CN120129702A - RNA composition targeting claudin-18.2 - Google Patents

Abstract

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

Description

RNA compositions targeting claudin-18.2

Background

Cancer is the second leading cause of death worldwide and is expected to lead to an estimated 960 tens of thousands of deaths in 2018 (Bray et al 2018). Typically, with few exceptions (e.g., germ cells and some carcinoid tumors), once a solid tumor metastasizes, 5-year survival rarely exceeds 25%.

Recent advances in conventional therapies such as chemotherapy, radiation therapy, surgery and targeted therapies have improved outcome in patients with advanced solid tumors. Over the past several years, the food and drug administration (Food and Drug Administration, FDA) and European drug administration (European MEDICINES AGENCY, EMA) have approved eight checkpoint inhibitors, one monoclonal antibody targeting the CTLA-4 pathway, ipilimumab (ipilimumab), and seven antibodies targeting the programmed death receptor/ligand [ PD/PD-L1], including atuzumab (atezolizumab), avilamab (avelumab) and Devaluzumab (durvalumab), nivolumab (nivolumab), cimapr Li Shan antibody (cemiplimab) and pembrolizumab), for treating patients with multiple cancer types, principally solid tumors. These approvals greatly alter the condition of cancer treatment. However, certain cancers, such as pancreatic adenocarcinoma or metastatic biliary tract cancer, still do not benefit from existing therapies including immunotherapy.

Disclosure of Invention

The poor prognosis of certain cancers, such as pancreatic and cholangiocarcinoma types, highlights the need for additional treatment methods.

The present disclosure provides, inter alia, insights and techniques for treating cancer, particularly cancer associated with claudin-18.2 (CLDN-18.2) expression. In some embodiments, the present disclosure provides techniques for treating a cancer selected from pancreatic cancer, gastric or gastroesophageal cancer, cholangiocarcinoma, ovarian cancer, and the like. In some embodiments, the present disclosure provides techniques for administering therapy to locally advanced tumors. In some embodiments, the present disclosure provides techniques for treating unresectable tumors. In some embodiments, the provided techniques provide techniques for treating metastatic tumors. Thus, for example, in some embodiments, the provided treatments can be administered to a subject or population of subjects suffering from or susceptible to a cancer (e.g., a cancer selected from pancreatic, gastric, or gastroesophageal cancer, biliary tract cancer, ovarian cancer, and/or additionally involving one or more pancreatic, gastric, gastroesophageal, biliary tract, and/or ovarian tumors), which can be or comprise one or more locally advanced tumors, one or more unresectable tumors, and/or one or more metastases.

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

Zolpidem-Bei Tuo-mab (Zolbetuximab) (development code IMAB 362) is a monoclonal antibody targeting claudin-18 isoform 2, which is being studied for the treatment of gastrointestinal adenocarcinoma and pancreatic tumor (Tu reci et al 2019).

The present disclosure also provides insight that, in some embodiments, CLDN-18.2-targeted therapies may usefully involve the administration of RNA (e.g., ssRNA, e.g., mRNA) encoding an antibody agent that targets CLDN-18.2, as described herein. Still further, the present disclosure provides the particular insight that delivery of RNA via lipid nanoparticles targeting hepatocytes can be a particularly beneficial strategy for delivering such antibody agents.

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

The present disclosure provides insight, inter alia, that administration of RNA (e.g., ssRNA, such as mRNA) encoding CLDN-18.2 targeting agents and in particular CLDN-18.2 targeting antibody agents and in particular IMAB362 can represent a particularly desirable strategy for CLDN-18.2 targeted therapies. Without wishing to be bound by any particular theory, the present disclosure suggests that such delivery modes may achieve one or more improvements, such as effective administration with reduced incidence (e.g., frequency and/or severity) of TEAE and/or improved relationship between efficacy levels and TEAE levels (e.g., improved therapeutic window) relative to those observed when the corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements can be achieved, inter alia, by delivering IMAB362 via administration of an RNA (e.g., ssRNA, e.g., mRNA) encoding IMAB 362.

In some embodiments, the present disclosure provides, inter alia, insight that mRNA encoding an antibody agent (e.g., IMAB 362) or a functional portion thereof, which is a lipid nanoparticle (lipid nanoparticle, LNP)/or formulated with a lipid nanoparticle for Intravenous (IV) administration, can be absorbed by a target cell (e.g., a hepatocyte) to effectively produce a therapeutically relevant plasma concentration of the encoded antibody agent (e.g., IMAB 362), e.g., as shown in fig. 14 for RiboMab targeting CLDN-18.2.

In some embodiments, the present disclosure utilizes RiboMab as a CLDN-18.2 targeting agent. In some embodiments, such RiboMab is an antibody agent encoded by, for example, mRNA engineered for minimal immunogenicity and/or formulated in Lipid Nanoparticles (LNPs).

Furthermore, the present disclosure provides insight, inter alia, that the ability of an antibody agent targeting CLDN-18.2 as described herein to induce Antibody Dependent Cellular Cytotoxicity (ADCC) and/or Complement Dependent Cytotoxicity (CDC) against target cells (e.g., tumor cells) while utilizing the immune system of a subject may enhance the cytotoxic effects of chemotherapy and/or other anti-cancer therapies. In some embodiments, such combination therapies may extend progression free and/or overall survival, e.g., relative to treatment of an individual administered alone and/or relative to another suitable reference.

Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, such as, for example, gemcitabine, oxaliplatin, and 5-fluorouracil, appear to up-regulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines, and furthermore, that these agents increase de novo expression in CLDN-18.2 negative cell lines. See, for example Türeci et al.(2019)"Characterization of zolbetuximab in pancreatic cancer models"In Oncoimmunology 8(1),pp.e1523096.

The present disclosure provides, inter alia, insight that CLDN-18.2-targeted therapies as described herein can be particularly useful and/or effective when administered to tumors (e.g., tumor cells, subjects suspected of and/or having detected such tumors and/or tumor cells, etc.) that are characterized (e.g., have been determined to exhibit and/or are expected or predicted to exhibit) increased expression and/or activity of CLDN-18.2 expression in tumor cells (e.g., can be or have been caused by exposure to one or more chemotherapeutic agents). Indeed, the present disclosure teaches, among other things, that the provided CLDN-18.2-targeted therapies (e.g., administration of RNA, and more particularly, mRNA encoding CLDN-18.2-targeted antibody agents) as described herein can provide synergistic therapy when administered in combination with one or more CDLN 18.2.2-enhancing agents (e.g., one or more specific chemotherapeutic agents) (e.g., administered to a subject that has received and/or is receiving or is otherwise exposed to the one or more CDLN 18.2.2-enhancing agents (e.g., one or more specific chemotherapeutic agents)). Thus, in some embodiments, a treatment targeting CLDN-18.2 as described herein may be useful in combination with other anti-cancer agents that are expected and/or have been shown to up-regulate expression of CLDN-18.2 in tumor cells.

In some aspects, provided herein are pharmaceutical compositions that target CLDN-18.2. In some embodiments, such pharmaceutical compositions comprise (a) at least one RNA (e.g., ssRNA) comprising one or more coding regions encoding an antibody agent that binds to a claudin-18.2 (CLDN-18.2) polypeptide (e.g., preferentially binds to claudin-18.2 (CLDN-18.2) polypeptide over claudin-18.1 (CLDN 18.1) polypeptide) ("CLDN-18.2 targeting antibody agent"), and (b) a lipid nanoparticle, wherein at least one single-stranded RNA is encapsulated within at least one lipid nanoparticle. In some embodiments, such pharmaceutical compositions may comprise and/or deliver one or more RNAs encoding antibodies that bind to CLDN-18.2 polypeptides (e.g., preferentially bind to CLDN-18.2 polypeptides over CLND18.1 polypeptides). In some embodiments, such pharmaceutical compositions may comprise and/or deliver one or more RNAs encoding antigen-binding fragments that bind to CLDN-18.2 polypeptides (e.g., preferentially bind to CLDN-18.2 polypeptides over CLND18.1 polypeptides).

In some embodiments, an antibody agent that targets CLDN-18.2 (and may be encoded by RNA, e.g., ssRNA, e.g., mRNA as described herein) specifically binds to a first extracellular domain (ECD 1) of a CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an epitope of exposed ECD1 in a cancer cell.

In some embodiments, at least one RNA (e.g., ssRNA, e.g., mRNA) encodes a variable heavy chain (V_H) domain of a CLDN-18.2 targeted antibody agent and a variable light chain (V_L) domain of the antibody agent. In some embodiments, such a V_H domain and V_L domain of a CLDN-18.2 targeted antibody agent may be encoded by a single RNA construct, or in some embodiments, it may be encoded by at least two separate RNA constructs separately. For example, in some embodiments, an RNA as used herein comprises two or more coding regions comprising a heavy chain coding region encoding at least the V_H domain of an antibody agent and a light chain coding region encoding at least the V_L domain of an antibody agent. In some alternative embodiments, the pharmaceutical composition may comprise (i) a first RNA comprising a heavy chain coding region encoding at least the V_H domain of an antibody agent, and (ii) a second RNA comprising a light chain coding region encoding at least the V_L domain of an antibody agent.

In some embodiments, the heavy chain coding region may also encode a constant heavy chain (C_H) domain, and/or the light chain coding region may also encode a constant light chain (C_L) domain. For example, in some embodiments, the heavy chain coding region may encode a V_H domain, a C_H1 domain, a C_H2 domain, and a C_H3 domain of an antibody agent in immunoglobulin G (IgG) form, and/or the light chain coding region may encode a V_L domain and a C_L domain of an antibody agent in IgG form. In some embodiments, the antibody agent in IgG form is IgG1.

In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full length heavy chain of zo Bei Tuo ximab or clausimab (Claudiximab). In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full length light chain of zoffiti Bei Tuo mab or clausimab.

In some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent can comprise a secretion signal encoding region. In some embodiments, such secretion signal encoding regions allow CLDN-18.2 targeted antibody agents encoded by one or more RNAs to be secreted after translation by cells (e.g., present in a subject to be treated), thus producing a plasma concentration of CLDN-18.2 targeted antibody agents that are biologically active.

Those of skill in the art will recognize the emerging field of nucleic acid therapeutics and, in addition, RNA (e.g., ssRNA, such as mRNA) therapeutics (see, e.g., mRNA encoding proteins and/or cytokines). Various embodiments of the technology provided herein can utilize specific features of RNA (e.g., ssRNA, such as mRNA) treatment techniques 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., without limitation, pseudouridine), nucleosides, and/or linkages. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) can comprise a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and/or translational efficiency. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, e.g., mRNA) can comprise a specific combination of at least two 3' utr sequences (e.g., a combination of sequence elements of an amino terminal enhancer of a split RNA with sequences derived from a mitochondrially encoded 12S RNA). Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) may comprise a' 5UTR sequence derived from human α -globin mRNA. Alternatively or additionally, in some embodiments, the RNA (e.g., ssRNA, such as mRNA) may comprise a 5' cap analog, e.g., for co-transcriptional capping. Alternatively or additionally, in some embodiments, an RNA (e.g., ssRNA, such as mRNA) may comprise a secretion signal coding region (e.g., a human secretion signal coding sequence) with reduced immunogenicity such that the encoded antibody agent is expressed and secreted. In some embodiments, the RNA can be formulated in or with one or more delivery vehicles (e.g., nanoparticles such as lipid nanoparticles, etc.). Alternatively or additionally, in some embodiments, the RNA can be formulated in or with liver-targeted lipid nanoparticles (e.g., cationic lipid nanoparticles).

In some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent can comprise at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, 3 'untranslated regions (untranslated region, UTR), 5' UTR, co-transcribed capped cap structures for mRNA, poly adenine (poly a) tails, and any combination thereof. For example, in some embodiments, the RNAs (e.g., the first RNA and/or the second RNA) each independently comprise (a) a 5'utr, (b) a secretion signal encoding region, (c) an antibody chain encoding region, (d) a 3' utr, and (e) a polyA tail in a 5 'to 3' direction. In some embodiments, the polyA tail coding region contained in the RNA is or comprises a modified polyA sequence.

In some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent can comprise a 5' cap.

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

In some embodiments in which the pharmaceutical composition comprises a first RNA encoding a variable heavy chain (V_H) domain of a CLDN-18.2 targeted antibody agent (e.g., a heavy chain of a CLDN-18.2 targeted antibody agent) and a second RNA encoding a variable light chain (V_L) domain of the antibody agent (e.g., a light chain of a CLDN-18.2 targeted antibody agent), such first RNA and second RNA may be present in a molar ratio of about 1.5:1 to about 1:1.5. In some embodiments, such first and second RNAs can be present in a ratio of about 1.30, about 1.29, about 1.28, about 1.27, about 1.26, about 1.25, about 1.24, about 1.23, about 1.22, about 1.21, about 1.20, about 1.19, about 1.18, about 1.17, about 1.16, about 1.15, about 1.14, about 1.13, about 1.12, about 1.11, about 1.10, about 1.09, about 1.08, about 1.07, about 1.06, about 1.05, about 1.04, about 1.03, about 1.02, about 1.01, about 1.00, about 0.99, about 0.98, about 0.97, about 0.96, about 0.95, about 0.94, about 0.93, about 0.92, about 0.91, about 0.90, about 0.89, about 0.88, about 0.84, about 0.83, about 0.82, or about 80. In some embodiments, such first RNA and second RNA may be present in a weight ratio of 3:1 to 1:1. In some embodiments, such first RNA and second RNA can be present in a weight ratio of about 2:1. In some embodiments, such first RNA and second RNA can 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.

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

In some embodiments, the lipid nanoparticle provided in the pharmaceutical compositions described herein is a liver-targeted lipid nanoparticle. In some embodiments, the lipid nanoparticle provided in the pharmaceutical compositions described herein is a cationic lipid nanoparticle. In some embodiments, the average size of the lipid plasmids provided in the pharmaceutical compositions described herein may be about 50 to 150nm.

In some embodiments, the lipid forming the lipid nanoparticle comprises a polymer conjugated lipid, a cationic lipid, and a neutral lipid. In some such embodiments, the polymer conjugated lipid is present at about 1mol% to 2.5mol% of the total lipid, the cationic lipid is present at 35mol% to 65mol% of the total lipid, and the neutral lipid is present at 35mol% to 65mol% of the total lipid.

A variety of lipids (which include, for example, polymer conjugated lipids, cationic lipids, and neutral lipids) are known in the art and may be used herein to form lipid nanoparticles, such as lipid nanoparticles targeted to a particular cell type (e.g., hepatocytes). In some embodiments, the polymer conjugated lipid included in the pharmaceutical compositions described herein can be a PEG conjugated lipid (e.g., 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylamide or a derivative thereof). In some embodiments, the cationic lipid included in the pharmaceutical compositions described herein may be ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octanoate) or a derivative thereof. In some embodiments, the neutral lipid included in the pharmaceutical compositions described herein may be or include a phospholipid or derivative thereof (e.g., 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC)) and/or cholesterol.

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

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

In some embodiments, the pharmaceutical compositions described herein are absorbable by target cells for use in producing a therapeutically relevant plasma concentration of the encoded CLDN-18.2 targeted antibody agent. In some embodiments, such pharmaceutical compositions described herein can induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against a target cell (e.g., a tumor cell).

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

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

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

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

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

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

The dosage of the pharmaceutical compositions described herein may vary depending on a number of factors including, for example, but not limited to, the weight of the subject to be treated, the type of cancer and/or the stage of the cancer, and/or monotherapy or combination therapy. In some embodiments, the pharmaceutical compositions described herein are administered to a subject having a CLDN-18.2 positive solid tumor in at least one or more (including, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more) dosing cycles. In some embodiments, each dosing cycle may be a three week dosing cycle. In some embodiments, the pharmaceutical compositions described herein are administered at least one dose per administration cycle. In some embodiments, the dosing period involves administering a set number and/or pattern of doses, in some embodiments, the dosing period involves administering a set cumulative dose, e.g., over a particular period of time, and optionally through multiple doses, which may be administered, e.g., at set intervals and/or according to a set pattern. In some embodiments, each dose or cumulative dose of the pharmaceutical compositions described herein may comprise one or more RNAs encoding CLDN-18.2-targeted antibody agents (whether encoded by a single RNA or two or more RNAs) in an amount ranging from 0.1mg/kg to 5mg/kg of body weight of the subject to be administered.

Another aspect of the present disclosure relates to certain improvements in methods of delivering CLDN-18.2 targeted antibody agents in a subject for use in cancer treatment, the methods comprising administering the provided pharmaceutical compositions to a cancer subject. In some embodiments, the pharmaceutical compositions described herein may achieve one or more improvements, such as effective administration with reduced TEAE (e.g., frequency and/or severity) and/or improved relationship between efficacy levels and TEAE levels (e.g., improved therapeutic window) relative to those observed when the corresponding (e.g., encoded) protein (e.g., antigen) agent itself is administered. In particular, the present disclosure teaches that such improvements can be achieved, inter alia, by delivering IMAB362 via administration of an RNA (e.g., ssRNA, e.g., mRNA) encoding IMAB 362.

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

In some embodiments, the cell is present in a cell culture.

In some embodiments, the cell is present in a subject. In some such embodiments, the pharmaceutical compositions described herein may be administered to a subject in need thereof. In some embodiments, such pharmaceutical compositions may be administered to a subject such that CLDN-18.2 targets an antibody agent to treat a related plasma concentration. In some embodiments, the therapeutically relevant plasma concentration is sufficient to mediate cancer cell death by Antibody Dependent Cellular Cytotoxicity (ADCC). For example, in some embodiments, the therapeutically relevant plasma concentration is from 0.3 to 28 μg/mL.

The present disclosure also provides, inter alia, methods of characterizing one or more characteristics of an RNA or a composition thereof, wherein the RNA encodes a portion or all of an antibody agent. In some embodiments, the method comprises the steps of determining one or more characteristics of an antibody expressed by at least one mRNA introduced into a cell, wherein such at least one mRNA comprises one or more characteristics of at least one or more RNAs comprising a coding region that encodes an antibody that binds to a claudin-18.2 (CLDN-18.2) polypeptide (e.g., preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide relative to a claudin-18.1 polypeptide), wherein such one or more characteristics comprise (i) the protein expression level of the antibody, (ii) the binding specificity of the antibody to the CLDN-18.2, (iii) the efficacy of the antibody in mediating target cell death by ADCC, and (iv) the efficacy of the antibody in mediating target cell death by Complement Dependent Cytotoxicity (CDC).

In some embodiments, provided herein are methods of characterizing a pharmaceutical composition that targets CLDN-18.2. Such methods comprise the steps of (a) contacting a cell with at least one composition or pharmaceutical composition described herein that encodes a part or all of a CLDN-18.2 targeted antibody agent, and detecting the antibody agent produced by the cell. In some embodiments, the cell may be or comprise a hepatocyte.

In some embodiments, such methods may further comprise determining one or more characteristics of the antibody agent expressed by one or more RNAs described herein, wherein such one or more characteristics comprise (i) a protein expression level of the antibody agent, (ii) a binding specificity of the antibody agent to the CLDN-18.2 polypeptide, (iii) an efficacy of the antibody agent in mediating target cell death by ADCC, and (iv) an efficacy of the antibody agent in mediating target cell death by Complement Dependent Cytotoxicity (CDC). In some embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein may comprise comparing such characteristics of a CLDN-18.2 targeted antibody agent to characteristics of a reference CLDN-18.2 targeted antibody.

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

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

In some embodiments, the provided methods of characterizing a CLDN-18.2-targeted pharmaceutical composition or component thereof may further comprise characterizing the antibody as a CLDN-18.2-targeted antibody if the antibody expressed by one or more RNAs described herein comprises (a) the protein level of the antibody expressed by the cell is above a threshold level, (b) the antibody preferentially binds to CLDN-18.2 relative to CLDN18.1, and (c) killing of at least 50% of target cells (e.g., cancer cells) is mediated by ADCC and/or CDC.

In some embodiments, if the test antibody characteristics are at least comparable to those of the adjuvant Bei Tuo mab or the clausimab, the provided methods of characterizing a CLDN-18.2-targeted pharmaceutical composition or component thereof may further comprise characterizing the antibody agent expressed by one or more RNAs described herein as an adjuvant Bei Tuo mab or a clausimab equivalent antibody.

In some embodiments involving the step of determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein, such step may include determining one or more of the following characteristics:

whether the cells express CLDN-18.2 targeted antibody agent encoded by at least one RNA when assessed 48 hours after contact or administration;

Whether the antibody agent expressed by the cell preferentially binds to CLDN 18.2 polypeptide relative to CLDN18.1 polypeptide;

Whether the antibody agent expressed by the cell exhibits target specificity for CLDN-18.2 comparable to the reference CLDN-18.2 targeted monoclonal antibody, as observed in flow cytometry binding assays;

When assessed after incubating immune effector cells (e.g., PBMC cells) with CLDN-18.2 positive cells or CLDN-18.2 negative control cells for 48 hours in the presence of an antibody, whether CLDN-18.2 positive cells, but not control cells, were lysed;

Whether the antibody agent expressed by the cell exhibits an ADCC profile of the targeted CLDN-18.2 positive cell at least comparable to that observed with the same concentration of reference CLDN-18.2 targeted monoclonal antibody, and

When assessed after incubation of CLDN-18.2 positive cells or CLDN-18.2 negative control cells with human serum for 2 hours in the presence of an antibody agent, whether CLDN-18.2 positive cells but not control cells were lysed.

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

Also included within the scope of the present disclosure are methods of manufacture comprising the steps of:

(A) Determining one or more characteristics of an RNA encoding a portion or all of an antibody agent or a composition thereof, the one or more characteristics selected from the group consisting of:

(i) The length and/or sequence of the RNA;

(ii) Integrity of RNA;

(iii) The presence and/or location of one or more chemical moieties in the RNA;

(iv) The degree of expression of the antibody agent when the RNA is introduced into the cell;

(v) Stability of RNA or a composition thereof;

(vi) Levels of antibody agents in a biological sample from an organism into which RNA has been introduced;

(vii) Binding specificity of the antibody agent expressed by RNA, optionally with CLDN-18.2 and optionally relative to CLDN 18.1;

(viii) The efficacy of antibody agents in mediating target cell death by ADCC;

(ix) The efficacy of antibody agents in mediating target cell death by Complement Dependent Cytotoxicity (CDC);

(x) Identity and amount/concentration of lipids within the composition;

(xi) Size of lipid nanoparticles in the composition;

(xii) Polydispersity of lipid nanoparticles in the composition;

(xiii) Amount/concentration of RNA in the composition;

(xiv) Encapsulation degree of RNA in lipid nanoparticle, and

(Xv) A combination thereof;

(B) Comparing such one or more characteristics of the RNA or composition thereof with characteristics of an appropriate reference standard, and

(C) (i) if the comparison indicates that the RNA or composition thereof meets or exceeds the reference standard, then designating the RNA or composition thereof for one or more additional steps of manufacture and/or dispensing, or

(Ii) If the comparison indicates that the RNA or composition thereof does not meet or exceed the reference standard, an alternative action is taken.

In some embodiments of the methods of manufacture, when an RNA (e.g., an RNA described herein) is evaluated and one or more characteristics of the RNA meet or exceed appropriate reference criteria, such RNA is designated for formulation, which in some embodiments involves, for example, formulation with lipid particles described herein.

In some embodiments of the methods of manufacture, when a composition comprising RNA (e.g., RNA as described herein) is evaluated and one or more characteristics of the composition meet or exceed appropriate reference criteria, such composition is designated for release and/or dispensing of the composition.

In some embodiments of the methods of manufacture, when RNA (e.g., RNA as described herein) is designated for formulation and/or a composition comprising RNA (e.g., RNA as described herein) is designated for release and/or dispensing of the composition, such methods can further comprise administering the formulation and/or composition to a group of animal subjects each bearing a CLDN-18.2 positive xenograft tumor to determine anti-tumor activity.

Also provided herein are methods of determining the dosing regimen of a pharmaceutical composition targeting CLDN-18.2. For example, in some embodiments, such methods comprise the steps of (A) administering a pharmaceutical composition (e.g., a pharmaceutical composition described herein) to a subject having a CLDN-18.2 positive solid tumor under a predetermined dosing regimen, (B) periodically monitoring or measuring the tumor size of the subject over a period of time, and (C) evaluating the dosing regimen based on the tumor size measurement. For example, if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is not therapeutically relevant, the dosage and/or frequency of administration may be increased, or if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant, but exhibits an adverse effect (e.g., a toxic effect) in a subject, the dosage and/or frequency of administration may be decreased. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show adverse effects (e.g., toxic effects) in the subject, no changes are made to the dosing regimen.

In some embodiments, such methods of determining a dosing regimen of a pharmaceutical composition that targets CLDN-18.2 can be performed in a group of animal subjects (e.g., mammalian non-human subjects) each bearing a CLDN-18.2 positive xenograft tumor. In some such embodiments, the dosage and/or frequency of administration may be increased if less than 30% of the animal subjects exhibit a decrease in tumor size and/or the extent of decrease in tumor size exhibited by the animal subjects is not therapeutically relevant after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein), or the dosage and/or frequency of administration may be decreased if the decrease in tumor size is therapeutically relevant after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein), but exhibits a significant adverse effect (e.g., toxic effect) in at least 30% of the animal subjects. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show significant adverse effects (e.g., toxic effects) in an animal subject, no change is made to the dosing regimen.

The present disclosure provides, inter alia, compositions or pharmaceutical formulations comprising:

(i) An RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and

(Ii) An RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), wherein

(I) The coding region comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO. 16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO. 16, and

(Ii) The coding region comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO. 17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO. 17.

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

The second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO. 4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO. 4.

The present disclosure also provides a composition or pharmaceutical formulation comprising:

(i) An RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and

(Ii) An RNA comprising a coding region encoding a second polypeptide chain 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.

In some embodiments, the RNA, e.g., each RNA, comprises a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20.

In some embodiments, the RNA, 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.

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.

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.

The present disclosure also provides a composition or pharmaceutical formulation comprising:

(i) An RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and

(Ii) An RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2),

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.

In some embodiments, the RNA, e.g., each RNA, comprises a 5'utr comprising the nucleotide sequence of SEQ ID No. 18 or 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 18 or 20 and a 3' utr comprising the nucleotide sequence of SEQ ID No. 19 or 21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 19 or 21.

In some embodiments, the RNA, e.g., each RNA, comprises a 5'utr comprising the nucleotide sequence of SEQ ID No. 18 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 18 and a 3' utr comprising the nucleotide sequence of SEQ ID No. 19 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 19.

In some embodiments, the RNA, e.g., each RNA, comprises a 5'utr comprising the nucleotide sequence of SEQ ID No. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 20 and a 3' utr comprising the nucleotide sequence of SEQ ID No. 21 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID No. 21.

In some embodiments, the RNA, 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.

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.

In some embodiments of the present invention, in some embodiments,

(A) The coding region described in (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO. 16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO. 16, and

(Ii) The coding region comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO. 17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO. 17, and/or

(B) The first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO.3 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO.3, and

The second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO. 4 or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO. 4.

In some embodiments of the present invention, in some embodiments,

(I) The coding region comprises the nucleotide sequence of SEQ ID NO. 16 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 16, and

(Ii) The coding region comprises the nucleotide sequence of SEQ ID NO. 17 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 17.

In some embodiments of the present invention, in some embodiments,

The first polypeptide chain comprises the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 3, and

The second polypeptide chain comprises the amino acid sequence of SEQ ID NO. 4 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO. 4.

In some embodiments, the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule.

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

In some embodiments, the antibody agent preferentially binds to CLDN-18.2 relative to claudin-18.1 (CLDN-18.1).

In some embodiments, the antibody agent binds to the first extracellular domain of CLDN-18.2 (ECD 1).

In some embodiments, the antibody agent binds to an epitope of ECD1 of CLDN-18.2 exposed in cancer cells.

In some embodiments, the antibody that binds to CLDN-18.2 comprises two binding arms, wherein each binding arm comprises a heavy chain of the antibody that binds to CLDN-18.2 and a light chain of the antibody that binds to CLDN-18.2.

In some embodiments, the antibody agent is IgG1.

In some embodiments, the IgG1 is human IgG1.

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

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

In some embodiments, the VH (CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO 14.

In some embodiments, the VH (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOS 5, 6 and 7, respectively.

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

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

In some embodiments, the VL (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOS 8, 9 and 10, respectively.

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

In some embodiments, the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH (CLDN-18.2)) comprising CDR1, CDR2 and CDR3 of the sequences shown in SEQ ID NO. 5, 6 and 7, respectively, and the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL (CLDN-18.2)) comprising CDR1, CDR2 and CDR3 of the sequences shown in SEQ ID NO. 8, 9 and 10, respectively.

In some embodiments, the first polypeptide chain comprises a variable domain (VH) comprising the amino acid sequence of SEQ ID NO. 14 that binds to CLDN-18.2 (VH (CLDN-18.2)), and the second polypeptide chain comprises a variable domain (VL) comprising the amino acid sequence of SEQ ID NO. 15 that binds to CLDN-18.2 (VL (CLDN-18.2)).

In some embodiments, the first polypeptide chain comprises a variable domain (VH) of a heavy chain of an antibody agent that binds to CLDN-18.2 (VH (CLDN-18.2)), and the second polypeptide chain comprises a variable domain (VL) of a light chain of an antibody agent that binds to CLDN-18.2 (VL (CLDN-18.2)), wherein the VH (CLDN-18.2) interacts with the VL (CLDN-18.2) to form a binding domain that binds to claudin-18.2 (CLDN-18.2).

In some embodiments, the first polypeptide chain comprises a variable domain of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH) (VH (CLDN-18.2)), a constant domain of the heavy chain of an antibody agent 1 (CH 1), a constant domain of the heavy chain of an antibody agent 2 (CH 2), and a constant domain of the heavy chain of an antibody agent 3 (CH 3).

In some embodiments, the VH (CLDN-18.2), CH1, CH2, and CH3 are present in the first polypeptide chain in immunoglobulin G (IgG) form.

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

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

In some embodiments, CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain.

In some embodiments, the first polypeptide chain and the second polypeptide chain each independently comprise a secretion signal, wherein the secretion signal is preferably located N-terminal to the first polypeptide chain and the second polypeptide chain.

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

In some embodiments, the coding region set forth in (i) comprises the nucleotide sequence of SEQ ID NO:16 and the coding region set forth in (ii) comprises the nucleotide sequence of SEQ ID NO: 17.

In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO.3 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO. 4.

In some embodiments, the RNA, e.g., each RNA, comprises a poly-A sequence.

In some embodiments, the poly-A sequence is a discontinuous sequence of A nucleotides.

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

In some embodiments, the poly-a sequence comprises or consists of nucleotide sequence a_x-L-A_y, wherein a_x is a sequence of at least 20 a nucleotides, a_y is a sequence of at least 60a nucleotides and L is a linker of 1 to 20 nucleotides that may comprise nucleotides other than a.

In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO. 23.

In some embodiments, the composition or pharmaceutical formulation contains:

(i) An RNA comprising the nucleotide sequence of SEQ ID NO. 24 or 26 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 24 or 26, and

(Ii) RNA comprising the nucleotide sequence of SEQ ID NO. 25 or 27 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 25 or 27.

In some embodiments, the composition or pharmaceutical formulation contains:

(i) RNA comprising the nucleotide sequence of SEQ ID NO. 24, and

(Ii) RNA comprising the nucleotide sequence of SEQ ID NO. 25.

In some embodiments, the composition or pharmaceutical formulation contains:

(i) RNA comprising the nucleotide sequence of SEQ ID NO. 26, and

(Ii) RNA comprising the nucleotide sequence of SEQ ID NO. 27.

The present disclosure also provides a composition or pharmaceutical formulation comprising:

(i) RNA comprising the nucleotide sequence of SEQ ID NO. 24 or 26, and

(Ii) RNA comprising the nucleotide sequence of SEQ ID NO. 25 or 27.

The present disclosure also provides a composition or pharmaceutical formulation comprising:

(i) RNA comprising the nucleotide sequence of SEQ ID NO. 24, and

(Ii) Comprising the nucleotide sequence of SEQ ID NO. 25.

The present disclosure also provides a composition or pharmaceutical formulation comprising:

(i) RNA comprising the nucleotide sequence of SEQ ID NO. 26, and

(Ii) RNA comprising the nucleotide sequence of SEQ ID NO. 27.

In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces uridine.

In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces each uridine.

In some embodiments, the modified nucleoside is pseudouridine (ψ) and/or N1-methyl-pseudouridine (m1ψ).

In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap.

In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap m₂^7,3'-OGppp(m₁^2'-O) ApG.

In some embodiments, the RNA, e.g., each RNA, is a single-stranded RNA.

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

In some embodiments, the RNA, e.g., each RNA, is formulated in a Lipid Nanoparticle (LNP), e.g., each RNA is co-formulated in a Lipid Nanoparticle (LNP).

In some embodiments, the lipid forming the lipid nanoparticle comprises a cationic lipid, a polymer conjugated lipid, and a neutral lipid.

In some embodiments of the present invention, in some embodiments,

A. the cationic lipid is present at 35mol% to 65mol% of the total lipid;

b. the polymer conjugated lipid is present at about 1mol% to 2.5mol% of the total lipid, and

C. The neutral lipids are present in 35mol% to 65mol% of the total lipids.

In some embodiments, the cationic lipid is ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate).

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

In some embodiments, the neutral lipid comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC) and/or cholesterol.

In some embodiments, the lipid nanoparticle has an average size of about 50 to 150nm.

In some embodiments, the lipid nanoparticle comprises ((3-hydroxypropyl) azetidinediyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

In some embodiments, the composition is a pharmaceutical composition.

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

In some embodiments, the pharmaceutical formulation is a kit.

In some embodiments, the RNAs, e.g., each RNA, and optionally the particle-forming components are in separate vials.

In some embodiments, the pharmaceutical formulation further comprises instructions for use of the composition or pharmaceutical formulation for treating or preventing cancer.

The present disclosure also provides a composition or pharmaceutical formulation as described herein for pharmaceutical use.

In some embodiments, the pharmaceutical use includes therapeutic or prophylactic treatment of a disease or disorder.

In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing cancer.

In some embodiments, the cancer comprises CLDN-18.2 positive cancer.

In some embodiments, the cancer comprises CLDN-18.2 positive solid tumors.

In some embodiments, the cancer comprises CLDN-18.2 positive pancreatic cancer.

In some embodiments, the cancer comprises CLDN-18.2 positive gastric cancer.

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

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

In some embodiments, the therapeutic or prophylactic treatment of the disease or disorder further comprises administration of an additional treatment.

In some embodiments, the additional treatment comprises one or more selected from (i) surgery to cut, resect or debulk a tumor, (ii) radiation therapy, and (iii) chemotherapy.

In some embodiments, the additional treatment comprises administration of an additional therapeutic agent.

In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent.

In some embodiments, the compositions or pharmaceutical formulations described herein are for administration to a human.

In some embodiments, the compositions or pharmaceutical formulations described herein are for intravenous administration.

The present disclosure also provides methods of treating cancer in a subject comprising administering to the subject a composition described herein.

In some embodiments, the cancer comprises CLDN-18.2 positive cancer.

In some embodiments, the cancer comprises CLDN-18.2 positive solid tumors.

In some embodiments, the cancer comprises CLDN-18.2 positive pancreatic cancer.

In some embodiments, the cancer comprises CLDN-18.2 positive gastric cancer.

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

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

In some embodiments, the methods described herein further comprise administering an additional treatment.

In some embodiments, the additional treatment comprises one or more selected from (i) surgery to cut, resect or debulk a tumor, (ii) radiation therapy, and (iii) chemotherapy.

In some embodiments, the additional treatment comprises administration of an additional therapeutic agent.

In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent.

In some embodiments, the subject is a human.

In some embodiments, the composition is administered intravenously.

The present disclosure also provides compositions described herein for use in methods described herein.

In some embodiments, after expression of the polypeptide chain of the antibody agent that binds to claudin-18.2 (CLDN-18.2), the polypeptide chain is secreted into the blood stream as a fully assembled antibody and/or as a functional antibody. Fully assembled antibodies are tetramers composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain of the antibody agent bound to claudin-18.2 (CLDN-18.2). A functional antibody is an antibody having the desired biological activity of the antibody, e.g., binding to a target of the antibody and/or recruiting and/or stimulating the immune system (e.g., ADCC), e.g., to the same or similar level as the corresponding antibody expressed in vitro.

In some embodiments, the compositions or pharmaceutical formulations described herein are used to introduce the RNA into a hepatocyte and express the polypeptide chain encoded by the RNA in the hepatocyte.

In some embodiments, the compositions or pharmaceutical formulations described herein are used for systemic delivery of the polypeptide chain.

In some embodiments, the compositions or pharmaceutical formulations described herein are used for systemic delivery of the polypeptide chain after expression of the polypeptide chain in hepatocytes.

The present disclosure also provides a method for expressing an antibody agent that binds to claudin-18.2 (CLDN-18.2) in a subject, the method comprising:

(a) Administering a composition described herein such that the RNA is introduced into hepatocytes, and

(B) Expressing in said liver cells the polypeptide chain encoded by said RNA.

The present disclosure also provides a method for expressing an antibody agent that binds to claudin-18.2 (CLDN-18.2) in a subject, the method comprising:

(a) Administering a composition described herein such that the RNA is introduced into hepatocytes, and

(B) Expressing the polypeptide chain encoded by said RNA in said liver cells,

Wherein, following expression, the polypeptide chain is secreted into the blood stream.

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, the method comprising:

(a) Administering a composition described herein such that the RNA is introduced into hepatocytes, and

(B) Expressing the polypeptide chain encoded by said RNA in said liver cells,

Wherein, following expression, the polypeptide chain is secreted into the blood stream.

In some embodiments, the administration is parenteral.

In some embodiments, the administration is intravenous administration.

The present disclosure also provides, inter alia, the following:

1. a pharmaceutical composition comprising:

a. At least one single stranded RNA comprising one or more codes for an antibody agent that preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide over a claudin-18.1 polypeptide

Zone and

B. Lipid nanoparticles;

Wherein the at least one single stranded RNA is encapsulated within at least one of the lipid nanoparticles.

2. The pharmaceutical composition of clause 1, wherein the antibody agent specifically binds to the first extracellular domain (ECD 1) of the CLDN-18.2 polypeptide.

3. The pharmaceutical composition of item 2, wherein the antibody agent specifically binds to an epitope of exposed ECD1 in the cancer cell.

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

5. The pharmaceutical composition of any one of items 1 to 4, wherein the at least one single stranded RNA encodes both a variable heavy chain (V_H) domain of the antibody agent and a variable light chain (V_L) domain of the antibody agent.

6. The pharmaceutical composition of item 5, wherein said at least one single stranded RNA is a first single stranded RNA comprising at least a heavy chain coding region encoding a V_H domain of said antibody agent, and

A. wherein said first single stranded RNA further comprises a light chain coding region encoding at least the V_L domain of said antibody agent, or

B. Wherein the pharmaceutical composition further comprises a second single stranded RNA comprising a light chain coding region encoding at least the V_L domain of the antibody agent.

7. The pharmaceutical composition of item 6, wherein the heavy chain coding region further encodes a constant heavy chain (C_H) domain and/or the light chain coding region further encodes a constant light chain (C_L) domain.

8. The pharmaceutical composition of item 6, wherein said heavy chain coding region encodes the V_H domain, C_H1 domain, C_H2 domain, and C_H3 domain of said antibody agent in immunoglobulin G (IgG) form, and/or said light chain coding region encodes the V_L domain and C_L domain of said antibody agent in IgG form.

9. The pharmaceutical composition of item 8, wherein the IgG is IgG1.

10. The pharmaceutical composition of any one of clauses 6-9, wherein the heavy chain coding region consists of or comprises a nucleotide sequence encoding a full length heavy chain of zoysizumab or clausimab, or comprises a nucleotide sequence encoding a full length heavy chain of zoysiab Bei Tuo.

11. The pharmaceutical composition of any one of clauses 6-9, wherein the light chain coding region consists of or comprises a nucleotide sequence encoding a full length light chain of zoysizumab or clausimab, or comprises a nucleotide sequence encoding a full length light chain of zoysimab or clausimab.

12. The pharmaceutical composition of any one of items 6 to 11, wherein the first single-stranded RNA and/or the second single-stranded RNA each independently comprises a secretion signal coding region.

13. The pharmaceutical composition of any one of clauses 6 to 12, wherein the first single-stranded RNA and/or the second single-stranded RNA each independently comprises at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency).

14. The pharmaceutical composition of item 13, wherein the at least one non-coding sequence element comprises a3 'untranslated region (UTR), a 5' UTR, a co-transcribed capped cap structure for mRNA, and/or a poly-adenine (polyA) tail.

15. The pharmaceutical composition of any one of items 6 to 14, wherein the first single stranded RNA comprises in a5 'to 3' direction:

a 5' UTR coding region;

b. a secretion signal coding region;

c. Heavy chain coding region;

d.3' UTR coding region, and

PolyA tail coding region.

16. The pharmaceutical composition of any one of items 6 to 15, wherein the second single stranded RNA comprises in a5 'to 3' direction:

a 5' UTR coding region;

b. a secretion signal coding region;

c. A light chain coding region;

d.3' UTR coding region, and

PolyA tail coding region.

17. The pharmaceutical composition of clause 14 or 15, wherein the polyA tail is or comprises a modified polyA sequence.

18. The pharmaceutical composition of any one of items 6 to 16, wherein the first single stranded RNA and/or the second single stranded RNA comprises a 5' cap.

19. The pharmaceutical composition of any one of items 6 to 18, wherein the first single stranded RNA and/or the second single stranded RNA comprises at least one modified ribonucleotide.

20. The pharmaceutical composition of item 19, wherein the modified ribonucleotide comprises pseudouridine.

21. The pharmaceutical composition of any one of items 6 to 20, wherein the at least one single stranded RNA comprises the first single stranded RNA and the second single stranded RNA.

22. The pharmaceutical composition of any one of items 6 to 21, wherein the first single stranded RNA and the second single stranded RNA are present in a weight ratio of 3:1 to 1:1.

23. The pharmaceutical composition of any one of clauses 1 to 22, wherein the lipid nanoparticle is a liver-targeted lipid nanoparticle.

24. The pharmaceutical composition of any one of clauses 1 to 23, wherein the lipid nanoparticle is a cationic lipid nanoparticle.

25. The pharmaceutical composition of item 24, wherein the lipid forming the lipid nanoparticle comprises:

-a polymer conjugated lipid;

-cationic lipid, and

-Neutral lipids.

26. The pharmaceutical composition of item 25, wherein:

a. The polymer conjugated lipid is present at about 1mol% to 2.5mol% of the total lipid;

b. The cationic lipid is present in 35 to 65 mole% of the total lipid, and

C. The neutral lipids are present in 35mol% to 65mol% of the total lipids.

27. The pharmaceutical composition of clause 25 or 26, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylamide).

28. The pharmaceutical composition of any one of clauses 25 to 27, wherein the cationic lipid is ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (2-octanoate).

29. The pharmaceutical composition of any one of clauses 25 to 28, wherein the neutral lipid comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC) and/or cholesterol.

30. The pharmaceutical composition of any one of clauses 1 to 29, wherein the lipid nanoparticle has an average size of about 50 to 150nm.

31. The pharmaceutical composition of any one of clauses 1 to 30, further comprising a cryoprotectant (e.g., sucrose).

32. The pharmaceutical composition of any one of clauses 1 to 31, comprising an aqueous buffer solution.

33. The pharmaceutical composition of item 32, wherein the aqueous buffer solution comprises sodium ions.

34. The pharmaceutical composition of any one of clauses 1 to 33, further comprising a chemotherapeutic agent.

35. The pharmaceutical composition of item 34, wherein the chemotherapeutic agent is a chemotherapeutic agent suitable for treating pancreatic cancer.

36. The pharmaceutical composition of any one of clauses 1 to 35, wherein the at least one single stranded RNA is present at a concentration of 0.5mg/mL to 1.5 mg/mL.

37. A method comprising administering the pharmaceutical composition of any one of items 1 to 36 to a subject having a CLDN-18.2 positive solid tumor.

38. The method of item 37, wherein the CLDN-18.2 positive tumor is a pancreatic tumor.

39. The method of item 37, wherein the CLDN-18.2 positive tumor is a gastric tumor.

40. The method of item 37, wherein the CLDN-18.2 positive tumor is a biliary tract tumor.

41. The method of any one of items 37 to 40, wherein the CLDN-18.2 positive solid tumor is locally advanced, unresectable or metastatic.

42. The method of any one of items 37 to 41, wherein the subject has received a pretreatment sufficient to increase CLDN-18.2 levels such that the subject has a solid tumor characterized as a CLDN-18.2 positive solid tumor.

43. The method of any one of clauses 37 to 42, wherein the CLDN-18.2 positive tumor is characterized by ≡50% of tumor cells exhibiting ≡2+ CLDN-18.2 protein staining intensity as assessed by immunohistochemical assay in formalin fixed paraffin embedded tumor tissue from the subject.

44. The method of any one of clauses 37 to 43, wherein the pharmaceutical composition is administered as a monotherapy.

45. The method of any one of items 37 to 44, wherein the pharmaceutical composition is administered as part of a combination therapy comprising the pharmaceutical composition and a chemotherapeutic agent.

46. The method of any one of items 37 to 45, wherein the subject has received the chemotherapeutic agent.

47. The method of item 45, further comprising administering the chemotherapeutic agent to the subject such that the subject receives the combination therapy.

48. The method of clause 47, wherein the chemotherapeutic agent is administered at least four hours after administration of the pharmaceutical composition.

49. The method of any one of clauses 45 to 48, wherein for a subject having a CLDN-18.2 positive pancreatic tumor, the chemotherapeutic agent is or comprises gemcitabine and/or paclitaxel (e.g., nab-paclitaxel).

50. The method of any one of clauses 45 to 48, wherein the chemotherapeutic agent is or comprises FOLFIRINOX for a subject having a CLDN-18.2-positive pancreatic tumor.

51. The method of any one of clauses 45 to 48, wherein for a subject having CLDN-18.2 positive biliary tract cancer, the chemotherapeutic agent is or comprises gemcitabine and/or cisplatin.

52. The method of any one of items 37 to 51, wherein the subject is an adult subject.

53. The method of any one of claims 37 to 52, wherein the administering is by intravenous injection.

54. The method of any one of claims 37 to 53, wherein the pharmaceutical composition is administered in at least one, at least two, at least three, or more dosing cycles.

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

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

57. The method of clause 55 or 56, wherein said one or more doses comprise said at least one single stranded RNA in the range of 0.1mg/kg to 5mg/kg of body weight of said subject.

