 score throughout the study, but IMAB362 plus EOX significantly delayed deterioration of the global health score by 2.6 months vs EOX alone (p = 0.008).

[0520]

[0181] IMAB362 is also tested by Astellas Pharma Inc. in a global development program in Phase 2 and 3 trials in patients with CLDN-18.2+ gastric / gastroesophageal and pancreatic cancer.

[0521]

[0182] IMAB362 has been tested in various clinical trials as shown in Table 1 below.

[0522] Table 1: Summary of certain clinical trials involving administration of IMAB362

[0523] [183| The safety profile of 1MAB362 in patients is well characterized and repeated doses up to 1000 mg / m2q3w (cmaxof up to 603 μg / mL) have been tolerated without dose limiting toxicities (Sahin et al. 2018; Tureci et al. 2019).

[0524]

[0184] Without wishing to be bound by a particular theory, a main pharmacological mode of action of IMAB362 for executing tumor cell killing involves antibody-dependent cellular cytotoxicity (ADCC). Based on dose-response curves obtained by in vitro ADCC testing the concentration of a drug that gives 95% response is observed at IMAB362 concentrations of 0.3-28 μg / mL in serum (Sahin et al. 2018). For example, efficient lysis of CLDN-18.2+ cells through ADCC with an EC95 of 0.3-28 μg / mL has been reported (Sahin et al. 2018).

[0525]

[0185] Across various trials, IMAB362 was well tolerated, with nausea and vomiting being the dominant adverse events (AE), with no observed dose limiting toxicity (DLT) and clinical activity as a single agent and in combination with chemotherapy.

[0526]

[0186] Among other things, the present disclosure provides an insight that IMAB362 or a variant thereof (e.g., a variant that shares one or more features of IMAB362, including, e.g., one or more (and in many embodiments all) CDR sequences, one or more (and in many embodiments all) FR sequences, and / or heavy and / or light chain variable sequences, etc., and / or that is a class variant such as IgGl, IgM, IgA, etc.) may represent a particularly desirable antibody for delivery via administration of a ribonucleic acid as described herein. Without wishing to be bound by any particular theory, the present disclosure proposes that such delivering modality may achieve effective administration with reduced incidence (e.g., frequency and / or severity) of IMAB362 treatment-related adverse events (TEAEs) relative to those observed when IMAB362 antibody itself is administered. In the Phase 2a MONO trial with IMAB362 (NCT01197885), TEAEs occurred in 82% (n = 44 / 54) of the patients; nausea (61 %), vomiting (50%) and fatigue (22%) were the most frequent TEAEs. Grade 3 vomiting was reported in 12 patients (22%) and grade 3 nausea in eight patients (15%). These patients received the 600 mg / m2dose. The nausea and vomiting observed in this study were managed by pausing or slowing infusion of IMAB362 indicating that the AEs are Cmax related (Tureci et al. 2019).

[0527]

[0187] In particular, the present disclosure, among other things, demonstrates that the pharmacokinetic (PK) profile of IMAB362 delivered as a ribonucleic acid (“RiboMabOl”) described herein showed a gradual increase in antibody concentrations and a notably lower Cmax than IMAB362 between 48-72 hours post administration. The altered PK profile of RiboMabOl may reduce the Craax-related AEs seen in patients after treatment with IMAB362. The present disclosure also provides non-human primate study data, which shows that no systemic side effects such as diarrhea were observed.

[0528]

[0188] Among other things, the present disclosure appreciates the favorable risk / benefit profile observed for administered IMB362 antibody, particularly in certain indications with high medical need, and proposes that delivery as described herein may be effective and / or particularly desirable. III. RNA technologies for delivery of antibody-based therapeutics

[0529] 1189] Recombinant protein antibodies are widely used biologies for the treatment of diseases or disorders (e.g., cancer) but show a number of limitations, including, e.g., lengthy manufacturing process development and, for antibody derivatives, short serum half-life. The present disclosure, among other things, provides technologies that address certain limitations of recombinant antibody technologies, including for example, lengthy manufacturing process development, and for antibody derivatives, short serum half-life, by utilizing RNA technologies as a modality to express antibody agents, called RiboMabs, directly in the patient’s cells as a novel class of antibody-based therapeutics. In some embodiments, the present disclosure, among other things, provides insights that RiboMabs that are formulated with lipid nanoparticles (LNP) for intravenous (IV) administration can be taken up by cells (e.g., liver cells) for efficient production of the encoded RiboMab antibody 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, mRNA that encodes an antibody agent may comprise modified nucleotides (e.g., but not limited to pseudouridine and / or 1 -methyl- pseudouridine).