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

59. A method of producing a CLDN-18.2 targeting antibody comprising administering the pharmaceutical composition of any one of items 1-35 to a cell such that the cell expresses and secretes a CLDN-18.2 targeting antibody encoded by at least one single stranded RNA in the pharmaceutical composition.

60. The method of clause 59, wherein the cell is a hepatocyte.

61. The method of clause 59 or 60, wherein the cell is in a subject.

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

63. The method of clause 62, wherein the treatment-related plasma concentration is sufficient to mediate cancer cell death by antibody-dependent cellular cytotoxicity (ADCC).

64. The method of clause 63, wherein the treatment-related plasma concentration is 0.3 to 28 μg/mL.

65. A method comprising the steps of:

Determining one or more characteristics of an antibody agent expressed by at least one mRNA introduced into a cell, wherein the at least one mRNA comprises one or more characteristics of at least one or more single stranded RNAs comprising a coding region encoding an antibody agent that preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide relative to a claudin-18.1 polypeptide, wherein the one or more characteristics comprise (i) a protein expression level of the antibody agent, (ii) a binding specificity of the antibody agent to CLDN-18.2, (iii) a potency of the antibody agent to mediate target cell death by ADCC, and (iv) a potency of the antibody agent to mediate target cell death by Complement Dependent Cytotoxicity (CDC).

66. A method of characterizing a CLDN-18.2-targeted pharmaceutical composition, the method comprising the steps of:

contacting a cell with at least one pharmaceutical composition according to any one of items 1 to 35, and

Detecting the antibody produced by the cell.

67. The method of item 66, further comprising determining one or more characteristics of the antibody agent, wherein the one or more characteristics comprise (i) a protein expression level of the antibody agent, (ii) a binding specificity of the antibody agent to a CLDN-18.2 polypeptide, (iii) an efficacy of the antibody agent in mediating target cell death by ADCC, and (iv) an efficacy of the antibody agent in mediating target cell death by Complement Dependent Cytotoxicity (CDC).

68. The method of any one of clauses 65 to 67, wherein the cell is a hepatocyte.

69. The method of clause 65 or 67, wherein the determining step comprises comparing one or more characteristics of the antibody agent to characteristics of a reference CLDN-18.2 targeted antibody.

70. The method of any one of clauses 65 and 67 to 69, wherein the determining step comprises assessing that the protein expression level of the antibody agent is above a threshold level.

71. The method of item 70, wherein the threshold level is a level sufficient to induce ADCC.

72. The method of any one of clauses 65 and 67 to 71, wherein the determining step comprises assessing the binding of the antibody agent to the CLDN-18.2 polypeptide.

73. The method of clause 72, wherein the evaluating comprises determining the binding of the antibody agent to the CLDN-18.2 polypeptide relative to the binding of the antibody agent to the CLDN18.1 polypeptide.

74. The method of clause 72 or 73, wherein the evaluating comprises determining that the binding preference profile of the antibody agent is at least comparable to the binding preference profile of the reference CLDN-18.2 targeting antibody.

75. The method of clause 69 or 74, wherein the reference CLDN-18.2 targeting antibody is zoffiti Bei Tuo mab or clausimab.

76. The method of any one of clauses 65 to 75, further characterized as a CLDN-18.2 targeted antibody if the antibody comprises the following features:

a. The cells express an antibody agent at a protein level above a threshold level sufficient to induce ADCC;

b. the antibody agent preferentially binds to CLDN-18.2 relative to CLDN 18.1;

And

C. Killing of at least 50% of target cells is mediated by ADCC and/or CDC.

77. The method of item 76, further characterized as an adjuvant Bei Tuo mab or a clausimab equivalent antibody if the antibody has at least comparable characteristics to those of adjuvant Bei Tuo mab or clausimab.

78. The method of any one of clauses 65 to 77, wherein the target cell is a cancer cell.

79. The method of any one of clauses 65 and 66 to 78, wherein the step of determining comprises determining whether the cell expresses an anti-CLDN 18-2 antibody agent encoded by the at least one single stranded RNA when assessed after 48 hours of contact.

80. The method of any one of items 65 and 66 to 79, wherein the determining step comprises determining one or more of the following features:

-whether the antibody agent expressed by the cell preferentially binds to CLDN 18.2 polypeptide over CLDN18.1 polypeptide;

Whether the antibody agent expressed by the cells exhibits a target specificity towards CLDN-18.2 according to what is observed in a flow cytometry binding assay with reference to a CLDN-18.2 targeted monoclonal antibody;

-incubating immune effector cells (e.g., PBMC cells) with CLDN-18.2 positive cells or CLDN-18.2 negative control cells in the presence of the antibody for 48 hours before assessing whether the CLDN-18.2 positive cells but not the control cells are lysed;

Whether the antibody agent expressed by said cells exhibits an ADCC profile targeting clDN-18.2 positive cells at least comparable to that observed with the same concentration of reference clDN-18.2 targeting monoclonal antibody, and

-Whether CLDN-18.2 positive cells, or CLDN-18.2 negative control cells, were lysed instead of the control cells when evaluated after incubating the CLDN-18.2 positive cells with human serum for 2 hours in the presence of the antibody agent.

81. The method of any one of clauses 66 to 80, wherein the cell is present in a subject (e.g., a mouse or monkey subject).

82. The method of item 81, wherein the one or more characteristics comprise antibody levels in one or more tissues of the subject.

83. The method of any one of items 66 to 82, further comprising:

If the pharmaceutical composition is characterized as CLDN-18.2 targeted, the pharmaceutical composition is administered to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor to determine anti-tumor activity.

84. A method of manufacture, the method comprising the steps of:

(A) Determining one or more characteristics of a single stranded RNA (ssRNA) or a composition thereof, the ssRNA encoding a portion or all of an antibody agent, the one or more characteristics selected from the group consisting of:

(i) The length and/or sequence of the ssRNA;

(ii) Integrity of the ssRNA;

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

(iv) The degree of expression of the antibody agent when the ssRNA is introduced into a cell;

(v) Stability of the ssRNA or composition thereof;

(vi) Antibodies in biological samples from organisms into which the ssRNA has been introduced

The level of agent;

(vii) Binding specificity of the antibody agent expressed by the ssRNA, optionally with CLDN-18.2 and optionally relative to CLDN 18.1;

(viii) The efficacy of the antibody agent in mediating target cell death by ADCC;

(ix) The antibody agents mediate target cells through Complement Dependent Cytotoxicity (CDC)

Efficacy of death;

(x) The identity and amount/concentration of lipids within the composition;

(xi) Size of lipid nanoparticles within the composition;

(xii) Polydispersity of lipid nanoparticles within the composition;

(xiii) The amount/concentration of ssRNA within the composition;

(xiv) The degree of encapsulation of the ssRNA within the lipid nanoparticle, and

(Xv) A combination thereof;

(B) Comparing one or more characteristics of the ssRNA or composition thereof to characteristics of an appropriate reference standard, and

(C) (i) if the comparison indicates that the ssRNA or composition thereof meets or exceeds the reference standard, then designating the ssRNA or composition thereof for one or more additional steps of manufacture and/or dispensing, or

(Ii) If the comparison indicates that the ssRNA or composition thereof does not meet or exceed the reference standard, an alternative action is taken.

85. The method of item 84, wherein the ssRNA is evaluated and the one or more additional steps of step (C) (i) are or include at least formulating the ssRNA.

86. The method of clause 84 or 85, wherein the composition is evaluated and the composition comprises lipid nanoparticles, and the one or more additional steps of step (C) (i) are or comprise release and dispensing comprising the composition.

87. The method of item 85, further comprising administering the formulation to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor to determine anti-tumor activity.

88. A method of determining a dosing regimen for a CLDN-18.2 targeted pharmaceutical composition, the method comprising the steps of:

(A) Administering the pharmaceutical composition of any one of items 1 to 35 to a group of animal subjects each bearing a human CLDN-18.2 positive xenograft tumor under a predetermined dosing regimen;

(B) Periodically measuring tumor size of the animal subject;

(C) (i) increasing the dose and/or frequency of administration if the decrease in tumor size after administration of the pharmaceutical composition is not therapeutically relevant, or

(Ii) If the decrease in tumor size after administration of the pharmaceutical composition is therapeutically relevant and shows a toxic effect in at least 30% of the animal subjects, the dose and/or the frequency of administration is decreased, or

(Iii) If the decrease in tumor size after administration of the pharmaceutical composition is treatment-related and does not show toxic effects in the animal subject, no change is made to the dosing regimen.

The present disclosure also provides insight that the 3' end region of mRNA is a very sensitive and specific region in terms of translation ability as well as mRNA function. Both in vitro and in vivo results indicate that single nucleotide substitutions upstream of the poly (a) tail have an effect on the translation ability and function of mRNA.

Thus, the present disclosure also provides, inter alia, compositions or pharmaceutical formulations comprising RNA,

Wherein the RNA comprises:

(i) A coding sequence encoding a polypeptide,

(Ii) The sequence of the 3' UTR,

(Iii) A poly-A sequence, and

(Iv) A nucleotide sequence linking the 3' utr sequence and the poly-a sequence comprising sequence CUXGAGCUAGC, wherein X is C, A or U.

In some embodiments, the nucleotide sequence linking the 3' utr sequence and the poly-a sequence comprises sequence CUCGAGCUAGC.

In some embodiments, the RNA comprises in a 5'→3' direction the coding sequence encoding the polypeptide, the 3'utr sequence, a nucleotide sequence linking the 3' utr sequence and the poly-a sequence, and the poly-a sequence.

In some embodiments, the 3' UTR sequence comprises the nucleotide sequence of SEQ ID NO. 22 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 22.

In some embodiments, the RNA comprises a 3' UTR comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36.

In some embodiments, the RNA comprises a 3' utr comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID No. 37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID No. 37.

In some embodiments, the poly-A sequence is a discontinuous sequence of A nucleotides.

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

In some embodiments, the poly-a sequence comprises or consists of nucleotide sequence a_x-L-A_y, wherein a_x is a sequence of at least 20 a nucleotides, a_y is a sequence of at least 60a nucleotides and L is a linker of 1 to 20 nucleotides that may comprise nucleotides other than a.

In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO. 23 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 23.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO. 20.

In some embodiments, the RNA comprises a 5' utr comprising the nucleotide sequence of nucleotides 14 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 X1 is any nucleotide, preferably a or C, X2 is any nucleotide, preferably a or C, X3 is any nucleotide, preferably C, U or G, and X4 is a or is deleted.

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 X1 is any nucleotide, preferably a or C, and X3 is any nucleotide, preferably C or U.

In some embodiments, the RNA comprises a 5' utr comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID No.20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID No.20, which is preceded by a sequence comprising nucleotide sequence AGAAUAAACUAGU.

In some embodiments, the RNA comprises a 5' utr comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID No.20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID No.20, which is preceded by a sequence comprising nucleotide sequence AGCACAAACUAGU.

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.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 38.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 20, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36, and a poly-A sequence.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 20 and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 36.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 38, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36, and a poly-A sequence.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 38 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 38 and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of SEQ ID NO. 36 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 36.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20 and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36, and a poly-A sequence.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 20 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36, and a poly-A sequence.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 20 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequence of SEQ ID NO. 36.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 38 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO. 36, and a poly-A sequence.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO:38 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequence of SEQ ID NO: 36.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO. 37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO. 37.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO. 20 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 20, and a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of 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 a sequence downstream of the coding sequence encoding the polypeptide comprising the nucleotide sequence of SEQ ID NO. 37 or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO. 37.

In some embodiments, the RNA comprises a 5' UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO. 20 and a sequence downstream of the coding sequence encoding the polypeptide comprising 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 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequences 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 and a sequence downstream of the coding sequence for the polypeptide comprising the nucleotide sequence of SEQ ID NO. 37.

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

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

In some embodiments, the polypeptide encoded by the coding sequence is an antibody or polypeptide chain thereof, e.g., an antibody or polypeptide chain thereof that binds CLDN-18.2. In some embodiments, the antibody that binds to CLDN-18.2 or a polypeptide chain thereof is as described herein. However, the polypeptides encoded by the coding sequences may be any polypeptide, including but not limited to pharmaceutically active polypeptides and peptides, particularly those described herein.

In some embodiments, the RNA does not encode a polypeptide that binds to claudin-6 (CLDN-6) and/or CD 3.

In some embodiments, the RNA does not encode one or more polypeptide chains of a binding agent that binds to claudin-6 (CLDN-6) and/or CD 3.

In some embodiments, the RNA does not encode a cytokine.

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

In some embodiments, the RNA does not encode a polypeptide that binds to HIV.

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

In some embodiments, the RNA does not encode a polypeptide that binds to claudin-18.2 (CLDN-18.2).

In some embodiments, the RNA does not encode one or more polypeptide chains of a binding agent that binds to claudin-18.2 (CLDN-18.2).

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

In some embodiments, the RNA comprises at least two, e.g., two, RNA molecules, and at least one of the RNA molecules, e.g., the entirety of the RNA molecules, comprises a 5' utr, 3' utr sequence, poly-a sequence, and/or a nucleotide sequence linking the 3' utr sequence and the poly-a sequence as defined herein.

In some embodiments, the RNA contains:

(i) An RNA comprising a coding sequence encoding a first polypeptide chain comprising a heavy chain of an antibody agent, and

(Ii) An RNA comprising a coding sequence encoding a second polypeptide chain comprising a light chain of an antibody agent.

In some embodiments, the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule.

In some embodiments, the antibody agent binds to claudin-18.2 (CLDN-18.2).

In some embodiments, the antibody agent that binds to CLDN-18.2 is as described herein. In some embodiments, the coding sequence encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to CLDN-18.2 and the coding sequence encoding 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, a first polypeptide chain comprising a heavy chain of an antibody that binds to CLDN-18.2 and a second polypeptide chain comprising a light chain of an antibody that binds to CLDN-18.2 are as described herein.

In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces uridine.

In some embodiments, the RNA, e.g., each RNA, comprises a modified nucleoside that replaces each uridine.

In some embodiments, the modified nucleoside is pseudouridine (ψ) and/or N1-methyl-pseudouridine (m1ψ).

In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap.

In some embodiments, the RNA, e.g., each RNA, comprises a 5' cap m₂^7,3'-OGpp(m₁^2'-O) ApG.

In some embodiments, the RNA, e.g., each RNA, is a single-stranded RNA.

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

In some embodiments, the RNA, e.g., each RNA, is formulated in a Lipid Nanoparticle (LNP), e.g., each RNA is co-formulated in a Lipid Nanoparticle (LNP).

In some embodiments, the lipid forming the lipid nanoparticle comprises a cationic lipid, a polymer conjugated lipid, and a neutral lipid.

In some embodiments of the present invention, in some embodiments,

A. the cationic lipid is present at 35mol% to 65mol% of the total lipid;

b. the polymer conjugated lipid is present at about 1mol% to 2.5mol% of the total lipid, and

C. The neutral lipids are present in 35mol% to 65mol% of the total lipids.

In some embodiments, the cationic lipid is ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate).

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

In some embodiments, the neutral lipid comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC) and/or cholesterol.

In some embodiments, the lipid nanoparticle has an average size of about 50 to 150nm.

In some embodiments, the lipid nanoparticle comprises ((3-hydroxypropyl) azetidinediyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

In some embodiments, the composition is a pharmaceutical composition.

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

In some embodiments, the pharmaceutical formulation is a kit.

In some embodiments, the RNAs, e.g., each RNA, and optionally the particle-forming components are in separate vials.

In some embodiments, the composition or pharmaceutical formulation is for intravenous administration.

In some embodiments, the composition or pharmaceutical formulation is used to introduce the RNA into a hepatocyte and express a polypeptide encoded by the RNA in the hepatocyte.

In some embodiments, the composition or pharmaceutical formulation is for systemic delivery of the polypeptide. In some embodiments, the composition or pharmaceutical formulation is used for systemic delivery of the polypeptide after expression of the polypeptide in hepatocytes.

The present disclosure also provides, inter alia, methods for expressing a polypeptide in a subject, the methods comprising:

(a) Administering a composition described herein such that RNA encoding the polypeptide is introduced into hepatocytes, and

(B) Expressing the polypeptide in the liver cells.

The present disclosure also provides, inter alia, methods for expressing a polypeptide in a subject, the methods comprising:

(a) Administering a composition described herein such that RNA encoding the polypeptide is introduced into hepatocytes, and

(B) Expressing the polypeptide in the liver cells,

Wherein, following expression, the polypeptide is secreted into the blood stream.

The present disclosure also provides, inter alia, methods for systemic delivery of polypeptides in a subject, the methods comprising:

(a) Administering a composition described herein such that RNA encoding the polypeptide is introduced into hepatocytes, and

(B) Expressing the polypeptide in the liver cells,

Wherein, following expression, the polypeptide is secreted into the blood stream.

In some embodiments, the administration is parenteral. In some embodiments, the administration is intravenous administration.

Drawings

FIG. 1 shows expression of a CLDN-18.2 targeting antibody encoded by two RNAs encoding the heavy and light chains, respectively, of the CLDN-18.2 targeting antibody in primary human hepatocytes and CHO-K1 cells (RiboMab 01). (Panel A) Primary human hepatocytes were lipofected with 0.22 to 55.50 μg/mL of a composition comprising two or more RNAs encoding the heavy and light chains, respectively, of a CLDN-18.2 targeting antibody (RB_ RMAB 01). ELISA assay at RiboMab01 concentration 48 hours after (left) transfection. Western blot analysis of cell culture supernatants from designated liposome transfection (right). Recombinant purified IMAB362 was used as a reference for Western blot analysis. Analysis was performed under non-reducing conditions and with HRP conjugated anti-human antibody. Mixtures of fcγ fragment-specific and anti-kappa light chain-specific antibodies were used to detect full length IgG, free Heavy Chain (HC) and free Light Chain (LC). Supernatant from untransfected primary human hepatocytes was used as a simulated control. (Panel B) Liposome transfection of CHO-K1 cells was performed with 2.00 to 182.00ng/mL RB_ RMAB 01. The RiboMab01 concentrations determined by ELISA 48 hours after transfection are shown. Error bars are standard error of the mean (n=3).

FIG. 2 shows RiboMab01 binding to a target specific for CLDN-18.2. The targeted binding of RiboMab01 to CLDN-18.2 was determined by a flow cytometry binding assay visualized using a fluorescent-labeled antibody directed against the F (ab') 2 fragment of human IgG (h+l). Diluted rows of CHO-K1 cell culture supernatants containing RiboMab a 01 (panels a and B, left) or IMAB362 reference proteins (panels a and B, right) were incubated with 5 x 10⁵ (panel a) CLDN-18.2+ or (panel B) CLDN18.1+ HEK293 transfectants.

Figure 3 shows high target-specific cytotoxicity mediated by RiboMab a 01 expressed in vitro. Cell culture supernatants containing RiboMab01 from CHO-K1 cells transfected with RB RMAB01 liposomes were subjected to (panel a) ADCC and (panel B) CDC assays. (Panel A) for the ADCC assay, CLDN-18.2+NUG-C4 transfectants were used as target cells and CLDN-18.2 negative MDA-MB-231 cells were used as control cells. Human PBMC from three different healthy donors were used as effector cells (E: T: 30:1). Target cells or control cells and effector cells were incubated with indicated RiboMab and IMAB362 reference protein concentrations for 48 hours. Specific cell lysis as determined in luciferase-based assays is shown. (Panel B) for CDC assay, clDN-18.2+CHO-K1 transfectants (solid line) were used as target cells and clDN-18.2-negative CHO-K1 (dotted line) were used as control cells. Target cells and control cells were incubated with human serum and RiboMab a 01 at the indicated concentrations for 2 hours. CDC determined in luciferase-based assays is shown. Error bars are standard error of the mean (n=3).

FIG. 4 shows RiboMab 01-mediated specific tumor cell lysis generated in mice. Plasma from mice dosed with 5 repeated injections of 1 μg (about 0.04 mg/kg), 3 μg (about 0.10 mg/kg), 10 μg (about 0.40 mg/kg) and 30 μg (about 1.20 mg/kg) RB_ RMAB01 or 80 μg (about 3.20 mg/kg) IMAB362 24 hours after the 5 th injection was sampled and used for luciferase-based in vitro ADCC assays. IMAB 362-doped plasma from untreated mice was used as a measurement reference. CLDN-18.2+nug-C4 transfectants were used as targets and human PBMCs were used as effector cells. (panel a) shows RiboMab 01-mediated ADCC of NUG-C4 cells after 48 hours incubation with 1% plasma. (Panel B) there was no non-specific lysis of target negative MDA-MB-231 cells. Error bars are standard error of the mean (n=3).

Figure 5 shows RiboMab that is expressed by non-human primates mediates dose-dependent ADCC. Non-human primate (Non-Human Primate, NHP) received three repeat doses of 0.1, 0.4 or 1.6mg/kg RB_ RMAB01 once a week. Serum containing RiboMab a 01 from all monkeys sampled 24 hours (black bars) and 168 hours (white bars) after the first injection was used for luciferase-based ex vivo ADCC assay. CLDN-18.2+nug-C4 transfectants were used as target cells. Human PBMCs from two different healthy donors (24 hours, donor 1,168 hours, donor 2) were used as effector cells. (panel a) shows RiboMab 01-mediated ADCC of NUG-C4 cells after 48 hours of incubation. (Panel B) shows nonspecific lysis of target negative MDA-MB-231 cells. Error bars are standard error of the mean (n=3). (Panel C) serum of NHP No.14 (1.6 mg/kg RB_ RMAB01, riboMab01 serum concentration 232. Mu.g/mL) collected 48 hours after the third dose was used for luciferase-based ex vivo ADCC assay. CLDN-18.2+nug-C4 transfectants (solid line) were used as target cells and CLDN-18.2 negative MDA-MB-231 cells (dotted line) as control cells. Human PBMCs of healthy donors were used as effector cells. ADCC is shown for NUG-C4 cells mediated by serum containing RiboMab (red solid line) or recombinant-IMAB 362 reference protein (black solid line), with EC50 of 66pM and 151pM, respectively. The red and black dashed lines represent weak non-specific lysis of MDA-MB-231 control cells. Incubation time was 48 hours. Error bars are standard error of the mean (n=3).

Fig. 6 shows that the systemic availability of RiboMab01 mediates tumor growth inhibition in vivo. Mice bearing subcutaneous CLDN-18.2+ nci-N87 xenograft tumors were subjected to IV injection of 1 μg (about 0.04 mg/kg), 3 μg (about 0.10 mg/kg), 10 μg (about 0.40 mg/kg) and 30 μg (about 1.20 mg/kg) of rb_ RMAB01, 800 μg (about 32 mg/kg) of IMAB362 reference protein, 30 μg (about 1.20 mg/kg) of luciferase mRNA or saline only on days 15, 22, 29, 36, 43 and 50 after tumor cell inoculation. Median tumor growth is shown for the treated and control groups. The dashed line indicates injection. Significance was calculated by two-way ANOVA. ns indicates no significance.

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

Fig. 8 shows the concentration-time curve of RiboMab01 in rat serum after a single administration. RjHan Wister rats received a single IV injection of 0.04, 0.10, 0.40 or 1.20mg/kg of RB RMAB01 and 3.60mg/kg of IMAB362 reference protein. Plasma was sampled at 2, 6, 8, 10, 22, 24, 27, 30, 48, 72, 96, 168, 216, 264 and 336 hours after administration. The RiboMab concentrations in plasma measured by ELISA are shown. Error bars are standard error of the mean (n=3).

Figure 9 shows the kinetics of RB RMAB1 expression in mice after weekly injections. On days 1, 8, 15, 21 and 29 of the test, balb/cJRj mice received IV injections of 1 μg (about 0.04 mg/kg), 3 μg (about 0.10 mg/kg), 10 μg (about 0.40 mg/kg) and 30 μg (about 1.20 mg/kg) of RB_ RMAB01 and 80 μg (about 3.2 mg/kg) of IMAB362 reference protein. Plasma was sampled 24 hours prior to dosing and 24 hours after dosing. The RiboMab concentrations in plasma measured by ELISA are shown. The dashed line indicates injection. Error bars are standard error of the mean (n=3).

FIG. 10 shows the kinetics of RB_ RMAB01 expression following repeated dosing in NHP. NHP received IV injections of 0.1, 0.4 or 1.6mg/kg RB_ RMAB01 on days 1,8 and 15 of the test. Plasma was sampled at 6, 24, 48, 72, 96 and 168 hours after the 1 st and 3 rd dosing, and at 48, 72 and 168 hours after the 2 nd dosing, and at 264, 336 and 504 hours after the 3 rd dosing. The RiboMab concentrations in plasma measured by ELISA are shown. Error bars are standard error of the mean (n=3).

Fig. 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 at 6, 24, 48, 72 and 144 hours after administration. (panel a) shows bioluminescence images of individual organs of (left) individual mice at ventral locations (n=5) and (right) mice #1 and 26 hours after administration. (panel B) shows quantification of luciferase signal (photons/sec) at all analysis time points (n=5 or 3, average). LN indicates a lymph node.

FIG. 12 illustrates some exemplary embodiments of RNA techniques and their applications that may be used to encode a variety of antibody agent forms ("RiboMab") and formulations thereof. (Picture A)The platform is suitable for providing RNA constructs encoding a variety of antibody formats including, for example, but not limited to, monospecific antibody IgG, bispecific antibody bi- (scFv)₂, and bispecific antibody Fab- (svFv)₂. (panel B) in some embodiments, a therapeutic antibody, such as IgG, may be encoded by a purified mRNA comprising modified ribonucleotide (e.g., uridine replaced with pseudouridine) mRNA and encapsulated in a lipid nanoparticle (mRNA/LNP). Such mRNA constructs may also include one or more non-coding sequence elements (e.g., to enhance RNA stability and/or translation efficiency). In some embodiments, exemplary non-coding sequence elements include, but are not limited to, cap structures, 5 'utrs, 3' utrs, poly a tails, and any combination thereof. In some embodiments, the lipid nanoparticle may comprise conjugated lipids (e.g., PEG-conjugated lipids), cationic lipids, and neutral helper lipids. Such mRNA/LNP pharmaceutical product formulations can be administered to a subject in vivo such that the mRNA is translated in vivo to express the antibody. (panel C) somatic cells of a patient administered the mRNA/LNP drug product formulation described herein are capable of producing an active drug encoded by the mRNA (e.g., igG RiboMab). For example, in some embodiments, after IV injection, the mRNA/LNP encoding the antibody is internalized and translated by hepatocytes, resulting in systemic plasma concentrations of biological activity RiboMab. Abbreviations A30L70, poly (A) tail, 100 adenosine eliminated by the linker at position 30, bi, bispecific, C-terminal, CDS, coding sequence, CH, constant heavy chain domain, CL, constant light chain domain, fab, antigen binding fragment, igG, immunoglobulin G, LNP, lipid nanoparticle, m1 ψ, 1-methyl pseudouridine, N-terminal, scFv, single chain variable fragment, TAA, tumor associated antigen, UTR, untranslated region, VH, variable heavy chain domain, VL, variable light chain domain.

FIG. 13 is a schematic diagram of exemplary RNA constructs encoding Heavy (HC) and Light (LC) chains of an antibody agent, respectively. As shown in fig. 13, such RNA constructs encoding HC and LC form an RNA composition (RB RMAB 01), which in some embodiments can be formulated as lipid nanoparticles to form an RNA/LNP drug product formulation. Abbreviations Poly A, polyadenylation tail, CH, constant heavy chain domain, CL, constant light chain domain, sec, secretion signal, UTR, untranslated region, VH, variable heavy chain domain, VL, variable light chain domain.

Fig. 14 is a graph showing the dose-exposure correlation of rb_ RMAB01 in cynomolgus monkeys at t_max. Cynomolgus monkey (n=3) received IV injection of 0.1, 0.4 or 1.6mg/kg RB RMAB01. Dose-dependent RiboMab01 concentrations (average, n=3) in plasma measured by ELISA at C_max are depicted. The green line indicates the dose that can be administered to a human subject and its corresponding expected serum concentration.

FIG. 15 is an exemplary electrophoretogram of an exemplary RNA mixture comprising a first RNA encoding an antibody Heavy Chain (HC) and a second RNA encoding an antibody Light Chain (LC). The electropherograms show two peaks of LC and HC, respectively. A is the area under the peak, h is the peak height.

FIG. 16 shows in vitro expression of anti-CLDN 18.2 RiboMab. HEK293T/17 cells were electroporated with mRNA all encoding anti-CLDN 18.2RiboMab with the same backbone but different coding sequences. The anti-cldn18.2ribomab concentration was measured by ELISA 48 hours after transfection. Error bars are standard error of the mean (n=2).

Figure 17 shows exposure of anti-CLDN 18.2RiboMab in mice following repeated RNA-LNP dosing. Balb/cJRj mice received twice weekly IV injections of 3 or 30 μg of RNA-LNP, each containing mRNA encoding HC and LC against CLDN18.2RiboMab in the case of backbone A or backbone B, respectively. Serum was sampled at the indicated time point after the first administration. The arithmetic mean (n=3) and standard error of the concentration of anti-CLDN 18.2RiboMab in serum measured by ELISA are shown. The limit of detection was 0.074ng/mL. The downward arrow corresponds to the first and second RNA-LNP injections. Luc-RNA-LNP was used as a negative control. Elisa=enzyme-linked immunosorbent assay; luc=luciferase.

Figure 18 shows the cytotoxic activity of anti-CLDN 18.2 RiboMab encoded by RNAs utilizing backbones a and B. Shown is in vitro ADCC mediated by anti-CLDN 18.2 RiboMab in mouse serum collected 24 hours after administration of RNA-LNP at the indicated dose. CLDN18.2 transduced NUGC-4 transfectants were used as target cells and human PBMCs from healthy donors were used as effector cells at an E: T ratio of 20:1 (upper panel). CLDN18.2 negative MDA-MB-231 was used as a negative control (bottom panel). The target cells and effector cells were incubated for 24 hours. The results for the control antibodies are shown in the right panel. Data are mean ± SD of three measurements per mouse. Ab=antibody, adcc=antibody dependent cytotoxicity, E: T ratio=effector to target cell ratio, m1=mouse No.1, pbmcs=peripheral blood mononuclear cells.

Fig. 19 shows that EPO mRNA transcribed from scaffold C was superior to EPO mRNA transcribed from scaffold a in vivo, but inferior to EPO mRNA derived from scaffold B/D cassette.

FIG. 20 shows a comparison of mRNA translations from backbones B, C and D with different coding sequences and shows that the differences in expression between backbone C and backbone B/D are independent of coding sequence. Firefly luciferase mRNA in A, backbones B (∈), C (■), and D () (solid and dashed lines) was electroporated twice in hiDC. Bright-Glo assays were performed at the indicated times. B, eGFP mRNA in backbones C (■) and D (#) was electroporated twice in hiDC (solid and dashed lines). Cells were harvested and eGFP expression was measured at the indicated times using FACS. C, liposome transfection of primary human hepatocytes with hIL-18mRNA in the backbones B (+), C (■) and D (∈). Supernatants from transfected cells were collected at designated times and assayed for the presence of hIL-18 by ELISA.

FIG. 21 shows translation of firefly luciferase mRNA derived from backbones B and C, which contain different nucleotides at position 9 upstream of polyA in hiDC, and indicate that the 3' UTR terminal sequence affects long-term translation in vitro. Firefly luciferase mRNA from backbones B (Panel A) and C (Panel B) with A (), G (■), T (), or C (∈) at position 9 upstream of the polyA sequence was electroporated in hiDC in two separate experiments and luciferase expression was determined at the indicated time points.

Figure 22 shows that the 3' utr termination sequence significantly affects long term translation in vivo using backbone B.

Figure 23 shows that the 3' utr termination sequence significantly affects long term translation in vivo using backbone D.

FIG. 24 shows that 5' sequences located upstream of the coding sequence also affect long-term translation in vivo.

FIG. 25 shows that the combination of 5 'and 3' sequence elements affects long-term translation in vivo.

Figure 26 shows that skeleton B performed significantly better than the earlier skeleton versions (skeletons a and G) for long-term translation in vivo.

Although the present disclosure is described further in greater detail below, it is to be understood that the present disclosure is not limited to the specific methods, 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 meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, elements of the present disclosure will be described in more detail. These elements are listed with some particular embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only some of the embodiments explicitly described. The description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred elements. Furthermore, any arrangement and combination of all described elements in this application are considered to be disclosed by the specification of the application unless the context clearly indicates otherwise.

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

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

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

Certain definitions

An unlimited number of terms as used herein should be construed to encompass both the singular and the plural unless otherwise stated herein or clearly contradicted by context.

About or about the terms "about" or "approximately" as used herein when applied to one or more destination values refer to values similar to the reference value. In general, those skilled in the art who are familiar with the context will understand the relative degree of variation that is covered by the context "about" or "approximately. For example, in some embodiments, the term "about" or "approximately" may be encompassed within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

Administration the term "administration" and variations thereof as used herein generally refers to administration of a composition to a subject to effect delivery of an agent as or contained in the composition to a target site or site to be treated. Those of ordinary skill in the art will recognize a variety of routes that may be used to administer to a subject, such as a human, where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some embodiments, administration may be transbronchial (e.g., by bronchial instillation), buccal (buccal), transdermal (which may be or include, for example, a surface for one or more of dermis, intradermal (interdermal), transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular (intraventricular), within a particular organ (e.g., intrahepatic), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, the administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the administration of a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses separated in time) and/or periodic (e.g., individual doses separated by a common period) dosing. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time.

And/or "as used herein is considered a particular disclosure of each of two specified features or components with or without the other. For example, "X and/or Y" is considered a particular disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually listed herein.

Antibody Agents the term "antibody agent" as used herein refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes an immunoglobulin structural element 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 a mouse, rabbit, primate, or human antibody. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, the antibody agents used in accordance with the present disclosure are in a form selected from, but not limited to, whole IgA, igG, igE or IgM antibodies, bispecific or multispecific antibodies (e.g.Etc.), antibody fragments, such as Fab fragments, fab ' fragments, F (ab ') 2 fragments, fd ' fragments, fd fragments and isolated complementarity determining regions (complementarity determining region, CDRs) or groups thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof), camel antibodies, masking antibodies (e.g.,) Small modular immunopharmaceuticals ("SMIPSTM"), single-chain or tandem diabodiesVHH;A minibody; ankyrin repeat protein orDART, TCR-like antibodies;MicroProteins; AndIn some embodiments, the term "antibody" or "antibody agent" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds. In some embodiments, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). In some embodiments, each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. 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 (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3), and the CDRs of VL are called LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3). The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the order from amino-to carboxy-terminus: CH1, CH2, CH 3). The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as C1q. The term "full length" when used in the context of an antibody means that the antibody is not a fragment, but rather comprises all domains of a particular isotype that are commonly found in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody. The term "Fab arm" or "arm" as used herein refers to a heavy chain-light chain pair, and is used interchangeably herein with "half molecule". In some embodiments, an antibody may lack covalent modifications (e.g., linkages of glycans) that it would have if naturally produced. In some embodiments, the antibody may comprise a covalent modification (e.g., attachment of a glycan, payload [ e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc. ] or other pendent group [ e.g., polyethylene glycol, etc. ]. In many embodiments, the antibody agent is a polypeptide or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as Complementarity Determining Regions (CDRs), and in some embodiments, the antibody agent is a polypeptide or comprises a polypeptide whose amino acid sequence comprises 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 a CDR present in a reference antibody. In some embodiments, the CDRs contained are substantially identical to the reference CDRs because they are identical in sequence or contain 1 to 5 amino acid substitutions as compared to the reference CDRs. In some embodiments, the CDRs contained are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the CDRs contained are substantially identical to the reference CDRs in that they exhibit at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the comprised CDRs are substantially identical to the reference CDRs in that at least one amino acid in the comprised CDRs is deleted, added, or substituted as compared to the reference CDRs, but the comprised CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDRs. In some embodiments, the comprised CDRs are substantially identical to the reference CDRs because 1 to 5 amino acids in the comprised CDRs are deleted, added, or substituted as compared to the reference CDRs, but the comprised CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, the comprised CDRs are substantially identical to the reference CDRs in that at least one amino acid in the comprised CDRs is replaced as compared to the reference CDRs, but the comprised CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDRs. In some embodiments, the comprised CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids in the comprised CDRs are deleted, added, or substituted as compared to the reference CDRs, but the comprised CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by one of skill in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

Antibody agents can be prepared by the skilled artisan using methods known in the art and commercially available services and kits. For example, methods of preparing monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Other antibodies suitable for use in the present disclosure are described, for example, in the following publications, ：Antibodies ALaboratory Manual,Second edition.Edward A.Greenfield.Cold Spring Harbor Laboratory Press(2013, 9, 30, );Making and Using Antibodies:APractical Handbook,Second Edition.Eds.Gary C.Howard and Matthew R.Kaser.CRC Press(2013, 7, 29, );Antibody Engineering:Methods and Protocols,Second Edition(Methods in Molecular Biology).Patrick Chames.Humana Press(2012, 8, 21, );Monoclonal Antibodies:Methods and Protocols(Methods in Molecular Biology).Eds.Vincent Ossipow and Nicolas Fischer.Humana Press(2014, 2, 12 days), and Human Monoclonal Antibodies:Methods and Protocols(Methods in Molecular Biology).Michael Steinitz.Humana Press(2013, 9, 30 days).

Antibodies can be produced by standard techniques (e.g., by immunization with the appropriate polypeptide or portion thereof, or by use of phage display libraries). If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing the desired epitope (optionally hapten with another polypeptide). Depending on the host species, a variety of adjuvants may be used to enhance the immune response. Such adjuvants include, but are not limited to, freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from immunized animals is collected and processed according to known procedures. If the serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies may be purified by immunoaffinity chromatography or any other method known in the art. Techniques for generating and processing polyclonal antisera are well known in the art.

In relation to the term as used herein, an event or entity is "related to" another event or entity if the presence, level, and/or form of the two events or entities are related to the presence, level, and/or form of the other event or entity. 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 the incidence and/or susceptibility or likelihood of response to treatment (e.g., in a related population).

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

Cancer As used herein, the term "cancer" generally refers to a disease or disorder in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled and/or autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, the cancer may comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, and/or non-metastatic cells. In some embodiments, the cancer may be characterized by a solid tumor. In some embodiments, the cancer may be characterized by a hematological tumor. Generally, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemia, lymphomas (hodgkin and non-hodgkin), myelomas and myeloproliferative diseases, sarcomas, melanomas, adenomas, solid tissue cancers, oral, laryngeal, pharyngeal and pulmonary squamous cell cancers, liver cancers, genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer, as well as renal cell cancer, bone cancer, pancreatic cancer, skin or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, ovarian cancer, breast cancer, glioblastoma, colorectal cancer, gastrointestinal cancer and nervous system cancer, benign lesions such as papilloma and the like.

Cap the term "cap" as used herein refers to a structure comprising or consisting essentially of nucleoside-5 ' -triphosphates typically attached to the 5' -end of uncapped RNA (e.g., uncapped RNA with 5' -diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, including, for example, but not limited to, a 7-methylguanosine cap having a structure designated "m 7G". In some embodiments, the cap is or comprises a synthetic cap analogue that resembles an RNA cap structure and has stable RNA

The ability to attach to it, including, for example, but not limited to, anti-reverse cap analogs (anti-REVERSE CAP analog, ARCA) known in the art. Those skilled in the art will appreciate that methods of ligating caps to the 5' end of RNA are known in the art. For example, in some embodiments, the capped RNA can be obtained by capping RNA having a5 'triphosphate group or RNA having a5' diphosphate group in vitro with a capping enzyme system (which includes, for example, but is not limited to, a vaccinia capping enzyme system or a saccharomyces cerevisiae capping enzyme system). Alternatively, the capped RNA may be obtained by in vitro transcription of a DNA template (in vitro transcription, IVT) using methods known in the art, wherein the IVT system comprises a dinucleotide cap analogue (which includes, for example, an m7GpppG cap analogue or an N7-methyl, 2 '-O-methyl-GPPPG ARCA cap analogue or an N7-methyl, 3' -O-methyl-GPPPG ARCA cap analogue) in addition to GTP. In some embodiments, the cap is cap 0, cap 1 or cap 2, preferably cap 1 or cap 2. The term "cap 0" as used herein means the structure "m7 gppppn", where N is any nucleoside bearing an OH moiety at the 2' position. The term "cap 1" as used herein means the structure "m7 gppppnm", where Nm is any nucleoside bearing an OCH3 moiety at the 2' position. The term "cap 2" as used herein means the structure "m⁷ GpppNmNm" wherein each Nm is independently any nucleoside bearing an OCH₃ moiety at the 2' position.

CLDN-18.2 positive the terms "CLDN-18.2 positive" or "CLDN-18.2+" as used herein refer to clinically relevant CLDN-18.2 expression and/or activity, e.g., can be correlated with a particular disease, disorder or condition and/or can be detected in or on a sample that can be or comprise one or more cell or tissue samples. In some embodiments, CLDN-18.2+ refers to cancers associated with clinically relevant CLDN-18.2 expression and/or activity. In certain exemplary embodiments, CLDN-18.2 positive expression and/or activity may be over-expressed from head CLDN-18.2 or include over-expression from head CLDN-18.2, e.g., in cancer cells, alternatively or additionally, in some embodiments CLDN-18.2 positive expression and/or activity may be or have been correlated with exposure to one or more agents or conditions, e.g., one or more chemotherapeutic agents (which include, e.g., gemcitabine and/or cisplatin). In some embodiments, CLDN-18.2 "positive" is assessed relative to an appropriate reference (e.g., a "negative control," such as a suitably comparable level and/or activity of CLDN-18.2 in non-cancerous cells and/or tissues, a "positive control," such as a level and/or activity of CLDN-18.2 as may have been determined for known CLDN-18.2 positive cells and/or tissues, and/or a given threshold value of CLDN-18.2 level and/or activity associated with a normal (e.g., healthy, non-cancerous) versus an abnormal (e.g., cancerous) state). In some embodiments, the term "CLDN-18.2+" is used herein to refer to a tumor sample from a cancer patient when the sample has been determined to exhibit an increase in detectable CLDN-18.2 protein expression relative to an appropriate reference (e.g., the level observed in a sample determined to be or otherwise known to be negative for CLDN-18.2 expression). In some embodiments, a sample is considered to be CLDN-18.2+ when it is determined that ≡50% of tumor cells in the sample have ≡2+cldn-18.2 protein staining intensity as assessed by immunohistochemical assay in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue, and those skilled in the art recognize that pathologists typically use such a scoring system for interpreting IHC data obtained with respect to tumor samples. See, e.g., fedchenko AND REIFENRATH, diagnostic Pathology (2014) 9:221, which describes different methods for interpreting and reporting IHC analysis results, including scoring systems. See also Zimmermann et al, cancer Cytopathology (2014) 48-58. Thus, pathologists will readily recognize that 2+ refers to a grading score of 2 or higher, which indicates that such immunohistochemical assay results are clear. More precisely, 2+ describes a medium or strong staining in a qualitative scale of negative "(0)," weak "(1)," medium "(2)," strong "(3).

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

Codon optimisation the term "codon optimisation" as used herein refers to altering codons in the coding region of a nucleic acid molecule to reflect typical codon usage of the host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, the coding region may be codon optimized for optimal expression in a subject to be treated with an RNA (particularly mRNA) as described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA appearance in cells. Thus, the sequence of an RNA (particularly an mRNA) can be modified such that codons for which frequently occurring tRNA's are available are inserted in place of "rare codons".

Combination therapy the term "combination therapy" as used herein refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. 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 the first regimen are administered prior to any dose administration of the second regimen), in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administering" of a combination therapy may include administering one or more agents or modes to a subject receiving other agents or modes in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even must be administered simultaneously), but in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition.

Rather, the term "comparable" as used herein refers to two or more agents, entities, conditions, sets of conditions, etc., that may not be identical to each other but that are sufficiently similar to allow comparison therebetween so that one of ordinary skill in the art will understand that a conclusion can be reasonably drawn based on the observed differences or similarities. In some embodiments, a group of comparable conditions, environments, individuals, or populations is characterized by a plurality of substantially identical features and one or a few varying features. Those of ordinary skill in the art will understand what degree of identity is required for two or more such agents, entities, situations, condition sets, etc. in any given instance to be considered equivalent. For example, one of ordinary skill in the art will understand that the environment, group of individuals, or groups of individuals, are comparable to each other in that they are characterized by having a sufficient number and type of substantially identical features to ensure a reasonable conclusion-differences in the results or observed phenomena obtained under or with different environments, groups of individuals, or groups of individuals are caused by or are indicative of the changes in the characteristics of those changes.

Complementary the term "complementary" as used herein is used to refer to oligonucleotide hybridization associated with the base pairing rules. For example, the sequence "C-A-G-T" is complementary to the sequence "G-T-C-A". Complementarity may be partial or complete. Thus, any degree of partial complementarity is intended to be included within the term "complementary" provided that the partial complementarity allows hybridization of the oligonucleotides. Partial complementarity is according to the base pairing rules in which one or more nucleobases are mismatched. Full or complete complementarity between nucleic acids is where each and every nucleic acid base matches another base under the base pairing rules.

The words "comprise/include," "comprising," and variations such as "comprises/comprising" are to 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, element, member, integer or step. The term "consisting essentially of limits the scope of the claims or the scope of the disclosure to specific features, elements, members, integers or steps, as well as those essential features and novel features that do not materially affect the scope of the claims or the disclosure. The term "consisting of" limits the scope of the claims or disclosure to the specified features, elements, members, integers or steps. The term "comprising" encompasses the term "consisting essentially of, and" consisting essentially of @, composition "in turn. The term" by the. Thus, the term "comprising/including" may be replaced with the term "consisting essentially of" or "consisting of" at each occurrence in the present application. Also, in the present application, the term "consisting essentially of the composition" may be replaced with the term "consisting of the composition" at each occurrence.

Contact the term "deliver" and variations thereof or "contact" as used interchangeably herein refers to exposing Guan Babiao (e.g., cells, tissues, organisms, etc.) to RNA or a composition comprising or delivering it (as described herein) such that the RNA is delivered into a target cell (e.g., the cytosol of the target cell). The target cells may be cultured in vitro or ex vivo or present in a subject (in vivo). Those of skill in the art will appreciate that different contact methods may be used to achieve such delivery to target cells in vitro, ex vivo, or in vivo applications. In some embodiments, the cells in the contact culture may be or include in vitro transfection. In some embodiments, the contacting can utilize one or more delivery vehicles (e.g., lipid nanoparticles described herein). In some embodiments, contacting may be or include administering to a subject a pharmaceutical composition described herein.

Detection the term "detection" is used broadly herein to include any suitable means of determining the presence or absence of an entity of interest in a sample or any form of measurement of an entity of interest. Thus, "detecting" may include determining, measuring, assessing or determining the presence or absence, level, quantity and/or location of an entity of interest. Including quantitative and qualitative assays, measurements or evaluations, which include semi-quantitative. Such determination, measurement or evaluation may be relative (e.g., when the entity of interest is detected relative to a control reference), or may be absolute. Thus, the term "quantization" when used in the context of quantizing a destination entity may refer to absolute or relative quantization. Absolute quantification may be accomplished by correlating the level of the detected entity of interest with a known control standard (e.g., by generation of a standard curve). Or relative quantification may be accomplished by comparing the detection levels or amounts between two or more different destination entities to provide a relative quantification (i.e., relative to each other) of each of the two or more different destination entities.

Disease the term "disease" as used herein refers to a disorder or condition that impairs the normal function of a tissue or system, typically in a subject (e.g., a human subject), and that is typically manifested as a characteristic sign and/or symptom. In some embodiments, the exemplary disease is cancer.

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

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

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

Fc region the term "Fc region" as used herein refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, wherein the Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.

5' Untranslated region (FIVE PRIME untranslated region): the term "5' untranslated region" or "5' UTR" as used herein refers to the sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (typically AUG) of the RNA coding region.

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

Identity the term "identity" as used herein 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 "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which needs to be introduced for optimal alignment of the two sequences). Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percentage 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, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences may be determined using the GAP program in the GCG software package using the nwsgapdna. In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some embodiments, consecutive nucleotides). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

"Immunogenicity" refers to the ability of a foreign substance (e.g., RNA) to elicit an immune response in a human or other animal. The innate immune system is a relatively nonspecific and immediate component of the immune system. Which, together with the adaptive immune system, is one of two major components of the vertebrate immune system.

Immunoglobulin the term "immunoglobulin" as used herein relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors, such as antibodies or B Cell Receptors (BCR). Immunoglobulins are characterized by structural domains (i.e., immunoglobulin domains) having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins and soluble immunoglobulins. Membrane-bound immunoglobulins, also known as surface immunoglobulins or membrane immunoglobulins, are typically part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies. The structure of immunoglobulins is well characterized. See, e.g., fundamental Immunology ch.7 (Paul, W., ed., second edition, RAVEN PRESS, N.Y. (1989)). Briefly, immunoglobulins typically comprise several chains, typically two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains consist essentially of immunoglobulin domains or regions, such as V_L or VL (variable light chain) domains/regions, C_L or CL (constant light chain) domains/regions, V_H or VH (variable heavy chain) domains/regions, C_H or CH (constant heavy chain) domains/regions C_H1(CH1)、C_H2(CH2)、C_H (CH 3) and C_H (CH 4). The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH 3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between two heavy chains in an IgG molecule. Each light chain is typically composed of VL and CL. The light chain constant region is typically composed of one domain CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions, which may be in the form of sequence hypervariability and/or structurally defined loops), also known as complementarity determining regions (complementarity determining region, CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). each VH and VL is typically composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the 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 using DomainGapAlign according to IMGT rules (Lefranc MP., nucleic ACIDS RESEARCH 1999;27:209-212 and EHRENMANN f., kaas q.and Lefranc m. -p.nucleic Acids res.,38, d301-307 (2010); see also internet http address www.imgt.org). However, it should be understood that the present disclosure is not limited to CDR sequences determined only according to IMGT rules. There are five types of mammalian immunoglobulin heavy chains, α, δ, ε, γ and μ, which constitute different classes of antibodies, igA, igD, igE, igG and IgM. In contrast to the heavy chain of soluble immunoglobulins, the heavy chain of membrane or surface immunoglobulins comprises a transmembrane domain and a short cytoplasmic domain at its carboxy-terminus. In mammals, there are two types of light chains, λ and κ. Immunoglobulin chains comprise a variable region and a constant region. The constant regions are substantially conserved across different isotypes of immunoglobulins, with the variable portions being highly diverse and leading to antigen recognition.

Isolated "means removed (e.g., purified) from a natural state or from an artificial composition (e.g., a composition from a manufacturing process). For example, a nucleic acid or polypeptide naturally occurring in a living animal is not "isolated," but the same nucleic acid, peptide or polypeptide, partially or completely isolated from its coexisting materials in its natural state, is "isolated. The isolated nucleic acid or polypeptide may be present in a substantially purified form, or may be present in a non-natural environment, such as a host cell.

Lipid the term "lipid" as used herein relates to a molecule comprising one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic portion and a hydrophilic portion are also often referred to as biphenols. Lipids are generally insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, amphiphilic properties allow molecules to self-assemble into organized structures and distinct phases. In general, lipids can be divided into eight classes, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from condensation of ketoester acyl subunits), sterol lipids, and prenyl alcohol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fat, fat is a subset of lipids known as triglycerides. Lipids also encompass molecules such as fatty acids and derivatives thereof (including triglycerides, diglycerides, monoglycerides and phospholipids), and steroids, i.e. metabolites comprising sterols, such as cholesterol or derivatives thereof. Some examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, stigmasterol, cholesteryl-2 '-hydroxyethyl ether, cholesteryl-4' -hydroxybutyl ether, tocopherol, and derivatives thereof, and mixtures thereof.

Locally advanced tumor the term "locally advanced tumor" or "locally advanced cancer" as used herein 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 larger but has not yet spread to another body part. In some embodiments, locally advanced tumors are used to describe cancers that have grown outside of their starting tissue or organ but have not spread to distant sites within the subject. By way of example only, in some embodiments locally advanced pancreatic cancer generally refers to stage III disease with tumor expansion to adjacent organs (e.g., lymph nodes, liver, duodenum, superior mesenteric artery, and/or dry abdominal cavity) without signs of metastatic disease, however, complete surgical excision with a negative pathological incisal margin (margin) is not possible.

Mol%: "Mol%" as used herein is defined as the ratio of the moles of one component to the total moles of all components multiplied by 100. "mole% of total lipids" as used in the present disclosure is defined as the ratio of the moles of one lipid component to the total moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid materials.

Non-immunogenic RNA the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") as used herein refers to RNA that does not induce a response by the immune system, or induces a response that is weaker than the response induced by the same RNA, when administered (e.g., to a mammal), except that the same RNA is not subjected to modification and treatment that renders the non-immunogenic RNA non-immunogenic, i.e., is weaker than that induced by standard RNA (STANDARD RNA, STDRNA).

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

Nucleotide the term "nucleotide" as used herein refers to its art-recognized meaning. When the number of nucleotides is used as an indication of the size (e.g., of a polynucleotide), the particular number of nucleotides refers to the number of nucleotides on a single strand (e.g., of a polynucleotide).

Optionally, 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 where it does not.

Patient the term "patient" as used herein refers to any organism having or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. In some embodiments, the patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of the disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition for which the provided technology is applicable is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is receiving or has received a particular treatment to diagnose and/or treat a disease, disorder, or condition. In some embodiments, the patient is a cancer patient.

Polypeptides the term "polypeptide" as used herein generally has its art-recognized meaning-a polymer of at least three or more amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently broad to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains (e.g., fragments, portions, or domains that retain at least one activity) of such complete polypeptides. In some embodiments, the polypeptide may comprise an L-amino acid, a D-amino acid, or both and/or may comprise any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

Pharmaceutically active polypeptide the term "pharmaceutically active polypeptide" as used herein means a peptide or polypeptide that is useful in treating an individual in which expression of the peptide or polypeptide would be beneficial, for example in ameliorating symptoms of a disease. Preferably, the pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to ameliorate, alleviate, mitigate, reverse a disease, delay the onset of a disease or reduce the severity of one or more symptoms of a disease. In some embodiments, the pharmaceutically active peptide or polypeptide has a positive or beneficial effect on a disorder or disease state of an individual when administered to the individual in a therapeutically effective amount. The pharmaceutically active peptide or polypeptide may have prophylactic properties and may be used to delay the onset of a disease or to reduce the severity of such a disease. The term "pharmaceutically active peptide" or "pharmaceutically active polypeptide" includes the complete peptide or polypeptide and may also refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active variants and/or analogues of the peptide or polypeptide.

Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, immunostimulants such as cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis modulators, transcription factors, tumor suppressors, structural proteins, reprogramming factors, genome engineering proteins and blood proteins. In some embodiments, pharmaceutically active peptides and polypeptides include replacement proteins.

An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any component of the immune system, in particular immune effector cells. Immunostimulants may be pro-inflammatory (e.g., when treating an infection or cancer) or anti-inflammatory (e.g., when treating an autoimmune disease).

According to one aspect, the immunostimulant is a cytokine or variant thereof. Some examples of cytokines include interferons such as interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma), interleukins such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factors. According to a further aspect, the immunostimulant comprises an adjuvant-type immunostimulant, such as an APC Toll-like receptor agonist or a co-stimulatory/cell adhesive membrane protein. Some examples of Toll-like receptor agonists include co-stimulatory/adhesion proteins such as CD80, CD86 and ICAM-1.

The term "cytokine" relates to a protein having a molecular weight of about 5 to 60kDa and involved in cell signaling (e.g., paracrine, endocrine, and/or autocrine signaling). In particular, when released, cytokines have an effect on the behavior of cells around their release site. Some examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (tumor necrosis factor, TNF). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they generally function at more variable concentrations than hormones, and (ii) are generally produced by a broad range of cells (almost all nucleated cells can produce cytokines). Specific examples of cytokines include Erythropoietin (EPO), colony stimulating factor (colony stimulating factor, CSF), granulocyte colony stimulating factor (granulocyte colony stimulating factor, G-CSF), granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF), tumor Necrosis Factor (TNF), bone morphogenic protein (bone morphogenetic protein, BMP), interferon alpha (IFNalpha), interferon beta (IFNbeta), interferon gamma (INFgamma), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as variants and derivatives thereof.

According to the present disclosure, the cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, in particular any mammal. One particularly preferred cytokine is interferon- α.

Interferon (IFN) is a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites and tumor cells. In a typical case, virus-infected cells will release interferon, resulting in nearby cells enhancing their antiviral defenses. Interferons are generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate transcription of intracellular genes by binding to interferon receptors on the surface of the regulated cells, thereby preventing replication of intracellular viruses.

Interferons are generally classified into three types, type I interferons (type I interferons present in humans are IFN alpha, IFN beta, IFN epsilon, IFN kappa and IFN omega), type II interferons (type III interferons in humans are IFN gamma) based on the type of receptor through which the interferon signals.

In accordance with the present disclosure, the type I interferon is preferably IFN alpha or IFN beta, more preferably IFN alpha.

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

Interleukins (IL) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major classes based on different structural features. However, their amino acid sequence similarity is quite weak (typically 15% to 25% identity). The human genome encodes more than 50 interleukins and related proteins.

According to the present disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be human interleukin and may be derived from any vertebrate, in particular any mammal.

The immunostimulatory polypeptides described herein can be prepared as fusion polypeptides or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). Immunostimulants may be fused to kinetic (PK) extending groups, thereby increasing circulatory half-life. Some examples of non-limiting PK prolongation groups are serum albumin or fragments thereof or variants of serum albumin or fragments thereof (e.g., HAS or fragments or variants thereof), immunoglobulin FC or FC fragments and variants thereof, transferrin and variants thereof, and human serum albumin (human serum albumin, HSA) binding agents (as disclosed in U.S. publication nos. 2005/0287153 and 2007/0003549). Further exemplary PK extension groups are disclosed in Kontermann, expert Opin Biol Ther,2016Jul;16 (7): 903-15, which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutically active peptide or polypeptide comprises a surrogate protein. In these embodiments, the present disclosure provides methods for treating a subject having a condition requiring protein substitution (protein replacement) (e.g., protein deficiency) comprising administering to the subject an RNA (particularly an mRNA) as described herein encoding a surrogate protein. The term "protein substitution" refers to the introduction of a protein (including functional variants thereof) into a subject lacking such a protein. The term also refers to the introduction of a protein into a subject in need of or benefited from providing the protein (e.g., a subject with insufficient protein) in other ways. The term "disorder characterized by protein deficiency" refers to any disorder that exhibits a pathological condition caused by protein deficiency or insufficient amounts of protein. The term encompasses protein folding disorders, i.e. conformational disorders, that result in a biologically inactive protein product. Protein deficiency may involve infectious diseases, immunosuppression, organ failure, gland problems, radiation, nutritional deficiencies, poisoning or other environmental or external damage.

The term "hormone" relates to a class of signaling molecules produced by the gland, wherein signaling generally comprises the steps of (i) synthesizing a hormone in a specific tissue, (ii) storing and secreting, (iii) transporting the hormone to its target, (iv) binding of a receptor to the hormone, (v) transmission and amplification of a signal, and (vi) decomposition of the hormone. Hormones differ from cytokines in that (1) hormones generally act at less varying concentrations, and (2) are generally produced by specific classes of cells. In some embodiments, the "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (adrenocorticotropic hormone, ACTH), thyroid hormone, growth hormone (e.g., human or bovine growth hormone), oxytocin, atrial Natriuretic Peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

The term "adhesion molecule" relates to a protein that is located on the surface of a cell and is involved in the binding of the cell to other cells or to the extracellular matrix (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). Some specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectins), and addressees.

Integrins are also involved in signal transduction. In particular, following ligand binding, integrins regulate cellular signaling pathways, such as those of transmembrane protein kinases, such as receptor tyrosine kinases (receptor tyrosine kinase, RTKs). Such modulation may result in cell growth, division, survival or differentiation or apoptosis. Some specific examples of integrins include ：α₁β₁,α₂β₁,α₃β₁,α₄β₁,α₅β₁,α₆β₁,α₇β₁,α_Lβ₂,α_Mβ₂,α_IIbβ₃,α_Vβ₁,α_Vβ₃,α_Vβ₅,α_Vβ₆,α_Vβ₈, and a₆β₄.

The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules that are involved in the recognition, binding, and/or adhesion process of cells. A common feature of molecules belonging to this superfamily is that they comprise a region called an immunoglobulin domain or fold. Some members of the immunoglobulin superfamily include antibodies (e.g., igG), T Cell Receptors (TCRs), major histocompatibility complex (major histocompatibility complex, MHC) molecules, co-receptors (e.g., CD4, CD8, CD 19), antigen receptor accessory molecules (e.g., CD-3 gamma, CD3 delta, CD-3 epsilon, CD79a, CD79 b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD 86), and the like.

The term "immunocompetent compound" relates to any compound that alters an immune response, for example by inducing and/or inhibiting immune cell maturation, inducing and/or inhibiting cytokine biosynthesis, and/or altering humoral immunity by stimulating B-cell production of antibodies. The immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and may also down-regulate other aspects of the immune response, such as transferring the immune response from a TH2 immune response, which is useful for treating a wide range of TH2 mediated diseases. The immunologically active compounds are useful as vaccine adjuvants. Some specific examples of immunologically active compounds include interleukins, colony Stimulating Factors (CSF), granulocyte colony stimulating factors (G-CSF), granulocyte-macrophage colony stimulating factors (GM-CSF), erythropoietin, tumor Necrosis Factors (TNF), interferons, integrins, addressees (addressin), selectins (seletin), homing receptors (home receptors), and antigens, particularly tumor-associated antigens, pathogen-associated antigens (e.g., antigens of bacteria, parasites, or viruses), allergens, and autoantigens. The immunologically active compound may be a vaccine antigen, i.e. an antigen that is inoculated into a subject to induce an immune response.

In some embodiments, the RNAs (particularly mrnas) described in the present disclosure comprise a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject. "peptide or polypeptide comprising an epitope for inducing an immune response in a subject against an antigen" is also referred to herein as "vaccine antigen", "peptide and protein antigen" or simply "antigen".

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

Vaccine antigens comprise epitopes for inducing an immune response against the antigen in a subject. Thus, a vaccine antigen comprises an antigen sequence for inducing an immune response against the antigen in a subject. Such an antigen sequence may correspond to a target antigen or a disease-associated antigen, such as a protein of an infectious agent (e.g., a viral antigen or a bacterial antigen) or a tumor antigen, or may correspond to an immunogenic variant thereof, or an immunogenic fragment of a target antigen or a disease-associated antigen or an immunogenic variant thereof. Thus, the antigen sequence may comprise at least an epitope of the target antigen or a disease-associated antigen or an immunogenic variant thereof.

The antigen sequence or processed product thereof (e.g., fragment thereof) may bind to an antigen receptor carried by an immune effector cell (e.g., a TCR or CAR). In some embodiments, the antigen sequence is selected from an antigen or fragment thereof expressed by a target cell targeted by an immune effector cell, or a variant of the antigen sequence or fragment.

In some embodiments, RNA encoding a vaccine antigen is expressed in cells of a subject to provide an antigen or processed product thereof for binding to an antigen receptor expressed by an immune effector cell, the binding resulting in stimulation, sensitization and/or expansion of the immune effector cell.

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

The term "self antigen" or "autoantigen" refers to an antigen that is derived from the body of a subject (i.e., self antigens may also be referred to as "autoantigens") and that produces an abnormally severe immune response against that normal portion of the body. Such a severe immune response against autoantigens may be responsible for "autoimmune diseases".

Any suitable antigen that is a candidate for an immune response may be used in accordance with the present disclosure, wherein the immune response may comprise a humoral immune response or a cellular immune response, or both. In the case of some embodiments of the present disclosure, the antigen is presented by cells, for example by antigen presenting cells (in the case of MHC molecules), which result in an immune response against the antigen. The antigen may be a product corresponding to or 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 the antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, such as a viral protein, or a portion thereof.

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 that comprises an epitope that will stimulate the immune system of the host to generate an antigen-specific cellular immune response and/or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by a microorganism, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer (typically a tumor), e.g., tumor antigens.

In some embodiments, the antigen is a tumor antigen, i.e., a portion of a tumor cell, particularly those that are predominantly present within the cell or as surface antigens of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e. an antigen derived from a pathogen, e.g. an antigen from a virus, a bacterium, a unicellular organism or a parasite, e.g. a viral antigen such as viral ribonucleoprotein or a coating protein. In some embodiments, the antigen should be presented by MHC molecules, which result in modulation of cells of the immune system (e.g., cd4+ and cd8+ lymphocytes), particularly activation of cells of the immune system (e.g., cd4+ and cd8+ lymphocytes), particularly through modulation of the activity of T cell receptors.

The term "epitope" refers to an antigenic determinant in a molecule (e.g. an antigen), i.e. a part or fragment of a molecule that is recognized by the immune system, e.g. by T cells or B cells, particularly when presented in the context of MHC molecules. Epitopes of a protein may comprise contiguous or non-contiguous portions of the protein and may be, for example, from about 5 to about 100 amino acids in length, from about 5 to about 50, from about 8 to about 30, or from about 10 to about 25 amino acids in length.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when present in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to the gene complexes present in all vertebrates.

According to some embodiments, the amino acid sequence that enhances antigen processing and/or presentation and/or disrupts immune tolerance is fused directly to an antigenic peptide or polypeptide (antigen sequence) or through a linker.

The terms "immune response" and "immune response" are used interchangeably herein in their conventional sense and refer to the overall physical response to an antigen, and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the term "immune response to" or "immune response to" with respect to a substance (e.g., an antigen, a cell, or a tissue) relates to an immune response, e.g., a cellular response, to the substance. The immune response may include one or more responses selected from the group consisting of the appearance of antibodies to one or more antigens and the expansion of antigen-specific T lymphocytes (e.g., CD4⁺ and CD8⁺ T lymphocytes, such as CD8⁺ T lymphocytes, which may be detected in various proliferation or cytokine production assays in vitro).

The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the administration of one or more immunogens or antigens as described herein or derivatives thereof, in particular in the form of RNAs (especially mrnas) encoding same, to an individual and the stimulation of an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

The term "allergen" refers to a class of antigens that originate from outside the body of a subject (i.e., an allergen may also be referred to as a "heterologous antigen") and that produce an abnormally severe immune response, wherein the subject's immune system is resistant to perceived threats that are otherwise harmless to the subject. An "allergy" is a disease caused by such a severe immune response against an allergen. Allergens are typically antigens capable of stimulating a type I hypersensitivity reaction in an atopic individual through an immunoglobulin E (immunoglobulin E, igE) response. Specific examples of the allergen include allergens derived from peanut proteins (e.g., ara h 2.02), ovalbumin, grass pollen proteins (e.g., phlp 5), and dust mite proteins (e.g., der p 2).

The term "growth factor" refers to a molecule capable of stimulating cell growth, proliferation, healing and/or cell differentiation. Generally, growth factors act as signaling molecules between cells. The term "growth factor" includes specific cytokines and hormones that bind to specific receptors on the surface of their target cells. Some examples of growth factors include Bone Morphogenic Proteins (BMP), fibroblast Growth Factors (FGF), vascular Endothelial Growth Factors (VEGF) (e.g., VEGFA), epidermal Growth Factors (EGF), insulin-like growth factors, ephrines, macrophage colony stimulating factors, granulocyte macrophage colony stimulating factors, neuregulin, neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), nerve Growth Factor (NGF)), placental Growth Factors (PGF), platelet-derived growth factors (PDGF), renin (RNLS) (anti-apoptotic survival factor), T-cell growth factors (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor α (TGF- α), transforming growth factor β (TGF- β)), and tumor necrosis factor- α (TNF- α). In some embodiments, a "growth factor" is a peptide or polypeptide growth factor.

The term "protease inhibitor" refers to a molecule, in particular a peptide or polypeptide, that inhibits the function of a protease. Protease inhibitors may be categorized by the protease being inhibited (e.g., aspartic protease inhibitor) or by its mechanism of action (e.g., suicide inhibitor, e.g., serine protease inhibitor). Specific examples of protease inhibitors include serine protease inhibitors such as alpha 1-antitrypsin, aprotinin and bestatin (bestatin).

The term "enzyme" refers to a macromolecular biocatalyst that accelerates chemical reactions. Like any catalyst, enzymes are not consumed in the reactions they catalyze and do not alter the equilibrium of the reactions. Unlike many other catalysts, the specificity of enzymes is much higher. In some embodiments, the enzyme is essential for the homeostasis of the subject, e.g., any dysfunction of the enzyme (in particular reduced activity, which may be caused by any one of mutation, deletion or reduced yield) results in a disease. Some examples of enzymes include herpes simplex virus type 1thymidine kinase (herpes simplex virus type 1thymidine kinase,HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

The term "receptor" refers to a protein molecule that receives a signal from outside the cell, in particular a chemical signal called a ligand. Binding of a signal (e.g., ligand) to a receptor causes some type of response by the cell, such as intracellular activation of a kinase. Receptors include transmembrane receptors (e.g., ion channel-linked (ion channel) receptors, G protein-linked (metabolic) receptors, and enzyme-linked receptors) and intracellular receptors (e.g., cytoplasmic and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors in which the ligand is a peptide), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to a growth factor.

The term "apoptosis modulator" refers to a molecule, particularly a peptide or polypeptide, that modulates apoptosis (i.e., activates or inhibits apoptosis). Apoptosis modulators can be divided into two broad categories, apoptosis modulators that regulate mitochondrial function and apoptosis modulators that regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) for maintaining mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or preventing release of pro-apoptotic proteins (e.g., cytochrome C) into the cytosol. Also included in this first class are pro-apoptotic proteins (e.g., BAX, BAK, BIM) that promote cytochrome C release. The second category includes proteins such as inhibitor of apoptosis proteins (e.g., XIAP) or FLIP that block caspase activation.

The term "transcription factor" relates to a protein which modulates the transcription rate 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, regulate cell migration and organization during embryonic development, and/or respond to signals from outside the cell, such as hormones. The transcription factor comprises at least one DNA binding domain that binds to a specific DNA sequence that is typically adjacent to a gene regulated by the transcription factor. Specific examples of transcription factors include MECP2, FOXP3, STAT protein family and HOX protein family.

The term "tumor suppressor protein" relates to a molecule, in particular a peptide or polypeptide, that protects a cell from one step on the oncogenic pathway. Tumor suppressor proteins (typically encoded by the corresponding tumor suppressor genes) exhibit attenuation or suppression in regulating the cell cycle and/or promote apoptosis. Its function may be one or more of inhibiting genes necessary for sustaining the cell cycle, coupling the cell cycle to DNA damage (no cell division should occur as long as damaged DNA is present in the cell), initiating apoptosis if damaged DNA is not repairable, metastasis inhibition (e.g., preventing tumor cell proliferation, preventing loss of contact inhibition and inhibiting metastasis), and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase AND TENSIN homolog, PTEN, SWI/SNF (SWItch/Non-fermentable Sucrose (SWItch/Sucrose Non-Fermentable)), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (adenomatous polyposis coli, APC), CD95, tumor suppressor protein 5 (suppression of tumorigenicity, ST5), tumor suppressor protein 5 (ST 5), tumor suppressor protein 14 (ST 14), and YIppee-like 3 (YIppee-like 3, YPELK 3).

The term "structural protein" refers to a protein that imparts rigidity and rigidity to a biological component that is otherwise fluid. Structural proteins are predominantly fibrous (e.g. collagen and elastin), but may also be globular (e.g. actin and tubulin). Generally, globular proteins can be dissolved as monomers, but polymerize to form long fibers, which can, for example, constitute the cytoskeleton. Other structural proteins are motor proteins (e.g. myosin, kinesins and kinesins) capable of generating mechanical forces, and surface-active proteins. Specific examples of structural proteins include collagen, surfactant protein a, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

The term "reprogramming factor" or "reprogramming transcription factor" relates to a molecule, in particular a peptide or polypeptide, which when expressed in a somatic cell (optionally together with other substances such as other reprogramming factors) causes the somatic cell to reprogram or dedifferentiate into a cell having stem cell properties, in particular multipotency. Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

The term "genome-engineered protein" relates to a protein capable of inserting, deleting or replacing DNA in the genome of a subject. Specific examples of genome engineering proteins include meganucleases, zinc finger nucleases (zinc finger nuclease, ZFN), transcription activator-like effector nucleases (transcription activator-like effector nuclease, TALENs) and clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9, CRISPR-Cas 9).

The term "blood protein" relates to a peptide or polypeptide present in the plasma of a subject, in particular of a healthy subject. Blood proteins have a variety of functions, such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), protection from pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., α1-antitrypsin), and the like. Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (G-CSF), modified factor VIII, and anticoagulants.

Thus, in some embodiments, the 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) adhesion molecules, in particular integrins, (iii) immunoglobulins, in particular antibodies, (iv) immunocompetent compounds, in particular antigens, such as viral antigens or bacterial antigens, such as antigens of SARS-CoV-2, such as spike (spike, S) proteins of SARS-CoV-2 or variants thereof, (v) hormones, in particular booster, insulin or growth hormone, (vi) growth factors, in particular VEGFA, (vii) protease inhibitors, in particular alpha 1-antitrypsin, (viii) enzymes, preferably selected from the group consisting of herpes simplex virus thymidine kinase type 1 (HSV 1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatin and lactase, (ix) receptors, in particular growth factor receptors, (x) apoptosis modulators, in particular BAX, (xi) transcription factors, in particular FOXP3, (xii) tumor suppressor proteins, in particular p53, (xiii) structural proteins, in particular surfactant proteins B, (xiv) reprogramming factors, for example, selected from the group consisting of 4, SOX2, c-MYC, KLF4, LING 28 and NAv gene engineering, in particular clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas 9), and (xvi) blood proteins, in particular fibrinogen.

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

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.

In some embodiments, the target antigen is a tumor antigen and the antigenic sequence (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen, which is generally known to be expressed in a variety of cancers. Tumor antigens may also be "neoantigens" that are specific to an individual's tumor and have not been previously recognized by the immune system. Neoantigens or neoepitopes may be caused by one or more cancer-specific mutations in the cancer cell genome, resulting in amino acid changes. If the tumour antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of said neoantigen comprising one or more amino acid changes.

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

In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. Thus, in some embodiments, the RNA (particularly mRNA) described in this disclosure encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

Recombinant the term "recombinant" as used herein means "prepared by genetic engineering". In some embodiments, a "recombinant" in the context of the present disclosure is not naturally occurring.

Reference/reference criteria "reference" as used herein describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and/or assayed substantially simultaneously with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, the reference or control is or comprises a set of specifications (e.g., associated acceptance criteria). Generally, as will be appreciated by those skilled in the art, a reference or control is assayed or characterized under conditions or conditions comparable to those under evaluation. When sufficient similarity exists to demonstrate reliance on and/or comparison with a particular possible reference or control, those skilled in the art will understand.

Ribonucleotides: the term "ribonucleotide" as used herein includes unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (a) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can include one or more modifications including, but not limited to, for example, (a) terminal modifications such as 5 'terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse ligation, etc.), 3' terminal modifications (e.g., conjugation, reverse ligation, etc.), base modifications (b) substitutions such as with modified bases, stabilized bases, destabilized bases, or bases base pairing to an extended pool of partners or conjugated bases, (c) sugar modifications (e.g., at the 2 'position or the 4' position) or sugar substitutions, and (d) internucleoside linkage modifications including modifications or substitutions of phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotides that include both modified and unmodified ribonucleotides that are triphosphate.

Ribonucleic acid (RNA): the term "RNA" as used herein refers to a polymer of ribonucleotides. In some embodiments, the RNA is single stranded. In some embodiments, the RNA is double stranded. In some embodiments, the RNA comprises both a single-stranded portion and a double-stranded portion. In some embodiments, the RNA can comprise a backbone structure as described in the definition of "nucleic acid/polynucleotide" above. The RNA may be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA

(MRNA). In some embodiments, the RNA is mRNA. In some embodiments wherein the RNA is mRNA, the RNA typically comprises a poly (a) region at its 3' end. In some embodiments in which the RNA is mRNA, the RNA typically comprises a cap structure at its 5' end that is recognized in the art, e.g., for recognizing the mRNA and ligating it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNAs include RNAs synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).

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

Selectively or specifically, as those skilled in the art understand, the term "selective" or "specific" as used herein in connection with an agent having activity means that the agent distinguishes between potential target entities, states or cells. For example, in some embodiments, an agent is said to "specifically" bind to one or more competing surrogate targets if it preferentially binds to its target in the presence of the target. In many embodiments, the specific interaction depends on the presence of specific structural features (e.g., epitope, groove (cleft), binding site) of the target entity. It should be understood that the specificity need not be absolute. In some embodiments, specificity can be assessed relative to the specificity of the target binding moiety of one or more other potential target entities (e.g., competitors). In some embodiments, the specificity is assessed relative to the specificity of a reference specific binding member. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding member. In some embodiments, a CLDN-18.2 targeted antibody agent encoded by one or more RNAs (e.g., RNAs described herein) does not detectably bind to a competing surrogate target (e.g., CLDN18.1 polypeptide) under conditions that bind to the CLDN-18.2 polypeptide. In some embodiments, CLDN-18.2 targeted antibody agents bind to CLDN-18.2 polypeptides with higher binding rates, lower dissociation rates, increased affinity, reduced dissociation, and/or increased stability compared to competing surrogate targets (including, e.g., CLDN18.1 polypeptides).

Specific binding the term "specific binding" as used herein refers to the ability to distinguish between potential binding partners in the environment in which binding occurs. An antibody agent that interacts with a particular target in the presence of other potential targets is referred to as "specifically binding" the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between a CDR of an antibody agent and its partner, in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of an antibody agent-partner complex, in some embodiments, specific binding is assessed by detecting or determining the ability of an antibody agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or assay over a range of concentrations.

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

An individual who is susceptible to a "predisposed" disease, disorder or condition is an individual at risk of developing the disease, disorder or condition. In some embodiments, an individual susceptible to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, or condition is an individual who has been exposed to an environment associated with the occurrence of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and/or condition is population-based (e.g., family members of an individual suffering from the disease, disorder, or condition; carriers of genetic markers or other biomarkers associated with the disease, disorder, or condition, etc.).

An individual having a "suffering from" a disease, disorder, and/or condition has been diagnosed with and/or exhibiting one or more symptoms of the disease, disorder, or condition.

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

Therapeutic agent the phrase "therapeutic agent" or "treatment" as used interchangeably herein refers to an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect when administered to a subject or patient. In some embodiments, a therapeutic agent or treatment is any substance that is useful for alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, the therapeutic agent or treatment is a medical intervention (e.g., surgery, radiation, phototherapy) that may be performed to alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.

3' Untranslated region the term "3' untranslated region" or "3' UTR" as used herein refers to the sequence of an mRNA molecule that begins after the stop codon of the coding region of the open reading frame sequence. In some embodiments, the 3' utr begins immediately after the stop codon of the coding region of the open reading frame sequence. In other embodiments, the 3' utr does not begin immediately after the stop codon of the coding region of the open reading frame sequence.

Threshold level (e.g., acceptance criteria): the term "threshold level" as used herein refers to a level that is used as a reference to obtain information about and/or classify a measurement (e.g., a measurement obtained in an assay). For example, in some embodiments, the threshold level means a value measured in an assay that defines a demarcation line between two subsets of a population (e.g., batches meeting quality control criteria versus batches not meeting quality control criteria). Thus, values at or above the threshold level define one subset of the population, and values below the threshold level define another subset of the population. The threshold level may be determined based on one or more control samples or between groups of control samples. The threshold level may be determined before, simultaneously with, or after the measurement of the destination is made. In some embodiments, the threshold level may be a range of values.

Transfection the term "transfection" as used herein relates to the introduction of nucleic acids, in particular RNA, into cells. For the purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acid into a cell or the uptake of nucleic acid by such a cell, wherein the cell may be present in a subject (e.g., a patient), or the cell may be in vitro (e.g., ex vivo). Thus, according to the present disclosure, cells for transfection of the nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue and/or body of a patient. Transfection may be transient or stable in accordance with the present disclosure. For some applications of transfection, it is sufficient if only the transfected genetic material is transiently expressed. RNA can be transfected into cells to transiently express the protein it encodes. Since the nucleic acid introduced during transfection will not normally integrate into the nuclear genome, the exogenous nucleic acid will be diluted or degraded by mitosis. Cells that allow free amplification of nucleic acids greatly reduce dilution rates. If it is desired that the transfected nucleic acid is actually maintained in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection may be achieved by transfection using, for example, a viral-based system or a transposon-based system. RNA can be transfected into cells to transiently express the protein it encodes.

Treatment the term "treatment" and variations thereof as used herein refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. The treatment may be administered to a subject that does not exhibit signs of the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject that exhibits only early signs of a disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathological condition associated with the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject at a later stage of the disease, disorder, and/or condition.

Unresectable tumor the term "unresectable tumor" as used herein generally refers to a tumor characterized by one or more characteristics that are considered, according to sound medical judgment, to indicate that the tumor cannot be removed safely (e.g., without undue damage to the subject) by surgery, and/or for which a medical professional having the ability has determined that the risk of tumor resection to the subject outweighs the benefits associated with such resection. In some embodiments, a non-resectable tumor refers to a tumor that involves and/or has grown into a necessary organ or tissue (including a blood vessel that may not be reconstructable) and/or is in a location that is not readily accessible by surgery that would otherwise pose an unreasonable risk of damage to one or more other critical or necessary organs and/or tissues (including a blood vessel). In some embodiments, "unresectability" of a tumor refers to the likelihood of achieving a negative-cut (R0) excision. In the case of pancreatic cancer, the presence of tumor wrapping of large blood vessels such as Superior Mesenteric Artery (SMA) MESENTERIC ARTERY or celiac axis (encasement), portal vein occlusion, and celiac or periaortic lymphadenopathy is generally considered to be a finding that precludes R0 surgery. Those skilled in the art will appreciate parameters that determine whether a tumor is unresectable.

Those skilled in the art who review this description will appreciate that in many embodiments, standard techniques are available and can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and/or transformation (e.g., electroporation, lipofection, transfection). The enzymatic reaction and/or purification techniques may generally be performed according to manufacturer's instructions or as commonly practiced in the art or as described herein. In many embodiments, the foregoing techniques and operations may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al.,Molecular Cloning:ALaboratory Manual(2d ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(1989)),, which is incorporated by reference herein for any purpose.

Detailed Description

The outcome of Standard of Care (SOC) treatment remains poor for many cancer patients, and particularly for patients with recurrent or refractory advanced solid tumors. Treatment options also typically include palliative chemotherapy (which may be less tolerant after previous repeated exposure to cytotoxic compounds) or optimal supportive care, as well as research treatments that do not demonstrate benefit. Treatment in this population is not curable, with overall survival expected to be months. Immunotherapy has become an effective treatment option for some cancers with highly unmet medical needs. In particular, immune checkpoint inhibitors are approved for the treatment of a variety of cancer indications and function by activating pre-existing anti-tumor specific T cells. The medical need for multiple cancer types remains high. The present disclosure provides, inter alia, insights and techniques for treating cancer (e.g., pancreatic and/or cholangiocarcinoma) with treatment targeting claudin-18.2 (CLDN-18.2).

In some embodiments, the present disclosure provides, inter alia, RNA techniques for delivering a CLDN-18.2-targeted monoclonal antibody that combines both effective anti-tumor features and excellent safety characteristics, bypassing the hurdles of slow and cumbersome antibody manufacturing processes. Without wishing to be bound by any particular theory, the present disclosure suggests that such RNA delivery patterns may achieve one or more improvements, such as effective administration with reduced incidence (e.g., frequency and/or severity) of therapeutic emergency adverse events ("TEAE") and/or improved relationship between efficacy levels and TEAE levels (e.g., improved therapeutic window), relative to those observed when the corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches such improvements, particularly as may be achieved by delivering IMAB362 via administration of an RNA (e.g., ssRNA, e.g., mRNA) encoding IMAB 362.

In some embodiments, the present disclosure provides, inter alia, insight that mRNA encoding an antibody agent (e.g., IMAB 362) 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 absorbed by a target cell (e.g., a hepatocyte) to effectively produce a therapeutically relevant plasma concentration of the encoded antibody agent (e.g., IMAB 362), e.g., as shown in fig. 14 for CLDN-18.2 targeted antibody agent expressed by RNA (e.g., RNA as described herein). In some embodiments, the antibody agent is expressed from mRNA engineered for minimal immunogenicity and/or formulated in Lipid Nanoparticles (LNPs), for example. In some embodiments, the mRNA encoding the antibody agent may comprise modified nucleotides (e.g., without limitation, pseudouridine and/or 1-methyl-pseudouridine).

Furthermore, the present disclosure provides insight, inter alia, that CLDN-18.2 targeted antibody agents delivered as described herein can enhance the cytotoxicity of chemotherapy and/or other anti-cancer therapies by utilizing the ability of the subject's immune system to induce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells). In some embodiments, such combination therapies may extend progression free and/or overall survival, e.g., relative to treatment of an individual administered alone and/or relative to another suitable reference.

Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, such as gemcitabine, oxaliplatin, and 5-fluorouracil, appear to up-regulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines, and furthermore, that these agents are not observed to increase de novo expression in CLDN-18.2 negative cell lines. See, for example Türeci et al.,(2019)"Characterization of Zolbetuximab in pancreatic cancer models."In Oncoimmunology 8(1),pp.e1523096.

The present disclosure provides, inter alia, insight that CLDN-18.2-targeted therapies as described herein can be particularly useful and/or effective when administered to tumors (e.g., tumor cells, subjects suspected of and/or having detected such tumors and/or tumor cells, etc.) that are characterized (e.g., have been determined to exhibit and/or are expected or predicted to exhibit) increased expression and/or activity of CLDN-18.2 expression in tumor cells (e.g., may or have resulted from exposure to one or more chemotherapeutic agents). Indeed, the present disclosure teaches, among other things, that the provided CLDN-18.2-targeted therapies (e.g., administration of RNA, and more particularly, mRNA encoding CLDN-18.2-targeted antibody agents) as described herein can provide synergistic therapy when administered in combination with one or more CDLN-18.2 enhancers (e.g., one or more specific chemotherapeutic agents) (e.g., administered to a subject that has received and/or is receiving or is otherwise exposed to the one or more CDLN 18.2.2 enhancers (e.g., one or more specific chemotherapeutic agents). Thus, in some embodiments, targeted CLDN-18.2 treatment as described herein may be useful in combination with other anti-cancer agents that are expected and/or have been demonstrated to up-regulate CLDN-18.2 expression and/or activity in tumor cells.

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

I. claudin-18.2 polypeptide

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

CLDN18 expression in healthy tissue. Claudin 18.2 is a 27.8kDa protein with four transmembrane domains and two small extracellular loops (Niimi et al 2001). CLDN-18.2 is a tightly linked molecule of gastric epithelial cells. The gastric tight junction is highly specialized in rejecting gastric acid, which can damage the gastric lining (lining).

CLDN-18.2 is a highly selective antigen of the gastric lineage (Sahin et al 2008). Typically, their expression is limited to short-lived differentiated gastric epithelial cells of the fossa (pit) and basal region of the gastric gland. The stem cell region of differentiated epithelial cells that constantly complement the gastric glands is CLDN-18.2 negative. Without wishing to be bound by theory, it is generally believed that no other normal human cell type expresses CLDN-18.2 at the transcriptional or protein level.

CLDN18 expression in cancer. CLDN-18.2 is useful in a variety of human cancers including gastric cancer, gastroesophageal cancer (GE) and Pancreatic Cancer (PC) (Karanjawala et al 2008; coati et al 2019) as well as pre-cancerous lesions2014; Tanaka et al 2011). Tumor-associated CLDN-18.2 expression was also detected in ovarian cancer (Sahin et al 2008), biliary tract cancer (Shinozaki et al 2011) and lung cancer (Micke et al 2014).

About 77% of primary gastric adenocarcinoma (gastric adenocarcinoma, GAC) is CLDN-18.2+.56% of GAC showed strong expression of CLDN-18.2 in at least 60% of tumor cells (defined by immunohistochemical analysis as staining intensity. Gtoreq.2+). CLDN-18.2 expression was more frequent in diffuse gastric cancer than in intestinal gastric cancer. CLDN-18.2 protein is also often detected in lymph node metastasis from gastric carcinoma and distant metastasis to the ovary (so-called Krukenberg tumor) Bei Geliu. Furthermore, 50% of esophageal adenocarcinomas showed significant CLDN-18.2 expression.

In pancreatic cancer, CLDN-18.2 is expressed in pancreatic ductal adenocarcinoma (PANCREATIC DUCTAL ADENOCARCINOMA, PDAC) at an incidence of 60% to 90% (Karanjawala et al.2008; et al 2014). PDAC accounts for over 80% of all pancreatic tumors, is the seventh most common cancer in Europe, and is the fourth most cancer-related cause of death in the European Union (Ferlay et al 2010; jemal et al 2011; seufferlein et et al 2012). Almost 60% of patients with PDAC express membrane-bound CLDN-18.2 and CLDN-18.2 is ectopically activated in 20% of patients with pancreatic neuroendocrine tumors. CLDN-18.2 expression in primary and metastatic PDAC lesionset al.2014)。

Down-regulation of CLDN-18.2 by siRNA technology has been shown to result in inhibition of gastric cancer cell proliferation (Niimi et al 2001), suggesting involvement in proliferation of CLDN-18.2+ tumor cells.

Exemplary sequences of CLDN-18.2 (SEQ ID NO: 32) and splice variant CLDN18.1 (SEQ ID NO: 33) are shown below:

pi. Exemplary targeting pad antibody against protein-18.2 polypeptide

In some embodiments, an antibody agent that targets CLDN-18.2 specifically binds to a CLDN-18.2 polypeptide. In some embodiments, an antibody agent that targets CLDN-18.2 specifically binds to a first extracellular domain (ECD 1) of a CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an exposed ECD1 epitope in a cancer cell. In some embodiments, such an antibody agent may have a binding affinity (e.g., as measured by dissociation constant) for an ECD1 epitope of a CLDN-18.2 polypeptide, e.g., CLDN-18.2 polypeptide, of at least about 10^-4 M, at least about 10^-5 M, at least about 10^-6 M, at least about 10^-7 M, at least about 10^-8 M, at least about 10^-9 M, or lower. Those skilled in the art will appreciate that in some cases, binding affinity (e.g., as measured by dissociation constants) can be affected by non-covalent intermolecular interactions (e.g., hydrogen bonding, electrostatic interactions, hydrophobic forces, and van der Waals forces) between two molecules. Alternatively or additionally, the 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 techniques for measuring binding affinity and/or dissociation constants in accordance with the present disclosure, including, for example, but not limited to ELISA, gel migration assay, pull down assay (pull down assay), equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (surface plasmon resonance, SPR), biolayer interferometry, grating coupled interferometry, and spectrometry.

In some embodiments, an antibody that targets CLDN-18.2 can specifically bind to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide. In some embodiments, antibodies that target CLDN-18.2 do not bind to any other claudin family member, including closely related claudin-18 splice variant 1 (CLDN 18.1) expressed primarily in tissues such as the lung.

In some embodiments, the antibody agent that targets CLDN-18.2 may be any of the CLDN-18.2 targeting antibodies described in WO 2007/059997, WO2008/145338, and WO2013/174510 (the respective contents of which are incorporated herein by reference in their entirety for the purposes described herein).

In some embodiments, an antibody agent that targets CLDN-18.2 comprises (a) a variable heavy domain having at least one CDR selected from (including, for example, 1 CDR, 2 CDRs, and 3 CDRs), (i) CDR1 represented by amino acid residue (GYTFTSYW), (ii) CDR2 represented by amino acid residue (IYPSDSYT), and (iii) CDR3 represented by amino acid residue (TRSWRGNSFDY), and/or (b) a variable light domain having at least one CDR selected from (including, for example, 1 CDR, 2 CDRs, and 3 CDRs), (i) CDR1 represented by amino acid residue (QSLLNSGNQKNY), (ii) CDR2 represented by amino acid residue (WAS), and (iii) CDR3 represented by amino acid residue (QNDYSYPFT).

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

In some embodiments, an antibody agent that targets CLDN-18.2 comprises (a) a variable heavy domain having at least one CDR selected from (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) CDR1 represented by amino acid residues 45 to 52 of SEQ ID NO: 1;

(ii) CDR2 represented by amino acid residues 70 to 77 of SEQ ID No. 1, and (iii) CDR3 represented by amino acid residues 116 to 126 of SEQ ID No. 1, and/or (b) a variable light chain domain having at least one CDR (including, for example, 1 CDR, 2 CDRs, and 3 CDRs) selected from (i) CDR1 represented by amino acid residues 47 to 58 of SEQ ID No. 2, (ii) CDR2 represented by amino acid residues 76 to 78 of SEQ ID No. 2, and (iii) CDR3 represented by amino acid residues 115 to 123 of SEQ ID No. 2.

In some embodiments, an antibody agent that targets CLDN-18.2 comprises a variable heavy domain comprising the amino acid sequence of SEQ ID No. 14 and a variable light domain comprising the amino acid sequence of SEQ ID No. 15.

In some embodiments, an antibody agent that targets 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.

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

In some embodiments, an antibody agent that targets CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID No. 3 (wherein the underlined amino acid sequence corresponds to a secretion signal sequence) and a light chain consisting of or comprising the amino acid represented by amino acid residues 27 to 246 of SEQ ID No. 4 (wherein the underlined amino acid sequence corresponds to a secretion signal sequence).