[0530]

[0190] RiboMab technology can be utilized to deliver various antibody formats. For example, in some embodiments, RiboMab technology can be used to express a full immunoglobulin (Ig), including, e.g., but not limited to IgG. In some embodiments, a full immunoglobulin (Ig) may be encoded by a single RNA comprising a first coding region that encodes a heavy chain of an antibody and a second coding region that encodes a light chain variable domain of the antibody, wherein the single RNA comprises or encodes either an internal ribosome entry sides (IRES) or another internal promoter or peptide sequence such as “selfcleaving” 2A or 2A-like sequences (see, e.g., Szymczak et al. Nat Biotechnol 22:589, May 2004; ePub April 42004) to yield a respective heavy chain and light chain, which can then be processed to form a full IgG. In some embodiments, a full Ig may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a heavy chain of an antibody; and a second RNA comprising a coding region that encodes a light chain of the antibody. Such first and second RNAs are then translated into respective chains of an antibody and form a full Ig antibody in target cells.

[0191] In some embodiments, RiboMab technology can be used to express a bispecific antibody variant, e.g., as illustrated in Figure 12 (Panel A) or described in Stadler et al. (2016) Oncoimmunology 5(3): el091555; and / or in Stadler et al. (2017) Nature Medicine 23(7): 815- 817. For example, in some embodiments, a bivalent antibody agent may be encoded by a single RNA comprising a first coding region that encodes a single-chain variable fragment (scFv) for a first target and a second coding region that encodes a scFv for a second target. In some embodiments, a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a scFv for a first target and a coding region that encodes a heavy chain antigen binding fragment (Fab) for a second target; and a second RNA comprising a coding region that encodes a scFv for the same first target and a coding region that encodes a light chain Fab for the same second target. Such first and second RNAs are then translated into subunits of an antibody and form a bispecific antibody in target cells.

[0531]

[0192] In some embodiments, RNA agents (e.g., ssRNAs described herein) may be delivered with a carrier. In some embodiments, RNA / LNP is intravenously (IV) administered and taken up by target cells (e.g., liver cells) for efficient production of the encoded RiboMab antibody at therapeutically relevant plasma concentrations.

[0532] A. Provided RNAs encoding antibody agents directed to Claudin-18.2 polypeptides and compositions thereof

[0533]

[0193] In some embodiments, at least one RNA comprises one or more coding regions that encode an antibody agent as described in the section entitled “Exemplary antibody agents targeting Claudin-18.2 polypeptides” above. In some embodiments, at least one RNA comprises one or more coding regions that encode an antibody agent IMAB362 as described above or exemplified herein.

[0534]

[0194] Without wishing to be bound by any particular theory, the present disclosure, among other things, provides an insight that, in some embodiments, an antibody agent IMAB362 may be particularly useful and / or effective at least in part because it binds specifically to CLDN- 18.2 and, moreover, binds preferentially to CLDN-18.2 relative to CLDN18.1. In some embodiments, teachings provided herein may be applicable to other antibody agents specific to CLDN-18.2, and in particular to such antibodies that bind preferentially to CLDN-18.2 even relative to CLDN 18.1. For example, in some embodiments, at least one RNA comprises one or more coding regions that encode an antibody agent that binds preferentially 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 higher than that for a CLDN18.1 polypeptide by at least 50% or more including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher. In some embodiments, such an antibody agent has a binding affinity for a CLDN-18.2 polypeptide higher than that for a CLDN18.1 polypeptide by at least 1.1-fold or more including, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50- fold, at least 75-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10,000-fold or higher. In some embodiments, such an antibody agent does not detectably bind to any other claudin family member including CLDN 18.1. In some embodiments, an antibody agent may be or comprise an antibody. In some embodiments, an antibody agent may be or comprise an antigen binding fragment.

[0535]

[0195] 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 as described herein) specifically binds to a 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 ECD 1 that is exposed in cancer cells.

[0536]

[0196] In some embodiments, at least one RNA encodes a variable heavy chain (VH) domain of a CLDN-18.2-targeting antibody agent and a variable light chain (VL) domain of the antibody agent. In some embodiments, such VH domain(s) and VL domain(s) of a CLDN- 18.2- targeting antibody agent may be encoded by a single RNA construct; alternatively in some embodiments they may be encoded separately by at least two individual RNA constructs. For example, in some embodiments, an RNA as utilized herein comprises two or more coding regions, which comprises a heavy chain-coding region that encodes at least a VH domain of a CLDN-18.2-targeting antibody agent; and a light chain-coding region that encodes at least a VL domain of a CLDN-18.2-targeting antibody agent. In alternative embodiments, a composition comprises (i) a first RNA comprising a heavy chain-coding region that encodes at least a VH domain of a CLDN-18.2-targeting antibody agent; and (ii) a second RNA comprising a light chain-coding region that encodes at least a VL domain of a CLDN-18.2-targeting antibody agent.