In some embodiments, an antibody agent that targets 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.

In some embodiments, an antibody agent that targets CLDN-18.2 comprises one or more Fc regions having a lysine at their C-terminus. The source of this lysine is the naturally occurring sequence present in humans from which these Fc regions are derived. During cell culture to produce recombinant antibodies, the terminal lysine can be cleaved proteolytically by endogenous carboxypeptidase to produce a constant region with the same sequence but lacking the C-terminal lysine. Antibodies produced from nucleic acid sequences encoding or not encoding terminal lysines are essentially identical in sequence and function, as the degree of processing of terminal lysines is typically high when using antibodies produced in CHO-based production systems, for example (Dick, l.w.et al biotechnol bioeng.2008; 100:1132-1143). Thus, it is understood that a protein (e.g., an antibody) according to the invention may be produced with or without encoding or having a terminal lysine. It is also understood according to the invention that sequences with terminal lysines (e.g. constant region sequences with terminal lysines) can be understood as corresponding sequences without terminal lysines, and that sequences without terminal lysines can also be understood as corresponding sequences with terminal lysines.

In some embodiments, the antibody that targets CLDN-18.2 is IMAB362 (also known as zol Bei Tuo mab, clausimab). IMAB362 (an antibody targeting CLDN-18.2) is in late clinical development (NCT 01630083, NCT03816163, NCT03653507, NCT03505320, NCT 03504397) and is known in the art (see, e.g., ,Sahin et al.2018;Sahin et al.2017;Al-Batran et al.2017a;Al-Batran et al.2017b;Türeci et al.2019;Trarbach et al.2014;Morlock et al.2018a;Schuler et al.2016;Lordick et al.2016;Morlock et al.2018b). whose target CLDN-18.2 is a highly selective tumor-associated surface marker.

IMAB362 developed by Ganymed Pharmaceuticals GmbH and purchased by ASTELLAS PHARMA inc. Is an intact IgG1 antibody targeting the claudin CLDN-18.2 and mediates cell death by antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). IMAB362 recognizes with high affinity and specificity the first extracellular domain (ECD 1) of CLDN-18.2 (Sahin et al 2008; tu reci et al 2011). This epitope is not accessible in the normal epithelial barrier of the antibody. Disruption of tight junctions and loss of cell polarization are early markers of cancer. During this process, an epitope of IMAB362 is exposed. IMAB362 does not bind to any other claudin family member, including closely related claudin 18 splice variant 1 (CLDN 18.1) expressed primarily in tissues such as the lung.

IMAB362 plus epirubicin, oxaliplatin and capecitabine (EOX) in a phase 2 FAST trial for EOX (NCT 01630083) in first line patients with gastric and gastroesophageal cancer were tested (Morlock et al.2018a;Schuler et al.2016;Al-Batran et al.2016;Lordick et al.2016;Morlock et al.2018b).FAST patient populations including patients whose tumors had greater than or equal to 40% of tumor cells expressing CLDN-18.2 with moderate to strong (. Gtoreq.2+) staining intensity. A subset of patients whose tumors had > 70% of tumor cells with a staining intensity of > 2+CLDN-18.2 obtained the greatest benefit from IMAB362 treatment at a dose of 800/600mg/kg², with nearly double the median total survival (OS) (Al-Batran et al.2016; lordick et al.2016). The OS benefit of IMAB362 expression (+33.1 weeks; p < 0.0005) at > 70% CLDN-18.2 was accompanied by a significant delay in center independent review progress (+14.5 weeks; p < 0.0005) and higher objective response rates (objective response rate, ORR) (35.1% versus 27.1%). The addition of IMAB362 to EOX does not negatively impact patient-related outcomes. Throughout the study, no significant differences between treatment groups were observed in the mixed effect model repeat measurements of global health status or total ST022 scores (Mixed effect Model Repeat Measurement), but the deterioration of global health scores was significantly delayed by 2.6 months (p=0.008) relative to EOX alone, IMAB362 plus EOX.

ASTELLAS PHARMA Inc IMAB362 was also tested in phase 2 and phase 3 trials of global development programs in patients with CLDN-18.2+ gastric/gastroesophageal cancer and pancreatic cancer.

IMAB362 has been tested in a variety of clinical trials as shown in table 1 below.

TABLE 1 summary of certain clinical trials involving the administration of IMAB362

The safety profile of IMAB362 in patients is well characterized and has been tolerated with repeated doses up to 1000mg/m² q3w (up to 603. Mu.g/mL of c_max) without dose limiting toxicity (Sahin et al 2018; tu reci et al 2019).

Without wishing to be bound by a particular theory, the primary pharmacological mode of action of IMAB362 for effecting tumor cell killing involves Antibody Dependent Cellular Cytotoxicity (ADCC). Based on the dose response curve obtained by the in vitro ADCC test, drug concentrations yielding a 95% response were observed at IMAB362 concentrations in serum of 0.3 to 28 μg/mL (Sahin et al 2018). For example, effective lysis of CLDN-18.2+ cells by ADCC has been reported with EC₉₅ of 0.3 to 28 μg/mL (Sahin et al 2018).

In various trials, IMAB362 was well tolerated, nausea and vomiting were major Adverse Events (AE), and dose limiting toxicity (dose limiting toxicity, DLT) and clinical activity as single agents and in combination with chemotherapy were not observed.

The present disclosure provides inter alia that IMAB362 or variants thereof (e.g., variants that share one or more characteristics 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 variants that are a class of variants, e.g., igG1, igM, igA, etc.), can represent particularly desirable antibodies for delivery by administration of ribonucleic acids as described herein. Without wishing to be bound by any particular theory, the present disclosure proposes that such modes of delivery provide for effective administration, as well as reduced incidence (e.g., frequency and/or severity) of IMAB362 treatment-related adverse events (TEAEs) relative to those observed when IMAB362 antibodies themselves are administered. In the MONO test at stage 2a of IMAB362 (NCT 01197885), 82% (n=44/54) of patients developed TEAE, nausea (61%), vomiting (50%) and fatigue (22%) were the most common TEAE. Grade 3 emesis was reported in 12 patients (22%) and grade 3 nausea was reported in 8 patients (15%). These patients received a dose of 600mg/m². Nausea and vomiting observed in this study were controlled by pausing or slowing the infusion of IMAB362, indicating that AE is associated with C_max (Tu reci et al 2019).

In particular, the present disclosure demonstrates that the Pharmacokinetic (PK) profile of IMAB362 delivered as ribonucleic acid ("RiboMab") described herein shows a gradual increase in antibody concentration and significantly lower C_max compared to IMAB362 at 48 to 72 hours after administration. RiboMab01 altered PK profile can reduce C_max related AEs seen in patients after treatment with IMAB 362. The present disclosure also provides non-human primate study data that shows that no systemic side effects such as diarrhea are observed.

The present disclosure particularly understands the favorable risk/benefit profile observed for the administered IMB362 antibody, particularly in certain indications with high medical requirements, and suggests that delivery as described herein may be effective and/or particularly desirable.

RNA techniques for delivering antibody-based therapeutics

Recombinant protein antibodies are widely used biologicals for the treatment of diseases or disorders (e.g., cancer), but exhibit many limitations including, for example, lengthy manufacturing process development and short serum half-lives for antibody derivatives. The present disclosure provides, among other things, techniques that address certain limitations of recombinant antibody technology (including, for example, lengthy manufacturing process development and short serum half-life for antibody derivatives) as a new class of antibody-based therapies by utilizing RNA technology as a mode of direct expression of antibody agents (referred to as RiboMab) in cells of patients. In some embodiments, the present disclosure provides insight, inter alia, that RiboMab formulated with Lipid Nanoparticles (LNP) for Intravenous (IV) administration can be taken up by cells (e.g., hepatocytes) effective to produce a therapeutically relevant plasma concentration of the encoded RiboMab antibody (fig. 14). In some embodiments RiboMab is an antibody agent encoded by, for example, mRNA engineered for minimal immunogenicity and/or formulated in Lipid Nanoparticles (LNPs). In some embodiments, the mRNA encoding the antibody agent may comprise modified nucleotides (e.g., without limitation, pseudouridine and/or 1-methyl-pseudouridine).

RiboMab technology can be used to deliver a variety of antibody formats. For example, in some embodiments, riboMab techniques may be used to express intact immunoglobulins (igs), including, for example, but not limited to, igG. In some embodiments, an intact immunoglobulin (Ig) may be encoded by a single RNA comprising a first coding region encoding an antibody heavy chain and a second coding region encoding an antibody light chain variable domain, wherein the single RNA comprises or encodes an internal ribosome entry side (internal ribosome ENTRY SIDE, IRES) or another internal promoter or peptide sequence, such as a "self-cleaving" 2A or 2A-like sequence (see, e.g., szymczak et al. Nat Biotechnol 22:589,May 2004;ePub April 42004) to produce the corresponding heavy and light chains, which may then be processed to form an intact IgG. In some embodiments, the intact Ig may be encoded by two separate RNAs, a first RNA comprising a coding region encoding an antibody heavy chain and a second RNA comprising a coding region encoding an antibody light chain. Such first and second RNAs are then translated into corresponding antibody chains and form complete Ig antibodies in target cells.

In some embodiments, riboMab techniques can be used to express bispecific antibody variants, e.g., as shown in fig. 12 (panel a) or as described in STADLER ET al (2016) Oncoimmunology (3): el091555 and/or 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 encoding a single-chain variable fragment (scFv) of a first target and a second coding region encoding a scFv of a second target. In some embodiments, the bivalent antibody agent may be encoded by two separate RNAs, a first RNA comprising a coding region encoding a scFv of a first target and a coding region encoding a heavy chain antigen binding fragment (Fab) of a second target, and a second RNA comprising a coding region encoding a scFv of the same first target and a coding region encoding a light chain Fab of the same second target. Such first and second RNAs are then translated into subunits of antibodies in target cells and form bispecific antibodies.

In some embodiments, an RNA agent (e.g., ssRNA described herein) can be delivered with a vector. In some embodiments, the RNA/LNP is administered Intravenously (IV) and is taken up by target cells (e.g., hepatocytes) effective to produce a therapeutically relevant plasma concentration of the encoded RiboMab antibody.

A. RNA encoding antibodies against claudin-18.2 polypeptide and compositions thereof are provided

In some embodiments, at least one RNA comprises one or more coding regions encoding an antibody agent as described in the section entitled "exemplary antibody agent targeting claudin-18.2 polypeptide" above. In some embodiments, at least one RNA comprises one or more coding regions encoding an antibody agent IMAB362 as described above or exemplified herein.

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

In some embodiments, an antibody agent that targets CLDN-18.2 (and may be encoded by RNA, e.g., ssRNA, e.g., mRNA as described herein) specifically binds to a first extracellular domain (ECD 1) of a CLDN-18.2 polypeptide. For example, in some embodiments, such an antibody agent specifically binds to an exposed ECD1 epitope in a cancer cell.

In some embodiments, at least one RNA encodes a variable heavy chain (V_H) domain of a CLDN-18.2 targeted antibody agent and a variable light chain (V_L) domain of the antibody agent. In some embodiments, such a V_H domain and V_L domain of a CLDN-18.2 targeted antibody agent may be encoded by a single RNA construct, or in some embodiments, it may be encoded by at least two separate RNA constructs separately. For example, in some embodiments, an RNA as used herein comprises two or more coding regions comprising a heavy chain coding region encoding at least the V_H domain of a CLDN-18.2 targeted antibody agent and a light chain coding region encoding at least the V_L domain of a CLDN-18.2 targeted antibody agent. In some alternative embodiments, the composition comprises (i) a first RNA comprising a heavy chain coding region encoding a V_H domain of at least a CLDN-18.2-targeted antibody agent, and (ii) a second RNA comprising a light chain coding region encoding a V_L domain of at least a CLDN-18.2-targeted antibody agent.

In some embodiments, the heavy chain coding region may also encode a constant heavy chain (C_H) domain, and/or the light chain coding region may also encode a constant light chain (C_L) domain. For example, in some embodiments, the heavy chain coding region can encode the V_H domain, the C_H1 domain, the C_H2 domain, and the C_H3 domain of an immunoglobulin form (e.g., igG) of a CLDN-18.2 targeting antibody agent, and/or the light chain coding region can encode the V_L domain and the C_L domain of an Ig form (e.g., igG) of a CLDN-18.2 targeting antibody agent. For example, in some embodiments, an intact immunoglobulin (Ig) may be encoded by a single RNA comprising a first coding region encoding a heavy chain of a CLDN-18.2Ig antibody (e.g., igG) and a second coding region encoding a light chain variable domain of the CLDN-18.2Ig antibody (e.g., igG), wherein the single RNA requires protein translation to produce a fusion protein comprising the heavy and light chains of the antibody, and the fusion protein is posttranslationally cleaved into the corresponding heavy and light chains by a suitable protease, which may then be processed to form the intact Ig (e.g., igG). In some embodiments, an intact Ig may be encoded by two separate RNAs, a first RNA comprising a coding region encoding a heavy chain of a CLDN-18.2Ig antibody (e.g., igG) and a second RNA comprising a coding region encoding a light chain of a CLDN-18.2Ig antibody (e.g., igG). Such first and second RNAs are then translated into corresponding antibody chains and form complete Ig antibodies (e.g., igG) in the target cells. In some embodiments, the antibody agent in the form of IgG encoded by the one or more RNAs is IgG1.

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

In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 20 to 467 of SEQ ID NO. 1. In some embodiments, one or more amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO. 1 (e.g., to reduce immunogenicity and/or stability). For example, in some embodiments, SEQ ID NO. 1 may comprise at least one or more (including, 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, for example, NO more than 40, NO more than 30, NO more than 20, NO more than 10, or NO more than 5 or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO. 1. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence represented by amino acid residues 21 to 240 of SEQ ID NO. 2. In some embodiments, one or more non-CDR regions of SEQ ID NO. 2 may have one or more amino acid modifications (e.g., to reduce immunogenicity and/or stability). For example, in some embodiments, SEQ ID NO. 2 may comprise at least one or more (including, 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, for example, NO more than 40, NO more than 30, NO more than 20, NO more than 10, or NO more than 5 or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO. 2.

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

In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID NO. 3. In some embodiments, one or more non-CDR regions of SEQ ID NO. 3 may have one or more amino acid modifications (e.g., to reduce immunogenicity and/or stability). For example, in some embodiments, SEQ ID NO 3 may comprise at least one or more (including, 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, for example, NO more than 40, NO more than 30, NO more than 20, NO more than 10, or NO more than 5 or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO. 3. In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding an amino acid sequence represented by amino acid residues 27 to 246 of SEQ ID NO. 4. In some embodiments, one or more non-CDR regions of SEQ ID NO. 4 can have one or more amino acid modifications (e.g., to reduce immunogenicity and/or stability). For example, in some embodiments, SEQ ID NO. 4 may comprise at least one or more (including, 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, for example, NO more than 40, NO more than 30, NO more than 20, NO more than 10, or NO more than 5 or less) amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO. 4.

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

In some embodiments, the heavy chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full length heavy chain of adjuvant Bei Tuo mab or clausimab (e.g., as described and/or exemplified herein) or comprises a nucleotide sequence encoding the full length heavy chain of adjuvant Bei Tuo mab or clausimab (e.g., as described and/or exemplified herein). In some embodiments, the light chain coding region of the RNA consists of or comprises a nucleotide sequence encoding the full length light chain of zoffiti Bei Tuo mab or clausimab.

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

Secretion signal coding region in some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent may comprise a secretion signal coding region. In some embodiments, such secretion signal encoding regions allow CLDN-18.2 targeted antibody agents encoded by one or more RNAs to be secreted, after translation, by, for example, cells present in a subject to be treated, thereby producing a plasma concentration of CLDN-18.2 targeted antibody agents having biological activity. In some embodiments, the secretion signal encoding region contained in the RNA consists of or comprises a nucleotide sequence encoding a non-human secretion signal. For example, in some embodiments, such a non-human secretion signal may be a murine secretion signal, which in some embodiments may be an amino acid sequence of MGWSCIILFLVATATGVHS or MESQTQVLMSLLFWVSGTCG or an amino acid sequence comprising MGWSCIILFLVATATGVHS or MESQTQVLMSLLFWVSGTCG. In some embodiments, the secretion signal encoding region comprised in the RNA consists of or comprises a nucleotide sequence encoding a human secretion signal, which in some embodiments may be an amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or an amino acid sequence comprising MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the secretion signal encoding region contained in the RNA encoding the heavy chain domain of the CLDN-18.2 targeting antibody agent may comprise (i) a nucleotide sequence encoding a murine secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MGWSCIILFLVATATGVHS or comprise the amino acid sequence of MGWSCIILFLVATATGVHS, or (ii) a nucleotide sequence encoding a human secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the secretion signal encoding region contained in the RNA encoding the light chain domain of the CLDN-18.2 targeting antibody agent may comprise (i) a nucleotide sequence encoding a murine secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MESQTQVLMSLLFWVSGTCG or comprise the amino acid sequence of MESQTQVLMSLLFWVSGTCG, or (ii) a nucleotide sequence encoding a human secretion signal amino acid sequence, which in some embodiments may be the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS.

In some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent can comprise at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency). Some examples of non-coding sequence elements include, but are not limited to, 3 'untranslated regions (UTRs), 5' UTRs, co-transcribed capped cap structures for mRNA, poly adenine (poly a) tails, and any combination thereof.

UTR (5 'UTR and/or 3' UTR) in some embodiments, the RNA provided may comprise a nucleotide sequence encoding a 5'UTR of interest and/or a 3' UTR of interest. Those of skill in the art will appreciate that untranslated regions of an mRNA sequence (e.g., the 3'utr and/or the 5' utr) may contribute to mRNA stability, mRNA localization, and/or translation efficiency.

In some embodiments, the provided RNAs can comprise a 5'utr nucleotide sequence and/or a 3' utr nucleotide sequence. In some embodiments, such 5'utr sequences may be operably linked to a 3' coding sequence (e.g., comprising one or more coding regions). Additionally or alternatively, in some embodiments, the 3'utr sequence may be operably linked to 5' of a coding sequence (e.g., that comprises one or more coding regions).

In some embodiments of any aspect described herein, the 5 'and 3' utr sequences contained in the RNA may consist of or comprise 5 'and 3' utr sequences naturally occurring or endogenous to the open reading frame of the gene of interest. Or in some embodiments, the 5 'and/or 3' UTR sequences contained in the RNA are not endogenous to the coding sequence (e.g., they contain one or more coding regions), and in some such embodiments, such 5 'and/or 3' UTR sequences may be used to modify the stability and/or translation efficiency of the transcribed RNA sequence. For example, the skilled artisan will appreciate that AU-rich elements in the 3' UTR sequence may reduce mRNA stability. Thus, as will be appreciated by the skilled artisan, the 3 'and/or 5' UTRs may be selected or designed to enhance the stability of transcribed RNA based on the characteristics of UTRs well known in the art.

For example, one of skill in the art will understand that in some embodiments, a nucleotide sequence consisting of or comprising a Kozak sequence of the open reading frame sequence of a gene or nucleotide sequence of interest may be selected and used as the nucleotide sequence encoding the 5' utr. As the skilled artisan will appreciate, kozak sequences are known to increase the translation efficiency of some RNA transcripts, but not all RNAs need to require Kozak sequences to achieve efficient translation. In some embodiments, provided RNA polynucleotides can comprise a nucleotide sequence encoding a 5' utr derived from an RNA virus whose RNA genome is stable in a cell. In some embodiments, a variety of modified ribonucleotides (e.g., as described herein) can be used in the 3 'and/or 5' utr, e.g., to prevent exonuclease degradation of transcribed RNA sequences.

In some embodiments, the 5' utr contained in the RNA may be derived from human α -globin mRNA in combination with a Kozak region.

In some embodiments, the RNA can comprise one or more 3' utrs. For example, in some embodiments, the RNA can comprise two copies of a 3' -UTR derived from globin mRNA (e.g., such as α2-globin, α1-globin, β -globin (e.g., human β -globin) mRNA). In some embodiments, two copies of the 3' UTR derived from human β -globin mRNA may be used, e.g., in some embodiments, placed between the coding sequence of the RNA and the poly (A) tail to increase protein expression levels and/or to extend RNA persistence. In some embodiments, the 3'UTR contained in the RNA may be or comprise one or more (e.g., 1,2, 3, or more) of the 3' UTR sequences disclosed in WO 2017/060314 (the entire contents of which are incorporated herein by reference for the purposes of the present description). In some embodiments, the 3' -UTR may be a 12S ribosomal RNA derived from "split amino terminal enhancer" (amino TERMINAL ENHANCER of split, AES) mRNA (designated F) and mitochondrial coding

A combination of at least two sequence elements (FI elements), referred to as I. These were identified by performing an ex vivo selection procedure on sequences that confer RNA stability and enhance total protein expression (see WO 2017/060314, incorporated herein by reference).

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

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

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

In some embodiments, no nucleotide other than the a nucleotide is flanking the 3 'end of the polyA tail, i.e., the polyA tail is not masked or followed at its 3' end by a nucleotide other than a.

5 'Cap in some embodiments, the RNAs described herein may comprise a 5' cap that may be incorporated into such RNAs during transcription, or linked to such RNAs after transcription. In some embodiments, the RNA can comprise a 5' cap structure for co-transcriptional capping of the RNA. Some examples of cap structures for co-transcribing capping are known in the art, including, for example, as described in WO 2017/053297, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the 5 'cap contained in the RNAs described herein is or comprises m7G (5') ppp (5 ') (2' ome a) pG. In some embodiments, the 5' cap included in the RNAs described herein is or includes a cap structure [ e.g., m₂^7,3'-OGppp(m₁^2'-O) ApG ]. When the RNA sequences described herein have a5 'end with the nucleotide 5' -AG, and it is described that the RNA comprises a5 'cap [ e.g., m₂^7,3'-OGppp(m₁^2'-O) ApG ] containing the second and third nucleotides a and G, respectively, it is understood that in some embodiments, the second and third nucleotides of the cap correspond to the nucleotide 5' -AG of the RNA sequence.

Chemical modification in some embodiments, the RNA encoding the CLDN-18.2 targeted antibody agent may comprise at least one modified ribonucleotide, e.g., in some embodiments to increase the stability of such RNA and/or reduce the immunogenicity of such RNA and/or reduce the cytotoxicity of such RNA. For example, in some embodiments, at least one of A, U, C and G ribonucleotides of the RNA can be replaced by a modified ribonucleotide. For example, in some embodiments, some or all of the cytidine residues present in the RNA can be replaced with a modified cytidine, which in some embodiments can be, for example, 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all of the uridine residues present in the RNA may be replaced with a modified uridine, which in some embodiments may be 3-methyl-uridine (m 3U), 5-methoxy-uridine (mo 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), Uridine 5-glycollic acid (cmo 5U), uridine 5-glycollic acid methyl ester (mcmo U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm U), 5-methoxycarbonylmethyl-uridine (mcm 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5s 2U), 5-aminomethyl-2-thio-uridine (nm 5s 2U), 5-methylaminomethyl-uridine (mcm 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mcm 5s 2U), 5-methylaminomethyl-2-seleno-uridine (mnm 5se 2U), 5-carbamoylmethyl-uridine (ncm U), 5-carboxymethyl aminomethyl-uridine (cmnm U), 5-carboxymethyl aminomethyl-2-thio-uridine (cmnm s 2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5s 2U), 1-methyl-4-thio-pseudouridine (m 1s 4. Phi.), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3. Phi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp 3U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp 3. Phi.), 5- (isopentenylaminomethyl) uridine (mm 5U), 5- (isopentenylaminomethyl) -2-thio-uridine (mm 5s 2U), alpha-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m 5 Um), 2 '-O-methyl-pseudouridine (. Phi.,) 2-thio-2' -O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2 '-O-methyl-uridine (mm 5 Um), alpha-thio-uridine, 2' -O-methyl-uridine (mm 5 Um), 5-carbamoylmethyl-2 ' -O-methyl-uridine (ncm Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm Um), 3,2' -O-dimethyl-uridine (m 3 Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm 5 Um), 1-thio-uridine, deoxythymidine, 2' -F-arabino-uridine, 2' -F-uridine, 2' -OH-arabino-uridine, 5- (2-methoxycarbonylvinyl) uridine, 5- [3- (1-E-propenyl) amino) uridine, or any other modified uridine known in the art. In some embodiments, some or all of the uridine residues present in the RNA may be replaced with a modified uridine selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), 5-methyl-uridine (m 5U) and combinations thereof. In some embodiments, some or all of the uridine residues present in the RNA may be replaced with pseudouridine or derivatives thereof (e.g., 1-methyl pseudouridine). In some embodiments, some or all of the uridine residues present in the RNA may be replaced with pseudouridine. In some embodiments, some or all of the uridine residues present in the RNA may be replaced with 1-methyl pseudouridine. In some embodiments, all uridine residues present in RNA are replaced with pseudouridine. In some embodiments, all uridine residues present in the RNA are replaced with 1-methyl pseudouridine.

Codon optimization and GC enrichment the codons of the RNAs (particularly mRNA) described in the present disclosure may be further optimized, for example to increase the GC content of the RNA and/or to replace codons rare in a cell (or subject) in which the peptide or polypeptide of interest is to be expressed with common codons synonymously in the cell (or subject). In some embodiments, the amino acid sequences encoded by the RNAs (particularly mrnas) described in the present disclosure are encoded by coding sequences that are codon optimized and/or have increased G/C content as compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon optimized and/or have an increased G/C content as compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, codon optimization and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. In some embodiments, the guanosine/cytosine (G/C) content of the coding region of an RNA (particularly an mRNA) described herein is increased compared to the G/C content of a corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably unmodified 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 region of RNA to be translated is important for efficient translation of the RNA. Sequence ratios with increased G (guanosine)/C (cytosine) content have increased A (adenosine)/U

The sequence of (uracil) content is more stable. With respect to the fact that several codons encode one and the same amino acid (so-called degeneracy of the genetic code), the most advantageous codons for stability (so-called substitution codon usage) can be determined. Depending on the amino acids encoded by the RNA, there are a number of possibilities for modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing a and/or U nucleotides can be modified by replacing these codons with other codons (which encode the same amino acid but do not contain a and/or U or contain a lower content of a and/or U nucleotides). In various embodiments, the G/C content of the coding region of an RNA (particularly mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% or even more as compared to the G/C content of the coding region of a wild-type RNA.

Non-immunogenic RNA in certain embodiments, by incorporating modified nucleosides into RNA that inhibit RNA-mediated activation of innate immune receptors and/or limit double-stranded RNA

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

In order to render non-immunogenic RNAs (particularly mRNA) non-immunogenic by incorporating modified nucleosides, any modified nucleoside may be used as long as it reduces or inhibits the immunogenicity of the RNA. Particularly preferred are modified nucleosides that inhibit RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises a substitution of one or more uridine with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising the modified nucleobase is selected from the group consisting of 3-methyl-uridine (m³ U), 5-methoxy-uridine (mo⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s² U), 4-thio-uridine (s⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-glycolic acid (cmo⁵ U), Uridine 5-glycollic acid methyl ester (mcmo⁵ U), 5-carboxymethyl-uridine (cm⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵ U), 5-Methoxycarbonylmethyl-uridine (mcm⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s² U), 5-aminomethyl-2-thio-uridine (nm⁵s² U), 5-methylaminomethyl-uridine (nm⁵ U), and, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (nm⁵s² U), 5-methylaminomethyl-2-seleno-uridine (nm⁵se² U), 5-carbamoylmethyl-uridine (ncm⁵ U), 5-carboxymethyl aminomethyl-uridine (cmnm⁵ U), 5-carboxymethyl aminomethyl-2-thio-uridine (cmnm⁵s² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm⁵ U), and, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m⁵s² U), 1-methyl-4-thio-pseudouridine (m¹s⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp³ U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp³. Phi.), 5- (isopentenylaminomethyl) uridine (inm⁵ U), 5- (prenylaminomethyl) -2-thio-uridine (inm⁵s² U), alpha-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m⁵ Um), 2 '-O-methyl-pseudouridine (ψm), 2-thio-2' -O-methyl-uridine (s² Um), 5-Methoxycarbonylmethyl-2 ' -O-methyl-uridine (mcm⁵ Um), 5-carbamoylmethyl-2 ' -O-methyl-uridine (ncm⁵ Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm⁵ Um), 3,2' -O-dimethyl-uridine (m³ Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm⁵ Um), 1-thio-uridine, deoxythymidine, 2' -F-arabino-uridine, 2' -F-uridine, 2' -OH-arabino-uridine, 5- (2-methoxycarbonylvinyl) uridine and 5- [3- (1-E-propenyl amino) uridine. In certain embodiments, the nucleoside comprising the modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), or 5-methyl-uridine (m 5U), particularly N1-methyl-pseudouridine.

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

During synthesis of mRNA by In Vitro Transcription (IVT) using T7 RNA polymerase, a number of abnormal products, including double-stranded RNA (dsRNA), are produced due to the unusual activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes, resulting in inhibition of protein synthesis. Formation of dsRNA may be limited during synthesis of mRNA by In Vitro Transcription (IVT), for example, by limiting the amount of uridine triphosphate (uridine triphosphate, UTP) during synthesis. Optionally, UTP may be added one or several times during mRNA synthesis. Likewise, dsRNA can be removed from RNA (e.g., IVT RNA) by ion-pair reverse phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using E.coli (E.coli) RNase III that specifically hydrolyzes dsRNA but does not hydrolyze ssRNA can be used, thereby eliminating dsRNA contaminants from the IVT RNA formulation. Furthermore, dsRNA can be isolated from ssRNA by using cellulosic material. In some embodiments, the RNA formulation is contacted with the cellulosic material and the ssRNA is separated from the cellulosic material under conditions that allow the dsRNA to bind to the cellulosic material but not allow the ssRNA to bind to the cellulosic material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524. In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, e.g., dsRNA (especially dsmRNA) is removed from the 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 (particularly mRNA) is free or substantially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.). Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with an antibody specific for dsRNA, and the amount of antibody bound to RNA may be used as a measure of the amount of dsRNA in the sample. Samples containing known amounts of dsRNA can be used as references. For example, RNA can be spotted onto a membrane, such as a nylon blotting membrane. The membrane may be blocked, for example in TBS-T buffer (20mM TRIS pH 7.4,137mM NaCl,0.1% (v/v) Tween-20) containing 5% (w/v) skim milk powder. For detection of dsRNA, the membrane may be incubated with a dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (engish & SCIENTIFIC CONSULTING, szir ak, 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. No. 715-035-150), and the signal provided by the secondary antibody may be detected, non-immunogenic RNAs (particularly mrnas) are more efficient in translation in cells than standard RNAs having the same sequence, the translation is enhanced by a factor of 2 relative to its unmodified counterpart, in some embodiments, in some embodiments, translation is enhanced to a factor of 4. In some embodiments, translation is enhanced to a factor of 5. In some embodiments, translation is enhanced to a factor of 6. In some embodiments, translation is enhanced to a factor of 7. In some embodiments, translation is enhanced to a factor of 8. In some embodiments, translation is enhanced to a factor of 9. In some embodiments, translation is enhanced to a factor of 10. In some embodiments, translation is enhanced to a factor of 15. In some embodiments, translation is enhanced to a factor of 20. In some embodiments, translation is enhanced to a factor of 50. In some embodiments, translation is enhanced to a factor of 100. In some embodiments, translation is enhanced to a factor of 200. In some embodiments, translation is enhanced to a factor of 500. In some embodiments, translation is enhanced to a factor of 1000. In some embodiments, translation is enhanced to a factor of 2000. In some embodiments, the factor is 10 to 1000 times. In some embodiments, the factor is 10 to 100 times. In some embodiments, the factor is 10 to 200 times. In some embodiments, the factor is 10 to 300 times. In some embodiments, the factor is 10 to 500 times. In some embodiments, the factor is 20 to 1000 times. In some embodiments, the factor is 30 to 1000 times. In some embodiments, the factor is 50 to 1000 times. In some embodiments, the factor is 100 to 1000 times. In some embodiments, the factor is 200 to 1000 times. In some embodiments, translation is enhanced to any other significant amount or range of amounts. In some embodiments, the non-immunogenic RNA (particularly mRNA) exhibits significantly lower inherent immunogenicity than a standard RNA having the same sequence. In some embodiments, the non-immunogenic RNA (particularly mRNA) exhibits an innate immune response that is one-half as low as its unmodified counterpart. In some embodiments, the inherent immunogenicity is reduced to a factor of 3. In some embodiments, the inherent immunogenicity is reduced to a factor of 4. In some embodiments, the inherent immunogenicity is reduced to a factor of 5. In some embodiments, the inherent immunogenicity is reduced to a factor of 6. In some embodiments, the inherent immunogenicity is reduced to a factor of 7. In some embodiments, the inherent immunogenicity is reduced to a factor of 8. In some embodiments, the inherent immunogenicity is reduced to a factor of 9. In some embodiments, the inherent immunogenicity is reduced to a factor of 10. In some embodiments, the inherent immunogenicity is reduced to a factor of 15. In some embodiments, the inherent immunogenicity is reduced to a factor of 20. In some embodiments, the inherent immunogenicity is reduced to a factor of 50. In some embodiments, the inherent immunogenicity is reduced to a factor of 100. In some embodiments, the inherent immunogenicity is reduced to a factor of 200. In some embodiments, the inherent immunogenicity is reduced to a fraction of 500. In some embodiments, the inherent immunogenicity is reduced to a fraction of 1000. In some embodiments, the inherent immunogenicity is reduced to a fraction of 2000. The term "exhibiting significantly lower intrinsic immunogenicity" refers to a detectable decrease in intrinsic immunogenicity. In some embodiments, the term refers to reducing such that an effective amount of non-immunogenic RNA (particularly mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to reducing such that non-immunogenic RNAs (particularly mrnas) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce the production of proteins encoded by the non-immunogenic RNAs. In some embodiments, the reduction allows for repeatable administration of non-immunogenic RNAs (particularly mRNA) without eliciting an innate immune response sufficient to eliminate detectable production of proteins encoded by the non-immunogenic RNAs.

In some embodiments, the RNA encoding the heavy chain of a CLDN-18.2 targeted antibody agent comprises in the 5' to 3' direction (a) a 5' UTR, (b) a secretion signal coding region, (c) a heavy chain coding region;

(d) 3' UTR, and (e) polyA tail. See, for example, fig. 13. In some embodiments, the 5' utr is or comprises a sequence derived from human α -globin mRNA in combination with a Kozak region. In some embodiments, the secretion signal encoding region is or comprises a nucleotide sequence encoding the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or a nucleotide sequence encoding the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the heavy chain coding region encodes the V_H domain, the C_H1 domain, the C_H2 domain, and the C_H3 domain of a CLDN-18.2 targeting antibody agent in IgG form (e.g., as described herein, such as IMAB 262), or an amino acid sequence represented by amino acid residues 27 to 474 of SEQ ID NO: 3. In some embodiments, the 3' UTR is or comprises a combination of at least two sequence elements (FI elements) derived from a "split amino terminal enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I). In some embodiments, the polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately after 30 consecutive adenosines). In some embodiments, such RNA comprises a 5' CAP structure comprising a CAP1 structure, or m₂^7,3'-OGppp(m₁^2'-O) ApG. In some embodiments, such RNA comprises all uridine replaced with N1-methyl pseudouridine.

In some embodiments, the RNA encoding the light chain of a CLDN-18.2 targeted antibody agent comprises in the 5 'to 3' direction (a) a 5'UTR, (b) a secretion signal encoding region, (c) a light chain encoding region, (d) a 3' UTR, and (e) a polyA tail. See, for example, fig. 13. In some embodiments, the 5' utr is or comprises a sequence derived from human α -globin mRNA in combination with a Kozak region. In some embodiments, the secretion signal encoding region is or comprises a nucleotide sequence encoding the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS or a nucleotide sequence encoding the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS. In some embodiments, the light chain coding region encodes the V_L domain and the C_L domain of a CLDN-18.2 targeting antibody agent in IgG form (e.g., as those described herein, e.g., IMAB 262), or the amino acid sequence represented by amino acid residues 27 to 246 of SEQ ID NO: 4. In some embodiments, the 3' UTR is or comprises the following, an mRNA (referred to as F) derived from "split amino terminal enhancer" (AES) and a mitochondrially encoded 12S ribosomal RNA

A combination of at least two sequence elements (FI elements), referred to as I. In some embodiments, the polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately after 30 consecutive adenosines). In some embodiments, such RNA comprises a 5' CAP structure comprising a CAP1 structure, or m₂^7,3'-OGppp(m₁^2'-O) ApG. In some embodiments, such RNA comprises all uridine replaced with N1-methyl pseudouridine.

In some embodiments, the RNA is or comprises one or more single-stranded RNAs (e.g., single-stranded mRNA).

In some embodiments, the composition comprises a single-stranded mRNA encoding a heavy chain (e.g., open reading frame, ORF) of an antibody agent that targets CLDN-18.2 (e.g., an antibody agent that targets CLDN-18.2 described herein) and a single-stranded mRNA encoding a light chain (e.g., open reading frame, ORF) of an antibody agent that targets CLDN-18.2 (e.g., an antibody agent that targets CLDN-18.2 described herein) that is translated into the corresponding subunit in the target cell after introduction into the target cell and forms an intact IgG antibody. An exemplary drug substance is schematically illustrated in fig. 13.

In some embodiments, the RNA drug substance is or comprises a combination of two RNAs encoding the Heavy Chain (HC) and the Light Chain (LC) of an IgG CLDN-18.2 targeted antibody, respectively. In some embodiments, each of such two RNAs may be manufactured separately, and the RNA drug substance may be prepared by mixing RNAs encoding HC and LC of an IgG CLDN-18.2 targeting antibody, respectively, in a suitable weight ratio (e.g., such that the resulting molar ratio of RNAs encoding HC and LC is about 1.5:1 to 1:1.5 to form a suitable weight ratio of IgG).

In some embodiments, the first RNA encoding a polypeptide comprising a heavy chain of a CLDN-18.2 targeted antibody agent and the second RNA encoding a polypeptide comprising a light chain of a CLDN-18.2 targeted antibody agent may be present in a molar ratio of about 1.5:1 to about 1:1.5. In some embodiments, such first and second RNAs can be present in a ratio of about 1.30, about 1.29, about 1.28, about 1.27, about 1.26, about 1.25, about 1.24, about 1.23, about 1.22, about 1.21, about 1.20, about 1.19, about 1.18, about 1.17, about 1.16, about 1.15, about 1.14, about 1.13, about 1.12, about 1.11, about 1.10, about 1.09, about 1.08, about 1.07, about 1.06, about 1.05, about 1.04, about 1.03, about 1.02, about 1.01, about 1.00, about 0.99, about 0.98, about 0.97, about 0.96, about 0.95, about 0.94, about 0.93, about 0.92, about 0.91, about 0.90, about 0.89, about 0.88, about 0.84, about 0.83, about 0.82, or about 80. In some embodiments, such first RNA and second RNA can be present in a weight ratio of 3:1 to 1:1. In some embodiments, such first RNA and second RNA can be present in a weight ratio of about 2:1. In some embodiments, such first RNA and second RNA can 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.

In some embodiments, the RNA encoding HC and/or LC of the CLDN-18.2 targeted IgG antibody may comprise one or more non-coding sequence elements, e.g., to enhance RNA stability and/or translation efficiency. For example, in some embodiments, such RNAs may comprise a cap structure, e.g., a cap structure that may increase resistance of RNA molecules to extracellular and intracellular rnase degradation and result in higher protein expression. In some embodiments, the exemplary cap structure is (m₂^7,3'-OGppp(m₁^2'-O) ApG (cap 1) or comprises (m₂^7,3'-OGppp(m₁^2'-O) ApG (cap 1). In some embodiments, such RNAs may comprise one or more non-coding sequence elements at one or both of the 5 'and 3' untranslated regions (UTRs), e.g., naturally occurring sequence elements at the 5 'and 3' UTRs, which may significantly increase the intracellular half-life and translation efficiency of the molecule (see, e.g., holtkamp et al 2006; orlandini von NIESSEN ET al 2019). In some embodiments, exemplary 5' utr sequence elements are or comprise a characteristic sequence from human α -globin and a Kozak consensus sequence. In some embodiments, an exemplary 3' UTR sequence element is or comprises a combination of two sequence elements (FI elements) derived from a "split amino terminal enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I). For sequence information of exemplary 3' UTR sequence elements see, e.g., WO 2017/060314, the entire contents of which are incorporated herein by reference. In some embodiments, such RNAs can comprise poly (a) tails, e.g., poly (a) tails designed to enhance RNA stability and/or translation efficiency. In some embodiments, an exemplary poly (a) tail is a modified poly (a) sequence of 110 nucleotides in length or a modified poly (a) sequence comprising a stretch of 110 nucleotides in length comprising a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence and another stretch of 70 adenosine residues (a 30L 70). In some embodiments, such RNAs may comprise one or more modified ribonucleotides. For example only, in some embodiments, uridine of RNA can be replaced with a modified analog (e.g., N1-methyl pseudouridine) to reduce and/or inhibit immunomodulatory activity and thus enhance translation of RNA.

In some embodiments, the RNA drug substance is or comprises a combination of a first RNA having an RNA-HC construct as disclosed in Table 2 below and a second RNA having an RNA-LC construct as disclosed in Table 2 below. In some such embodiments, the RNA drug substance can be prepared by mixing the first RNA and the second RNA in a weight ratio of about 2:1.

TABLE 2 exemplary constructs encoding RNAs for CLDN-18.2 targeted IgG antibodies

It is shown herein that the expression intensity of an antibody agent encoded by RNA depends on the choice of specific sequences in the coding region as well as in the non-coding region. Thus, it has been shown that the open reading frames shown in SEQ ID NO. 16 and SEQ ID NO. 17 result in a strong expression of the encoded antibody agent when cells are transfected with RNA containing these open reading frames.

It is also shown that the antibody agent is expressed after in vivo administration of the RNA encoding it. In particular, it is shown that the antibody agent is expressed after intravenous administration of the RNA encoding it.

RNA comprising a 5' UTR comprising the sequence shown in SEQ ID NO. 18 or SEQ ID NO. 20 has been shown to be suitable.

Furthermore, RNAs comprising a 3' UTR comprising the FI element, in particular the sequence shown in SEQ ID NO. 22, have been shown to be suitable.

RNA comprising a 3' UTR comprising the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 21 has been shown to be suitable.

Thus, RNAs comprising a 5'UTR comprising the sequence shown in SEQ ID NO. 18 or SEQ ID NO. 20 and a 3' UTR comprising the sequence shown in SEQ ID NO. 19 or SEQ ID NO. 21 have been shown to be suitable.

In particular, RNAs comprising a 5'UTR comprising the sequence shown in SEQ ID NO. 18 and a 3' UTR comprising the sequence shown in SEQ ID NO. 19 have been shown to be suitable.

In particular, RNAs comprising a 5'UTR comprising the sequence shown in SEQ ID NO. 20 and a 3' UTR comprising the sequence shown in SEQ ID NO. 21 have been shown to be suitable.

RNA comprising polyA sequences, in particular the sequences shown in SEQ ID NO. 23, have been shown to be suitable.

RNAs comprising a first RNA comprising the sequence shown in SEQ ID NO. 24 or SEQ ID NO. 26 and a second RNA comprising the sequence shown in SEQ ID NO. 25 or SEQ ID NO. 27 have been shown to be suitable.

RNAs comprising a first RNA comprising the sequence set forth in SEQ ID NO. 24 and a second RNA comprising the sequence set forth in SEQ ID NO. 25 have been shown to be suitable.

In particular, RNAs comprising a first RNA comprising the sequence set forth in SEQ ID NO. 26 and a second RNA comprising the sequence set forth in SEQ ID NO. 27 have been shown to be suitable.

Furthermore, RNA formulated in lipid nanoparticles has been shown to be suitable.

B. exemplary manufacturing methods

The individual RNAs can be produced by methods known in the art. For example, in some embodiments, RNA can be produced by in vitro transcription, e.g., using a DNA template. Plasmid DNA used as an in vitro transcription template to generate RNA described herein is also within the scope of the present disclosure.

DNA templates are used for in vitro RNA synthesis in the presence of suitable RNA polymerases (e.g., recombinant RNA polymerase, e.g., T7 RNA polymerase) and ribonucleotides triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, RNA (e.g., an RNA described herein) can be synthesized in the presence of modified ribonucleotides triphosphates. For example only, in some embodiments, N1-methyl pseudouridine triphosphate (^m1 ψTP) may be used in place of Uridine Triphosphate (UTP). As will be apparent to those of skill in the art, during in vitro transcription, RNA polymerase (e.g., as described and/or used herein) typically passes through at least a portion of the DNA template in the 3'→5' direction to produce complementary RNA in the 5'→3' direction.

In some embodiments in which the RNA comprises a polyA tail, those skilled in the art will appreciate that such polyA tail may be encoded in a DNA template, for example, by using appropriate tailed PCR primers, or it may be added to the RNA after in vitro transcription, for example, by enzymatic treatment (for example, using a Poly (a) polymerase, such as e.g. escherichia coli Poly (a) polymerase).

In some embodiments, one of skill in the art will appreciate that adding a 5' cap to RNA (e.g., mRNA) can aid in RNA recognition and ligation of RNA to ribosomes to initiate translation and enhance translation efficiency. Those skilled in the art will also appreciate that the 5 'cap may also protect the RNA product from 5' exonuclease mediated degradation and thus increase half-life. Methods of capping are known in the art, and one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, e.g., capping enzymes such as vaccinia virus). In some embodiments, the cap and the plurality of ribonucleoside triphosphates can be introduced during in vitro transcription such that the cap is incorporated into the RNA during transcription (also referred to as co-transcription capping). In some embodiments, a 5 'cap analog (e.g., a 5' cap analog as described herein, e.g., as m₂^7,3'-OGppp(m^2'-O) ApG) for co-transcription capping may be used during in vitro transcription. During polymerization, the RNA is capped at the 5 'end with a 5' cap analog (e.g., m₂^7,3'-OGppp(m^2'-O) ApG. In some embodiments, a multiple addition GTP fed-batch operation during the reaction may be used to maintain a low concentration of GTP to effectively cap the RNA.

After RNA transcription, the DNA template is digested. In some embodiments, digestion may be accomplished under appropriate conditions using dnase I.

In some embodiments, the in vitro transcribed RNA may be provided in a buffer solution, e.g., in a buffer such as HEPES, phosphate buffer, citrate buffer, acetate buffer, in some embodiments such a solution may be buffered to a pH in the range of, e.g., about 6.5 to about 7.5, in some embodiments, about 7.0. In some embodiments, the generation of RNA may further comprise one or more of the steps of purification, mixing, filtration, and/or packing.