[0537]

[0197] In some embodiments, a heavy chain-coding region can further encode a constant heavy chain (CH) domain; and / or a light chain-coding region can further encode a constant light chain (C ) domain. For example, in some embodiments, a heavy chain-coding region may encode a VH domain, a CHI domain, aCn2 domain, and a CH3 domain of a CLDN-18.2-targeting antibody agent in an immunoglobulin form (e.g., IgG); and / or a light chain-coding region may encode a VL domain and a CL domain of a CLDN-18.2-targeting antibody agent in an Ig form (e.g., IgG). For example, in some embodiments, a full immunoglobulin (Ig) maybe encoded by a single RNA comprising a first coding region that encodes a heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG) and a second coding region that encodes a light chain variable domain of the CLDN-18.2 Ig antibody (e.g. ,IgG), which single RNA requires protein translation to yield a fusion protein comprising a heavy chain and a light chain of the antibody and post-translational cleavage of the fusion protein by a suitable protease into respective heavy chain and light chain, which can then be processed to form a full Ig (e.g., IgG). In some embodiments, a full Ig may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG); and a second RNA comprising a coding region that encodes a light chain of the CLDN-18.2 Ig antibody (e.g., IgG). Such first and second RNAs are then translated into respective chains of an antibody and form a full Ig antibody (e.g. , IgG) in target cells. In some embodiments, an antibody agent encoded by one or more RNAs in an IgG form is IgGl .

[0538]

[0198] In some embodiments, a heavy chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 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, a light chaincoding region of an RNA consists of or comprises a nucleotide sequence that encodes at least one CDR (including, e.g. , 1 CDR, 2 CDRs, and 3 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).

[0539]

[0199] In some embodiments, a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an 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 to one or more non-CDR regions of SEQ ID NO: 1. For example, in some embodiments, SEQ ID NO: 1 may comprise at least one or more (including, e.g., 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 (including, e.g., amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, no more than 50 (including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 1. In some embodiments, a light-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an 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) may be present to one or more non-CDR regions of SEQ ID NO: 2. For example, in some embodiments, SEQ ID NO: 2 may comprise at least one or more (including, e.g., 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 (including, e.g., amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, no more than 50 (including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 2.

[0540]

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

[0541]

[0201] In some embodiments, a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an 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 to one or more non-CDR regions of SEQ ID NO: 3. For example, in some embodiments, SEQ ID NO: 3 may comprise at least one or more (including, e.g., 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 (including, e.g., amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, no more than 50 (including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 3. In some embodiments, a light-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an 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 to one or more non-CDR regions of SEQ ID NO: 4. For example, in some embodiments, SEQ ID NO: 4 may comprise at least one or more (including, e.g., 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 (including, e.g., amino acid insertions, deletions, and / or substitutions) to one or more non-CDR regions. In some embodiments, no more than 50 (including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 4.

[0542]

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

[0543]

[0203] In some embodiments, a heavy chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes a full-length heavy chain of Zolbetuximab or Claudiximab (e.g., as described and / or exemplified herein). In some embodiments, a light chaincoding region of an RNA consists of or comprises a nucleotide sequence that encodes a full- length light chain of Zolbetuximab or Claudiximab.

[0544]

[0204] In some embodiments, one or more RNAs can be used to encode a bispecific or multispecific antibody agent, which binds to two or more target molecules, e.g., one of which is a CLDN-18.2 polypeptide. For example, Figure 12A illustrates exemplary bispecific antibody encoded by one or more RNAs. See also, e.g., Stadler et al. (2016) Oncoimmunology 5(3): el091555; and / or in Stadler et al. (2017) Nature Medicine 23(7): 815-817. In some embodiments, a bivalent antibody agent may be encoded by a single RNA comprising a first coding region that encodes a single-chain variable fragment (scFv) that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a second coding region that encodes a scFv for a second target (e.g., in some embodiments which 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 that encodes a scFv that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a coding region that encodes a heavy chain antigen binding fragment (Fab) for a second target (e.g., in some embodiments which may be a T cell receptor); and a second RNA comprising a coding region that encodes a scFv targeting the CLDN-18.2 polypeptide and a coding region that encodes a light chain Fab for 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 that encodes a scFv for a first target e.g., in some embodiments which may be a T cell receptor) and a coding region that encodes a heavy chain antigen binding fragment (Fab) that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide); and a second RNA comprising a coding region that encodes a scFv for the same first target and a coding region that encodes a light chain Fab targeting the CLDN-18.2 polypeptide. Such first and second RNAs are then translated into subunits of an antibody and form a bispecific antibody in target cells.

[0545]

[0205] Secretion signal-encoding region: In some embodiments, RNA(s) that encode a

[0546] CLDN-18.2-targeting antibody agent may comprise a secretion signal-encoding region. In some embodiments, such a secretion signal-encoding region allows a CLDN-18.2-targeting antibody agent encoded by one or more RNAs to be secreted upon translation by cells, e.g., present in a subject to be treated, thus yielding a plasma concentration of a biologically active a CLDN-18.2- targeting antibody agent. In some embodiments, a secretion signal-encoding region included in an RNA consists of or comprises a nucleotide sequence that encodes a non-human secretion signal. For example, in some embodiments, such a non-human secretion signal may be a murine secretion signal, which may in some embodiments be or comprises the amino acid sequence of MGWSCIILFLVATA GVHS or MESQTQVLMSLLFWVSGTCG. In some embodiments, a secretion signal-encoding region included in an RNA consists of or comprises a nucleotide sequence that encodes a human secretion signal, which may in some embodiments be or comprises the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, a secretion signalencoding region included in an RNA encoding a heavy chain domain of a CLDN-18.2-targeting antibody agent may comprise a nucleotide sequence (i) that encodes a murine secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MGWSCIILFLVATATGVHS; or that (ii) encodes a human secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS . In some embodiments, a secretion signal-encoding region included in an RNA encoding a light chain domain of a CLDN-18.2-targeting antibody agent may comprise a nucleotide sequence (i) that encodes a murine secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MESQTQVLMSLLFWVSGTCG; or that (ii) encodes a human secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS .

[0547]

[0206] In some embodiments, RNA(s) that encode a CLDN-18.2-targeting antibody agent may comprise at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding sequence elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure for co-transcriptional capping of mRNA, a poly adenine (polyA) tail, and any combinations thereof.

[0548]

[0207] UTRs (5’ UTRs and / or 3 ' UTRs): In some embodiments, a provided RNA can comprise a nucleotide sequence that encodes a 5’UTR of interest and / or a 3’ UTR of interest. One of skill in the art will appreciate that untranslated regions (e.g., 3’ UTR and / or 5’ UTR) of a mRNA sequence can contribute to mRNA stability, mRNA localization, and / or translational efficiency.

[0549]

[0208] “ In some embodiments, a provided RNA can comprise a 5’ UTR nucleotide sequence and / or a 3’ UTR nucleotide sequence. In some embodiments, such a 5’ UTR sequence can be operably linked to a 3’ of a coding sequence (e.g., encompassing one or more coding regions). Additionally or alternatively, in some embodiments, a 3’ UTR sequence can be operably linked to 5’ of a coding sequence e.g., encompassing one or more coding regions).

[0550]

[0209] In some embodiments of any aspects described herein, 5' and 3' UTR sequences included in an RNA can consist of or comprise naturally occurring or endogenous 5' and 3' UTR sequences for an open reading frame of a gene of interest. Alternatively, in some embodiments, 5’ and / or 3’ UTR sequences included in an RNA are not endogenous to a coding sequence (e.g., encompassing one or more coding regions); in some such embodiments, such 5’ and / or 3’ UTR sequences can be useful for modifying the stability and / or translation efficiency of an RNA sequence transcribed. For example, a skilled artisan will appreciate that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, as will be understood by a skilled artisan, 3’ and / or 5’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0551]

[0210] For example, one skilled in the art will appreciate that, in some embodiments, a nucleotide sequence consisting of or comprising a Kozak sequence of an 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 a skilled artisan, Kozak sequences are known to increase the efficiency of translation of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation. In some embodiments, a provided RNA polynucleotide can comprise a nucleotide sequence that encodes 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' UTRs, for example, to impede exonuclease degradation of the transcribed RNA sequence.

[0552]

[0211] In some embodiments, a 5’ UTR included in an RNA may be derived from human a-globin mRNA combined with Kozak region.

[0553]

[0212] In some embodiments, an RNA may comprise one or more 3 ’UTRs. For example, in some embodiments, an RNA may comprise two copies of 3'-UTRs derived from a globin mRNA, such as, e.g., alpha2-globin, alpha 1 -globin, beta-globin (e.g., a human beta-globin) mRNA. In some embodiments, two copies of 3’UTR derived from a human beta-globin mRNA may be used, e.g., in some embodiments which may be placed between a coding sequence of an RNA and a poly(A)-tail, to improve protein expression levels and / or prolonged persistence of an RNA. In some embodiments, a 3’ UTR included in an RNA may be or comprise one or more (e.g., 1, 2, 3, or more) of the 3’UTR sequences disclosed in WO 2017 / 060314, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, a 3‘-UTR may be a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called 1). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017 / 060314, herein incorporated by reference).

[0213] In some embodiments, a 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.

[0554]

[0214] In some embodiments, a 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.

[0555]

[0215] PolyA tail: In some embodiments, a provided RNA can comprise a nucleotide sequence that encodes a polyA tail. A polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenosine nucleotides. In some embodiments, a polyA tail is a nucleotide sequence comprising at least 30 adenosine nucleotides or more, including, 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, a polyA tail is or comprises a polyA homopolymeric tail. In some embodiments, a polyA tail may comprise one or more modified adenosine nucleosides, including, but not limited to, cordiocipin and 8-azaadenosine. In some embodiments, a polyA tail may comprise one or more non-adensoine nucleotides. In some embodiments, a polyA tail may be or comprise a disrupted or modified polyA tail as described in WO 2016 / 005324, the entire content of which is incorporated herein by reference for the purpose described herein. For example, in some embodiments, a polyA tail included in an RNA described herein may be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 20 A consecutive nucleotides, which is 3’ of the linker sequence. In some embodiments, a modified polyA sequence may comprise: a linker sequence which is not a polyA sequence comprising at least ten nucleotides (e.g., T, G, and / or C nucleotides); a first sequence of at least 30 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 70 A consecutive nucleotides, which is 3’ of the linker sequence.