In some embodiments, the RNA can be purified (e.g., in some embodiments, after an in vitro transcription reaction), for example, to remove components used or formed during production, such as, for example, proteins, DNA fragments, and/or nucleotides. A variety of nucleic acid purifications known in the art may be used in accordance with the present disclosure. Some purification steps may be or include, for example, one or more of precipitation, column chromatography (which includes but is not limited to anion, cation, hydrophobic interaction chromatography (hydrophobic interaction chromatography, HIC)), solid matrix-based purification (e.g., magnetic bead-based purification). In some embodiments, the RNA can be purified using magnetic bead-based purification, which in some embodiments can be or include magnetic bead-based chromatography. In some embodiments, hydrophobic Interaction Chromatography (HIC) and/or diafiltration may be used to purify RNA. In some embodiments, HIC followed by diafiltration may be used to purify RNA.

In some embodiments, the dsRNA may be obtained as a byproduct during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, a cellulosic material (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, e.g., in some embodiments in chromatographic form. In some embodiments, the cellulosic material (e.g., microcrystalline cellulose) may be pretreated to inactivate potential rnase contamination, for example in some embodiments by autoclaving followed by incubation with an aqueous alkaline solution (e.g., naOH). In some embodiments, the RNA can be purified using cellulosic material according to the methods described in WO 2017/182524 (the entire contents of which are incorporated herein by reference).

In some embodiments, the RNA batch may be further processed by one or more filtration and/or concentration steps. For example, in some embodiments, RNA (e.g., after removal of dsRNA contamination) may also be diafiltered (e.g., in some embodiments, by tangential flow filtration), e.g., to adjust the concentration of RNA to a desired RNA concentration and/or to replace the buffer with a drug substance buffer.

In some embodiments, wherein the CLDN-18.2 targeted antibody agent is encoded by a first RNA encoding the heavy chain of the CLDN-18.2 targeted antibody agent and a second RNA encoding the light chain of the CLDN-18.2 targeted antibody agent such that upon translation and expression, the first RNA batch and the second RNA batch each can be mixed in a suitable ratio after purification (e.g., as described herein). For example, in some embodiments, such first and second RNA batches may be mixed in a molar ratio of about 1:1.5 to about 1.5:1, e.g., in some embodiments in a molar ratio of about 1:1.

In some embodiments, the RNA can be treated by 0.2 μm filtration before it is filled into a suitable container.

In some embodiments, the RNA and compositions thereof can be made according to methods as described herein or as otherwise known in the art.

In some embodiments, the RNA and compositions thereof can be manufactured in large scale. For example, in some embodiments, RNA batches can be manufactured on a scale of greater than 1g, greater than 2g, greater than 3g, greater than 4g, greater than 5g, greater than 6g, greater than 7g, greater than 8g, greater than 9g, greater than 10g, greater than 15g, greater than 20g, or greater.

In some embodiments, RNA quality control may be performed and/or monitored at any time during the production process of RNA and/or compositions comprising the same. For example, in some embodiments, RNA quality control parameters, including one or more of RNA identity (e.g., sequence, length, and/or RNA properties), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or some steps of the RNA manufacturing process, e.g., after in vitro transcription and/or after each purification step.

In some embodiments, the stability of an RNA (e.g., produced by in vitro transcription) and/or a composition comprising two or more RNAs (e.g., one HC encoding an antibody and another LC encoding an antibody) can be assessed over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or more) under a variety of test storage conditions, such as at room temperature and refrigerator or subzero temperatures. In some embodiments, RNA (e.g., RNA as described herein) and/or compositions thereof can be stably stored at refrigerator temperatures (e.g., about 4 ℃ to about 10 ℃) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, RNA (e.g., RNA as described herein) and/or compositions thereof can be stably stored at subzero temperatures (e.g., -20 ℃ or less) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, the RNA (e.g., RNA described herein) and/or compositions thereof can be stable for storage for at least 1 month or more at room temperature (e.g., at about 25 ℃).

In some embodiments, one or more of the evaluations as described in example 11 can be used during manufacture of the RNA or other preparation or use (e.g., as a release test).

In some embodiments, one or more quality control parameters may be evaluated to determine whether the RNAs described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for dispensing). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art, for example, one skilled in the art will recognize that in some embodiments, RNA quality assessment may be performed using one or more analytical tests. Some examples of such certain analytical tests may include, but are not limited to, gel electrophoresis, UV absorption, and/or PCR assays.

In some embodiments, one or more characteristics of the RNA batch as described herein may be evaluated to determine the next course of action. For example, if an RNA quality assessment indicates that an RNA batch meets or exceeds relevant acceptance criteria, such an RNA batch may be designated for one or more additional steps of manufacture and/or formulation and/or distribution. Otherwise, if such an RNA lot does not meet or exceed the acceptance criteria, then an alternative action may be taken (e.g., discard the lot).

In some embodiments, RNA batches meeting the evaluation results can be used for one or more additional steps of manufacturing and/or formulation and/or dispensing.

RNA delivery techniques

The provided RNA (e.g., mRNA) can be delivered for therapeutic applications described herein using any suitable method known in the art, including, for example, as naked RNA delivery, or by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vector-mediated delivery. See, e.g., ,Wadhwa et al."Opportunities and Challenges in the Delivery of mRNA-Based Vaccines"Pharmaceutics(2020)102(, page 27), the contents of which are incorporated herein by reference to obtain information about the various methods that can be used to deliver the RNAs described herein.

In some embodiments, one or more RNAs may be formulated with lipid nanoparticles for delivery (e.g., in some embodiments, by intravenous injection).

In some embodiments, the lipid nanoparticle may be designed to protect RNA (e.g., mRNA) from extracellular rnases and/or engineered for systemic delivery of RNA to target cells (e.g., hepatocytes). In some embodiments, such lipid nanoparticles may be particularly useful for delivering RNA (e.g., mRNA) when the RNA is administered intravenously to a subject in need thereof.

A. Lipid nanoparticles

In some embodiments, provided RNAs (e.g., mrnas) can be formulated with lipid nanoparticles. In various embodiments, such lipid nanoparticles may have an average size (e.g., average diameter) of about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 70 to about 90nm, or about 70nm to about 80nm. In some embodiments, the average size (e.g., average diameter) of lipid nanoparticles that can be used in accordance with the present disclosure can be about 50nm to about 100nm. In some embodiments, the average size (e.g., average diameter) of the lipid nanoparticle may be about 50nm to about 150nm. In some embodiments, the average size (e.g., average diameter) of the lipid nanoparticle may be about 60nm to about 120nm. In some embodiments, the average size (e.g., average diameter) of lipid nanoparticles that can be used in accordance with the present disclosure can be about 30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150nm.

In certain embodiments, when RNA (e.g., RNA) is present in the provided lipid nanoparticle, it is resistant to degradation with a nuclease in aqueous solution.

In some embodiments, the lipid nanoparticle is a liver-targeted lipid nanoparticle.

In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., the cationic lipids described herein). In some embodiments, the cationic lipid nanoparticle may comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

1. Helper lipids

In some embodiments, the lipid nanoparticle for delivery of RNA described herein comprises at least one helper lipid, which may be a neutral lipid, a positively charged lipid, or a negatively charged lipid. In some embodiments, the helper lipid is a lipid that can be used to increase the effectiveness of delivering the lipid-based particles (e.g., cationic lipid-based particles) to the target cell. In some embodiments, the helper lipid may be or comprise a structural lipid at a concentration selected to optimize LNP particle size, stability, and/or encapsulation.

In some embodiments, the lipid nanoparticle for delivering RNA described herein comprises a neutral helper lipid. Some examples of such neutral helper lipids include, but are not limited to, phosphatidylcholine, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), phosphatidylethanolamine (e.g., 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), sphingomyelin (sphingomyelin, SM), ceramides, cholesterol, steroids (e.g., sterols), and derivatives thereof.

In some embodiments, the lipid nanoparticle for delivery of RNA described herein comprises at least two helper lipids (e.g., helper lipids described herein). In some such embodiments, the lipid nanoparticle may comprise DSPC and cholesterol.

2. Cationic lipids

In some embodiments, the lipid nanoparticle for delivery of RNA described herein comprises a cationic lipid. Cationic lipids are typically lipids with a net positive charge. In some embodiments, the cationic lipid may comprise one or more positively charged amine groups. In some embodiments, the cationic lipid may comprise a cationic headgroup, meaning a positively charged headgroup. In some embodiments, the cationic lipid can have a hydrophobic domain (e.g., one or more domains of a neutral lipid or an anionic lipid), provided that the cationic lipid has a net positive charge. In some embodiments, the cationic lipid comprises a polar head group, in some embodiments it may comprise one or more amine derivatives, such as primary, secondary and/or tertiary amines, quaternary amines, various amine combinations, ammonium salts or guanidine and/or imidazole groups, as well as pyridine, piperazine and amino acid head groups (e.g., lysine, arginine, ornithine and/or tryptophan). In some embodiments, the polar head group of the cationic lipid comprises one or more amine derivatives. In some embodiments, the polar head group of the cationic lipid comprises a quaternary amine. In some embodiments, the headgroup of the cationic lipid may comprise a plurality of cationic charges. In some embodiments, the headgroup of the cationic lipid comprises one cationic charge. Some examples of monocationic lipids include, but are not limited to, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine (DMEPC), 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and/or 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP), 1, 2-dimyristoyl-3-trimethylammoniopropane (dmtpap), 2, 3-ditetradecyloxy) propyl- (2-hydroxyethyl) -dimethylazo-bromide (dmriie), didodecyl (dimethyl) azo-gen bromide (DDAB), 1, 2-dioleyloxy-propyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 3P- [ N- (n\n' -dimethylamino-ethane) carbamoyl ] cholesterol (DC-Choi), and/or dioleylether phosphatidylcholine (DOEPC).

In some embodiments, positively charged lipid structures described herein may also comprise one or more other components that may be generally used to form vesicles (e.g., for stabilization). Some examples of such other components include, but are not limited to, fatty alcohols, fatty acids and/or cholesterol esters, or any other pharmaceutically acceptable excipient that can affect surface charge, membrane fluidity, and facilitate incorporation of the lipid into the lipid assembly. Some examples of sterols include cholesterol, cholesterol hemisuccinate, cholesterol sulfate, or any other cholesterol derivative. Preferably, the at least one cationic lipid comprises DMEPC and/or DOTMA.

In some embodiments, the cationic lipid is ionizable such that it can exist in a positively charged form or a neutral form depending on pH. Such ionization of cationic lipids can affect the surface charge of the lipid particles at different pH conditions, which in some embodiments can affect plasma protein absorption, blood clearance, and/or tissue distribution, as well as the ability to form endosomal non-bilayer structures. Thus, in some embodiments, the cationic lipid may be or comprise a pH-responsive lipid. In some embodiments, the pH-responsive lipid is a fatty acid derivative or other amphiphilic compound capable of forming a readily soluble lipid phase and having a pKa value of from pH 5 to pH 7.5. This means that the lipid is uncharged at pH above the pKa value and positively charged at pH below the pKa value. In some embodiments, the pH-responsive lipids can be used to supplement (addition) or replace cationic lipids, for example, by binding one or more RNAs to a lipid or a mixture of lipids at a low pH. pH responsive lipids include, but are not limited to, 1, 2-dioleyloxy-3-dimethylamino-propane (DODMA).

In some embodiments, the lipid nanoparticle may comprise one or more cationic lipids, as described in WO 2017/075531 (e.g., as shown in tables 1 and 3 therein) and WO 2018/081480 (e.g., as shown in tables 1-4 therein), each of which is incorporated herein by reference in its entirety for the purposes described herein.

In some embodiments, cationic lipids that can be used in accordance with the present disclosure are amino lipids comprising a titratable tertiary amino headgroup linked to at least two saturated alkyl chains via an ester linkage that can be readily hydrolyzed to facilitate rapid degradation and/or excretion via the renal pathway. In some embodiments, such amino lipids have an apparent pK_a of about 6.0 to 6.5 (e.g., an apparent pK_a of about 6.25 in one embodiment), resulting in a molecule that is substantially fully positively charged at an acidic pH (e.g., pH 5). In some embodiments, such amino lipids, when incorporated into LNP, can confer different physicochemical properties that regulate particle formation, cellular uptake, fusibility (fusogenicity), and/or endosomal release of RNA. In some embodiments, introducing an aqueous RNA solution to a lipid mixture comprising such amino lipids at pH 4.0 can result in electrostatic interactions between the negatively charged RNA backbone and the positively charged cationic lipids. Without wishing to be bound by any particular theory, such electrostatic interactions result in particle formation consistent with efficient encapsulation of the RNA drug substance. After RNA encapsulation, the pH of the medium surrounding the resulting LNP is adjusted to a more neutral pH (e.g., pH 7.4) resulting in neutralization of LNP surface charge. When all other variables are kept constant, such charge neutral particles exhibit a longer in vivo circulation life and better delivery to hepatocytes than charged particles that are rapidly cleared by the reticuloendothelial system. After endosomal uptake, the low pH of the endosome fuses LNP comprising such amino lipids and allows release of RNA into the cytosol of the target cell.

In some embodiments, cationic lipids that can be used in accordance with the present disclosure have one of the structures shown in table 3 below:

TABLE 3 exemplary cationic lipids

In certain embodiments, a cationic lipid that can be used in accordance with the present disclosure is ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octoate) or comprises ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octoate), the chemical structure of which is shown in example 14.

The cationic lipids can be used alone or in combination with neutral lipids (e.g., cholesterol and/or neutral phospholipids), or in combination with other known lipid assembly components.

3. Polymer conjugated lipids

In some embodiments, the lipid nanoparticle for delivering RNA may comprise at least one polymer conjugated lipid. The polymer conjugated lipid is typically a molecule comprising a lipid moiety and a polymer moiety conjugated thereto.

In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid. In some embodiments, PEG conjugated lipids are designed to sterically stabilize the lipid particle by forming a protective hydrophilic layer that protects the hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, PEG conjugated lipids may reduce their association with serum proteins and/or uptake of the resulting reticuloendothelial system.

A variety of PEG conjugated lipids are known in the art and include, but are not limited to, pegylated diacylglycerol (PEGYLATED DIACYLGLYCEROL, PEG-DAG) (e.g., 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEGYLATED PHOSPHATIDYLETHANOLOAMINE, PEG-PE)), PEG diacylglycerol succinate (PEG succinate diacylglycerol, PEG-S-DAG) (e.g., 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) diethyl succinate (PEG-S-DMG)), pegylated ceramide (PEGYLATED CERAMIDE, PEG-cer), or PEG dialkoxypropyl carbamate (e.g., omega-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl-N- (omega methoxy (polyethoxy) ethyl) carbamate), and the like.

Certain PEG conjugated lipids (also known as pegylated lipids) are clinically approved and exhibit safety in clinical trials. PEG conjugated lipids are known to affect cellular uptake, which is a prerequisite for endosomal localization and payload delivery. The present disclosure provides, inter alia, insight that pharmacology of encapsulated nucleic acids can be controlled in a predictable manner by adjusting the alkyl chain length of the PEG-lipid anchors. In some embodiments, the present disclosure provides insight, inter alia, that such PEG conjugated lipids can be selected for RNA/LNP drug product formulations to provide optimal delivery of RNA to the liver. In some embodiments, such PEG conjugated lipids may be designed and/or selected based on reasonable solubility characteristics and/or molecular weights thereof to effectively perform the function of a spatial barrier. For example, in some embodiments, such pegylated lipids do not exhibit significant surfactant or permeability enhancement or interference effects on biological membranes. In some embodiments, PEG in such PEG conjugated lipids can be linked to the diacyl lipid anchors with biodegradable amide linkages, thereby facilitating rapid degradation and/or excretion. In some embodiments, LNP comprising PEG conjugated lipids retains a full complement of pegylated lipids. In the blood compartment, such pegylated lipids dissociate from the particles over time, showing more fused particles that are more easily taken up by the cells, ultimately resulting in release of the RNA payload.

In some embodiments, the lipid nanoparticle may comprise one or more PEG conjugated or pegylated lipids, as described in WO 2017/075531 and WO 2018/081480, each of which is incorporated herein by reference in its entirety for the purposes described herein. For example, in some embodiments, PEG conjugated lipids that may be used according to the present disclosure may have the following structure as described in WO 2017/075531 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

Wherein R₈ and R₉ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages, and w has an average value in the range of from 30 to 60. In some embodiments, R8 and R9 are each independently a straight saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 43 to 53. In other embodiments, the average w is about 45. In some embodiments, the PEG conjugated lipid is or comprises 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, the chemical structure of which is shown in example 14.

In some embodiments, the lipids forming the lipid nanoparticles described herein comprise a polymer conjugated lipid, a cationic lipid, and a helper neutral lipid. In some such embodiments, the total polymer conjugated lipid may be present at about 0.5mol% to 5mol%, about 0.7mol% to 3.5mol%, about 1mol% to 2.5mol%, about 1.5mol% to 2mol%, or about 1.5mol% to 1.8mol% of the total lipid. In some embodiments, the total polymer conjugated lipids may be present at about 1mol% to 2.5mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipid to total polymer conjugated lipid (e.g., PEG conjugated lipid) may be about 100:1 to about 20:1, or about 50:1 to about 20:1, or about 40:1 to about 20:1, or about 35:1 to about 25:1. In some embodiments, the molar ratio of total cationic lipid to total polymer conjugated lipid may be from about 35:1 to about 25:1.

In some embodiments involving polymer conjugated lipids, cationic lipids, and helper neutral lipids in lipid nanoparticles described herein, the total cationic lipids are present at about 35mol% to 65mol%, about 40mol% to 60mol%, about 41mol% to 49mol%, about 41mol% to 48mol%, about 42mol% to 48mol%, about 43mol% to 48mol%, about 44mol% to 48mol%, about 45mol% to 48mol%, about 46mol% to 48mol%, or about 47.2mol% to 47.8mol% of the total lipids. In certain embodiments, the total cationic lipids are present at about 47.0mol%, 47.1mol%, 47.2mol%, 47.3mol%, 47.4mol%, 47.5mol%, 47.6mol%, 47.7mol%, 47.8mol%, 47.9mol%, or 48.0mol% of the total lipids.

In some embodiments involving polymer conjugated lipids, cationic lipids, and helper neutral lipids in the lipid nanoparticles described herein, the total neutral lipids are present at about 35mol% to 65mol%, about 40mol% to 60mol%, about 45mol% to 55mol%, or about 47mol% to 52mol% of the total lipids. In some embodiments, the total neutral lipids are present at 35mol% to 65mol% of the total lipids. In some embodiments, the total non-steroid neutral lipids (e.g., DPSC) are present at about 5mol% to 15mol%, about 7mol% to 13mol%, or 9mol% to 11mol% of the total lipids. In some embodiments, the total non-steroid neutral lipids are present at about 9.5mol%, 10mol%, or 10.5mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipid to non-steroid neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to 4.8:1.0. In some embodiments, total steroid neutral lipids (e.g., cholesterol) are present at about 35mol% to 50mol%, about 39mol% to 49mol%, about 40mol% to 46mol%, about 40mol% to 44mol%, or about 40mol% to 42mol% of the total lipids. In certain embodiments, total steroid neutral lipids (e.g., cholesterol) are present at about 39mol%, 40mol%, 41mol%, 42mol%, 43mol%, 44mol%, 45mol%, or 46mol% of the total lipids. In certain embodiments, the molar ratio of total cationic lipid to total steroid neutral lipid is about 1.5:1 to 1:1.2 or about 1.2:1 to 1:1.2.

In some embodiments, a lipid composition comprising cationic lipids, polymer conjugated lipids, and neutral lipids may have individual lipids present in a particular molar percentage of total lipids or in a particular molar ratio (relative to each other) as described in WO 2018/081480, each of which is incorporated herein by reference in its entirety for the purposes described herein.

In some embodiments, the lipid forming the lipid nanoparticle comprises a polymer conjugated lipid (e.g., a PEG conjugated lipid), a cationic lipid, and a neutral lipid, wherein the polymer conjugated lipid is present at about 1mol% to 2.5mol% of the total lipid, the cationic lipid is present at 35mol% to 65mol% of the total lipid, and the neutral lipid is present at 35mol% to 65mol% of the total lipid. In some embodiments, the lipid forming the lipid nanoparticle comprises a polymer conjugated lipid (e.g., a PEG conjugated lipid), a cationic lipid, and a neutral lipid, wherein the polymer conjugated lipid is present at about 1mol% to 2mol% of the total lipid, the cationic lipid is present at 45mol% to 48.5mol% of the total lipid, and the neutral lipid is present at 45mol% to 55mol% of the total lipid. In some embodiments, the lipid forming the lipid nanoparticle comprises a polymer conjugated lipid (e.g., PEG conjugated lipid), a cationic lipid, and a neutral lipid (comprising a non-steroid neutral lipid and a steroid neutral lipid), wherein the polymer conjugated lipid is present at about 1mol% to 2mol% of the total lipid, the cationic lipid is present at 45mol% to 48.5mol% of the total lipid, the non-steroid neutral lipid is present at 9mol% to 11mol% of the total lipid, and the steroid neutral lipid is present at about 36mol% to 44mol% of the total lipid. In many such embodiments, the PEG-conjugated lipid is or comprises 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide or a derivative thereof. In many such embodiments, the cationic lipid is ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (2-octanoate) or a derivative thereof or comprises ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (2-octanoate) or a derivative thereof. In many such embodiments, the neutral lipid comprises DSPC and cholesterol, wherein DSPC is a non-steroid neutral lipid and cholesterol is a steroid neutral lipid.

In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic ionizable lipid (e.g., a cationic ionizable lipid as shown above), a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polymer conjugated lipid as shown in the above table. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising cationic lipid a, neutral lipid, steroid, and polymer conjugated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer conjugated lipid is a pegylated lipid, such as PEG conjugated lipid a. In some embodiments, the RNAs described herein are formulated in lipid nanoparticle compositions comprising a cationic ionizable lipid (e.g., a cationic ionizable lipid as shown above), a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a pegylated lipid as shown in the above table. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising cationic lipid a, neutral lipid, steroid, and pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is PEG conjugated lipid a. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic ionizable lipid (e.g., a cationic ionizable lipid as shown above), DSPC, cholesterol, and a pegylated lipid. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationically ionizable lipid, DSPC, cholesterol, and a pegylated lipid as shown in the above table. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic lipid A, DSPC, cholesterol, and a pegylated lipid. In some embodiments, the pegylated lipid is PEG conjugated lipid a. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationic ionizable lipid (e.g., a cationic ionizable lipid as shown above), DSPC, cholesterol, and PEG conjugated lipid a. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising a cationically ionizable lipid, DSPC, cholesterol, and PEG conjugated lipid a as shown in the above table. In some embodiments, the RNAs described herein are formulated in a lipid nanoparticle composition comprising cationic lipid A, DSPC, cholesterol, and PEG conjugated lipid a.

Cationic lipid A (((3-hydroxypropyl) azanediyl) bis (nonane-9, 1-diyl) bis (2-octanoate)

PEG conjugated lipid A2- [ (polyethylene glycol) -2000] -N, N-Bitetradecylacetamide/2- [2- (. Omega. -methoxy (polyethylene glycol 2000) ethoxy ] -N, N-Bitetradecylacetamide

DSPC 1, 2-distearoyl-sn-glycero-3-phosphorylcholine

Cholesterol:

the N/P value is preferably at least about 4. In some embodiments, the N/P value is 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

B. Exemplary method of preparing lipid nanoparticles

Lipid nanoparticles and lipids comprising nucleic acids and methods of making the same are known in the art and include, for example, as described in U.S. Pat. Nos. 8,569,256, 5,965,542 and U.S. patent publications

No.2016/0199485,2016/0009637,2015/0273068,2015/0265708,2015/0203446,2015/0005363,2014/0308304,2014/0200257,2013/086373,2013/0338210,2013/0323269,2013/0245107,2013/0195920,2013/0123338,2013/0022649,2013/0017223,2012/0295832,2012/0183581,2012/0172411,2012/0027803,2012/0058188,2011/0311583,2011/0311582,2011/0262527,2011/0216622,2011/0117125,2011/0091525,2011/0076335,2011/0060032,2010/0130588,2007/0042031,2006/0240093,2006/0083780,2006/0008910,2005/0175682,2005/017054,2005/0118253,2005/0064595,2004/0142025,2007/0042031,1999/009076 And PCT publication No.WO 99/39741,WO 2018/081480,WO 2017/004143,WO 2017/075531,WO 2015/199952,WO 2014/008334,WO 2013/086373,WO 2013/086322,WO 2013/016058,WO 2013/086373,W02011/141705, and WO 2001/07548, the entire disclosures of which are incorporated herein by reference in their entirety for the purposes described herein.

For example, in some embodiments, the cationic lipid, neutral lipid (e.g., DSPC and/or cholesterol), and polymer conjugated lipid may be dissolved in ethanol at a predetermined molar ratio (e.g., the molar ratios described herein). In some embodiments, the Lipid Nanoparticle (LNP) is prepared at a total lipid to RNA weight ratio of about 10:1 to 30:1. In some embodiments, such RNA can be diluted to 0.2mg/mL in acetate buffer.

In some embodiments, using ethanol injection techniques, a colloidal lipid dispersion comprising RNA can be formed by injecting an ethanol solution comprising lipids such as cationic lipids, neutral lipids, and polymer-conjugated lipids into an aqueous solution comprising RNA (e.g., RNA described herein).

In some embodiments, the lipid and RNA solutions may be mixed at room temperature by pumping each solution into a mixing unit at a controlled flow rate (e.g., using a piston pump). In some embodiments, the flow rates of the lipid solution and the RNA solution into the mixing unit are maintained at a ratio of 1:3. After mixing, nucleic acid-lipid particles are formed as the ethanol lipid solution is diluted by the aqueous RNA. Lipid solubility decreases, while positively charged cationic lipids interact with negatively charged RNAs.

In some embodiments, the solution comprising the RNA-encapsulated lipid nanoparticle may be treated by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.

In some embodiments, the RNA-encapsulated lipid nanoparticle may be treated by filtration (e.g., 0.2 μm filtration).

In some embodiments, the particle size and/or internal structure of the lipid nanoparticle may be monitored by suitable techniques such as, for example, small-angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (transmission electron cryomicroscopy, cryoTEM).

V. pharmaceutical composition provided for targeting claudin-18.2

In some embodiments, the compositions comprise provided RNAs encoding CLDN-18.2 targeted antibody agents. In some embodiments, such RNAs can be formulated with lipid nanoparticles (e.g., lipid nanoparticles described herein) for administration to a subject in need thereof. Accordingly, one aspect provided herein relates to a pharmaceutical composition comprising an RNA encoding a CLDN-18.2 targeted antibody agent provided and a lipid nanoparticle (e.g., a lipid nanoparticle described herein), wherein such RNA is encapsulated by the lipid nanoparticle.

In some embodiments in which the pharmaceutical composition comprises a first RNA encoding a variable heavy chain (V_H) domain of a CLDN-18.2 targeted antibody agent (e.g., a CLDN-18.2 targeted antibody agent described herein) and a second RNA encoding a variable light chain (V_L) domain of an antibody agent (e.g., an antibody agent described herein), such first RNA and second RNA may be present in a molar ratio of about 1.5:1 to about 1:1.5, or in some embodiments in a molar ratio of about 1.2:1 to about 1:1.2, or in some embodiments in a molar ratio of about 1:1. In some embodiments, a first RNA encoding a variable heavy chain (V_H) domain of a CLDN-18.2 targeted antibody agent (e.g., a CLDN-18.2 targeted antibody agent described herein) and a second RNA encoding a variable light chain (V_L) domain of an antibody agent (e.g., an antibody agent described herein) may be present in a weight ratio of 3:1 to 1:1, or in some embodiments, in a weight ratio of about 2:1.

In some embodiments, the RNA content of the pharmaceutical compositions described herein (e.g., one or more RNAs encoding CLDN-18.2 targeted antibody agents) is present at a concentration of about 0.5mg/mL to about 1.5mg/mL or about 0.8mg/mL to about 1.2 mg/mL.

Pharmaceutical formulations may additionally comprise pharmaceutically acceptable excipients, as used herein, which include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, such as those .Remington's The Science and Practice of Pharmacy,21st Edition,A.R.Gennaro(Lippincott,Williams&Wilkins,Baltimore,MD,2006; suitable for the particular dosage form desired, are incorporated herein by reference) disclose a variety of excipients for formulating pharmaceutical compositions, as well as known techniques for their preparation. Unless any conventional excipient medium is incompatible with the substance or derivative thereof, such as by producing any undesirable biological effect or in other cases interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present disclosure.

In some embodiments, the excipient is approved for human and for veterinary use. In some embodiments, the excipient is approved by the U.S. food and drug administration (United States Food and Drug Administration). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the united states pharmacopeia (United States Pharmacopoeia, USP), the european pharmacopeia (European Pharmacopoeia, EP), the british pharmacopeia (British Pharmacopoeia), and/or the international pharmacopeia (International Pharmacopoeia).

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or fragrances may be present in the composition at the discretion of the formulator.

General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21st ed., lippincott Williams & Wilkins,2005 (incorporated herein by reference).

In some embodiments, the pharmaceutical compositions provided herein may be formulated according to conventional techniques with one or more pharmaceutically acceptable carriers or diluents and any other known excipients and excipients, such as those disclosed in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21st ed., lippincott Williams & Wilkins,2005 (incorporated herein by reference).

The pharmaceutical compositions described herein may be administered by any suitable method known in the art. As will be appreciated by those of skill in the art, the route and/or mode of administration may depend on a variety of factors including, for example, but not limited to, the stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, including modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticle, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption (e.g., monostearates and gelatins).

Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration. In some embodiments, the pharmaceutical compositions may be prepared as described herein and/or by methods known in the art.

In some embodiments, the dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Some examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preserving, wetting, emulsifying and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the sterilization procedure and by the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, and the like). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption (e.g., aluminum monostearate and gelatin).

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or hereafter developed. Generally, such preparation methods include the steps of associating the active ingredient with a diluent or another excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, shaping, and/or packaging the product into the desired single or multiple dose units.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and/or methods described herein.

The relative amounts of RNA encapsulated in the LNP, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical composition can vary depending on the subject, target cell, disease or disorder to be treated, and can further depend on the route of administration of the composition.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. The actual dosage level of the active ingredient (e.g., RNA encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein can be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition being employed, the age, sex, weight, condition, general health and past history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian may begin with a dose of the active ingredient (e.g., RNA encapsulated in the lipid nanoparticle) below the level required in the pharmaceutical composition to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. For example, exemplary dosages as described in example 8 may be used to prepare pharmaceutically acceptable dosage forms.

In some embodiments, the pharmaceutical compositions described herein are formulated (e.g., for intravenous administration) to deliver an active amount that confers a plasma concentration of CLDN-18.2 targeted antibody encoded by RNA (e.g., the RNA described herein) that mediates pharmacological activity through its primary mode of action ADCC. The dose response correlation of ADCC was well characterized clinically for IMAB362 and it was reported that CLDN-18.2+ cells were effectively lysed by ADCC with EC₉₅ at 0.3 to 28 μg/mL (Sahin et al 2018). Thus, in some embodiments, the pharmaceutical compositions described herein are formulated (e.g., for intravenous administration) to deliver an active dose that confers a plasma concentration of about 0.3 to 28 μg/mL of a CLDN-18.2 targeted antibody encoded by RNA (e.g., RNA described herein) that mediates pharmacological activity through its primary mode of action ADCC.

In some embodiments, the pharmaceutical compositions described herein are formulated (e.g., for intravenous administration) to desirably achieve delivery of one or more RNAs (e.g., mrnas) described herein encoding an antibody agent against CLDN-18.2 at levels above about 0.1 μg/mL of antibody levels (e.g., plasma levels and/or tissue levels), in some embodiments, above about 0.2μg/mL、0.3μg/mL、0.4μg/mL、0.5μg/mL、0.6μg/mL、0.7μg/mL、0.8μg/mL、0.9μg/mL、1μg/mL、1.5μg/mL、2μg/mL、5μg/mL、8μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL or having ranges up to or above that observed with antibody administration.

In some embodiments, the pharmaceutical composition is formulated (e.g., for intravenous administration) to deliver a dose of 0.15mg RNA/kg, corresponding to about 7 μg/mL CLDN-18.2 targeted antibody agent at Cmax. Figure 14 shows dose-exposure correlation of RNA drug substance encoding CLDN-18.2 targeted antibody agent at tmax (48 hours) in cynomolgus monkeys. As will be appreciated by those of skill in the art, given that LNP transfection efficacy and mRNA translation are comparable between cynomolgus monkey and human (Coelho et al.2013), in some embodiments, the pharmaceutical composition is formulated (e.g., for intravenous administration) to deliver a suitable dose corresponding to the desired plasma level of CLDN-18.2 targeted antibody agent encoded by RNA, as shown in fig. 14.

In some embodiments, the pharmaceutical compositions described herein are formulated (e.g., for intravenous administration) to deliver a dose of one or more RNAs (e.g., mRNA) encoding an antibody agent against CLDN-18.2, at a dose as described in example 8, including, for example, at a dose of 0.15mg/kg、0.2mg/kg、0.225mg/kg、0.25mg/kg、0.3mg/kg、0.35mg/kg、0.4mg/kg、0.45mg/kg、0.5mg/kg、0.55mg/kg、0.6mg/kg、0.65mg/kg、0.7mg/kg、0.75mg/kg、0.80mg/kg、0.85mg/kg、0.9mg/kg、0.95mg/kg、1.0mg/kg、1.25mg/kg、1.5mg/kg、1.75mg/kg、2.0mg/kg、2.25mg/kg、2.5mg/kg、2.75mg/kg、3.0mg/kg、3.25mg/kg、3.5mg/kg、4mg/kg、5mg/kg or higher. In some embodiments, the pharmaceutical compositions described herein are formulated (e.g., for intravenous administration) to deliver a dose of one or more RNAs (e.g., mRNA) encoding an antibody agent to CLDN-18.2 at a dose of 1.5 mg/kg. In some embodiments, the pharmaceutical compositions described herein are formulated to deliver a dose of one or more RNAs (e.g., mRNA) encoding an antibody agent against CLDN-18.2 at a dose of 5 mg/kg.

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, which, for example, may in some embodiments enhance the stability of such compositions under specific conditions. Some examples of additives may include, but are not limited to, salts, buffer substances, preservatives, and carriers. For example, in some embodiments, the pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffer solution, which in some embodiments may comprise one or more salts, including, for example, alkali metal salts or alkaline earth metal salts, e.g., such as sodium, potassium, and/or calcium salts.

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more active agents other than RNA (e.g., ssRNA, e.g., mRNA) encoding a CLDN-18.2 targeting agent (e.g., an antibody agent). For example, in some embodiments, such other active agents may be or include chemotherapeutic agents. In some embodiments, exemplary chemotherapeutic agents may be or include chemotherapeutic agents suitable for treating pancreatic cancer including, for example, but not limited to, gemcitabine and/or paclitaxel (e.g., nab-paclitaxel), folinic acid, fluorouracil, irinotecan, oxaliplatin, and the like. In some embodiments, exemplary chemotherapeutic agents may be or include chemotherapeutic agents suitable for treating biliary tract cancers, including, for example, but not limited to, gemcitabine and/or cisplatin.

In some embodiments, the active agent that may be included in the pharmaceutical compositions described herein is or includes a therapeutic agent that is administered in a combination therapy described herein. The pharmaceutical compositions described herein may be administered as a combination therapy, i.e., in combination with other agents. For example, in some embodiments, a combination therapy may comprise a provided pharmaceutical composition with at least one anti-inflammatory agent or at least one immunosuppressant. Some examples of such therapeutic agents include, but are not limited to, one or more anti-inflammatory agents such as a steroid or NSAID (non-steroidal anti-inflammatory drug), aspirin (aspirin) and other salicylates, cox-2 inhibitors such as rofecoxib (Vioxx) and celecoxib (Celebrex), NASID such as ibuprofen ibuprofen (Motrin, advil), fenoprofen fenoprofen (Nalfon), naproxen Naprosyn, sulindac sulindac (Clinoril), diclofenac Voltaren, piroxicam piroxicam (Feldene), ketoprofen ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (nabumetone) (refen), etodolac etodolac (Lodine), oxaprozine oxaprozin) (Daypro) and indomethacin Indocin, in some embodiments such agents may result in low CTLA-dose or IL-2-receptor inactivation of anti-IL antibodies, such as CTLA-2.

In some embodiments, such therapeutic agents may include one or more chemotherapeutic agents, such as paclitaxel (Taxol) derivatives, taxotere (taxotere), paclitaxel (e.g., nab-paclitaxel), gemcitabine, 5-fluorouracil, doxorubicin (doxorubicin) (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan, procytox, neosar), folinic acid, irinotecan, oxaliplatin. In some embodiments, the pharmaceutical compositions described herein may be administered in combination with one or more chemotherapeutic agents that may increase CLDN-18.2 expression levels in tumors of a patient to be treated for cancer, e.g., by at least 10% or more, including, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more.

In some embodiments, the pharmaceutical compositions described herein may be administered in combination with radiation therapy and/or autologous peripheral stem cell or bone marrow transplantation.

In some embodiments, the pharmaceutical compositions described herein may be administered in combination with one or more antibodies selected from the group consisting of anti-CD 25 antibodies, anti-EPCAM antibodies, anti-EGFR, anti-Her 2/neu and anti-CD 40 antibodies.

In some embodiments, the pharmaceutical compositions described herein may be administered in combination with an anti-C3 b (i) antibody to enhance complement activation.

In some embodiments, the pharmaceutical compositions provided herein are preservative-free sterile RNA-LNP dispersions in aqueous buffers for intravenous administration. In some embodiments, the RNA drug substance (e.g., RNA described herein) contained in the pharmaceutical composition is filled to a nominal fill volume of 5.0mL at 0.8 to 1.2 mg/mL. The pharmaceutical composition is stored at-80 ℃ to-60 ℃.

Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to all kinds of animals. It is well understood that modifications are made to pharmaceutical compositions suitable for administration to humans to a variety of animals, and that a veterinarian of ordinary skill can design and/or make such modifications by merely ordinary (if any) experimentation.

A. identification and/or characterization of useful components

To ensure proper quality of the components useful in the pharmaceutical compositions described herein (e.g., RNA encoding CLDN-18.2 targeted antibody agents), one or more quality assessments and/or relevant criteria (e.g., as described in examples 11-12) can be performed and/or monitored.

Furthermore, the present disclosure provides methods of characterizing one or more characteristics of an RNA encoding a portion or all of an antibody agent or a composition thereof.

In some embodiments, RNA (e.g., in some embodiments, a composition comprising at least two RNAs each encoding a heavy or light chain of a CLDN-18.2 targeted antibody agent) can be evaluated by modulating a capillary gel electrophoresis assay. In some embodiments, the proportion of longer HC-encoding RNAs is evaluated to describe the integrity of two RNAs encoding different strands of a CLDN-18.2 targeted antibody agent. For example, an RNA composition comprising two or more RNAs can be analyzed by capillary gel electrophoresis, which results in an electropherogram. By way of example only, RNA compositions comprising two different RNAs elute in two separate peaks, e.g., each corresponding to an RNA encoding a different chain (e.g., heavy or light chain) of an antibody. See, for example, fig. 15.

The present disclosure provides insight, inter alia, that the molecular ratio strength affects this parameter and sets the specification of the mixture according to the molecular ratio measured by microdroplet digital PCR. The specification depends on the given mixture defined by the exact sequence and weight ratio.

Additionally or alternatively, in some embodiments, the RNA ratio of RNA encoding CLDN-18.2 targeted antibody heavy chain to RNA encoding CLDN-18.2 targeted antibody light chain can be measured by microdroplet digital PCR.

Additionally or alternatively, in some embodiments, the residual DNA template and residual dsRNA are measured as in-process controls according to an acceptance criterion for drug substance intermediate levels to ensure quality of individual RNAs prior to mixing to drug substance (e.g., prior to mixing two RNAs encoding different strands of CLDN-18.2 targeted antibody agent). In some embodiments, the relevant acceptance criteria are used for quality control in the process of RNA alone.

Additionally or alternatively, in some embodiments, residual host cell DNA and/or host cell protein may be measured in a composition comprising RNA.

B. characterization of effective delivery (e.g., plasma concentration)

In some embodiments, the compositions and components thereof may be evaluated to determine their efficacy. In some embodiments, the principal pharmacodynamics and/or pharmacokinetics of the pharmaceutical compositions described herein in vitro and/or in vivo can be determined. Some examples of useful pharmacokinetic metrics may include one or more of the following parameters:

C_max corresponds to the maximum (or peak) plasma/serum concentration achieved by the drug in a particular compartment or test area of the body after administration of the drug and before administration of the second dose. The relevant pharmacokinetic parameter t_max is the time at which Cmax was observed.

C_min corresponds to the minimum plasma/serum concentration achieved by the drug after administration.

C_trough corresponds to the trough plasma concentration at the end of the dosing interval at steady state (usually collected directly before the next administration)

Area under the curve (area under the curve, AUC) is the constant integral of the curve describing the change in drug concentration in plasma as a function of time. AUC (from zero to infinity) represents total drug exposure over time.

In some embodiments, functional assembly of an RNA-encoded CLDN-18.2 targeted antibody agent can be determined in a dose-dependent manner in vitro and in vivo, e.g., as described in example 6.

In some embodiments, the binding specificity, mediating and/or antitumor activity against ADCC and CDC of CLDN-18.2 targeted antibody agents encoded by RNAs described herein can be determined, e.g., as described in examples 1-4.

The present disclosure provides, inter alia, methods comprising the steps of determining one or more characteristics of an antibody expressed by at least one mRNA introduced into a cell, wherein such at least one mRNA comprises one or more characteristics of at least one or more RNAs comprising a coding region encoding an antibody that binds to a claudin-18.2 (CLDN-18.2) polypeptide (e.g., preferentially binds to a claudin-18.2 (CLDN-18.2) polypeptide relative to a claudin-18.1 polypeptide), wherein such one or more characteristics comprise (i) a protein expression level of the antibody, (ii) a binding specificity of the antibody to CLDN-18.2, (iii) an efficacy of the antibody in mediating target cell death by ADCC, and (iv) an efficacy of the antibody in mediating target cell death by Complement Dependent Cytotoxicity (CDC).

In some embodiments, provided herein are methods of characterizing a pharmaceutical composition that targets CLDN-18.2. Such methods comprise the steps of (a) contacting a cell with at least one composition or pharmaceutical composition described herein that encodes a part or all of a CLDN-18.2 targeted antibody agent, and detecting the antibody agent produced by the cell. In some embodiments, the cell may be or comprise a hepatocyte.

In some embodiments, such methods may further comprise determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein, wherein such one or more characteristics comprise (i) a protein expression level of the antibody agent, (ii) a binding specificity of the antibody agent to a CLDN-18.2 polypeptide, (iii) an efficacy of the antibody agent in mediating target cell death by ADCC, and (iv) an efficacy of the antibody agent in mediating target cell death by Complement Dependent Cytotoxicity (CDC). In some embodiments, the step of determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein may comprise comparing such characteristics of a CLDN-18.2 targeted antibody agent to characteristics of a reference CLDN-18.2 targeted antibody.

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

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

In some embodiments, the provided methods of characterizing a CLDN-18.2-targeted pharmaceutical composition or a component thereof may further comprise characterizing an antibody expressed by one or more RNAs described herein as a CLDN-18.2-targeted antibody if the antibody comprises (a) a protein level of the antibody expressed by the cell that is above a threshold level, (b) preferential binding of the antibody to CLDN-18.2 relative to CLDN18.1, and (c) killing of at least 50% of target cells (e.g., cancer cells) is mediated by ADCC and/or CDC.

In some embodiments, the provided methods of characterizing a CLDN-18.2-targeted pharmaceutical composition or component thereof may further comprise characterizing an antibody agent expressed by one or more RNAs described herein as an adjuvant Bei Tuo mab or a clausimab equivalent antibody if the test characteristics of the antibody are at least comparable to the test characteristics of adjuvant Bei Tuo mab or clausimab.

In some embodiments involving the step of determining one or more characteristics of an antibody agent expressed by one or more RNAs described herein, such step may include determining one or more of the following characteristics:

At 48 hours after contact or administration, whether the cells express CLDN-18.2 targeted antibody agent encoded by at least one RNA;

Whether the antibody agent expressed by the cell preferentially binds to CLDN 18.2 polypeptide relative to CLDN18.1 polypeptide;

Whether the antibody agent expressed by the cell exhibits target specificity for CLDN-18.2 comparable to the reference CLDN-18.2 targeted monoclonal antibody, as in a flow cytometry binding assay

Observed;

When assessed after incubating immune effector cells (e.g., PBMC cells) with CLDN-18.2 positive cells or CLDN-18.2 negative control cells for 48 hours in the presence of an antibody, whether CLDN-18.2 positive cells, but not control cells, were lysed;

Whether the antibody agent expressed by the cell exhibits a positive for the targeted CLDN-18.2 at least comparable to that observed with the same concentration of reference CLDN-18.2 targeted monoclonal antibody

ADCC profile of cells, and

When assessed after incubation of CLDN-18.2 positive cells or CLDN-18.2 negative control cells with human serum for 2 hours in the presence of an antibody agent, whether CLDN-18.2 positive cells, but not control cells, were lysed.

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

Also included within the scope of the present disclosure are methods of manufacture comprising the steps of:

(A) Determining one or more characteristics of an RNA or a composition thereof, the RNA encoding a portion or all of an antibody agent, the one or more characteristics selected from the group consisting of:

(i) The length and/or sequence of the RNA;

(ii) Integrity of RNA;

(iii) The presence and/or location of one or more chemical moieties in the RNA;

(iv) The degree of expression of the antibody agent when the RNA is introduced into the cell;

(v) Stability of RNA or a composition thereof;

(vi) Levels of antibody agents in a biological sample from an organism into which RNA has been introduced;

(vii) Binding specificity of antibody agent expressed by RNA, optionally with CLDN-18.2 and

Optionally binding specificity relative to CLDN 18.1;

(viii) The efficacy of antibody agents in mediating target cell death by ADCC;

(ix) The efficacy of antibody agents in mediating target cell death by Complement Dependent Cytotoxicity (CDC);

(x) Identity and amount/concentration of lipids within the composition;

(xi) Size of lipid nanoparticles in the composition;

(xii) Polydispersity of lipid nanoparticles in the composition;

(xiii) Amount/concentration of RNA in the composition;

(xiv) Encapsulation degree of RNA in lipid nanoparticle, and

(Xv) A combination thereof;

(B) Comparing such one or more characteristics of the RNA or composition thereof with characteristics of an appropriate reference standard, and

(C) (i) if the comparison indicates that the RNA or composition thereof meets or exceeds a reference standard, then designating the RNA or composition thereof for one or more additional steps of manufacture and/or dispensing, or

(Ii) If the comparison indicates that the RNA or composition thereof does not meet or exceed the reference standard,

Then an alternative action is taken.

In some embodiments, the reference criteria may be any quality control criteria including, for example, historical references, regulatory specifications. As will be appreciated by those skilled in the art, in some embodiments, no direct comparison is required. In some embodiments, the reference standard is based on an acceptance criteria such as physical appearance, lipid identity and/or content, LNP size, LNP polydispersity, RNA encapsulation, RNA length, identity (as RNA), integrity, sequence and/or concentration, pH, osmolarity (osmolality), RNA ratio (e.g., HC RNA to LC RNA ratio), potency, bacterial endotoxin, bioburden, residual organic solvent, osmolarity, pH, and combinations thereof.

In some embodiments, the pharmaceutical compositions described herein may be determined by one or more potency assays, i.e., such as, but not limited to, in vitro translation, enzyme-linked immunosorbent assay (ELISA), and/or T cell activation bioassays. For example, in some embodiments, expression of a CLDN-18.2 targeted antibody encoded by an RNA composition (e.g., an RNA composition described herein) in a cell can be measured by ELISA in culture supernatants of liposome transfected producer cells. In some such embodiments, the supernatant of the liposome-transfected producer cells may be added to a co-culture of CLDN-18.2 expressing target cells and FcRIIIa-positive luciferase reporter cells as effector cells. Simultaneous binding of the antibody to the CLDN-18.2 and FcgammaRIIIa receptors results in activation of effector cells and in luciferase expression, which is quantified by luminescent readout.

In some embodiments of the methods of manufacture, when an RNA (e.g., an RNA described herein) is evaluated and one or more characteristics of the RNA meet or exceed suitable reference criteria, such RNA is designated for formulation, e.g., in some embodiments, involving formulation with lipid particles described herein.

In some embodiments of the methods of manufacture, when a composition comprising RNA (e.g., RNA as described herein) is evaluated and one or more characteristics of the composition meet or exceed suitable reference criteria, such composition is designated for release and/or dispensing of the composition.

In some embodiments of the methods of manufacture, when RNA (e.g., RNA as described herein) is designated for formulation and/or a composition comprising RNA (e.g., RNA as described herein) is designated for release and/or dispensing of the composition, such methods can further comprise administering the formulation and/or composition to a group of animal subjects each bearing huma CLDN-18.2 positive xenograft tumors to determine anti-tumor activity.

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

In some embodiments, the cell is present in a cell culture.

In some embodiments, the cell is present in a subject. In some such embodiments, the pharmaceutical compositions described herein may be administered to a subject in need thereof. In some embodiments, such pharmaceutical compositions may be administered to a subject such that CLDN-18.2 targeted antibody agents are produced at therapeutically relevant plasma concentrations. In some embodiments, the therapeutically relevant plasma concentration is sufficient to mediate cancer cell death by Antibody Dependent Cellular Cytotoxicity (ADCC). For example, in some embodiments, the therapeutically relevant plasma concentration is from 0.3 to 28 μg/mL.

Exemplary cancers associated with high CLDN-18.2 expression

A. Solid tumors

Cancer is the second leading cause of death worldwide and is expected to lead to an estimated 960 tens of thousands of deaths in 2018 (brain et al 2018). Generally, in addition to a few (e.g., germ cell tumors and some carcinoid tumors), once a solid tumor metastasizes, 5 years rarely survive more than 25%.

Treatment of advanced and metastatic solid tumors

Recent advances in conventional therapies (e.g., chemotherapy, radiation therapy, surgery, and targeted therapies) have improved the outcome of patients with advanced solid tumors. Over the past several years, the Food and Drug Administration (FDA) and European Medicines Administration (EMA) have approved eight checkpoint inhibitors (one monoclonal antibody that targets the CTLA-4 pathway, ipilimumab, and seven antibodies that target the programmed death receptor/ligand [ PD/PD-L1], including atuzumab, avilamab, divarlimumab, nivolumab, cimip Li Shan antibody, and pembrolizumab) for the treatment of patients with multiple cancer types, principally solid tumors. These approvals have greatly changed the profile of cancer treatment (land slope). However, certain cancers, such as pancreatic adenocarcinoma or metastatic biliary tract cancer, have not yet benefited from existing immunotherapy. This phenomenon is multifactorial, due to the systemic and invasive nature of Pancreatic Ductal Adenocarcinoma (PDAC), its complex mutational environment, its connective tissue-promoting matrix, and the powerful immunosuppressive tumor microenvironment.

The poor prognosis of both cancer types highlights the need for additional treatment methods. The present disclosure provides, inter alia, insight that CLDN-18.2 represents a tumor-associated antigen that is particularly useful for targetable therapy. To date, no CLDN-18.2 targeted therapies have been approved for any cancer indication. Thus, in some embodiments, the present disclosure provides insight that an antibody that targets an RNA encoding CLDN-18.2 can induce ADCC and/or CDC and/or enhance the cytotoxicity of chemotherapy and/or other anti-cancer therapies, thereby converting to prolonged progression free and/or overall survival, e.g., relative to treatment of an individual administered alone and/or relative to another suitable reference.

B. pancreatic ductal adenocarcinoma

Pancreatic Ductal Adenocarcinoma (PDAC) is the most common pancreatic neoplastic disease, accounting for more than 90% (Kleeff et al 2016) of all pancreatic malignancies. To date, PDAC is the fourth most common cause of cancer-related death worldwide, with overall survival of less than 8% for 5 years (Siegel et al 2018). The incidence of PDACs is expected to rise further in the future, and predictions suggest that the number of cases in the united states and european countries will be more than 2 times higher in the next 10 years, both in terms of new diagnostics and PDAC related deaths (Quante et al 2016; rahib et al 2014; CANCER RESEARCH UK).

The efficacy and outcome of PDAC treatment is largely dependent on the stage of the disease at the time of diagnosis. Surgical resection followed by adjuvant chemotherapy is the only possible curative treatment available, but only 10% to 20% of PDAC patients are in resectable PDAC stages, with the remaining 80% to 90% showing locally advanced, unresectable stages or (in most cases) distal metastases (gillin et al 2010; werner et al 2013). Systemic chemotherapy is commonly used as a first line treatment in patients with unresectable or borderline resectable tumors. This encompasses nucleoside analogs including gemcitabine and capecitabine, or the pyrimidine analog 5-fluorouracil, either in a monotherapy setting or in combination with other modes of treatment (e.g., radiation therapy) (Werner et al 2013; manji et al 2017; teague et al 2015). The median survival of the multi-chemotherapy regimen FOLFIRINOX consisting of folinic acid, 5-fluorouracil, irinotecan and oxaliplatin in the transfer phase was reported to be almost double compared to gemcitabine alone (Conroy et al 2011), and the combination of gemcitabine and nanoparticulate albumin-bound paclitaxel (nanoparticle albumin-bound paclitaxel, nab-paclitaxel) also showed a significant improvement in overall survival (Von Hoff et al 2013). These treatments are associated with relatively high toxicity, thus generally preventing their use in elderly patients and/or in ill physical performance patients, however, overall quality of life is reported to be improved during use (Gourgou-Bourgade et al.2013).

The epidermal growth factor receptor inhibitor erlotinib (eriotinib) is the only targeted therapy approved in the united states in combination with gemcitabine for first line therapy in patients with locally advanced, unresectable, or metastatic pancreatic cancer. A random control experiment comparing erlotinib with placebo showed 0.4 month middle OS benefit and 0.3 month middle PFS benefit. The treatment described herein targeting CLDN-18.2+ subpopulations of PDACs can potentially address the rather high unmet medical needs of the population.

C. biliary tract cancer

Biliary tract cancer constitutes an epithelial malignancy of the biliary tract system (biliary tree) and includes gallbladder cancer, fatt ampulla (ampulla of Vater) cancer (extrahepatic bile duct and intrahepatic bile duct). In the past, this term covered extrahepatic bile ducts and intrahepatic bile ducts, excluding gall bladder cancer and Fatt's ampulla cancer (de Groen et al 1999).

Biliary tract cancer accounts for about 3% of all gastrointestinal malignancies (Charbel et al 2011) and is the most common hepatobiliary cancer following hepatocellular carcinoma (HENNEDIGE ET al 2014). Unfortunately, mortality (3.58 out of every 100,000) is very high. This is comparable to the incidence in the uk (3.64 per 100,000 people) (National CANCER INTELLIGENCE Network) 2015 and to 2% of 5-year survival in metastatic environments (National cancer institute (National Cancer Institute) Seer data 2015; seer data 2014). Global BTC prevalence has increased by 22% and 150,000 patients were diagnosed with BTC in 2015

(Vos et al 2015). Overall, the incidence varies greatly, and some areas show high prevalence (e.g., japan and korea). This can be explained by infection with liver flukes (thailand liver flukes (Opisthorchis viverrini) and clonorchiasis sinensis (Clonorchiasis sinensis)) in areas (north eastern and china) where cholangiocarcinoma is more common (Parkin et al 1991; kahn et al 2008). Areas with high prevalence of cholelithiasis correspond to areas with high prevalence of gallbladder cancer, such as india and chile (Randi et al 2009; khan et al 1999; KIRSTEIN AND vogel 2016). Geographical areas where the risk factors described above are unusual have fewer cases of BTC (Kahn et al 1999).

In addition to the risk factors described above, primary sclerosing cholangitis, primary biliary cirrhosis, cirrhosis due to other causes, hepatitis c, and congenital malformations such as choledocholithiasis and multiple bile duct papillomatosis are also associated with increased risk of developing BTC (Kahn et al 2008; lee et al 2004; chapman 1999). In addition, patients with germline mutations that result in a genetic abnormality of the Lynch syndrome (BRCA 1 and BRCA2 (breast cancer genes 1 and 2) are also susceptible to BTC. In BRCA2 carriers, the lifetime risk of developing BTC and Linked's syndrome is 2%, and the relative risk of developing cholangiocarcinoma is 4.97% (Golan et al 2017; shigeyasu et al 2014).

Treatment for BTC is stratified according to disease stage, where surgery remains the mainstay of early stage cure, although this accounts for a small fraction (10% to 40%) of the patient (Cidon 2016). For first line treatment of advanced disease, phase 3 trial ABC-02 established that the combination of gemcitabine and cisplatin is superior to the single agent gemcitabine. The median OS reported was 11.7 months vs 8.1 months (risk ratio [ hazard ratio, HR ]0.64;95% confidence interval [ confidence interval, CI ]0.52 to 0.80; p < 0.001), respectively (Valle et al 2010), and since then this has become the global standard of care for advanced BTC. Although the modest survival benefits obtained from this regimen have not been exceeded in the randomized phase 3 trial, in one phase 3 trial of gemcitabine in combination with oral fluoropyrimidine S-1, a median OS of 15.1 months vs. 13.4 months in the gemcitabine/cisplatin group was reported for gemcitabine with S-l group (HR 0.95;90% CI 0.78 to 1.15; p=0.046, non-inferior efficacy) (Morizane et al 2018). This regimen may be considered as an alternative treatment for patients in which complications limit the use of platinum-based agents. Preliminary results with a median OS of 19.2 months in a phase 2 trial evaluating a combination of gemcitabine, cisplatin, and nab-paclitaxel in a first line background in patients with advanced BTC reported a median PFS that was superior to the historically relevant standard gemcitabine/cisplatin regimen (11.4 months versus 8.0 months). This test (NCT 02392637) was performed in 2019 (Shroff et al.2017; shroff et al.2018).

Patient population

The techniques provided herein are useful for treating diseases or disorders associated with increased expression and/or activity of CLDN-18.2. In some embodiments, the techniques provided herein can be used to treat CLDN-18.2 positive solid tumors. In some embodiments, CLDN-18.2 positive solid tumors can be determined by immunohistochemical analysis according to the practice of a skilled pathologist with a staining intensity score of 2 or higher.

The present disclosure recognizes, among other things, that pancreatic and cholangiocarcinomas typically have high CLDN-18.2 expression. Thus, in some embodiments, the techniques provided herein can be used to treat pancreatic cancer. For example, in some embodiments, the techniques provided herein may be used to treat Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the techniques provided herein may be used to treat cholangiocarcinoma.

In some embodiments, the techniques provided herein can be used to treat gastroesophageal cancer that is determined to be CLDN-18.2 positive, for example, by immunohistochemical analysis. In some embodiments, the techniques provided herein can be used to treat non-small cell lung cancer (NSCLC) that is determined to be CLDN-18.2 positive, e.g., by immunohistochemical analysis.

In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with metastatic CLDN-18.2+ solid tumors. In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with unresectable CLDN-18.2+ solid tumors, e.g., in some embodiments in which surgical resection may result in a severe incidence. In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with locally advanced CLDN-18.2+ solid tumors. Additionally or alternatively, in some embodiments, the cancer in such patients may have progressed after treatment, or such cancer patients may not have satisfactory replacement therapy.

In some embodiments, the techniques provided herein can be used to treat adult patients with locally advanced, unresectable, or metastatic CLDN-18.2+ pancreatic cancer. In some embodiments, the techniques provided herein can be used to treat adult patients with locally advanced, unresectable, or metastatic CLDN-18.2+ biliary tract cancer. In some embodiments, a patient undergoing treatment as described herein may have received other cancer treatments, such as, but not limited to, chemotherapy.

In some embodiments, a subject with a CLDN-18.2 positive solid tumor may have received sufficient pretreatment to increase CLDN-18.2 levels/activity such that his/her solid tumor is characterized as a CLDN-18.2 positive solid tumor (e.g., a CLDN-18.2 positive solid tumor as described herein). For example, in some embodiments, such cancer patients may have received chemotherapy that is expected or predicted to increase expression and/or activity of CLDN-18.2 or that may or may have resulted in expression and/or activity of CLDN-18.2. For example, in some embodiments, such chemotherapy may be expected or predicted to increase expression and/or activity of CLDN-18.2 or may result in or have resulted in an increase in expression and/or activity of CLDN-18 of at least 50% or more, including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, when compared to expression and/or activity of CLDN-18.2 in the absence of such chemotherapy. In some embodiments, such chemotherapy may be expected or predicted to increase expression and/or activity of CLDN-18.2 or may result in or have resulted in an increase in expression and/or activity of CLDN-18 of at least 2-fold or more, including, for example, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more when compared to expression and/or activity of CLDN-18.2 in the absence of such chemotherapy. Some examples of such chemotherapeutic agents include, but are not limited to nab-paclitaxel, gemcitabine, cisplatin, and/or FOLFIRINOX.

In some embodiments, cancer patients that meet one or more of the disease-specific inclusion criteria as described in example 16 are suitable for treatment described herein (e.g., receiving a provided pharmaceutical composition as a monotherapy or as part of a combination therapy). In some embodiments, such cancer patients administered with the treatment described herein may also meet one or more of the other inclusion criteria as described in example 16.

In some embodiments, cancer patients that meet one or more of the disease-specific inclusion criteria as described in example 16 are suitable for treatment described herein (e.g., receiving a provided pharmaceutical composition as a monotherapy or as part of a combination therapy). In some embodiments, such cancer patients administered with the treatment described herein may also meet one or more of the other inclusion criteria as described in example 16.

In some embodiments, a cancer patient whose tumor does not express CLDN-18.2 or is determined to be non-CLDN-18.2 positive (e.g., according to the disclosure described herein) is not administered a treatment described herein.

In some embodiments, a cancer patient having a CLDN-18.2 positive tumor but meeting one or more of the exclusion criteria as described in example 17 is not administered the treatment described herein.

Treatment (e.g., dosing regimen)

In some embodiments, the pharmaceutical compositions described herein can be taken up by target cells to produce encoded CLDN-18.2 targeted antibody agents at therapeutically relevant plasma concentrations. In some embodiments, such pharmaceutical compositions described herein can deliver the encoded CLDN-18.2 targeted antibody agent at a plasma concentration sufficient to induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against a target cell (e.g., a tumor cell).

Accordingly, another aspect of the present disclosure relates to methods of using the pharmaceutical compositions described herein. For example, one aspect provided herein is a method comprising administering the provided pharmaceutical composition to a subject having a CLDN-18.2 positive solid tumor. In some embodiments, the provided pharmaceutical compositions are administered by intravenous injection or infusion. Some examples of CLDN-18.2 positive solid tumors include, but are not limited to, biliary tract tumors, gastric tumors, gastroesophageal tumors, ovarian tumors, pancreatic tumors, and tumors that express or exhibit CLDN-18.2 polypeptide levels above a threshold level (e.g., CLDN-18.2 levels observed in normal tissue), e.g., at least 50% or more, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, or at least 2-fold or more, including, e.g., at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more, in some embodiments.

Another aspect of the present disclosure relates to certain improvements in methods of delivering CLDN-18.2 targeted antibody agents for cancer treatment in a subject, the methods comprising administering the provided pharmaceutical compositions to a cancer subject. In some embodiments, the pharmaceutical compositions described herein may achieve one or more improvements, such as effective administration with reduced incidence (e.g., frequency and/or severity) of TEAE and/or improved relationship between efficacy levels and TEAE levels (e.g., improved therapeutic window) relative to those observed when the corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered. In particular, the present disclosure teaches that such improvements can be achieved, in particular, by delivering IMAB362 via administration of RNA (e.g., ssRNA, e.g., mRNA) encoding the same.

Dosing regimen one skilled in the art knows that cancer therapeutic agents are typically administered in a dosing cycle. In some embodiments, the pharmaceutical compositions described herein are administered in one or more dosing cycles.

In some embodiments, one dosing cycle is at least 3 days or more (including, for example, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 days).

In some embodiments, one dosing cycle may involve multiple doses, e.g., according to such a pattern, e.g., one dose may be administered daily, e.g., as in one cycle, or one dose may be administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, in one cycle.

In some embodiments, multiple cycles may be applied. For example, in some embodiments, at least 2 cycles (including, for example, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, or more) may be administered. In some embodiments, the number of dosing cycles to be administered can vary with the type of treatment (e.g., monotherapy vs. combination therapy). In some embodiments, at least 3 to 8 dosing cycles may be administered.

In some embodiments, there may be "rest periods" between periods, and in some embodiments, there may be no rest periods between periods. In some embodiments, there may be periods of inactivity between periods and there may be no periods of inactivity.

In some embodiments, the length of the rest period may be in the range of days to months. For example, in some embodiments, the length of the resting period may be at least 3 days or more, including, for example, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more. In some embodiments, the length of the resting period may be at least 1 week or more, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.

In some embodiments, for example, a pharmaceutical composition described herein for monotherapy can be administered in at least three cycles, wherein in some embodiments, each cycle is 21 days. In some embodiments, a pharmaceutical composition described herein, e.g., for combination therapy, may be administered in at least eight cycles, with each cycle being 21 days in some embodiments.

In some embodiments, the pharmaceutical compositions provided herein may be administered on day 1 of each 3 week dosing cycle (21 days/Q3W). In some embodiments, a cancer patient with CLDN-18.2+ solid tumor can receive up to three cycles of treatment. In some embodiments, a cancer patient with CLDN-18.2+ solid tumor can receive up to eight cycles.

Dosage the dosage of the pharmaceutical compositions described herein may vary depending on a number of factors including, for example, but not limited to, the weight of the subject to be treated, the type of cancer and/or the stage of the cancer, and/or monotherapy or combination therapy. In some embodiments, the dosing cycle involves administering a set number and/or pattern of doses. For example, in some embodiments, the pharmaceutical compositions described herein are administered in at least one dose/dosing cycle, including, for example, at least two doses/dosing cycles, at least three doses/dosing cycles, at least four doses/dosing cycles, or more.

In some embodiments, the dosing cycle involves a cumulative dose being set, e.g., over a specific period of time and optionally by multi-dose administration, which may be administered, e.g., at set intervals and/or according to a set pattern. In some embodiments, the set cumulative dose may be administered at set intervals with multiple doses such that there is at least some temporal overlap in the biological and/or pharmacokinetic effects produced by such multiple doses on the target cells or on the subject being treated. In some embodiments, the set cumulative dose may be administered at set intervals by multiple doses, such that the biological and/or pharmacokinetic effects produced by such multiple doses on the target cells or on the subject being treated may be additive. By way of example only, in some embodiments, a set cumulative dose of X mg may be administered by two doses and each dose is X/2mg, where such two doses are administered in close enough time that the biological and/or pharmacokinetic effects produced by each X/2mg dose on the target cells or on the subject being treated may be additive.

In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level such that CLDN-18.2 targeted antibody agent expressed by the provided RNA is expected to achieve a level (e.g., plasma level and/or tissue level) high enough to trigger antibody-dependent cytotoxicity against the target cells (e.g., cancer cells) throughout the administration cycle. For IMAB362, the dose response correlation against ADCC is well characterized clinically, and it has been reported that CLDN-18.2+ cells are effectively lysed by ADCC with 0.3 to 28 μg/mL of EC₉₅ (Sahin et al 2018). Thus, in some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered in an amount that confers a plasma concentration of about 0.3 to 28 μg/mL of CLDN-18.2 targeted antibody agent encoded by RNA (e.g., RNA described herein).

In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level such that CLDN-18.2 targeted antibody agent expected to be expressed by the provided RNA achieves a level (e.g., plasma level and/or tissue level) comparable to the therapeutically relevant level (e.g., plasma level and/or tissue level) observed with administration of IMAB 362. In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level such that CLDN-18.2 targeted antibody agent intended to be expressed by the provided RNA achieves a level (e.g., plasma level and/or tissue level) of greater than about 0.05 to 3 μg/mL, in some embodiments greater than about 0.1 to 10 μg/mL, in some embodiments greater than about 0.2 to 15 μg/mL, in some embodiments greater than about 0.3 to 30 μg/mL, in some embodiments greater than about 0.3 to 28 μg/mL. In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level such that a CLDN-18.2 targeted antibody agent intended to be expressed by the provided RNA achieves a C_trough level (e.g., plasma level and/or tissue level) of greater than about 5 μg/mL, in some embodiments greater than about 10 μg/mL, in some embodiments greater than about 15 μg/mL.

In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level that is expected to achieve a level (e.g., plasma level and/or tissue level) of greater than about 0.1 μg/mL, in some embodiments, greater than about 0.2μg/mL、0.3μg/mL、0.4μg/mL、0.5μg/mL、0.6μg/mL、0.7μg/mL、0.8μg/mL、0.9μg/mL、1μg/mL、1.5μg/mL、2μg/mL、5μg/mL、8μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL or up to and above the range observed with IMAB362 antibody administration to deliver one or more RNAs (e.g., mrnas) described herein that encode CLDN-18.2 targeted antibody agents.

Without wishing to be bound by any particular theory, the present disclosure provides insight that when applied to an antibody encoded by an mRNA, the AUC of IMAB362 may not accurately elucidate the concentration of pharmacological activity over a dosing period (e.g., over a 21 day dosing period). In some embodiments, AUC is monitored or measured at least once. In some embodiments, AUC is not monitored or measured. Regardless, in many embodiments, the amount and/or frequency of administration can be independent of the AUC of IMAB 362.

Without wishing to be bound by any particular theory, the present disclosure provides insight, inter alia, that it may not be necessary to reach C_max reported for IMAB362, and that the risk of toxicity induced by the pharmaceutical compositions described herein and the corresponding antibody agents expressed thereby may be increased. For example, in some embodiments, the pharmaceutical compositions described herein may have an improved pharmacokinetic profile that maintains the biologically active dose of the antibody for an extended period of time due to sustained expression from RNA. Thus, in some embodiments, the pharmaceutical compositions described herein can be administered at a level such that RiboMab of the targeted CLDN-18.2 expressed by the provided RNAs is expected to achieve a level (e.g., plasma level and/or tissue level) lower than C_max reported for IMAB 362. In some embodiments, the amount and/or frequency of administration may be independent of C_max reported for IMAB 362.

In some embodiments, each dose or cumulative dose of the pharmaceutical compositions described herein (e.g., for intravenous administration) may comprise one or more RNAs encoding CLDN-18.2 targeted antibody agents (whether encoded by a single RNA or by two or more RNAs) in an amount ranging from 0.1mg RNA/kg to 5mg RNA/kg of the subject's body weight to be administered. In some embodiments, each dose or cumulative dose can comprise RNA (e.g., RNA)：0.1mg RNA/kg、0.15mg RNA/kg、0.2mg RNA/kg、0.225mg RNA/kg、0.25mg RNA/kg、0.3mg RNA/kg、0.35mg RNA/kg、0.4mg RNA/kg、0.45mg RNA/kg、0.5mg RNA/kg、0.55mg RNA/kg、0.6mg RNA/kg、0.65mg RNA/kg、0.7mg RNA/kg、0.75mg RNA/kg、0.80mg RNA/kg、0.85mg RNA/kg、0.9mg RNA/kg、0.95mg RNA/kg、1.0mg RNA/kg、1.25mg RNA/kg、1.5mg RNA/kg、1.75mg RNA/kg、2.0mg RNA/kg、2.25mg RNA/kg、2.5mg RNA/kg、2.75mg RNA/kg、3.0mg RNA/kg、3.25mg RNA/kg、3.5mg RNA/kg、4mg RNA/kg、5mg RNA/kg or more as described herein), in some embodiments, each dose or cumulative dose can comprise RNA (e.g., as described herein) in an amount of 1.5mg RNA/kg, in some embodiments, each dose or cumulative dose can comprise RNA (e.g., as described herein) in an amount of 5mg RNA/kg.

In some embodiments, each dose or cumulative dose of the provided pharmaceutical compositions (e.g., for intravenous administration) is administered to deliver a dose of 0.15mg RNA/kg, which in some embodiments may correspond to about 7 μg/mL CLDN-18.2 targeted antibody agent at Cmax. FIG. 14 shows that RNA drug substance encoding CLDN-18.2 targeted antibody agent is at tmax in cynomolgus monkey

Dose-exposure correlation at (48 hours). As will be appreciated by those of skill in the art, given that LNP-transfection efficacy and mRNA translation are comparable between cynomolgus monkey and human (Coelho et al.2013), in some embodiments, each dose or cumulative dose of the provided pharmaceutical composition may be administered (e.g., for intravenous administration) to deliver the appropriate dose corresponding to the desired plasma level of CLDN-18.2 targeted antibody agent encoded by RNA, as shown in fig. 14.

In some embodiments, the administration may be adjusted based on the response of the subject receiving the treatment. For example, in some embodiments, administration may involve administration of a higher dose followed by a lower dose if one or more parameters for safety pharmacology assessment (e.g., as described in example 5) indicate that the previous dose may not meet medical safety requirements according to a physician. In some embodiments, the dose escalation may be performed at one or more of the levels shown in table 13 of example 8, in some embodiments, the dose escalation may involve administration of at least one lower dose from table 13 followed by administration of at least one higher dose from table 13. Without wishing to be bound by any particular theory, the present disclosure provides, inter alia, insight that a pharmaceutically directed dose escalation (pharmaceutically guided dose escalation, PGDE) method can be applied to determine the appropriate dose of the pharmaceutical composition described herein. An exemplary dose escalation study is provided in example 8.

Also provided herein are methods of determining the dosing regimen of a pharmaceutical composition targeting CLDN-18.2. For example, in some embodiments, such methods comprise the steps of (A) administering a pharmaceutical composition (e.g., a pharmaceutical composition described herein) to a subject having a CLDN-18.2 positive solid tumor under a predetermined dosing regimen, (B) periodically monitoring or measuring the tumor size of the subject over a period of time, and (C) evaluating the dosing regimen based on the tumor size measurement. For example, if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is not therapeutically relevant, the dosage and/or frequency of administration may be increased, or if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant, but exhibits an adverse effect (e.g., a toxic effect) in a subject, the dosage and/or frequency of administration may be decreased. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show adverse effects (e.g., toxic effects) in the subject, no change is made to the dosing regimen.

In some embodiments, such methods of determining a dosing regimen of a pharmaceutical composition targeting CLDN-18.2 can be performed in a group of animal subjects (e.g., mammalian non-human subjects) each bearing huma CLDN-18.2 positive xenograft tumors. In some such embodiments, the dosage and/or frequency of administration may be increased if less than 30% of the animal subjects exhibit a decrease in tumor size after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein) and/or the extent of decrease in tumor size exhibited by the animal subjects is not therapeutically relevant, or the dosage and/or frequency of administration may be decreased if the decrease in tumor size after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein) is therapeutically relevant but exhibits a significant adverse effect (e.g., toxic effect) in at least 30% of the animal subjects. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show significant adverse effects (e.g., toxic effects) in an animal subject, no change is made to the dosing regimen.

Although the dosing regimens (e.g., dosing schedules and/or dosages) provided herein are primarily suitable for administration to humans, those skilled in the art will appreciate that dosage equivalents for administration to all species of animals can be determined. A veterinarian of ordinary skill can design and/or make such a determination using only ordinary experimentation if present.

In some embodiments, the pharmaceutical compositions described herein may be administered as monotherapy to a patient suffering from CLDN-18.2+ solid tumors.

Combination therapy the present disclosure provides, inter alia, the insight that the ability of the CLDN-18.2-targeted pharmaceutical compositions described herein to induce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) when utilizing the immune system of a recipient subject can enhance the cytotoxic effects of chemotherapy and/or other anti-cancer therapies. In some embodiments, such combination treatments may prolong progression free and/or overall survival, e.g., relative to treatment of an individual administered alone and/or relative to another suitable reference. Thus, in some embodiments, the pharmaceutical compositions described herein may be administered in combination with other anti-cancer agents to patients suffering from CLDN-18.2+ solid tumors.

Without wishing to be bound by a particular theory, the present disclosure observes that certain chemotherapeutic agents, such as, for example, gemcitabine, oxaliplatin, and 5-fluorouracil, appear to up-regulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines, and furthermore, that these agents increase de novo expression in CLDN-18.2 negative cell lines. See, for example ,Türeci et al.(2019)"Characterization of Zolbetuximab in pancreatic cancer models"In Oncoimmunology 8(1),pp.e1523096.

The present disclosure provides, inter alia, the insight that CLDN-18.2 targeted therapies described herein can be particularly useful and/or effective when administered to tumors (e.g., tumor cells, subjects suspected of having and/or having been detected) characterized by (e.g., determined to exhibit and/or expected or predicted to exhibit) elevated expression and/or activity of CLDN-18.2 expression in tumor cells (e.g., which may or may not have resulted from exposure to one or more chemotherapeutic agents). Indeed, the present disclosure teaches, among other things, that CLDN-18.2 targeted therapies (e.g., administration of RNA, and more particularly, mRNA encoding CLDN-18.2 targeted antibody agents) as provided herein can provide synergistic therapy when administered in combination with one or more CDLN-18.2 enhancers (e.g., one or more certain chemotherapeutic agents) (e.g., administered to a subject that has received and/or is receiving or is otherwise exposed to the one or more CDLN-18.2 enhancers). Thus, in some embodiments, CLDN-18.2 targeted therapies as described herein can be used in combination with other anti-cancer agents that are expected and/or have been shown to up-regulate CLDN-18.2 expression and/or activity in tumor cells. For example, in some embodiments, the pharmaceutical compositions described herein may be combined with already effective but not durable cytotoxic therapies.

In some embodiments, provided pharmaceutical compositions may be administered as part of a combination therapy comprising such pharmaceutical compositions and a chemotherapeutic agent. Thus, in some embodiments, provided pharmaceutical compositions may be administered to a subject having CLDN-18.2+ solid tumors that has received a chemotherapeutic agent. In some embodiments, provided pharmaceutical compositions can be co-administered with a chemotherapeutic agent to a subject having CLDN-18.2+ solid tumors. In some embodiments, the provided pharmaceutical compositions and chemotherapeutic agents may be administered simultaneously or sequentially. For example, in some embodiments, the first dose of the chemotherapeutic agent may be administered after administration of the provided pharmaceutical composition (e.g., at least four hours later). In some embodiments, the chemotherapeutic agent and the provided pharmaceutical composition are administered simultaneously.

In some embodiments in which a chemotherapeutic agent is expected to increase expression and/or activity of CLDN-18.2 in a cancer subject, such chemotherapeutic agent may be administered prior to administration of the provided pharmaceutical composition. In some embodiments, the pharmaceutical compositions described herein can be administered once such that the CLDN-18.2 targeted antibody agent expressed by the RNAs described herein reaches its therapeutically relevant plasma concentration (e.g., as described herein) during an increase in CLDN-18.2 expression and/or activity in response to administration of such chemotherapeutic agents. In some embodiments, the pharmaceutical compositions described herein can be administered at one time such that expression and/or activity of CLDN-18.2 is increased by at least 50% or more, including, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, when compared to expression and/or activity of CLDN-18.2 in the absence of such chemotherapeutic agent, the CLDN-18.2 targeted antibody agent expressed by RNA described herein reaches its therapeutically relevant plasma concentration (e.g., as described herein). In some embodiments, the pharmaceutical compositions described herein can be administered once such that expression and/or activity of CLDN-18.2 is increased to at least 1.5-fold, at least 2-fold or more, including, for example, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more, when compared to expression and/or activity of CLDN-18.2 in the absence of such a chemotherapeutic agent, the CLDN-18.2 targeted antibody expressed by RNA described herein reaches its therapeutically relevant plasma concentration (e.g., as described herein). Some examples of such chemotherapeutic agents include, but are not limited to nab-paclitaxel, gemcitabine, cisplatin, and/or FOLFIRINOX.

Combination therapy with anticancer therapy comprising gemcitabine in some embodiments, the therapy comprising administration of the provided pharmaceutical compositions may be co-administered or overlapped with an anticancer therapy comprising gemcitabine. Gemcitabine kills cells undergoing deoxyribonucleic acid (DNA) synthesis and blocks cell progression through the G1/S phase boundary. Gemcitabine is metabolized by nucleoside kinases to diphosphate and triphosphate (dCTP) nucleosides. Gemcitabine diphosphate inhibits ribonucleotide reductase (the enzyme responsible for catalyzing the reaction that produces deoxynucleoside triphosphates for DNA synthesis), resulting in a decrease in deoxynucleotide concentration (including dCTP). Gemcitabine triphosphate competes with dCTP for incorporation into DNA. The effect of the diphosphate on the reduction of intracellular dCTP concentration enhances the incorporation of gemcitabine triphosphate into DNA (self-enhancement). After the gemcitabine nucleotide is incorporated into the DNA, only one additional nucleotide is added to the growing DNA strand, which ultimately leads to the initiation of apoptotic cell death.

Combination therapy with anticancer therapy comprising nab-paclitaxel in some embodiments, the therapy comprising administration of the provided pharmaceutical composition may be co-administered or overlapped with the anticancer therapy comprising nab-paclitaxel. nab-paclitaxel is an albumin bound form of paclitaxel with an average particle size of about 130nm. It is a microtubule inhibitor that promotes microtubule assembly from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in inhibition of the normal dynamic recombination of the microtubule network critical for important interphase and mitotic cell function. Paclitaxel induces abnormal microtubule arrays or "bundles" throughout the cell cycle, as well as multiple microtubule stars during mitosis.

Combination therapy with anticancer therapy comprising cisplatin in some embodiments, the therapy comprising administration of the provided pharmaceutical composition may be co-administered or overlapped with an anticancer therapy comprising cisplatin. Cisplatin is a heavy metal complex comprising a central platinum atom surrounded in cis position by two chlorine atoms and two ammonia molecules. Without wishing to be bound by theory, cisplatin is thought to kill cancer cells by binding to DNA and interfering with its repair mechanisms, ultimately leading to cell death.

Combination therapy with anticancer therapy comprising FOLFIRINOX in some embodiments, the therapy comprising administration of the provided pharmaceutical composition may be co-administered or overlapped with an anticancer therapy comprising FOLFIRINOX, a combination of cancer drugs comprising FOLFIRINOX-folinic acid (also known as leucovorin, calcium folinate, or FA), F-fluorouracil (also known as 5 FU), irin-irinotecan, ox-oxaliplatin.

Leucovorin is a mixture of diastereomers of the 5-formyl derivative of leucovorin. The biologically active compound of the mixture is the (-) -l-isomer, known as orange factor (citrovorum factor) or (-) -folinic acid. Leucovorin does not require reduction by the enzyme dihydrofolate reductase to participate in reactions that utilize folic acid as a source of "single carbon" moieties. l-leucovorin (l-5-formyltetrahydrofolate) is rapidly metabolized (by 5, 10-methyltetrahydrofolate, then 5, 10-methylenetetrahydrofolate) to l, 5-methyltetrahydrofolate. l, 5-methyltetrahydrofolate can in turn be metabolized back to 5, 10-methyltetrahydrofolate by other pathways, which are converted to 5-methyltetrahydrofolate by irreversible enzyme-catalyzed reduction using the cofactors flavin adenine dinucleotide and nicotinamide-adenine dinucleotide phosphate.

Leucovorin can enhance the therapeutic and toxic effects of fluoropyrimidines (e.g., 5-fluorouracil) used in cancer therapy. The simultaneous administration of leucovorin did not show alterations to the plasma PK of 5-fluorouracil. 5-fluorouracil is metabolized to fluorodeoxyuridylate, which binds to and inhibits the enzyme thymidylate synthase (an important enzyme in DNA repair and replication). Leucovorin is readily converted to another reduced folic acid, 5, 10-methylenetetrahydrofolate, which acts to stabilize the binding of fluorodeoxynucleotides to thymidylate synthase and thereby enhance inhibition of this enzyme.

Fluorouracil is an inhibitor of nucleoside metabolism that interferes with DNA synthesis and to a lesser extent inhibits RNA formation, which affects rapidly growing cells and can lead to cell death. Fluorouracil is converted to three major active metabolites, 5-fluoro-2 ' -deoxyuridine-5 ' -monophosphate, 5-fluorouridine-5 ' -triphosphate and 5-fluoro-2 ' -deoxyuridine-5 ' -triphosphate. These metabolites have several roles, including inhibition of thymidylate synthase by 5-fluoro-2 ' -deoxyuridine-5 ' -monophosphate, incorporation of 5-fluorouridine-5 ' -triphosphate into RNA and incorporation of 5-fluoro-2 ' -deoxyuridine-5 ' -triphosphate into DNA.

Irinotecan is a derivative of camptothecin. Camptothecins interact specifically with the enzyme topoisomerase I, which relieves torsional strain in DNA by inducing reversible single strand breaks. Irinotecan and its active metabolite SN-38 bind to the topoisomerase I-DNA complex and prevent the religation of these single strand breaks. Current studies indicate that the cytotoxicity of irinotecan is due to double stranded DNA damage generated during DNA synthesis when replicase interacts with the ternary complex formed by topoisomerase I, DNA, and either irinotecan or SN-38. Mammalian cells are unable to repair these double strand breaks effectively.

Oxaliplatin is non-enzymatically converted in physiological solution to a reactive derivative by displacement of labile oxalic acid ligands. Several transiently reactive species are formed, including single-water and double-water DACH platinum, which are covalently bound to macromolecules. Both inter-and intra-chain plasma tumor DNA crosslinks are formed. A crosslink is formed between two adjacent guanines, adjacent adenine-guanines, and the N7 positions of guanines separated by an intervening nucleotide. These crosslinks inhibit DNA replication and transcription. Cytotoxicity is cell cycle non-specific.

In some embodiments, the techniques provided herein can be used for administration to a subject having a CLDN-18.2 positive pancreatic tumor. In some embodiments, such subjects may be receiving the provided pharmaceutical compositions as monotherapy or as part of a combination therapy comprising such provided pharmaceutical compositions and a chemotherapeutic agent suitable for treating pancreatic tumors. In some embodiments, such chemotherapeutic agents may be or comprise FOLFIRINOX, which is a combination of cancer drugs comprising folinic acid (FOL), fluorouracil (F), irinotecan (IRIN), and Oxaliplatin (OX). In some embodiments, such chemotherapeutic agents may be gemcitabine and/or paclitaxel (e.g., nab-paclitaxel) or comprises gemcitabine and/or paclitaxel (e.g., nab-paclitaxel). In some embodiments, the pharmaceutical compositions described herein can be administered in combination with gemcitabine as described in example 18 according to approved dosages and treatment regimens of Gemzar (e.g., gemzar) as monotherapy for the treatment of pancreatic cancer. In some embodiments, the pharmaceutical compositions described herein can be administered in combination with gemcitabine at lower doses (e.g., less than 10%, less than 20%, less than 30% or more) and/or at less aggressive treatment regimens (e.g., every 10 days, or once every two weeks, etc.) than approved doses and treatment regimens (as described above) of gemcitabine as monotherapy for the treatment of pancreatic cancer. In some embodiments, the pharmaceutical compositions described herein can be administered in combination with gemcitabine and nab-paclitaxel according to approved dosages and treatment regimens for nab-paclitaxel/gemcitabine combination treatment, as described in example 18. In some embodiments, provided pharmaceutical compositions described herein can be administered in combination with nab-paclitaxel and gemcitabine, at least one of which is administered at a lower dose (e.g., less than 10%, less than 20%, less than 30% or more) and/or in a less aggressive treatment regimen (e.g., every 10 days, or once every two weeks, etc.) than approved doses and treatment regimens of the nab-paclitaxel/gemcitabine combination treatment, as described in example 18. In some embodiments, the pharmaceutical compositions described herein provided can be administered in combination with nab-paclitaxel and gemcitabine according to the dosing regimen described in table 17 (example 18).

In some embodiments, the techniques provided herein may be used for administration to a subject having a CLDN-18.2 positive biliary tract tumor. In some embodiments, such subjects may be receiving provided compositions as monotherapy or as part of a combination therapy comprising such provided pharmaceutical compositions and chemotherapeutic agents suitable for the treatment of biliary tract tumors. In some embodiments, such chemotherapeutic agents may be or comprise gemcitabine and/or cisplatin.

Efficacy monitoring in some embodiments, patients receiving the provided therapy may be monitored periodically during the dosing regimen to assess the efficacy of the administered therapy. For example, in some embodiments, the efficacy of an administered therapy may be assessed by in-treatment imaging at regular intervals, e.g., every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer. In some embodiments, one or more efficacy assessments as described in example 19 may be performed.

In some embodiments, for example, in the case of standard of care, one or more of a variety of pharmacokinetic and pharmacodynamic markers (e.g., as described in example 6) can be evaluated, which can be used as an indicator of antitumor and safety activity of the provided pharmaceutical composition (e.g., as monotherapy or as combination therapy).

Example

Example 1 in vitro characterization of CLDN-18.2 targeted antibody agents expressed from one or more exemplary mrnas

This example demonstrates the in vitro characterization of an exemplary CLDN-18.2 targeted antibody expressed from one or more mRNA encoding the CLDN-18.2 targeted antibody after introduction into a cell.

Complete IgG assembly after RNA transfection of hepatocytes. This example shows translation, assembly, and secretion of CLDN-18.2 targeted antibody agents (hereinafter referred to as "CLDN-18.2 targeted RiboMab") expressed by one or more exemplary mrnas (e.g., mrnas described herein) after uptake of the corresponding mrnas by cells in vitro. In this example, two different expression systems were used, similar to primary human hepatocytes and chinese hamster ovary cells (CHO-K1) targeted to the liver in vitro. Liposome transfection of cells is performed with a composition comprising mRNA encoding a CLDN-18.2 targeted antibody agent as described herein. For example, cell supernatants containing secreted CLDN-18.2 targeted RiboMab were harvested after 48 hours and analyzed, e.g., by Western blot and ELISA. Fully assembled CLDN-18.2 targeting RiboMab (e.g., CLDN-18.2 targeting IgG antibodies) was generated in both expression systems (fig. 1).

Exemplary CLDN-18.2 targets the binding specificity of RiboMab. To determine the target specificity of an exemplary CLDN-18.2 targeting antibody agent expressed from one or more exemplary mrnas (e.g., mRNA as described herein) for a CLDN-18.2 polypeptide, a flow cytometry binding assay was performed using cell culture supernatants comprising CLDN-18.2+hek293 transfectants expressed in CHO-K1 cells and CLDN-18.2 targeting RiboMab as target cells. To assess the cross-reactivity of CLDN-18.2 targeting RiboMab with the closely related splice variant CLDN18.1, CLDN-18.2 targeting RiboMab was tested for binding to CLDN18.1 transfected cells. CLDN-18.2 targeting RiboMab expressed by one or more exemplary mrnas (e.g., the mrnas described herein) preferentially binds to the tightly-engaged polypeptide CLDN-18.2 polypeptide relative to the CLDN18.1 polypeptide. In some embodiments, CLDN-18.2 targeting RiboMab expressed by one or more exemplary mrnas (e.g., the mrnas described herein) is restricted in binding or specific for CLDN-18.2 polypeptides and exhibits a concentration dependence comparable to reference protein IMAB362 (or referred to as adjuvant Bei Tuo mab or clausimab) (fig. 2).

Analysis of modes of action in vitro Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC). Assessment of one or more CLDN-18.2-encoding targets RiboMab by analysis of antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)

(E.g., CLDN-18.2 targeting RiboMab described herein) mRNA is targeted to the biological activity of RiboMab by CHO-K1 cells expressed after in vitro translation. Exemplary ADCC assays are performed, for example, using CLDN-18.2+ gastric cancer transfectants (e.g., NUG-C4) and target-negative breast cancer cell lines (e.g., MDA-MB-231) to assess specific lysis. For the exemplary CDC assay, clDN-18.2+ transfectants (e.g., CHO-K1) and clDN-18.2 negative (e.g., CHO-K1) cell lines were used. To mimic in vivo conditions, human PBMCs from three different healthy donors were used as effector cells in an ADCC assay at a 30:1 effector to target (effector to target, E: T) ratio, and human serum (e.g., commercially available human serum) was used as a source of complement in a CDC assay. CLDN-18.2 targeting RiboMab effectively mediates target-specific and dose-dependent cytotoxicity comparable to reference protein IMAB362 in ADCC [ fig. 3, panel a; EC₅₀ to 127ng/mL (CLDN-18.2 targeting RiboMab), 14 to 265ng/mL

(IMAB 362) ] and CDC assays (FIG. 3, panel B).