[0556]

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

[0217] 5’ cap: In some embodiments, an RNA described herein may comprise a 5’ cap, which may be incorporated into such an RNA during transcription, or joined to such an RNA post-transcription. In some embodiments, an RNA may comprise a 5’ cap structure for co- transcriptional capping of RNA. Examples of a cap structure for co-transcriptional capping are known in the art, including, e.g., as described in WO 2017 / 053297, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, a 5’ cap included in an RNA described herein is or comprises m7G(5')ppp(5')(2'OMeA)pG. In some embodiments, a 5’ cap included in an RNA described herein is or comprises a cap1 structure [e.g., m27’3’°Gppp(m12-0)ApG]. When an RNA sequence described herein has a 5' end with the nucleotides 5'-AG, and it is described that the RNA comprises a 5’ cap containing as second and third nucleotides A and G, respectively, [e.g., m27’3-0Gppp(mi2-0)ApG], it is to be understood that in some embodiments, the second and third nucleotides of the cap correspond to the nucleotides 5'-AG of the RNA sequence.

[0557]

[0218] Chemical modification: In some embodiments, RNA(s) that encode a CLDN- 18.2-targeting antibody agent may comprise at least one modified ribonucleotide, for example, in some embodiments to increase the stability of such RNA(s) and / or decrease immunogenicity of such RNA(s) and / or to decrease cytotoxicity of such RNAs. For example, in some embodiments, at least one of A, U, C, and G ribonucleotide of RNA(s) may be replaced by a modified ribonucleotide. For example, in some embodiments, some or all of cytidine residues present in an RNA may be replaced by a modified cytidine, which in some embodiments may be, e.g., 5- methylcytidine. Alternatively or additionally, in some embodiments, some or all of uridine residues present in an RNA may be replaced by a modified uridine, which in some embodiments may be 3 -methyl -uridine (m3U), 5 -methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5 -hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo- uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethyl -pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5 -carboxyhydroxymethyl -uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio- uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1 -ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1 -prop ynyl -pseudouridine, 5-taurinomethyl-uridine (rm5U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4- thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4\| / ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N 1 -methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 \| / ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio- uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O- dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, some or all of uridine residues present in an RNA may be replaced by a modified uridine selected from the group consisting of pseudouridine (\| / ), Nl- methyl-pseudouridine (ml y), 5-methyl-uridine (m5U), and combinations thereof. In some embodiments, some or all of uridine residues present in an RNA may be replaced by pseudouridine or a derivative thereof, e.g., 1 -methylpseudouridine. In some embodiments, some or all of uridine residues present in an RNA may be replaced by pseudouridine. In some embodiments, some or all of uridine residues present in an RNA may be replaced by 1 - methylpseudouridine. In some embodiments, all uridine residues present in an RNA are replaced by pseudouridine. In some embodiments, all uridine residues present in an RNA are replaced by 1 -methylpseudouridine.

[0558]

[0219] Codon optimization and GC enrichment: The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and / or to replace codons which are rare in the cell (or subject) in which a peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and / or increased in the G / C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence. In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine) / C (cytosine) content are more stable than sequences having an increased A (adenosine) / U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so- called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleotides. In various embodiments, the G / C content of the coding region of the RNA (in particular, 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.

[0559]

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

[0560]

[0221] For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement 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 selected from the group consisting of 3-methyl-uridine (m3U), 5 -methoxy-uridine (mo5U), 5-aza- uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5-iodo-uridine or 5 -bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5- oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl -uridine (mnm5U), 1 -ethyl -pseudouridine, 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl- uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl -uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio- uridine (m5s2U), l-methyl-4-thio-pseudouridine (m’s4^), 4-thio-l-methyl-pseudouridine, 3- methyl-pseudouridine (m3Ψ) , 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2 -methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ ),

[0561] 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2 '-O-methyl -uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- methyl-pseudouridine (Ψm) , 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'- O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'- F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (Ψ), Nl-methyl-pseudouridine (m1Ψ) or 5-methyl-uridine (m5U), in particular N 1 -methyl -pseudouridine.

[0562]

[0222] In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement 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.

[0563]

[0223] During 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 unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524. In some embodiments, the amount of doublestranded RNA (dsRNA) is limited, e.g., dsRNA (especially dsmRNA) is removed from non- immunogenic RNA , such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single- stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, , at least 99.993%,, at least 99.994%, , at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.). Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dsRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference. For example, RN A may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in 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 detection of dsRNA, the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected. In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2- fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3- fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10- fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1 OOO-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 (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3 -fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by an 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor. The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0564]

[0224] In some embodiments, an RNA encoding a heavy chain of a CLDN-18.2- targeting antibody agent comprises, in a 5’ to 3’ direction: (a) a 5’UTR; (b) a secretion signalcoding region; (c) a heavy chain-coding region; (d) a 3’ UTR; and (e) a polyA tail. See, for example, Figure 13. In some embodiments, a 5’UTR is or comprises a sequence derived from human a-globin mRNA combined with Kozak region. In some embodiments, a secretion signalcoding region is or comprises a nucleotide sequence that encodes the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS . In some embodiments, a heavy chain-coding region encodes a VH domain, a Cm domain, aCH2 domain, and a CH3 domain of a CLDN-18.2-targeting antibody agent in an IgG form (e.g., ones as described herein, such as IMAB262, or an amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO: 3. In some embodiments, a 3’ UTR is or comprises a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). In some embodiments, a polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines). In some embodiments, such an RNA comprises a 5’ cap structure comprising a CAP1 structure, or m?7’3’ °Gppp(mi2‘°)ApG. In some embodiments, such an RNA comprises all uridines replaced by Nl- methylpseudouridine.

[0225] In some embodiments, an RNA encoding a light chain of a CLDN-18.2-targeting antibody agent comprises, in a 5’ to 3’ direction: (a) a 5’UTR; (b) a secretion signal-coding region; (c) a light chain-coding region; (d) a 3’ UTR; and (e) a polyA tail. See, for example, Figure 13. In some embodiments, a 5’UTR is or comprises a sequence derived from human a- globin mRNA combined with Kozak region. In some embodiments, a secretion signal-coding region is or comprises a nucleotide sequence that encodes the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS . In some embodiments, a light chain-coding region encodes a VL domain and a CL domain of a CLDN-18.2-targeting antibody agent in an IgG form (e.g., ones as described herein, such as IMAB262, or an amino acid sequence represented by amino acid residues 27-246 of SEQ ID NO: 4. In some embodiments, a 3’ UTR is or comprises a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). In some embodiments, a polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines). In some embodiments, such an RNA comprises a 5’ cap structure comprising a CAP1 structure, or m27-3-0Gppp(m12-0)ApG. In some embodiments, such an RNA comprises all uridines replaced by N1 -methylpseudouridine.

[0565]

[0226] In some embodiments, RNA(s) is or comprises one or more single-stranded RNA(s), e.g., single-stranded mRNAs.

[0566]

[0227] In some embodiments, a composition comprises a single- stranded mRNA encoding a heavy chain (...

Claims

Claims1. A composition or medical preparation comprising:(i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and(ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17.

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

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

4. The composition or medical preparation of any one of claims 1 to 3, wherein 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.

5. The composition or medical preparation of any one of claims 1 to 4, wherein 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.

6. The composition or medical preparation of any one of claims 1 to 5, wherein 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.

7. The composition or medical preparation of any one of claims 1 to 6, wherein the RNA, e.g., 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. The composition or medical preparation of any one of claims 1 to 7, wherein the RNA, e.g., 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. A composition or medical preparation comprising:(i) an RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and(ii) an RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein 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.

10. The composition or medical preparation of any one of claims 1 to 9, wherein 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.

11. The composition or medical preparation of any one of claims 1 to 10, wherein 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.

12. The composition or medical preparation of any one of claims 1 to 11, wherein 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.

13. The composition or medical preparation of any one of claims 1 to 12, wherein the RNA, e.g., 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. The composition or medical preparation of any one of claims 1 to 12, wherein the RNA, e.g., 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. The composition or medical preparation of any one of claims 9 to 14, wherein:(a) the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and / or(b) the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.

16. The composition or medical preparation of any one of claims 1 to 15, wherein the coding region under (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, and the coding region under (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.

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

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

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

20. The composition or medical preparation of any one of claims I to 19, wherein the antibody agent binds preferentially to CLDN-18.2 relative to Claudin-18.1 (CLDN-18.1).

21. The composition or medical preparation of any one of claims 1 to 20, wherein the antibody agent binds to a first extracellular domain (ECD1) of CLDN-18.2.

22. The composition or medical 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 in cancer cells.

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

24. The composition or medical preparation of any one of claims 1 to 23, wherein the antibody agent is IgGl.

25. The composition or medical preparation of claim 24, wherein the IgGl is human IgGl.

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

28. The composition or medical 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 medical preparation of claim 27 or 28, wherein the VH(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ. ID NO: 5, 6, and 7, respectively.

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

31. The composition or medical 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 medical preparation of claim 30 or 31, wherein the VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 8, 9, and 10, respectively.

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

34. The composition or medical preparation of any one of claims 1 to 33, wherein the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising CDR1, CDR2 and CDR3 comprising thesequences as set forth in SEQ ID NO: 5, 6, and 7, respectively, and the the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising CDR1, CDR2 and CDR3 comprisingthe sequences as set forth in SEQ ID NO: 8, 9, and 10, respectively.

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

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

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

38. The composition or medical preparation of claim 37, wherein the VH(CLDN-18.2), CHI, CH2 and CH3 are present in the first polypeptide chain in an immunoglobulin G (IgG) form.