Example 2 characterization of CLDN-18.2 targeted antibody agents expressed in vivo from one or more exemplary mrnas in rodents

The biological activity of CLDN-18.2 targeted RiboMab expressed in vivo by exemplary mRNA (e.g., mRNA described herein) was evaluated in an ex vivo ADCC assay. ADCC assays were performed using Balb/cJRj mouse plasma containing CLDN-18.2 targeted RiboMab or IMAB362, sampled 24 hours after administration of 1 μg (about 0.04 mg/kg), 3 μg (about 0.10 mg/kg), 10 μg (about 0.40 mg/kg), and 30 μg (about 1.20 mg/kg) of a pharmaceutical composition comprising at least one or more mRNAs encoding CLDN-18.2 targeted antibody or 80 μg (about 3.20 mg/kg) of IMAB362 at IV administration 5

("CLDN-18.2 targeted RNA composition"). IMAB 362-doped plasma from untreated mice was used as a measurement reference. CLDN-18.2+ gastric cancer transfectants (e.g., NUG-C4) were used as targets and human PBMCs from healthy donors were used as effector cells. Target cells and effector cells were incubated at a 30:1 ratio of E:T (effector to target) with plasma containing 1% of CLDN-18.2 targeted RiboMab for 48 hours and ADCC was determined in a luciferase-based assay. CLDN-18.2 targeting RiboMab expressed in rodents showed high and dose-dependent target cell lysis similar to 80 μg (about 3.20 mg/kg) of reference protein IMAB362 (fig. 4, panel a). No nonspecific lysis was observed for the target negative breast cancer cell line MDA-MB-231 used as a control, indicating target specificity of CLDN-18.2 targeting RiboMab (fig. 4, panel B). The results indicate that CLDN-18.2 targeting RiboMab expressed in rodents can mediate high ADCC to targeted tumor cells.

Example 3 characterization of CLDN-18.2 targeting antibody Agents expressed in vivo from one or more exemplary mRNAs in non-human primate

To determine the biological activity of CLDN-18.2 targeting RiboMab in organisms closely related to human phylogenetic and physiology, ADCC studies were performed with non-human primate (NHP) (e.g., cynomolgus monkey) sera comprising CLDN-18.2 targeting RiboMab sampled 24 hours and 168 hours after IV administration of 0.1mg/kg, 0.4mg/kg and 1.6mg/kg CLDN-18.2 targeting RNA compositions. ADCC assays were performed as described in example 2. CLDN-18.2 targeting RiboMab expressed in NHP showed high and dose-dependent target cell lysis (fig. 5, panel a). Low effect, donor-dependent non-specific lysis was observed on the target negative breast cancer cell line MDA-MB-231 (fig. 5, panel B). Serum from monkey No. 14 (the animal with the highest established CLDN-18.2 targeted RiboMab concentration (232 μg/mL)) collected 48 hours after injection of CLDN-18.2 targeted RNA composition at 3 rd was subjected to luciferase-based ADCC assay in 10-point dilution rows (10-point dilution row). Purified IMAB362 was used as an assay reference protein. CLDN-18.2 targeting RiboMab expressed by NHP mediated high and specific lysis of NUG-C4 target cells at 10ng/mL (66 pM) EC₅₀ (fig. 5, panel C). These results indicate that CLDN-18.2 targeting RiboMab expressed by NHP mediates ADCC that is potent and target specific.

EXAMPLE 4 intravenous administration of a CLDN-18.2 targeting RNA composition mediates tumor growth inhibition in vivo

To determine the anti-tumor activity of Intravenous (IV) administration of CLDN-18.2 targeted RNA compositions in a CLDN-18.2+ human gastric cancer xenograft tumor model Hsd nude-Foxn 1nu/nu mice were inoculated subcutaneously with 5X 10⁶ CLDN-18.2+ NCI-N87 transfectants. On test days 15, 22, 29, 36, 43 and 50, mice with established tumors (average >30mm³) were subjected to six single IV bolus injections of 3 μg, 10 μg and 30 μg CLDN-18.2 targeted RNA composition, 30 μg control mRNA encoding luciferase, saline or 800 μg reference protein IMAB362. Significant tumor growth inhibition compared to the control was observed after the 3 rd dosing cycle with 30 μg CLDN-18.2 targeted RNA composition. The antitumor activity of 30 μg CLDN-18.2 targeted RNA composition was comparable to the tumor growth retardation obtained with 800 μg of reference protein IMAB362 (fig. 6). EXAMPLE 5 safety pharmacological assessment of CLDN-18.2 targeting RNA compositions

GLP-compliant CNS and respiratory safety assessments were performed in mice after repeated dosing. The potential effect of CLDN-18.2 targeted RNA compositions on non-human primate (NHP) blood pressure after repeated dosing was evaluated in a non-GLP PK/tolerance study. All studies were designed in a manner consistent with ICH S7A (table 4).

Table 4. Overview of exemplary safety pharmacology studies.

Central nervous and respiratory safety. A GLP-compliant sub-chronic toxicity study was performed to evaluate the effect of repeated intravenous bolus injections of CLDN-18.2 targeted RNA compositions in male and female mice. The study included a safety pharmacology assessment of the companion animal (SATELLITE ANIMAL) as shown below (table 5).

TABLE 5 evaluation of neurological and respiratory safety in GLP sub-chronic repeat dose toxicity studies

^a Dose level expressed as total mRNA dose

To assess respiratory safety of CLDN-18.2 targeted RNA compositions, plethysmography was performed before, 4 hours and 24 hours after administration of the second and fourth injections. Respiratory rate, tidal volume, minute ventilation, peak inspiratory flow, peak expiratory flow, inspiratory time, expiratory time, and airway resistance index were assessed every 10 minutes from 10 minutes to 60 minutes (before and after dosing), and measured every 30 minutes from 1 to 4 hours (after dosing) after test item administration to give an average value for each time period.

Animals were subjected to neurological tests prior to dosing and 48 hours after the first and fourth injections. Consciousness, emotion, motor activity, CNS excitation, posture, muscle tone, reflex and voluntary body temperature, hindleg deployment, grip strength and voluntary activity were tested.

A statistically significant change in one parameter (p≤0.05) was seen in that male mice receiving 100. Mu.g of the CLDN-18.2 targeted RNA composition/animal showed a decrease in grip strength (p≤0.01) 48 hours after the first administration, and female mice receiving 100. Mu.g of the CLDN-18.2 targeted RNA composition/animal showed a similar decrease in grip strength (p≤0.05) after the first injection.

Gastric safety. Without wishing to be bound by theory, CLDN-18.2 target is expressed in healthy stomach tissue of humans and mice (Tu reci et al 2011). Macroscopic and histopathological evaluation of the stomach was included in GLP-compliant repeat dose toxicity studies performed in mice (see example 7).

Cardiovascular system safety. In the PK/tolerability study, blood pressure measurements were taken before the first dose and 24 hours after the third dose to the animals (the designed study is described in example 7).

Peripheral arterial systolic and diastolic blood pressure and the resulting mean blood pressure are within normal physiological limits of the animals treated with the test item.

EXAMPLE 6 pharmacokinetic assessment of CLDN-18.2 targeting RNA compositions

The pharmacokinetics of Lipid Nanoparticle (LNP) formulated RNA can be divided into two phases, i.e., systemic distribution of LNP in the circulation after intravenous injection and delivery of RNA to the intended target organ liver. Second, hepatocytes are transfected with the LNP preparation, translating the RNA and secreting the encoded protein.

The pharmacokinetic profile of CLDN-18.2 targeting RiboMab was characterized in three different species following single dose administration [ in mice (fig. 7) and in rats (fig. 8) and repeated dose administration [ in mice (fig. 9) and in non-human primates (fig. 10) ].

Table 6 summary of exemplary studies of pharmacokinetics.

To evaluate the CLDN-18.2-targeted RiboMab PK translated from the CLDN-18.2-targeted RNA composition, a single dose PK study was performed in Balb/c JRj mice. Treatment groups received IV bolus injections of 1 μg (about 0.040 mg/kg), 3 μg (about 0.10 mg/kg), 10 μg (about 0.40 mg/kg), or 30 μg (about 1.20 mg/kg) of CLDN-18.2 targeting RNA composition and 40 μg

(About 1.60 mg/kg) IMAB362 reference protein as an internal control. Plasma was sampled 6, 24, 96, 168, 264, 336 and 504 hours after administration and CLDN-18.2 targeted RiboMab concentrations were assessed by ELISA. The CLDN-18.2 targeted RiboMab concentration showed concentration-dependent expression of CLDN-18.2 targeted RNA compositions, with peaks at 24 hours after administration and gradually decreasing after that. Peak concentrations of about 450 μg/mL were reached at the highest dose, and CLDN-18.2 targeted RiboMab concentrations could be detected up to 504 hours after administration (fig. 7). The results indicate that CLDN-18.2 targeting RiboMab is expressed in a dose-dependent manner in mice after a single administration.

To evaluate CLDN-18.2-targeted RiboMab PK translated from CLDN-18.2-targeted RNA compositions in larger rodent organisms, a single dose study was performed in RjHan:wister rats. Treatment groups received an IV bolus dose of 0.04mg/kg, 0.10mg/kg, 0.40mg/kg, or 1.20mg/kg of the CLDN-18.2 targeting RNA composition and 3.60mg/kg of the IMAB362 reference protein. Plasma was sampled at 2,6, 8, 10, 22, 24, 27, 30, 48, 72, 96, 168, 216, 264 and 336 hours after administration and CLDN-18.2 targeted RiboMab concentrations were determined by ELISA. CLDN-18.2 targeting RiboMab showed a dependent expression of CLDN-18.2 targeting RNA composition concentration, with peaks reached 24 hours after administration and gradually decreasing after that. Peak concentrations of about 450 μg/mL similar to mice (fig. 7) were reached at the highest dose, and CLDN-18.2 targeted RiboMab concentrations were detectable in all dose groups until 336 hours post administration study termination (fig. 8). The results indicate that CLDN-18.2 targeted RiboMab expression levels in rats can be similar to mice.

Repeated dose PK studies were performed in Balb/cJRj mice to assess whether CLDN-18.2 targeted RiboMab concentrations were maintained by weekly administration of CLDN-18.2 targeted RNA compositions. Treatment groups received five IV bolus doses of 1 μg (about 0.04 mg/kg), 3 μg at weekly intervals

(About 0.10 mg/kg), 10 μg (about 0.40 mg/kg) or 30 μg (about 1.20 mg/kg) of CLDN-18.2 targeting RNA composition and 80 μg (about 3.20 mg/kg) of IMAB362 reference protein as an internal control. Plasma was sampled 24 hours prior to and 24 hours after dosing (C_max), respectively, and the CLDN-18.2 targeted RiboMab concentration was determined by ELISA. Repeated administration of CLDN-18.2 targeted RNA compositions resulted in sustained CLDN-18.2 targeted RiboMab levels with peak concentrations as high as about 1000 μg/mL (30 μg CLDN-18.2 targeted RNA composition) without translational loss (fig. 9). The results indicate that sustained CLDN-18.2 targeted RiboMab concentrations can be achieved in mice by weekly administration of CLDN-18.2 targeted RNA compositions.

Repeated dose PK studies of CLDN-18.2 targeted RNA compositions were performed in NHPs that are organisms closely related to human genetics and physiology (see table 7 for a description of exemplary study designs).

TABLE 7 exemplary study design of PK/tolerability study in NHP

^a Dose level expressed as total mRNA dose

The treatment group received 3 IV bolus injections of 0.1mg/kg, 0.4mg/kg or 1.6mg/kg at weekly intervals. As a control, saline or empty LNP was also administered. Serum was sampled at 6, 24, 48, 72, 96 and 168 hours after the 1 st and 3 rd dosing, and at 48, 72 and 168 hours after the 2 nd dosing, and at 264, 336 and 504 hours after the 3 rd dosing. The concentration of CLDN-18.2 targeted RiboMab was analyzed by ELISA. CLDN-18.2 targeting RiboMab showed dose-dependent expression of CLDN-18.2 targeted RNA compositions, with peaks between 48 and 72 hours after administration and gradually decreasing after that. The peak serum concentration of 231.7 μg/mL was reached 48 to 72 hours after the 3 rd administration of CLDN-18.2 targeting RNA composition at the highest dose, and CLDN-18.2 targeting RiboMab was detectable until 840 hours after the 1 st administration (fig. 10). The results indicate that weekly administration of CLDN-18.2 targeted RNA compositions in NHPs can result in sustainable CLDN-18.2 targeted RiboMab expression.

Distribution following single IV injection, the biodistribution of CLDN-18.2 targeted RNA compositions was studied in mice. Messenger RNA and Lipid Nanoparticles (LNP) in murine tissues were quantified by digital droplet PCR (mRNA) or liquid scintillation spectroscopy (radiolabeled LNP), respectively. Organ targeting and expression of LNP encapsulating luciferase-encoding mRNA was studied by bioluminescence imaging.

MRNA distribution a single dose of 100 μg CLDN-18.2 targeted RNA composition per animal IV was administered to Balb/c mice (3/sex/time point) and blood and tissues (spleen, lung, liver, kidney, heart and brain) were sampled 0.083 (5 minutes), 0.5, 6, 24, 72 and 168 hours after administration.

TABLE 8 exemplary design of mRNA biodistribution studies

^a Dose level expressed as total mRNA dose

LNP distribution hepatic targeting and kinetics of in vivo translated mRNA were assessed by assessing the biodistribution of Lipid Nanoparticles (LNP) by formulating modified mRNA encoding firefly luciferase with Lipid Nanoparticles (LNP). After IV administration, luciferase proteins showed time-dependent translation of high bioluminescence signals located mainly in the liver (fig. 11). The results indicate that LNP-encapsulated mRNA can target and be expressed in the liver.

Tissue distribution profile of LNP of CLDN-18.2 targeted RNA compositions was studied in CD-1 mice (4/sex/time point) after single IV bolus injection at 1 mg/kg. The [³ H ] -CLDN-18.2 targeted RNA composition was used for this analysis, and the particles contained the non-exchangeable, non-metabolizable LNP marker [³ H ] -cholesterol cetyl ether ([³H]-cholesteryl hexadecyl ether,[³ H ] -CHE). An exemplary study design is shown in table 9 below.

TABLE 9 exemplary study design of LNP biodistribution study

A represents the dose level as the total mRNA dose

B at bold time points, tissue was collected in addition to blood and plasma

Mice were euthanized and blood and plasma were collected 0.083 (5 minutes), 0.25, 0.5, 1, 2, 4, 8 and 24 hours after dosing. Tissues were sampled only 0.25, 1, 4 and 24 hours after dosing. Radioactivity in all samples was determined by standard liquid scintillation counting (liquid scintillation counting, LSC) and the resulting values were used to calculate total and relative lipid concentrations.

The [³ H ] -CLDN-18.2 targeted RNA composition shows biphasic kinetics in the blood and plasma of mice, where the initial drop in blood/plasma concentration is rapid, followed by a slower elimination phase. The distribution of the [³ H ] -CLDN-18.2 targeting RNA composition into tissues was rapid, with peak levels observed in all tissues 0.5 to 2 hours after dosing. The major tissues/organs of the [³ H ] -CLDN-18.2 targeted RNA composition were liver and spleen (at 4 hours post injection, the injected doses present in the liver and spleen were about 70% to 74% and about 0.8% to 1.2%, respectively), and minimal distribution in other tissues was observed. A summary of calculated total lipid concentrations (i.e., calculated total lipid concentrations of all 4 lipids administered) and calculated injection doses (%) for the [³ H ] -CLDN-18.2 targeted RNA composition in various tissues is shown in table 10.

TABLE 10 tissue levels of total lipid (from CLDN-18.2 targeted RNA composition) 4 hours after IV bolus injection in CD-1 mice

Metabolism and excretion messenger RNA (including pseudo-uridine modified mRNA) is generally susceptible to degradation by cellular RNase enzymes and undergoes nucleic acid metabolism. Nucleotide metabolism occurs continuously in cells, where nucleosides are degraded into waste products and excreted or recovered for nucleotide synthesis.

In some embodiments of CLDN-18.2 targeted RNA compositions described herein, such compositions comprise a plurality of lipids, some of which may be naturally occurring (e.g., in some embodiments, neutral lipids such as cholesterol and DSPC, for example). The skilled artisan reading the present disclosure may expect that the metabolism and excretion of naturally occurring lipids may be similar to the metabolism and excretion of endogenous lipids. Those of skill in the art will further appreciate that methods known in the art may be used to characterize the metabolism and excretion of other lipids (e.g., conjugated lipids and cationic lipids) within CLDN-18.2 targeted RNA compositions.

In some embodiments, the expressed CLDN-18.2 targeting RiboMab structure is based on an IgG1 antibody. In some such embodiments, the metabolism may be similar to the metabolism of endogenous IgG1 molecules. Exemplary metabolism includes, but is not limited to, degradation into small peptides and amino acids.

Example 7 toxicology assessment of cldn-18.2 targeted RNA compositions

Toxicology assessment of CLDN-18.2 targeted RNA compositions can include in vitro studies using human blood components and in vivo studies in mice and cynomolgus monkeys. The blood compatibility of the drug product with human blood can be assessed in vitro, while toxicity mediated by CLDN-18.2 targeted RNA compositions (RNA and LNP) and by translated CLDN-18.2 targeted RiboMab (protein) can be detected in selected in vivo models. A summary of certain features evaluated in non-clinical studies is given in table 11 below.

Table 11 exemplary non-clinical safety and toxicology studies for CLDN-18.2 targeted RNA compositions and encoded antibodies.

In some embodiments, the relevant species for assessing antibody (CLDN-18.2 targeted RiboMab) mediated toxicity are mice and cynomolgus monkeys, because of the highly conserved protein sequence and identical expression pattern of CLDN-18.2 targets in these species (Tu reci et al 2011).

Single dose toxicology. Single dose toxicity studies were performed in male and female CD-1 mice to i) characterize the potential toxicity of CLDN-18.2 targeted RNA compositions, ii) compare the toxicity of CLDN-18.2 targeted RNA compositions with corresponding control items (e.g., empty lipid nanoparticles), and iii) evaluate reversibility, progression, and/or potential delay effects of CLDN-18.2 targeted RNA compositions after 4 weeks of observation (termination on day 29).

Mice were subjected to single IV doses of CLDN-18.2 targeted RNA composition (at total mRNA dose levels of 1, 2 or 4 mg/kg) or control items (e.g., empty nanoparticles or saline controls) by IV administration on day 1. Animals were euthanized on day 3 (primary animals) and day 29 (recovery/delay survey results). Endpoints of study included mortality, clinical observations, weight changes, clinical chemistry, autopsy observations, organ weights and histopathology (liver, spleen and stomach).

CLDN-18.2 targeted RNA compositions or empty LNP at single IV doses of 1, 2 and 4mg/kg were generally well tolerated in male and female CD-1 mice. There was no death during the 28 day observation period. On day 3, secondary findings were noted regarding liver parameters and spleen weight increase. Secondary findings in microscopic evaluation of liver and spleen were considered non-adverse. After the recovery period on day 29, all survey results were resolved.

Repeated dose toxicology. Repeated dose toxicity studies in Balb/c mice for 21 days in compliance with GLP were performed with weekly intravenous bolus administration of CLDN-18.2 targeted RNA compositions followed by a 2-week recovery period (see table 12 for exemplary study designs). Study readouts include, but are not limited to, intolerant clinical signs (e.g., eyelid sagging, erectile wool, reduced motility and/or coolness to the touch), mortality, weight and food consumption, local tolerance, hematology, clinical chemistry (e.g., globulin, albumin, cholesterol, creatinine, total protein, blood glucose, alkaline phosphatase (aP), lactate Dehydrogenase (LDH) and aspartate aminotransferase (ALAP) and glutamate dehydrogenase (GLDH) blood levels), urinalysis, ophthalmic and auditory systems, macroscopic post-mortem findings, organ weight, bone marrow, histopathology and cytokines (e.g., IL-6, TNF- α, IFN- γ, IL-1 β, IL-2, IL-10 and/or IL-12p 70).

TABLE 12 exemplary design of GLP-compliant repeat dose toxicity study

Immunotoxicity-determination of blood compatibility of CLDN-18.2 targeted RNA compositions in human serum and blood, drug product mediated complement activation and cytokine release, respectively, were tested. In addition, in vivo immunotoxicity was assessed as part of repeat dose toxicity studies and cynomolgus monkey pharmacokinetic studies in mice. All studies were designed according to the ICH S8 guidelines (human drug immunotoxicity study).

Preliminary results of toxicity studies showed transient increases in IL-6 and TNF- α at 6 hours post-administration in the empty LNP control group and in both dose groups (30 and 100 μg of CLDN-18.2 targeted RNA composition/animal), while transient increases in IFN- α and IFN- γ were seen in both dose groups. Plasma levels returned to baseline 48 hours after administration. No increase in IL-1β, IL-2, IL-10 or IL-12p70 was observed in any of the groups.

In the PK/tolerance study performed in cynomolgus monkeys as described in example 6, no cytokine elevation was observed in any of the groups.

In vitro complement activation of human serum. The potential of CLDN-18.2 targeted RNA compositions to activate human complement in vitro was assessed by incubation in normal human serum, with drug product concentrations selected based on plasma C_max levels at doses related to toxicity of similar lipid nanoparticle products (e.g., comprising siRNA) administered to humans (Fitzgerald et al 2014; coelho et al 2013; tabernero et al 2013; PATISARAN FDA approves 2017). Complement activation was assessed using a multiplex flow microbead array by assessing the levels of complement cleavage products C3a, C4a, C5a and the levels of terminal complement complex SC5b-9 using an enzyme immunoassay.

In vitro incubation of CLDN-18.2 targeted RNA compositions with normal human serum complement did not result in an increase in complement cleavage products or terminal complement complexes when compared to negative controls, whereas positive controls induced the expected activation. In summary, CLDN-18.2 targeted RNA compositions did not activate human complement in vitro under the conditions tested.

Whole blood cytokine release. In some embodiments, CLDN-18.2 targeted RNA compositions described herein can be administered parenterally. In some such embodiments, the CLDN-18.2 targeted RNA composition can be contacted with peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs) during circulation in the blood. Those of ordinary skill in the art having read this disclosure will appreciate that interactions between the drug product and blood components may result in induction of cytokine secretion. Thus, in vitro tolerability of exemplary CLDN-18.2 targeted RNA compositions was studied using human whole blood. For example, secretion of pro-inflammatory cytokines (e.g., without limitation, IFN- α, IFN- γ, IL-1β, IL2, IL-6, IL-8, IL-12p70, IP-10, and/or TNF- α) is evaluated after incubation at a dilution range representing the desired concentration in human blood. No induction of cytokine secretion associated with the test item was detected in this assay and in vitro tolerance could be demonstrated.

Example 8 exemplary administration (e.g., up-dosing)

In some embodiments, the pharmaceutical compositions provided herein may be administered as monotherapy and/or in combination with other anti-cancer therapies to patients suffering from CLDN-18.2 positive cancer.

In some embodiments, the administration involves one or more cycles. In some embodiments, the pharmaceutical compositions provided herein may be administered for at least 3 to 8 cycles.

In some embodiments, the dosing regimen, and in particular the monotherapy dosing regimen, may be every 21 days (Q3W) or include every 21 days (Q3W) of administration.

In some embodiments, dose escalation may be performed. In some such embodiments, administration may be at one or more of the levels shown in Table 13 below, and in some embodiments, the dose escalation may involve administration of at least one lower dose from Table 13 followed by administration of at least one higher dose from Table 13.

TABLE 13 exemplary drug delivery

¹ As shown in table 13, "dose" refers to total RNA dose.

² Dose increments relative to the immediately preceding dose are presented in table 13, starting with the indicated exemplary starting dose

In some embodiments, additional or alternative dosage levels may be evaluated, including, for example, dosage levels of 0.2, 0.225, 0.25, 0.35, 0.4, 0.45, 0.5, 0.55, 0.65, 0.7, 0.75, 0.80, 0.85, 0.9, 0.95, 1.25, 1.75, 2.25, 2.75, 3.25, 3.5, and 4 mg/kg.

Efficacy of treatment can be assessed by imaging in treatment, for example, at week 6 (+7 days), for 24 weeks every 6 weeks (±7 days), and thereafter every 12 weeks (±7 days).

EXAMPLE 9 approval treatment for treatment of certain cancers

Approved treatments are useful for certain cancers associated with CLDN-18.2 expression. For example, the epidermal growth factor receptor (EPIDERMAL GROWTH FACTOR RECEPTOR, EGFR) inhibitor erlotinib is the only targeted therapy approved in the united states in combination with gemcitabine for first line therapy in patients with locally advanced, unresectable, or metastatic pancreatic cancer. However, the random control trial (randomized controlled trial, RCT) comparing erlotinib with placebo showed a median Overall Survival (OS) benefit of 0.4 months and a median progression-free survival (PFS) benefit of 0.3 months.

In some embodiments, the recommended daily dose of erlotinib (e.g., erlotinib hydrochloride) for treating pancreatic cancer is about 109mg taken in combination with gemcitabine at least one hour prior to ingestion of food or at least two hours after ingestion of food. In some embodiments, the recommended dose of gemcitabine (Gemzar) for treating pancreatic cancer is 1000mg/m² in 30 minutes, once a week for the first 7 weeks, then a week rest, once a week for 3 weeks every 28 day cycle.

Example 10 exemplary adverse events

In some embodiments, one or more indicators of potential adverse events of a subject administered a pharmaceutical composition as described herein may be monitored over a period of time of a treatment regimen. For example, in some embodiments, a subject may be monitored for one or more of hematological toxicity (e.g., presence of neutropenia, thrombocytopenia, and/or anemia, etc.) and/or non-hematological toxicity (e.g., elevation of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), bilirubin, etc.).

Example 11 exemplary evaluation and/or Standard for Single-stranded RNA as described herein

In some embodiments, one or more of the evaluations as described herein (e.g., as a release test) may be used during the manufacture or other preparation or use of single stranded RNA.

In some embodiments, one or more quality control parameters may be evaluated to determine whether the single stranded RNAs described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for partitioning). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Methods for assessing RNA quality are known in the art, for example, one skilled in the art will recognize that in some embodiments, one or more analytical tests as described in Table 14 may be used for RNA quality assessment.

In some embodiments, single stranded RNA batches may be evaluated for the features listed below in table 14 to determine the next action step. For example, if the RNA quality assessment indicates that a single-stranded RNA batch meets or exceeds the acceptance criteria set forth in table 14, such single-stranded RNA batch may be designated for one or more additional steps of manufacture and/or formulation and/or dispensing. Otherwise, if such a single-stranded RNA batch does not meet or exceed the acceptance criteria, then an alternative action may be taken (e.g., discard the batch).

In some embodiments, single stranded RNA batches with exemplary evaluation results as shown in table 14 can be used for one or more additional steps of manufacturing and/or formulation and/or dispensing.

TABLE 14 exemplary testing and Specifications of RNA alone.

Example 12 exemplary evaluation and/or Standard of compositions comprising two or more RNAs

In some embodiments, one or more of the evaluations as described herein may be used during manufacture or other preparation or use of the drug substance (e.g., as a release test).

In some embodiments, a first single stranded RNA batch encoding a CLDN-18.2 targeted antibody heavy chain and a second single stranded RNA batch encoding a CLDN-18.2 targeted antibody light chain are evaluated for one or more of the characteristics as described in example 11. In some such embodiments, the first ssRNA and the second ssRNA batch that meet or exceed the acceptance criteria as set forth in table 14 are then mixed together, for example, in a molar ratio of about 1.5:1 to about 1:1.5, to form the RNA drug substance. In some embodiments, such RNA drug substances may be evaluated for one or more quality control parameters (e.g., for release and/or for further manufacture), including, for example, but not limited to, physical appearance, RNA length, identity (as RNA), integrity, sequence and/or concentration, pH, osmolarity, RNA ratio (e.g., HC RNA to LC RNA ratio), potency, bacterial endotoxin, bioburden, and combinations thereof. Such quality control parameters may be assessed by one or more specific analytical methods known in the art, such as, for example, visual inspection, gel electrophoresis (e.g., agarose gel electrophoresis, capillary gel electrophoresis), enzymatic degradation, sequencing, UV absorption spectrophotometry, PCR methods, bacterial endotoxin testing (e.g., limulus Amoebocyte Lysate (LAL) testing).

Example 13 exemplary RNA product formulation

In some embodiments, the exemplary RNA product formulation is a sterile RNA-lipid nanoparticle (RNA-LNP) dispersion in an aqueous buffer, e.g., for intravenous administration. For example, in some embodiments, such RNA product formulations can be filled to a nominal fill volume of 5.0mL at about 0.8 to about 1.2 mg/mL. In some embodiments, each vial may be intended for a single use. In some embodiments, the RNA product formulation (e.g., as described herein) can be stored frozen at-80 to-60 ℃.

In some embodiments, such exemplary RNA product formulations can comprise two or more different RNAs each encoding a portion of a CLDN-18.2 targeting antibody (e.g., an RNA encoding a CLDN-18.2 targeting antibody heavy chain and an RNA encoding a CLDN-18.2 targeting antibody light chain), at least one cationic lipid, at least one conjugated lipid, at least one neutral lipid, and an aqueous buffer comprising one or more salts. In some embodiments, the polymer conjugated lipid (e.g., PEG conjugated lipid, for example as in some embodiments, PEG conjugated lipid is 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide or comprises 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide) may be present at about 1mol% to 2.5mol% of the total lipid. In some embodiments, the cationic lipid (e.g., in some embodiments ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octoate) or a cationic lipid comprising ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octoate) may be present at about 35mol% to 65mol% of the total lipid. In some embodiments, the neutral lipid (e.g., in some embodiments, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and/or synthetic cholesterol or neutral lipid comprising 1, 2-distearoyl-sn-glycero-3-phosphorylcholine and/or synthetic cholesterol) may be present at about 35mol% to 65mol% of the total lipid. In some embodiments, the composition of an exemplary RNA product formulation can be characterized as shown in table 15.

TABLE 15 quantitative composition of exemplary RNA product formulations

^[1] Cationic lipid a= ((3-hydroxypropyl) azanediyl) bis (nonane-9, 1-diyl) bis (2-octanoate)

^[2] PEG conjugated lipid a=2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide

^[3] Dspc=1, 2-distearoyl-sn-glycero-3-phosphorylcholine

Q.s. =sufficient (quantum satis) (as much as possible)

Example 14 exemplary lipid excipients in RNA/LNP pharmaceutical product formulations described herein

Materials used in the manufacturing process of pharmaceutical products may be purchased, inspected, sampled, identified, tested, and released from qualified suppliers. The excipient tests were performed according to predetermined specifications or according to ph.eur/USP.

In some embodiments, the RNA/LNP pharmaceutical product formulation comprises four lipid excipients shown in table 16, table 16 providing further information about the lipid excipients. All excipients are provided as GMP grade materials.

TABLE 16 lipid excipients in exemplary RNA/LNP drug products described herein

Cationic lipid A (((3-hydroxypropyl) azanediyl) bis (nonane-9, 1-diyl) bis (2-octanoate)

In some embodiments, the amino lipid ((3-hydroxypropyl) azetidinyl) bis (nonane-9, 1-diyl) bis (butyl 2-octanoate)) is a functional cationic lipid component of an RNA/LNP pharmaceutical product formulation described herein. It aims at promoting biodegradation, metabolism and clearance in vivo. The amino lipids comprise a titratable tertiary amino headgroup linked to two saturated alkyl chains via an ester linkage that, when incorporated into LNP, imparts different physicochemical characteristics that regulate particle formation, cellular uptake, fusion, and/or endosomal release of RNA. The ester bonds can be readily hydrolyzed to promote rapid degradation and excretion via the renal pathway. The amino lipid has an apparent pK_a of about 6.25, yielding a substantially fully positively charged molecule at pH 5. During the manufacturing process, the introduction of an aqueous RNA solution into an ethanol lipid mixture comprising the amino lipids at pH 4 results in electrostatic interactions between the negatively charged RNA backbone and the positively charged cationic lipids. This electrostatic interaction results in particle formation consistent with efficient encapsulation of the RNA drug substance. After RNA encapsulation, adjusting the pH of the medium surrounding the resulting LNP to 7.4 results in neutralization of the surface charge of the LNP. When all other variables were kept constant, the charge neutral particles showed longer in vivo circulation life and better delivery to hepatocytes than the charged particles that were cleared rapidly by the reticuloendothelial system. After endosomal uptake, the low pH of the endosome fuses the LNP and allows release of RNA into the cytosol of the target cell.

PEG conjugated lipid A2- [ (polyethylene glycol) -2000] -N, N-Bitetradecylacetamide

In some embodiments, the RNA/LNP pharmaceutical product formulation described herein comprises the functional lipid excipient 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide. The pegylated lipids are similar in structure to other clinically approved pegylated lipids, and their safety is demonstrated in clinical trials. The primary function of the pegylated lipids is to sterically stabilize the particle by forming a protective hydrophilic layer of a protective hydrophobic lipid layer. Furthermore, when the particles are administered in vivo, the pegylated lipids reduce association with serum proteins and the uptake of the reticuloendothelial system resulting therefrom. PEG lipids are known to affect cellular uptake, which is a prerequisite for endosomal localization and payload delivery. It has been found that pharmacology of encapsulated nucleic acids can be controlled in a predictable manner by modulating the alkyl chain length of the PEG-lipid anchors. In some embodiments, such pegylated lipids are selected for RNA/LNP pharmaceutical product formulations to provide optimal delivery of RNA to the liver. In some embodiments, such selection is also based on reasonable solubility characteristics and molecular weights thereof to effectively function as a spatial barrier. Such pegylated lipids do not show significant surfactant or permeability enhancement or interference effects on biological membranes. In addition, PEG in such pegylated lipids is linked to the diacyl lipid anchors via biodegradable amide linkages, facilitating rapid degradation and excretion. In the vial, the particles retained a full complement of pegylated lipids. In the blood compartment, such pegylated lipids dissociate from the particles over time, showing more fused particles that are more easily taken up by the cells, ultimately resulting in release of the RNA payload.

Neutral lipid DSPC and cholesterol

In some embodiments, the RNA/LNP pharmaceutical product formulation comprises two or more neutral lipids. In some such embodiments, the RNA/LNP pharmaceutical product formulation may comprise two or more neutral lipids, including DSPC and/or cholesterol. In some embodiments, such neutral lipids (e.g., DSPC and/or cholesterol) may be referred to as structural lipids, at concentrations selected to optimize LNP particle size, stability, and encapsulation. For example, DSPC and cholesterol have been used in approved pharmaceutical products, e.g., DSPC is used asAndIs a compound of the formula (I). Cholesterol is used asAndIs a compound of the formula (I).Both DSPC and cholesterol are included.

Example 15 exemplary evaluation and/or criteria for RNA/LNP pharmaceutical product formulations described herein

In some embodiments, one or more of the evaluations as described herein may be used during manufacture or other preparation or use of the pharmaceutical product (e.g., as a release test).

In some embodiments, the RNA/LNP drug product can be evaluated for one or more quality control parameters (e.g., for release and/or for further processing), including, but not limited to, physical appearance, lipid characteristics and/or content, LNP size, LNP polydispersity, RNA encapsulation, RNA length, identity (as RNA), integrity, sequence and/or concentration, pH, osmolarity, RNA ratio (e.g., HC RNA to LC RNA ratio), potency, bacterial endotoxin, bioburden, residual organic solvent, osmolarity, pH, and combinations thereof. Such quality control parameters may be assessed by one or more specific analytical methods known in the art, such as, for example, visual inspection, gel electrophoresis (e.g., agarose gel electrophoresis, capillary gel electrophoresis), enzymatic degradation, sequencing, UV absorbance spectrophotometry, RNA labeling dyes, PCR methods, bacterial endotoxin testing (e.g., limulus Amoebocyte Lysate (LAL) testing), dynamic light scattering, liquid chromatography with charged aerosol detector, gas chromatography, and/or in vitro translation systems (e.g., rabbit reticulocyte lysate translation system and³⁵ S-methionine).

In some embodiments, the RNA/LNP pharmaceutical product formulation (e.g., the RNA/LNP pharmaceutical product formulation described herein) lot can be evaluated for quality control parameters (e.g., the quality control parameters described herein) to determine the next step of action. For example, if the quality assessment indicates that a batch of RNA/LNP pharmaceutical product formulation (e.g., an RNA/LNP pharmaceutical product formulation described herein) meets or exceeds the listed relevant release criteria, such batch may be designated for one or more additional steps of manufacture and/or distribution. Otherwise, if such a lot does not meet or exceed the release criteria, then an alternative action may be taken (e.g., discard the lot).

Example 16 exemplary inclusion criteria

In some embodiments, a cancer patient whose tumor expresses CLDN-18.2 can be selected for treatment with the compositions and/or methods described herein. In some embodiments, the cancer patient is a pancreatic cancer patient. In some embodiments, the cancer patient is a cholangiocarcinoma patient.

In some embodiments, cancer patients selected to meet one or more of the following disease-specific inclusion criteria are treated with the compositions and/or methods described herein:

Cldn-18.2 positive tumors (regardless of tumor histology), defined as ≡50% of tumor cells with ≡2+cldn-18.2 protein staining intensity as assessed by central test using validated immunohistochemical assays in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue;

ffpe tumor tissue samples were available for CLDN-18.2 testing. Allowing new biopsies and archival biological samples. If archival tissue samples at several time points are available, then the most recent archival tissue sample is preferred;

3. histological recordings of the original primary tumor by pathology reporting.

A. such histologically determined solid tumor being metastatic or unresectable and for which no standard treatment is available that would likely confer clinical benefit, or for which the patient is not a candidate for such available treatment, and optionally a disease that is measurable or evaluable according to RECIST 1.1, or

B. histologically-determined unresectable locally advanced or metastatic pancreatic ductal adenocarcinoma without prior palliative chemotherapy, and optionally a measurable or evaluable disease according to RECIST 1.1, or

C. Histologically determined unresectable locally advanced or metastatic PDAC meeting the conditions for treatment with nab-paclitaxel + gemcitabine or FOLFIRINOX without prior palliative chemotherapy, and optionally a disease which is measurable or evaluable according to RECIST 1.1, or

D. Histologically-determined locally advanced or metastatic BTC meeting the conditions for treatment with cisplatin+gemcitabine without prior palliative chemotherapy, and optionally a measurable or evaluable disease according to RECIST 1.1.

In some embodiments, cancer patients that meet at least one of the above disease-specific inclusion criteria and further meet at least one of the following additional inclusion criteria are selected for treatment with the compositions and/or methods described herein:

1. The age is more than or equal to 18 years old.

2. Eastern tumor cooperative group (Eastern Cooperative Oncology Group, ECOG) exhibited states of 0 to 1.

3. Sufficient clotting function upon screening, as determined by:

a. International normalized ratio (International normalized ratio, INR) or prothrombin time +.1.5×upper normal limit (upper limit normal, ULN; except for therapeutic anticoagulant, its value is within the therapeutic window).

B. activating a portion of the thromboplastin time (ACTIVATED PARTIAL thromboplastin time, aPTT) to less than or equal to 1.5 XULN (except for therapeutic anticoagulants, values within the therapeutic window).

4. Sufficient hematologic function at the time of screening, as determined by:

a. White blood cell count (white blood count, WBC). Gtoreq.3×⁹/L.

B. Absolute neutrophil count (absolute neutrophil count, ANC). Gtoreq.1.5X10⁹/L (patients could not use granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor to achieve these WBC and ANC levels during the last 7 days).

C. the platelet count is more than or equal to 100 multiplied by 10⁹/L.

D. hemoglobin is 9.0g/dL or more (erythropoietin cannot be infused or used to achieve this level over the last 7 days).

5. Sufficient liver function at the time of screening, as determined by:

a. Total bilirubin is less than or equal to 1.5mg/dL (or less than or equal to 2.0mg/dL for patients with known Gilbert's syndrome) or metastatic carcinoma of the liver.

B. Aspartic acid Aminotransferase (AST) and alanine Aminotransferase (ALT) are less than or equal to 2.5 XULN, and for patients with metastatic liver cancer, less than or equal to 3ULN.

6. Sufficient kidney function upon screening, as determined by:

a. Glomerular filtration rate ≡45 mL/min/1.73 m² -according to the equation for simplified renal disease diet Modification (Modification of DIET IN RENAL DISEASE):

Gfr=186× (S _Creatinine^-1.154) × (age^-0.203)

(Wherein serum creatinine levels are expressed in mg/dL; multiplied by 0.742 if the patient is female, and by 1.212 if the patient is african americans (level et al 1999).

7. Women with fertility potential (women of childbearing potential, WOCBP) must have a negative serum (β -human chorionic gonadotrophin) test/value at the time of screening. Postmenopausal or permanently sterile patients may be considered to have no reproductive potential.

8. Women with fertility potential must agree not to donate eggs (egg cells, oocytes) for assisted reproduction purposes during the treatment regimen until 6 months after the last CLDN-18.2 targeted treatment described herein.

9. During the trial and 6 months after receiving the last dose of CLDN-18.2 targeted therapy described herein, men active with WOCBP and not undergoing vasectomy must agree to use a fertility controlled barrier method, e.g., use a condom with spermicidal foam/gel/film/cream/suppository, or a partner with an occlusion cap (septum or cervical/vault cap) with spermicidal foam/gel/film/cream/suppository.

10. Men must agree not to donate sperm during the treatment regimen and 6 months after receiving the last dose of CLDN-18.2 targeted therapy described herein.

Example 17 exemplary exclusion criteria

In some embodiments, cancer patients whose tumors do not express CLDN-18.2 are not suitable for the compositions and/or methods described and/or used herein.

In some embodiments, (i) has recently received cancer treatment, (ii) has received systemic steroid treatment, (iii) has recently received major surgery, (iv) has an active infection and is being treated with anti-infective therapy, and/or (v) is diagnosed with developing brain or pia mater metastasis cancer is not suitable for use in the compositions and/or methods described and/or used herein.

In some embodiments, the following cancer patients may not be recommended for CLDN-18.2 targeted therapy described herein (e.g., administration of the compositions described herein and/or the methods of treatment described herein).

Previous and concomitant treatments

1. Radiation therapy, chemotherapy or molecular targeted agents or tyrosine kinase inhibitors are received 2 weeks or 5 half-lives (whichever is longer) from the start of CLDN-18.2 targeted therapy described herein, immunotherapy/monoclonal antibodies are received 3 weeks from the start of CLDN-18.2 targeted therapy described herein, nitrosoureas, antibody-drug conjugates or radioisotopes are received 6 weeks from the start of CLDN-18.2 targeted therapy described herein.

2. Systemic (oral or IV) steroid therapy or equivalent thereof is administered daily with >10mg of prednisone for the underlying condition.

3. Major surgery was performed within 4 weeks prior to the first dose of CLDN-18.2 targeted therapy described herein.

4. Ongoing or active infections requiring IV therapy with anti-infective therapy administered less than 2 weeks prior to the first dose of CLDN-18.2 targeted therapy described herein.

5. The side effects of any prior treatment or manipulation of any medical condition did not return to the general term Standard of national cancer institute AE (National Cancer Institute Common Terminology Criteria for AE, NCI CTCAE) grade.5≤1. It should be noted that a peripheral neuropathy level of < 2 is permissible, and any level of hair loss is permissible.

Medical conditions

6. Current evidence of new or growing brain or pia metastatic cancer during screening. Patients with known brain or pia metastases may be eligible if they have:

a. Radiation therapy, surgery or stereotactic surgery for brain or pial metastatic cancer.

B. no neurological symptoms (excluding neuropathy with grade No. 2).

C. within 4 weeks before signing the informed consent form, computed tomography (computer)

A tomograph, CT) or magnetic resonance imaging (magnet resonance imaging, MRI) scan shows that brain or pia disease is stable.

D. No acute corticosteroid treatment or steroid fade-in was performed.

It should be noted that patients with central nervous system symptoms should undergo brain CT scan or MRI to exclude new or progressive brain metastases. Spinal metastatic cancer is permitted unless a fracture is predicted in which spinal cord compression is imminent.

7. History of convulsions during childhood except isolated febrile convulsions, history of cerebrovascular accidents or transient ischemic attacks less than 6 months prior to screening.

8. Effusion (pleural, pericardial or ascites) requiring drainage.

9. Active or past history of autoimmune disease including, but not limited to, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, or multiple sclerosis.

10. Active immune conditions requiring immunosuppression with steroids or other immunosuppressants (e.g., azathioprine, cyclosporin a) are excluded from patients with isolated vitiligo, resolved childhood asthma or atopic dermatitis, controlled adrenal or hypopituitary hypofunction, and normal thyroid with a history of graves' disease. Patients with controlled hyperthyroidism must be negative for thyroglobulin, thyroid peroxidase antibodies, and thyroid stimulating immunoglobulins prior to administration of the trial treatment.

11. It is known that human immunodeficiency virus has a seropositive history of cd4+ T cell count <350 cells/μl and a history of opportunistic infections as defined by acquired immunodeficiency syndrome.

12. Hepatitis b history/positive serology is known to require active antiviral treatment (unless immunized due to vaccination or resolved natural infection or unless passively immunized due to immunoglobulin treatment). Patients with positive serology must have a hepatitis b viral load below the limit of quantification.

13. Active Hepatitis C Virus (HCV) infection, and allows for curative antiviral treatment of patients with HCV loads below the limit of quantification.

14. It is known to have hypersensitivity to components of CLDN-18.2 targeted therapies described herein.

15. Other primary malignancies that have not been alleviated for at least 2 years, except for those with negligible risk of metastasis or death (e.g., fully treated cervical carcinoma in situ, basal or squamous cell skin carcinoma, localized prostate cancer, or ductal carcinoma in situ).

Other common diseases

16. Clinically significant abnormal electrocardiography, such as Fridericia corrected QT interval is prolonged by >480 milliseconds.

17. It is within the knowledge of the treatment practitioner that there are any complications that can cause excessive medical hazards or interfere with interpretation of the treatment results, including but not limited to:

a. Ongoing or active infections requiring antibiotic/antiviral/antifungal therapy.

B. and congestive heart failure (new york heart association functional grade (New York Heart Association Functional Classification Class) grade III or IV).

C. And unstable angina pectoris is complicated.

D. The treatment of a concurrent arrhythmia is required (excluding asymptomatic atrial fibrillation).

E. acute coronary syndromes occur within the past 6 months.

F. significant lung disease (shortness of breath at rest or in mild exercise), for example due to complications of severe obstructive pulmonary disease.

18. Cognitive, psychological or social mental disorders that would impair the patient's ability to receive treatment according to the regimen, or adversely affect the patient's ability to follow the informed consent process and follow-up regimen necessary visits and manipulations.

19. Pregnancy or nursing.

Example 18 exemplary dosing regimen of CLDN-18.2 targeting compositions described herein in combination with nab-paclitaxel and/or gemcitabine

In some embodiments, the pharmaceutical compositions provided herein may be administered to a patient suffering from CLDN-18.2 positive cancer in combination with other anti-cancer therapies. In some embodiments, the administration involves one or more cycles. In some embodiments, the pharmaceutical compositions provided herein may be administered for at least 3 to 8 cycles.

In some embodiments, administration of the CLDN-18.2 targeted compositions described herein may be performed at one or more of the levels shown in table 13 above (see example 8), in some embodiments, administration may involve administration of at least one lower dose from table 13 followed by administration of at least one higher dose from table 13.