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

40. The composition or medical 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. The composition or medical preparation of claim 39 or 40, wherein the CHI on the first polypeptide chain interacts with the CL on the second polypeptide chain.

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

43. The composition or medical preparation of claim 42, wherein the secretion signal of the first polypeptide chain and / or the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13.

44. The composition or medical preparation of any one of claims 1 to 43, wherein the coding region under (i) comprises the nucleotide sequence of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of SEQ ID NO: 17.

45. The composition or medical preparation of any one of claims 1 to 44, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4.

46. The composition or medical preparation of any one of claims 1 to 45, wherein the RNA, e.g., each RNA, comprises a poly-A sequence.

47. The composition or medical preparation of claim 46, wherein the poly-A sequence is an interrupted sequence of A nucleotides.

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

49. The composition or medical preparation of any one of claims 46 to 48, wherein the poly-A sequence comprises or consists of the nucleotide sequence Ax-L-Av, wherein Ax is a sequence of at least 20 A nucleotides, Ayis 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.

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

51. The composition or medical preparation of any one of claims 1 to 50, which 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) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 25 or 27.

52. The composition or medical preparation of claim 51, which comprises:(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and(ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25.

53. The composition or medical preparation of claim 51, which comprises:(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and(ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27.

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

55. A composition or medical preparation comprising:(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and(ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25.

56. A composition or medical preparation comprising:(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and(ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 27.

57. The composition or medical preparation of any one of claims 1 to 56, wherein the RNA, e.g., each RNA, comprises a modified nucleoside in place of uridine.

58. The composition or medical preparation of any one of claims 1 to 57, wherein the RNA, e.g., each RNA, comprises a modified nucleoside in place of each uridine.

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

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

61. The composition or medical preparation of any one of claims 1 to 60, wherein the RNA, e.g., each RNA, comprises a 5' cap.

62. The composition or medical preparation of any one of claims 1 to 61, wherein the RNA, e.g., each RNA, comprises the 5' cap m27,3'0Gppp(m12-0)ApG.

63. The composition or medical preparation of any one of claims 1 to 62, wherein the RNA, e.g., each RNA, is single-stranded RNA.

64. The composition or medical preparation of any one of claims 1 to 63, wherein the RNA, e.g., each RNA, is mRNA.

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

66. The composition or medical preparation of claim 65, wherein lipids that form the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid.

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

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

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

71. The composition or medical preparation of any one of claims 65 to 70, wherein the lipid nanoparticles have an average size of about 50-150 nm.

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

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

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

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

76. The medical preparation of claim 75, wherein the RNA, e.g., each RNA, and optionally the particle forming components are in separate vials.

77. The medical preparation of claim 75 or 76, further comprising instructions for use of the composition or medical preparation for treating or preventing cancer.

78. The composition or medical preparation of any one of claims 1 to 77 for pharmaceutical use.

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

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

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

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

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

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

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

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

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

88. The composition or medical preparation of claim 87, wherein the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) radiotherapy, and (iii) chemotherapy.

89. The composition or medical preparation of claim 87 or 88, wherein the further therapy comprises administering a further therapeutic agent.

90. The composition or medical preparation of claim 89, wherein the further therapeutic agent comprises an anti-cancer therapeutic agent.

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

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

93. A method of treating cancer in a subject comprising administering to the subject the composition of any one of claims 1 to 74.

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 a CLDN-18.2- positive pancreatic cancer.

97. The method of any one of claims 93 to 96, wherein the cancer comprises a 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 a CLDN-18.2- positive locally advanced, unresectable, or metastatic cancer.

100. The method of any one of claims 93 to 99, which further comprises administering a further therapy.

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

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

103. The method of claim 102, wherein the furthertherapeutic 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. The method of any one of claims 93 to 104, wherein the composition is administered intravenously.

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

107. The composition or medical preparation of any one of claims 1 to 92, which is for introducing the RNA into liver cells and expressing the polypeptide chains encoded by the RNA in liver cells.

108. The composition or medical preparation of any one of claims I to 92, which is for systemic delivery of the polypeptide chains.

109. The composition or medical preparation of any one of claims I to 92, which is for systemic delivery of the polypeptide chains following expression of the polypeptide chains in liver cells.

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

111. A method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising:(a) administering a composition of any one of claims 1 to 74 such that the RNA is introduced into liver cells; and(b) expressing the polypeptide chains encoded by the RNA in the liver cells, wherein, following expression, the polypeptide chains are secreted into the bloodstream.

112. A method for systemic delivery of an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising:(a) administering a composition of any one of claims 1 to 74 such that the RNA is introduced into liver cells; and(b) expressing the polypeptide chains encoded by the RNA in the liver cells, wherein, following expression, the polypeptide chains are secreted into the bloodstream.

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

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

115. A composition or medical preparation comprising RNA, wherein the RNA comprises:(i) a coding sequence that encodes a polypeptide,(ii) a 3' UTR sequence,(iii) a poly-A sequence, and(iv) a nucleotide sequence linking the 3' UTR sequence and the poly-A sequence comprising the sequence CUXGAGCUAGC, wherein X is C, A, or U.

116. The composition or medical preparation of claim 115, wherein the nucleotide sequence linking the 3' UTR sequence and the poly-A sequence comprises the sequence CUCGAGCUAGC.

117. The composition or medical preparation of claim 115 or 116, wherein the RNA comprises in the 5' — 3' direction the coding sequence that encodes a polypeptide, the 3' UTR sequence, the nucleotide sequence linking the 3' UTR sequence and the poly-A sequence, and the poly- A sequence.

118. The composition or medical 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. The composition or medical 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. The composition or medical 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 SEQID 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. The composition or medical preparation of any one of claims 115 to 120, wherein the poly-A sequence is an interrupted sequence of A nucleotides.

122. The composition or medical preparation of any one of claims 115 to 121, wherein the poly-A sequence comprises at least 100 nucleotides.

123. The composition or medical preparation of any one of claims 115 to 122, wherein the poly-A sequence comprises or consists of the nucleotide sequence Ax-L-Ay, wherein Axis a sequence of at least 20 A nucleotides, Ayis 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. The composition or medical preparation of any one of claims 115 to 123, wherein the poly-A 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. The composition or medical 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. The composition or medical preparation 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 which is preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4AACUAGU, wherein XI is any nucleotide, preferably A or C, X2 is any nucleotide, preferably A or C, X3 is any nucleotide, preferably C, U or G, and X4 is A or is missing.

127. The composition or medical preparation 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 XI is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.

128. The composition or medical 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 which is preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU.

129. The composition or medical 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 which is preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU.

130. The composition or medical 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. The composition or medical 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. The composition or medical 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 medical 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 that encodes 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 poly-A sequence.

134. The composition or medical 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 that encodes 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 poly-A sequence.

135. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.

136. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, or anucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

137. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.

138. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

139. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

140. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36.

141. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.

142. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36.

143. The composition or medical 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 that encodes 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 poly-A sequence.

144. The composition or medical 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 that encodes 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 poly-A sequence.

145. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 37.

146. The composition or medical 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 that encodes a polypeptide, a sequencecomprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a poly-A sequence.

147. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ. ID NO: 37, and a poly-A sequence.

148. The composition or medical 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 that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 37.

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

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

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

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

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

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

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

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

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

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

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

160. The composition or medical preparation of any one of claims 115 to 159, wherein 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 poly-A sequence, and / or a nucleotide sequence linking a 3' UTR sequence and a poly-A sequence as defined.

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

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

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

164. The composition or medical preparation of any one of claims 115 to 163, wherein the RNA, e.g., each RNA, comprises a modified nucleoside in place of uridine.

165. The composition or medical preparation of any one of claims 115 to 164, wherein the RNA, e.g., each RNA, comprises a modified nucleoside in place of each uridine.

166. The composition or medical preparation of claim 164 or 165, wherein the modified nucleoside is pseudouridine (Ψ) and / or Nl-methyl-pseudouridine (m1Ψ) .

167. The composition or medical preparation of any one of claims 164 to 166, wherein the modified nucleoside is Nl-methyl-pseudouridine (m1Ψ) .

168. The composition or medical preparation of any one of claims 115 to 167, wherein the RNA, e.g., each RNA, comprises a 5' cap.

169. The composition or medical preparation of any one of claims 115 to 168, wherein the RNA, e.g., each RNA, comprises the 5' cap m27,3’0Gppp(m12'0)ApG.

170. The composition or medical preparation of any one of claims 115 to 169, wherein the RNA, e.g., each RNA, is single-stranded RNA.

171. The composition or medical preparation of any one of claims 115 to 170, wherein the RNA, e.g., each RNA, is mRNA.

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

173. The composition or medical preparation of claim 172, wherein lipids that form the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid.

174. The composition or medical preparation of claim 173, wherein: a. the cationic lipid is present in 35-65 mol% of the total lipids; a. the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; and c. the neutral lipid is present in 35-65 mol% of the total lipids.

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

176. The composition or medical 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. The composition or medical preparation of any one of claims 173 to 176, wherein the neutral lipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

178. The composition or medical preparation of any one of claims 172 to 177, wherein the lipid nanoparticles have an average size of about 50-150 nm.

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

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

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

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

183. The medical preparation of claim 182, wherein the RNA, e.g., each RNA, and optionally the particle forming components are in separate vials.

184. The composition or medical preparation of any one of claims 115 to 183, which is for intravenous administration.

185. The composition or medical preparation of any one of claims 115 to 184, which is for introducing the RNA into liver cells and expressing the polypeptide encoded by the RNA in liver cells.

186. The composition or medical preparation of any one of claims 115 to 185, which is for systemic delivery of the polypeptide.

187. The composition or medical preparation of any one of claims 115 to 186, which is for systemic delivery of the polypeptide following expression of the polypeptide in liver cells.

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

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

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

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

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

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