When administered in combination with nab-paclitaxel and gemcitabine, in some embodiments, the CLDN-18.2 targeting composition may be administered prior to the first infusion of the cytotoxic treatment. For example, in some embodiments, CLDN-18.2 targeted compositions can be administered a minimum of 4 hours prior to the first infusion of a cytotoxic treatment (e.g., nab-paclitaxel and gemcitabine). In some embodiments, CLDN-18.2 targeted compositions can be administered at Q3W and chemotherapy will follow a protocol approved according to the local guidelines. For example, in some embodiments, a combination therapy comprising a CLDN-18.2 targeting composition and nab-paclitaxel and/or gemcitabine may be administered for at least eight cycles, e.g., in some embodiments, according to the regimen as shown in table 17.

TABLE 17 exemplary dosing regimen for administration of CLDN-18.2 targeting composition and nab-paclitaxel and gemcitabine

As shown in table 17, the cycle length for CLDN-18.2 targeted therapy was defined as 21 days (q 3 w) and CLDN-18.2 targeted compositions were administered on day 1 of each cycle. Nab-paclitaxel and gemcitabine were administered every 28 days on days 1, 8 and 15. The bold "x" is highlighted when administration of nab-paclitaxel/gemcitabine on day 1 is matched to administration of the anti-CLDN 18.1 composition.

Gemcitabine alone has been used to treat pancreatic cancer. For example, a recommended dose of gemcitabine (e.g., gemzar) is 1000mg/m² within 30 minutes intravenously. In some embodiments, the recommended treatment regimen is:

week 1 to week 8, weekly administration for the first 7 weeks followed by 1 week rest.

Weekly administration after week 8 on days 1, 8 and 15 of the 28 day cycle.

In some embodiments, CLDN-18.2 targeted compositions described herein can be administered in combination with gemcitabine as a monotherapy for the treatment of pancreatic cancer (e.g., gemzar) according to approved dosages and treatment regimens, as described above. In some embodiments, CLDN-18.2 targeted compositions described herein can be administered in combination with gemcitabine at lower doses (e.g., less than 10%, less than 20%, less than 30% or more) and/or at less aggressive treatment regimens (e.g., every 10 days, or once every two weeks, etc.) than approved doses and treatment regimens (as described above) of gemcitabine as monotherapy for the treatment of pancreatic cancer.

Nab-paclitaxel in combination with gemcitabine is known for the treatment of metastatic pancreatic adenocarcinoma. For example, nab-paclitaxel is administered as an IV infusion over 30 to 40 minutes on days 1, 8 and 15 of each 28 day cycleHowever, gemcitabine should be administered immediately after the 1 st, 8 th and 15 th day of each 28 th day cycle nab-paclitaxel, 125mg/m².

In some embodiments, CLDN-18.2 targeted compositions described herein can be administered in combination with gemcitabine and nab-paclitaxel according to approved dosages and treatment regimens for nab-paclitaxel/gemcitabine combination treatment, as described above. In some embodiments, CLDN-18.2 targeted compositions described herein can be administered in combination with nab-paclitaxel and gemcitabine, at least one of which is administered at a lower dose (e.g., less than 10%, less than 20%, less than 30% or more) and/or at a less aggressive treatment regimen (e.g., every 10 days, or once every two weeks, etc.) than the approved dose and treatment regimen (as described above) of nab-paclitaxel/gemcitabine combination treatment.

In some embodiments, pre-and post-operative administration of antipyretics (e.g., acetaminophen, non-steroidal anti-inflammatory drugs), anti-emetics, proton pump inhibitors, and anxiolytics may be allowed according to drug/regulatory guidelines. In some embodiments, the patient should be suitably pre-hydrated prior to administration of the CLDN-18.2 targeting composition described herein. In some embodiments, the corticosteroid is not used as a pre-operative drug of the CLDN-18.2 targeting composition described herein.

Example 19 exemplary efficacy assessment and/or monitoring

In some embodiments, cancer patients administered CLDN-18.2 targeting compositions described herein as monotherapy or in combination with additional anti-cancer therapies may be monitored periodically for adjustment of therapeutic efficacy and/or therapeutic dose/schedule.

In some embodiments, the efficacy of the treatment may be assessed by computed tomography and/or magnetic resonance imaging scans. In some embodiments, MRI scans may be performed using a 3 tesla whole body instrument. In some embodiments, when evaluating lesions for efficacy assessment, one or more of the following criteria may be used:

Complete response-all target lesions disappeared. The short axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to <10mm.

Partial response the sum of the diameters of target lesions was reduced by at least 30% with the baseline total diameter as reference.

Progressive disease °a sum of diameters of target lesions is increased by at least 20% with the minimum sum in the study as a reference (if this is the minimum sum in the study, it includes the baseline sum). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5mm. The appearance of one or more new lesions is also considered progress.

Stable disease o neither met sufficient reduction of PR nor sufficient increase of progressive disease, taking as reference the minimum sum diameter in the study.

Example 20 HEK293T/17 transfection to test in vitro production of anti-CLDN 18.2 RiboMab based on different RNA coding sequences

RiboMab expression of different mRNA constructs encoding anti-CLDN 18.2 RiboMab Light (LC) and Heavy (HC) chains was tested. These constructs all contain the same RNA backbone (all RNA sequences in mRNA except the coding sequence) and encode the same LC or HC, but differ in that they have different LC or HC RNA coding sequences (optimizations 1, 3, or 10). Optimization 1 corresponds to the sequences shown in SEQ ID No. 16 and 17, respectively. Optimization 3 and optimization 10 are as follows:

Optimizing 3, hc-coding sequences:

Optimizing 3, lc-coding sequence:

Optimizing 10, hc-coding sequence:

optimization 10, lc-coding sequence:

48 hours prior to transfection, 1X 10⁵ HEK 293T/17 cells/cm² were plated in 25mL of complete Dulbecco's modified Eagle's medium (DMEM+GlutaMax supplemented with 10% non-heat inactivated FBS) in T175 cm² tissue culture flasks. By adding, prior to transfectionThe solution was used to detach the cells, which were washed twice with X-Vivo-15 medium and resuspended in X-Vivo-15 medium to determine the cell count. RNA coding sequences of HC and LC constructs with different optimized coding sequences (optimizations 1,3 and 10) are shown above. In each case, mRNA encoding HC was mixed with mRNA encoding the corresponding LC in a 1:1 molar ratio of HC to LC, and 25. Mu.g of the RNA mixture was added to 250. Mu.L of cell suspension (8X 10⁶/mL) in a 0.4cm cuvette. HEK293T/17 cells were electroporated (250V, 2 pulses, 5 ms) and inoculated into serum-free expression medium at a density of 2X 10⁶/mL. Cell culture supernatants were harvested after 48 hours and the concentration of anti-CLDN 18.2 RiboMab was determined by ELISA. As can be seen in fig. 16, the highest levels of anti-CLDN 18.2 RiboMab were detected in the supernatant of cells transfected with HC and LC mRNA encoding sequences optimized 1.

Example 21 in vivo translation efficacy against CLDN18.2 RiboMab with different mRNA backbones

Translation efficacy against CLDN18.2 RiboMab HC and LC optimization 1 coding sequences in two different mRNA backbones (backbone a (SEQ ID NOs: 18, 19) and backbone B (SEQ ID NOs: 20, 21)) was tested.

To determine the in vivo exposure of mRNA encoded anti-CLDN 18.2 RiboMab, the mouse strain Balb/cJRj (CHARLES RIVER, sulzfeld, germany) was selected. In this study, 30 female Balb/c mice, 8 to 12 weeks old, were treated with twice weekly intravenous (i.v.) bolus injection treatment or control. The treatment groups received 3 or 30 μg of mRNA formulated as Lipid Nanoparticles (LNP) encoding HC and LC against CLDN18.2 RiboMab (optimization 1) in either backbone a or backbone B, respectively. LNP was diluted in 150. Mu.l DPBS. The control group received a non-specific control mRNA encoding firefly luciferase formulated as LNP. Serum samples were analyzed for anti-CLDN 18.2 RiboMab concentration by ELISA.

The anti-CLDN 18.2 antibody concentration was dose-dependent and time-dependent, with t_max occurring 24 to 72 hours after the first administration (fig. 17). The peak anti-CLDN 18.2 RiboMab concentration (C_max) was 743.3 μg/mL (30 μg backbone a) and 1818 μg/mL (30 μg backbone B), respectively. Thus, RNA-LNP using backbone B gave the highest level of anti-CLDN 18.2 RiboMab. The same was observed in the results obtained for the 3 μg dose group (skeleton a=44 μg/mL vs. skeleton b=127 mg/mL).

Example 22 cytotoxic Activity of anti-CLDN18.2RiboMabs encoded by optimized 1RNA Using either backbone A or B

The activity of anti-CLDN 18.2 RiboMab expressed in mice dosed with RNA-LNP using either backbone a or backbone B, also tested in example 21, was assessed by an Antibody Dependent Cellular Cytotoxicity (ADCC) assay (figure 18). ADCC assays were performed with the CLDN18.2 positive gastric cancer cell line NUG-C4. The target negative cell line MDA-MB-231_luc_tom cells served as negative controls. Human PBMCs from healthy donors were used as effector cells with a ratio of effector cells to target cells of 20:1. The reference anti-CLDN 18.2 antibody was used as a positive control. The cytotoxic activity of CLDN18.2 RiboMab encoded by the two RNA-LNP samples was comparable, with average EC₅₀ values of 4.27ng/mL (backbone a) and 4.21ng/mL (backbone B) for mice treated with 30 μg RNA-LNP. Equivalent values were obtained for mice treated with lower doses. It can thus be concluded that antibodies generated with both framework a and framework B constructs are functional.

Example 23 improvement of the translatability of mRNA by optimization of cloning vector to ensure prolonged persistence of encoded protein

One of the key advantages of In Vitro Transcription (IVT) mRNA based technology is the in vivo synthesis of therapeutic proteins (Qin et al 2022). However, the long-term persistence of the protein of interest is strongly dependent on the stability, immunogenicity, and translational capacity of the IVT mRNA. It is well known that these properties can be greatly improved and balanced by optimizing the coding sequence (CDS), by incorporating nucleoside modifications into the mRNA, and by removing aberrant products generated during in vitro transcription (loosis et al 2015).

Comparative studies (WO 2021/214204) on the performance of mRNA transcribed from DNA constructs that differ in terms of transcription initiation site and short non-coding intermediate sequences underscores the importance of non-translated regions that do not belong to the CDS but are inserted due to individual cloning strategies. It has been previously found that even small changes in the untranslated region of the RNA backbone can greatly affect and enhance translational activity in vivo. Specifically, for example, the first transcribed nucleotide nt4+5AGACG (backbone a) is replaced with AGAAT (backbone C), and additionally the Lig3 motif directly upstream of the Poly (a) tail is removed (WO 2021/214204).

Given that variations in the nucleotide content of the mRNA intermediate sequence can affect the persistence of the encoded protein, we performed comparative studies on mRNA transcribed from different DNA constructs including backbone a, backbone C, and backbone B, and backbone D (fig. 19A). The backbone comprises, upstream and downstream from the coding sequence, the following sequences, backbone A18/34, backbone B20/36, backbone C20/35, and backbone D38/36, respectively. Frameworks B and D contain previously optimized transcription initiation sites AGAAT (framework B) or AGCAC (framework D), which show the highest translational capacity. The only differences between backbone C and backbone B/backbone D vectors are (i) the intermediate sequence between the termination codon of the coding sequence and the 3'-UTR, and (ii) the intermediate sequence between the 3' -UTR and the Poly-A tail. The results herein show that not only the transcription initiation site can affect its translational activity, but also the nucleotide composition of the non-coding region at the 3' end of the mRNA. Here, the present invention demonstrates how the mRNA optimization studies of the present invention lead to the selection of novel leader cloning vector backbones B and D.

Skeleton A (SEQ ID NO 18+34)

Framework B (SEQ ID NO 20+36)

Framework C (SEQ ID NO 20+35)

Skeleton D (SEQ ID NO 38+36)

Backbone E (SEQ ID NO 20+37), given for comparison

In vivo study

For the template, four plasmids corresponding to framework a, framework B, framework C and framework D encoding codon optimized murine erythropoietin (murine erythropoietin, mEPO) were used and linearized by enzymatic restriction with BspQI (NEW ENGLAND Biolabs, catalog No. R0712L) or by PCR. mRNA starting from AGACG (backbone A), AGAAU (backbones B and C) or AGCAC (backbone D) was designed to contain the 5 'untranslated region (5' UTR) sequence of human alpha-globin (hAg) mRNA, the FI element (SEQ ID NO: 22) as the 3'UTR and the 100nt long 3' Poly (A) tail (SEQ ID NO: 23) flanking the insert coding sequence. MEGASCRIPT T7 RNA polymerase kit (Thermo FISHER SCIENTIFIC, catalog No. AMB 1334-5) was used for transcription and UTP was replaced with N1-methylpseuduride triphosphate (m 1 ψ) (TriLink, catalog No. N-1081). mRNA was co-transcribed using the reverse transcription resistant cap 1 analogue CleanCap413 (TriLink, catalog number N-7413) at a final concentration of 3 mM. To reduce the initial byproducts and obtain the desired transcript produced with the cap analogue, the initial GTP and m1ψTP concentrations in the transcription reaction were reduced from 7.5mM to 1.5mM (Triana-Alonso et al 1995) and incubated in the hybridization chamber for 30 min at 37 ℃. The initial concentration of additional nucleotides, including ATP and CTP, corresponds to a final concentration of 7.5 mM. After incubation for 30, 60, 90 and 120 minutes, the mixture was added with an additional 1.5mM GTP and m1ψTP and incubated for a further 30 minutes at 37 ℃. To remove the template, 1/10 volume of DNA Turbo DNase (Thermo FISHER SCIENTIFIC, cat. AM 1907) was added to the reaction mixture and the mixture was incubated for 15 min at 37 ℃. The synthesized mRNA was isolated from the reaction mixture by precipitation with half the reaction volume of 8M LiCl solution (Sigma-Aldrich, cat. L7026). After cooling at-20 ℃ for at least 1 hour, RNA precipitate was collected by centrifugation at 17.000×g for 5 minutes at 4 ℃. After washing the RNA pellet twice with at least 200. Mu.l ice-cold 75% ethanol solution, it was dissolved in nuclease-free water. The concentration and quality of the mRNA transcribed in vitro were measured on a NanoDrop2000C spectrophotometer (Thermo FISHER SCIENTIFIC, catalog ND-2000C). Aliquots of denatured IVT mRNA were analyzed by electrophoresis in agarose gels containing 0.005% (v/v) GelRed^TM nucleic acid gel stain (Masek et al.2005). a small aliquot of the mRNA sample was stored in a siliconized tube at-20 ℃. All mRNA was purified as described for celluloseet al.2019)。

In order to measure the translational efficiency of EPO m1 ψ -mRNA transcribed from different DNA constructs comprising scaffold a, scaffold C and scaffold B and scaffold D, in vivo comparative studies were performed. 8 to 10 week old female BALB/c mice from Janvier laboratories (14 Route des Ch E nes Secs,53940Genest Saint Isle,France) were used for in vivo experiments according to the federal policy on animal research (ethical approval number: G18-12-027). Mice (n=3/group) were injected intravenously (i.v.) with 3 μg of TransIT-complexed (Mirus Bio, cat. No. MIR 2255) EPO m1 ψ -mRNA in Dulbecco's modified Eagle medium, DMEM, dulbecco's modified Eagle's medium, final volume of 200 μl. Mice used as controls were injected with the TransIT reagent diluted in DMEM, but without RNA. To quantify plasma EPO levels, 20 μl of blood was collected from each mouse at 6, 24, 48 and 72 hours after injection, and EPO levels were analyzed by the mouse erythropoietin DuoSet ELISA kit (R & D Systems, minneapolis, MN, USA, catalog No. DY 959). Flat bottom 96-well plates were pre-coated with 2 μg/ml rat anti-mouse EPO capture antibody (100 μl/well) and incubated overnight at Room Temperature (RT). plates were washed three times with PBS containing 0.05% tween 20 and incubated with 1% BSA (bovine serum albumin) (Sigma-Aldrich, cat 2153) solution for 2 hours at room temperature to prevent non-specific binding of antibodies and washed again. A7-point standard curve using 2-fold serial dilutions and a 4000pg/ml high standard was applied. Plasma samples with final volumes of 50 μl and standards diluted in 1% BSA solution were added to the appropriate wells and incubated for 2 hours at RT. After washing the plates, 100 μl of 1 μg/ml rat biotinylated anti-mouse EPO detection antibody in 1% BSA solution was assigned to each well and incubated for 2 hours at RT. the plate was washed and then incubated with 100. Mu.l of streptavidin conjugated with horseradish peroxidase (diluted in 1% BSA solution (1:200)) for 20 minutes at room temperature. After washing, TMB 2 component microporous peroxidase substrate solution (Medac Gmbh, catalog number 50-76-11) was added to each well (100. Mu.l/well). Samples were incubated at room temperature for 5 minutes and 2M sulfuric acid (R & D Systems, catalog number DY 994) (50 μl/well) was added to terminate the reaction and absorbance was measured at 450nm and 570nm using an Infinite 200Pro reader (Tecan).

The presented data show that EPO mRNA transcribed from scaffold C is superior to scaffold a but inferior to that derived from both scaffold B and scaffold D cassettes (fig. 19a, B). The translation of EPO mRNA transcribed from backbone C was significantly better than the construct containing the lower transcription start site AGACG and Lig3 motif (backbone a) 48 hours and 72 hours after injection, but was 2.5 and 10 times lower than that of EPO mRNA prepared from backbone B and backbone D, respectively (fig. 19a, B). Subsequently, the reason for the difference in the long-term translational efficacy of the encoded protein was investigated. Considering that the previously selected backbone C differs at the 3 '-end of the CDS downstream region and at the 9 th position of the Poly (A) tail upstream region compared to backbone B and backbone D, it was determined that the 9 th nucleotide of the Poly (A) tail may have an effect on translation (FIG. 22A) because it has been previously shown that altering the sequence downstream of the coding sequence and upstream of the 3' -UTR has no effect on the translation ability of mRNA (WO 2021/214204). The scaffold C has G at position 9 of the mRNA 3' end, while the scaffold B has C, and the modified scaffold B appears to be significantly better than the original scaffold C-box, especially at later time points (fig. 22A). To analyze the effect of the nucleotide at position 9 on translation, four different backbone B constructs were designed that contained G, T, A or C nucleotides at position 9 upstream of the Poly (a) tail. The translations of EPO m1 ψ -mRNA containing G in this position were 1.5-fold, 3.3-fold and 8-fold lower at 6 to 24 hours, 48 hours and 72 hours respectively than those using C at the same position (fig. 22B). When EPO m1 ψ -mRNA contains U or a in a given position, the difference in EPO levels was 1.3 and 2-fold lower at 6 to 24 hours and 48 to 72 hours, respectively, compared to those with C at the same position (fig. 22B). These data are very consistent with the results obtained from independent in vitro experiments (fig. 20, 21).

To ensure that this one nucleotide exchange has no effect on mRNA translation efficiency independent of the transcription start site, EPO-encoding m1 ψ -mRNA containing four different SNPs (G, U, A or C) at the same 9 th position upstream of the encoded Poly (a) tail transcribed from the backbone D box using AGCAC start site instead of AGAAT was injected intravenously into mice. According to the results of the EPO-specific ELISA, the translation of EPO m1 ψ -mRNA containing G at position 9 upstream of Poly (a) tail was 2-fold, 3-fold and 8-fold lower than those using A, U or C at the same position, respectively, at 24, 48 and 72 hours, whether or not cassettes containing different initiation sites were contained (fig. 23A). Two new cassettes containing C at position 9 upstream of the encoded Poly (a) tail (framework B and framework D) perform best (fig. 22B and 23A). To test whether this SNP effect at position 9 interferes with mRNA function, hematocrit levels were determined in individual mice injected with EPO mRNA complexed with TransIT reagent by collecting 18. Mu.L of whole blood (total blood) at the indicated time (FIG. 23B) into tubes containing 2. Mu.L of EDTA solution, followed by centrifugation in Drummond microcaps glass capillaries (20. Mu.L volume, merck, germany) as described (Mahiny et al.2016). SNPs located at the 3' end of the mRNA construct have a significant effect on the function of mRNA encoding murine EPO. On day 7 after injection, the in vitro transcribed mRNA containing C at position 9 upstream of Poly (a) tail resulted in the highest hematocrit level (58%) (fig. 23B), which was significantly higher than those carrying G at this position in the case of both backbones B (51%) and D (53.5%). On day 7 after injection, EPO m1 ψ -mRNA with A, U or C at position 9 upstream of Poly (a) tail showed an elevated hematocrit at least 4% to 5% higher than those with G at the same position, whichever backbone was used (fig. 23B).

In vitro study

To compare frameworks B, C and D, we produced mRNAs encoding hIL-18, firefly luciferase, and eGFP in the context of these frameworks. In immature human dendritic cells or primary human hepatocytes, in vitro transcribed m1Y modified mRNA capped with CC413 cap analogs is lipofected or electroporated. When electroporation was performed in hiDC (FIG. 20A), firefly luciferase was more strongly expressed when encoded by frameworks B and D compared to framework C. Similarly, when hiDC was electroporated with EGFP containing backbone C and D mRNA (fig. 20B), the latter showed stronger EGFP expression. When primary human hepatocytes were lipofected with mRNA encoding human IL-18 in scaffolds B, C and D (FIG. 20C), higher titers of hIL-18 were detected in the medium in the case of scaffolds B and D compared to scaffold C. Thus, regardless of the coding sequence, it was observed that either framework B or D provided the same enhancement performance as compared to framework C.

To investigate the effect of single nucleotide changes between backbones B and D and C at position 9 upstream of the polyA sequence, firefly luciferase mRNA was produced in backbones B and C containing A, G, T or C nucleotides at that position. In vitro transcribed m1Y mRNA capped with CC413 cap analogues was electroporated in hiDC and luciferase expression was determined (fig. 21a, b). For both backbones, G at position 9 of polyA has a negative effect on luciferase expression, indicating that SNP at position 9 upstream of polyA sequence has a major effect on mRNA translation.

For electroporation experiments, m1Y modified mRNA capped with CC413 cap analogue transcribed in vitro was electroporated in hiDC cells with 30mM RNA and in duplicate with a single pulse of 300V/12 ms. Cells were plated and cultured in 12 or 96 well plates in RPMI medium supplemented with hIL-4/GM-CSF (Miltenyi). For firefly luciferase mRNA, luciferase expression was determined using Bright-Glo assay (Promega) 2 to 48 hours after electroporation. For eGFP mRNA, cells were harvested at 6 to 96 hours and analyzed with FACS CantoII (BD).

For the liposome transfection experiments, primary human hepatocytes (BioIVT) were transfected with TransIT (Mirus) according to the manufacturer's protocol. Supernatants were collected at 16 to 168 hours and analyzed for hIL-18 translation by ELISA (R & D Systems).

Conclusion(s)

In summary, the presented data indicate that the 3' -terminal region of mRNA is a very sensitive and specific region in terms of translation capacity as well as function of mRNA. This study evaluated and directly compared the quality, translational efficiency and function of m1ψ modified EPO mRNA derived from 4 DNA constructs including backbone a, backbone B, backbone C and backbone D. M1ψ -mRNA transcribed from the previously selected backbone C was significantly better than backbone A, which contained the inferior transcription start site AGACG and Lig3 motif at the 3' end (FIG. 19A). However, the further adjusted framework B and framework D cassettes perform significantly better in vitro and in vivo than the framework C cassette, especially at later time points (fig. 19A, 19B, 20A, 20B, 20C). Both in vitro and in vivo results unexpectedly demonstrate that single nucleotide substitutions at position 9 upstream of the encoded Poly (a) tail (e.g., guanine (G) vs. cytosine (C) in scaffold B and scaffold D) in scaffold C) provide improved expression of scaffold B and scaffold D compared to scaffold C. The use of other nucleotides than C at this position, in particular G, has a negative effect on the translation capacity and function of the mRNA (FIGS. 21A, 21B, 22B, 23A, 23B), regardless of the start site.

Additional comparative study

Comparative studies were also performed to assess how other changes in mRNA intermediate nucleotide content could affect the persistence of the encoded protein. Comparative studies were also performed on mRNA transcribed from different DNA constructs including scaffold a, scaffold G, scaffold I and scaffold B (fig. 24). The backbone comprises, upstream and downstream from the coding sequence, the following sequences, backbone A18/34, backbone B20/36, backbone G43/36, and backbone I44/45, respectively. The results herein show that not only the transcription initiation site can affect its translational activity, but also the nucleotide composition of the non-coding region at the 3' end of the mRNA. Here, the present invention demonstrates how the mRNA optimization studies of the present invention lead to the selection of novel leader cloning vector backbones B and D.

Skeleton A (SEQ ID NO 18+34)

Framework B (SEQ ID NO 20+36)

Skeleton G (SEQ ID NO 43+36)

Skeleton I (SEQ ID NO 44+45)

In vitro transcribed mRNA comprising backbone G corresponding to TEV 5'UTR has a beneficial effect on the long term translation activity of mRNA and has been previously found to have superior performance to that of in vitro transcribed hAg' UTR specific mRNA comprising backbone A. However, these two constructs differ not only from each other in the 5' UTR, but also in other non-coding intermediate regions, including transcription initiation sites, kozak sequences, restriction sites relevant from the point of view of cloning strategies, and non-coding stuffer sequences located at the 3' end of mRNA between the 3' UTR and the poly-A sequence (ligation 3, lig 3). To find out if the backbone G construct can improve the effect on the long-term persistence of the encoded protein, four other DNA constructs were designed by combining the mentioned non-coding sequences of backbone a and backbone G, which resulted in backbone I, backbone I and backbone B constructs without Kozak sequences (fig. 24). mRNA encoding EPO containing m1 ψ was prepared from each DNA construct. After purification, 3 μg of TransIT-complexed mRNA was injected intravenously into Balb/c mice to determine EPO levels using murine EPO-specific ELISA at 6, 24 and 48 hours after administration (FIG. 24). This shows that the amount of EPO levels translated from the DNA construct transcribed backbone G mRNA was 4-fold and 10-fold higher than those produced by DNA template backbone a, respectively, 24 and 48 hours after injection (fig. 24). However, this also shows that EPO mRNA from the newly created DNA construct scaffold B (no lig, having AGAAUA transcription start site, hAg' utr and Kozak sequence) at 24 and 48 hours after injection, exhibited significantly better than those of scaffold a and scaffold I starting at AGACGA at each time point, and also better than those of scaffold G (fig. 24). This finding suggests that the long term translation capacity of mRNA is determined not only by the 5' utr, but also by other non-coding intermediate sequences (e.g., transcription initiation sites, kozak sequences, and restriction and stuffer sequences for cloning procedures).

Subsequently, it was determined that not only the 5 'terminal sequence could affect the translational capacity of the IVT RNA encoding murine EPO, but also the 3' terminal sequence could affect the translational capacity of the IVT RNA encoding murine EPO. For efficient cloning, bamHI (GGAUCC) and XhoI (CUCGAG) sites were incorporated downstream of CDS and upstream of 3'utr and polyA tail, respectively, in the newly created scaffold B with transcription initiation sites (AGACG and AGAAT) and with 3' ends from the scaffold a and scaffold G constructs (with or without ligation 3) (fig. 25). Mu.g of TransIT-complexed m1 ψ -modified EPO mRNA transcribed from each DNA construct variant was injected intravenously into mice. Thereafter, EPO levels in plasma collected from mice were measured at 6, 24, 48 and 72 hours after administration to compare the translational activity of each mRNA construct (fig. 25). At each time point, the translational efficiency of EPO mRNA from backbone B (AGAAU +kozak+ hAg 5' utr+bamhi/XhoI, no ligation 3) was significantly better than that of backbone a or G, regardless of which restriction sites (BamHI and XhoI) were used downstream of CDS (fig. 25). This result suggests that backbone B and DNA template encoding backbone B were used as novel cloning vectors and templates for IVT reactions for the preparation of mRNA, rather than backbone G.

In direct comparative experiments, EPO mRNA with backbone B was found to translate much better than those with backbone a or G after intravenous injection of the TransIT complexed EPO mRNA (fig. 26). To confirm that this effect is independent of formulation, each EPO RNA was individually encapsulated in the same baseline Lipid Nanoparticle (LNP) formulation and its performance was determined in vivo. Plasma EPO levels were measured at 6, 24, 48 and 72 hours after intravenous administration ((LNP)) using a murine EPO-specific ELISA for 3 μg of mice injected with mRNA each formulated as LNP. This suggests that backbone B has a strong beneficial effect on long-term translation of EPO mRNA formulated as LNP, since at 24 and 48 hours after injection, backbone B mRNA is translated at least 4-fold and 14-fold more than those transcribed from backbone a and backbone G, respectively (fig. 26). EPO ELISA showed that the differences between constructs were more pronounced when LNP formulations were used instead of the TransIT reagent, possibly due to differences in the target cell types that ingest mRNA. Taken together, the mRNA translation of the backbone B construct is optimal and provides a long duration of encoded protein, independent of the formulation used, indicating that this effect is sequence dependent and that the combination of non-encoding intermediate sequences in backbone B is particularly advantageous.

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Equivalent solution

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations and permutations in which one or more of the limitations, elements, clauses, descriptive terms, etc. of one or more of the listed claims are introduced into another claim (or as related to any other claim) that is dependent on the same base claim, unless otherwise indicated or unless it would be apparent to one of ordinary skill in the art that contradiction or inconsistency would arise. Furthermore, it should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a particular exclusion is set forth in the specification. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims (192)

Translated fromChinese

1.组合物或药物制剂，其含有：1. A composition or pharmaceutical preparation comprising:(i)包含编码第一多肽链的编码区的RNA，所述第一多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的重链，和(i) an RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and(ii)包含编码第二多肽链的编码区的RNA，所述第二多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的轻链，其中(ii) an RNA comprising a coding region encoding a second polypeptide chain, wherein the second polypeptide chain comprises a light chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), wherein(i)中所述的编码区包含SEQ ID NO:16的第79至1422位核苷酸的核苷酸序列或者与SEQ ID NO:16的第79至1422位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，并且The coding region in (i) comprises a nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and(ii)中所述的编码区包含SEQ ID NO:17的第79至738位核苷酸的核苷酸序列或者与SEQ ID NO:17的第79至738位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。The coding region described in (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:17 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO:17.2.权利要求1所述的组合物或药物制剂，其中2. The composition or pharmaceutical preparation of claim 1, wherein所述第一多肽链包含SEQ ID NO:3的第27至474位氨基酸的氨基酸序列或者与SEQ IDNO:3的第27至474位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列，并且The first polypeptide chain comprises an amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity with the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and所述第二多肽链包含SEQ ID NO:4的第27至246位氨基酸的氨基酸序列或者与SEQ IDNO:4的第27至246位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列。The second polypeptide chain comprises an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence that is at least 90% identical to an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4.3.组合物或药物制剂，其含有：3. A composition or pharmaceutical preparation comprising:(i)包含编码第一多肽链的编码区的RNA，所述第一多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的重链，和(i) an RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and(ii)包含编码第二多肽链的编码区的RNA，所述第二多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的轻链，其中(ii) an RNA comprising a coding region encoding a second polypeptide chain, wherein the second polypeptide chain comprises a light chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), wherein所述第一多肽链包含SEQ ID NO:3的第27至474位氨基酸的氨基酸序列或者与SEQ IDNO:3的第27至474位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列，并且The first polypeptide chain comprises an amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity with the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and所述第二多肽链包含SEQ ID NO:4的第27至246位氨基酸的氨基酸序列或者与SEQ IDNO:4的第27至246位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列。The second polypeptide chain comprises an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence that is at least 90% identical to an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4.4.权利要求1至3中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。4. The composition or pharmaceutical formulation of any one of claims 1 to 3, wherein the RNA, e.g., each RNA, comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20.5.权利要求1至4中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR，所述5’UTR包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第7至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。5. The composition or pharmaceutical formulation of any one of claims 1 to 4, wherein the RNA, e.g., each RNA, comprises a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20.6.权利要求1至5中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR，所述5’UTR包含SEQ ID NO:18或20的核苷酸序列或者与SEQ ID NO:18或20的核苷酸序列具有至少90％同一性的核苷酸序列。6. The composition or pharmaceutical formulation 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 with the nucleotide sequence of SEQ ID NO: 18 or 20.7.权利要求1至6中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含3’UTR，所述3’UTR包含SEQ ID NO:22的核苷酸序列或者与SEQ ID NO:22的核苷酸序列具有至少90％同一性的核苷酸序列。7. The composition or pharmaceutical 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 with the nucleotide sequence of SEQ ID NO: 22.8.权利要求1至7中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含3’UTR，所述3’UTR包含SEQ ID NO:19或21的核苷酸序列或者与SEQ ID NO:19或21的核苷酸序列具有至少90％同一性的核苷酸序列。8. The composition or pharmaceutical formulation 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 with the nucleotide sequence of SEQ ID NO: 19 or 21.9.组合物或药物制剂，其含有：9. A composition or pharmaceutical preparation comprising:(i)包含编码第一多肽链的编码区的RNA，所述第一多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的重链，和(i) an RNA comprising a coding region encoding a first polypeptide chain comprising a heavy chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2), and(ii)包含编码第二多肽链的编码区的RNA，所述第二多肽链包含与密蛋白-18.2(CLDN-18.2)结合的抗体剂的轻链，(ii) an RNA comprising a coding region encoding a second polypeptide chain comprising a light chain of an antibody agent that binds to claudin-18.2 (CLDN-18.2),其中所述RNA，例如每种RNA，包含5’UTR和/或3’UTR，所述5’UTR包含SEQ ID NO:18或20的核苷酸序列或者与SEQ ID NO:18或20的核苷酸序列具有至少90％同一性的核苷酸序列，所述3’UTR包含SEQ ID NO:19或21的核苷酸序列或者与SEQ ID NO:19或21的核苷酸序列具有至少90％同一性的核苷酸序列。wherein the RNA, e.g., each RNA, comprises a 5'UTR and/or a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO: 18 or 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18 or 20, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO: 19 or 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19 or 21.10.权利要求1至9中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR和3’UTR，所述5’UTR包含SEQ ID NO:18或20的核苷酸序列或者与SEQ ID NO:18或20的核苷酸序列具有至少90％同一性的核苷酸序列，所述3’UTR包含SEQ ID NO:19或21的核苷酸序列或者与SEQ ID NO:19或21的核苷酸序列具有至少90％同一性的核苷酸序列。10. The composition or pharmaceutical formulation of any one of claims 1 to 9, wherein the RNA, e.g., each RNA, comprises a 5'UTR and a 3'UTR, wherein the 5'UTR comprises the nucleotide sequence of SEQ ID NO: 18 or 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18 or 20, and the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 19 or 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19 or 21.11.权利要求1至10中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR和3’UTR，所述5’UTR包含SEQ ID NO:18的核苷酸序列或者与SEQ ID NO:18的核苷酸序列具有至少90％同一性的核苷酸序列，所述3’UTR包含SEQ ID NO:19的核苷酸序列或者与SEQ ID NO:19的核苷酸序列具有至少90％同一性的核苷酸序列。11. The composition or pharmaceutical formulation of any one of claims 1 to 10, wherein the RNA, e.g., each RNA, comprises a 5'UTR and a 3'UTR, wherein the 5'UTR comprises the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 18, and the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 19.12.权利要求1至11中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR和3’UTR，所述5’UTR包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列，所述3’UTR包含SEQ ID NO:21的核苷酸序列或者与SEQ ID NO:21的核苷酸序列具有至少90％同一性的核苷酸序列。12. The composition or pharmaceutical formulation of any one of claims 1 to 11, wherein the RNA, e.g., each RNA, comprises a 5'UTR and a 3'UTR, wherein the 5'UTR comprises the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 20, and the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 21.13.权利要求1至12中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR和3’UTR，所述5’UTR包含SEQ ID NO:18的核苷酸序列，所述3’UTR包含SEQ ID NO:19的核苷酸序列。13. The composition or pharmaceutical preparation of any one of claims 1 to 12, wherein the RNA, e.g., each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO: 18, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO: 19.14.权利要求1至12中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’UTR和3’UTR，所述5’UTR包含SEQ ID NO:20的核苷酸序列，所述3’UTR包含SEQ ID NO:21的核苷酸序列。14. The composition or pharmaceutical preparation of any one of claims 1 to 12, wherein the RNA, e.g., each RNA, comprises a 5'UTR and a 3'UTR, the 5'UTR comprising the nucleotide sequence of SEQ ID NO: 20, and the 3'UTR comprising the nucleotide sequence of SEQ ID NO: 21.15.权利要求9至14中任一项所述的组合物或药物制剂，其中：15. The composition or pharmaceutical formulation of any one of claims 9 to 14, wherein:(a)(i)中所述的编码区包含SEQ ID NO:16的第79至1422位核苷酸的核苷酸序列或者与SEQ ID NO:16的第79至1422位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，并且(a)(i) The coding region comprises a nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and(ii)中所述的编码区包含SEQ ID NO:17的第79至738位核苷酸的核苷酸序列或者与SEQ ID NO:17的第79至738位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，以及/或者(ii) the coding region comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and/or(b)所述第一多肽链包含SEQ ID NO:3的第27至474位氨基酸的氨基酸序列或者与SEQID NO:3的第27至474位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列，并且(b) the first polypeptide chain comprises an amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3 or an amino acid sequence having at least 90% identity with the amino acid sequence of amino acids 27 to 474 of SEQ ID NO:3, and所述第二多肽链包含SEQ ID NO:4的第27至246位氨基酸的氨基酸序列或者与SEQ IDNO:4的第27至246位氨基酸的氨基酸序列具有至少90％同一性的氨基酸序列。The second polypeptide chain comprises an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4 or an amino acid sequence that is at least 90% identical to an amino acid sequence of amino acids 27 to 246 of SEQ ID NO:4.16.权利要求1至15中任一项所述的组合物或药物制剂，其中16. The composition or pharmaceutical preparation of any one of claims 1 to 15, wherein(i)中所述的编码区包含SEQ ID NO:16的核苷酸序列或者与SEQ ID NO:16的核苷酸序列具有至少90％同一性的核苷酸序列，并且The coding region in (i) comprises the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 16, and(ii)中所述的编码区包含SEQ ID NO:17的核苷酸序列或者与SEQ ID NO:17的核苷酸序列具有至少90％同一性的核苷酸序列。The coding region described in (ii) comprises the nucleotide sequence of SEQ ID NO:17 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:17.17.权利要求1至16中任一项所述的组合物或药物制剂，其中17. The composition or pharmaceutical preparation of any one of claims 1 to 16, wherein所述第一多肽链包含SEQ ID NO:3的氨基酸序列或者与SEQ ID NO:3的氨基酸序列具有至少90％同一性的氨基酸序列，并且The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 3, and所述第二多肽链包含SEQ ID NO:4的氨基酸序列或者与SEQ ID NO:4的氨基酸序列具有至少90％同一性的氨基酸序列。The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:4 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4.18.权利要求1至17中任一项所述的组合物或药物制剂，其中(i)中所述的RNA是第一RNA分子并且(ii)中所述的RNA是第二RNA分子。18. The composition or pharmaceutical formulation of any one of claims 1 to 17, wherein the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule.19.权利要求1至18中任一项所述的组合物或药物制剂，其中至少90％是至少95％、96％、97％、98％、99％。19. The composition or pharmaceutical formulation of any one of claims 1 to 18, wherein at least 90% is at least 95%, 96%, 97%, 98%, 99%.20.权利要求1至19中任一项所述的组合物或药物制剂，其中所述抗体剂相对于密蛋白-18.1(CLDN-18.1)优先与CLDN-18.2结合。20. The composition or pharmaceutical formulation of any one of claims 1 to 19, wherein the antibody agent preferentially binds to CLDN-18.2 relative to CLDN-18.1.21.权利要求1至20中任一项所述的组合物或药物制剂，其中所述抗体剂与CLDN-18.2的第一胞外结构域(ECD1)结合。21. The composition or pharmaceutical formulation of any one of claims 1 to 20, wherein the antibody agent binds to the first extracellular domain (ECD1) of CLDN-18.2.22.权利要求1至21中任一项所述的组合物或药物制剂，其中所述抗体剂与在癌细胞中暴露的CLDN-18.2的ECD1的表位结合。22. The composition or pharmaceutical formulation of any one of claims 1 to 21, wherein the antibody agent binds to an epitope of ECD1 of CLDN-18.2 exposed in cancer cells.23.权利要求1至22中任一项所述的组合物或药物制剂，其中与CLDN-18.2结合的所述抗体剂包含两个结合臂，其中每个结合臂包含与CLDN-18.2结合的抗体剂的重链和与CLDN-18.2结合的抗体剂的轻链。23. The composition or pharmaceutical formulation of any one of claims 1 to 22, wherein the antibody agent that binds to CLDN-18.2 comprises two binding arms, wherein each binding arm comprises a heavy chain of the antibody agent that binds to CLDN-18.2 and a light chain of the antibody agent that binds to CLDN-18.2.24.权利要求1至23中任一项所述的组合物或药物制剂，其中所述抗体剂是IgG1。24. The composition or pharmaceutical formulation of any one of claims 1 to 23, wherein the antibody agent is IgG1.25.权利要求24所述的组合物或药物制剂，其中所述IgG1是人IgG1。25. The composition or pharmaceutical formulation of claim 24, wherein the IgG1 is human IgG1.26.权利要求1至25中任一项所述的组合物或药物制剂，其中所述第一多肽链与所述第二多肽链相互作用以形成与CLDN-18.2结合的结合结构域。26. The composition or pharmaceutical formulation 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.权利要求1至26中任一项所述的组合物或药物制剂，其中所述第一多肽链包含与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))。27. The composition or pharmaceutical formulation of any one of claims 1 to 26, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)).28.权利要求27所述的组合物或药物制剂，其中所述VH(CLDN-18.2)包含SEQ ID NO:14的氨基酸序列的CDR1、CDR2和CDR3。28. The composition or pharmaceutical formulation 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.权利要求27或28所述的组合物或药物制剂，其中所述VH(CLDN-18.2)包含分别含有SEQ ID NO:5、6和7中所示序列的CDR1、CDR2和CDR3。29. The composition or pharmaceutical formulation of claim 27 or 28, wherein the VH (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6 and 7, respectively.30.权利要求1至29中任一项所述的组合物或药物制剂，其中所述第二多肽链包含与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))。30. The composition or pharmaceutical formulation of any one of claims 1 to 29, wherein the second polypeptide chain comprises the variable domain (VL) of a light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)).31.权利要求30所述的组合物或药物制剂，其中所述VL(CLDN-18.2)包含SEQ ID NO:15的氨基酸序列的CDR1、CDR2和CDR3。31. The composition or pharmaceutical formulation 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.权利要求30或31所述的组合物或药物制剂，其中所述VL(CLDN-18.2)包含分别含有SEQ ID NO:8、9和10中所示序列的CDR1、CDR2和CDR3。32. The composition or pharmaceutical formulation of claim 30 or 31, wherein the VL (CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences shown in SEQ ID NOs: 8, 9 and 10, respectively.33.权利要求1至32中任一项所述的组合物或药物制剂，其中所述第一多肽链包含含有氨基酸序列SEQ ID NO:14的CDR1、CDR2和CDR3之与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))，并且所述第二多肽链包含含有SEQ ID NO:15的氨基酸序列的CDR1、CDR2和CDR3之与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))。33. The composition or pharmaceutical formulation of any one of claims 1 to 32, wherein the first polypeptide chain comprises a variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 14 (VH (CLDN-18.2)), and the second polypeptide chain comprises a variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 comprising CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 15 (VL (CLDN-18.2)).34.权利要求1至33中任一项所述的组合物或药物制剂，其中所述第一多肽链包含分别含有SEQ ID NO:5、6和7中所示序列的CDR1、CDR2和CDR3之与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))，并且所述第二多肽链包含分别含有SEQ ID NO:8、9和10中所示序列的CDR1、CDR2和CDR3之与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))。34. The composition or pharmaceutical formulation of any one of claims 1 to 33, wherein the first polypeptide chain comprises a variable domain (VH) of a heavy chain of an antibody agent that binds to CLDN-18.2, comprising CDR1, CDR2, and CDR3 of the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain (VL) of a light chain of an antibody agent that binds to CLDN-18.2, comprising CDR1, CDR2, and CDR3 of the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively (VL(CLDN-18.2)).35.权利要求1至34中任一项所述的组合物或药物制剂，其中所述第一多肽链包含含有氨基酸序列SEQ ID NO:14之与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))，并且所述第二多肽链包含含有SEQ ID NO:15的氨基酸序列之与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))。35. The composition or pharmaceutical formulation of any one of claims 1 to 34, wherein the first polypeptide chain comprises a variable domain (VH) of a heavy chain of an antibody agent that binds to CLDN-18.2 comprising the amino acid sequence of SEQ ID NO: 14 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain (VL) of a light chain of an antibody agent that binds to CLDN-18.2 comprising the amino acid sequence of SEQ ID NO: 15 (VL(CLDN-18.2)).36.权利要求1至35中任一项所述的组合物或药物制剂，其中所述第一多肽链包含与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))，并且所述第二多肽链包含与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))，其中所述VH(CLDN-18.2)与所述VL(CLDN-18.2)相互作用以形成与密蛋白-18.2(CLDN-18.2)结合的结合结构域。36. The composition or pharmaceutical formulation of any one of claims 1 to 35, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)), wherein the VH(CLDN-18.2) interacts with the VL(CLDN-18.2) to form a binding domain that binds to claudin-18.2 (CLDN-18.2).37.权利要求1至36中任一项所述的组合物或药物制剂，其中所述第一多肽链包含与CLDN-18.2结合的抗体剂的重链的可变结构域(VH)(VH(CLDN-18.2))、抗体剂的重链的恒定结构域1(CH1)、抗体剂的重链的恒定结构域2(CH2)和抗体剂的重链的恒定结构域3(CH3)。37. The composition or pharmaceutical formulation of any one of claims 1 to 36, wherein the first polypeptide chain comprises the variable domain (VH) of the heavy chain of an antibody agent that binds to CLDN-18.2 (VH (CLDN-18.2)), the constant domain 1 (CH1) of the heavy chain of the antibody agent, the constant domain 2 (CH2) of the heavy chain of the antibody agent, and the constant domain 3 (CH3) of the heavy chain of the antibody agent.38.权利要求37所述的组合物或药物制剂，其中所述VH(CLDN-18.2)、CH1、CH2和CH3以免疫球蛋白G(IgG)形式存在于所述第一多肽链中。38. The composition or pharmaceutical formulation of claim 37, wherein the VH (CLDN-18.2), CH1, CH2 and CH3 are present in the first polypeptide chain in the form of immunoglobulin G (IgG).39.权利要求1至38中任一项所述的组合物或药物制剂，其中所述第二多肽链包含与CLDN-18.2结合的抗体剂的轻链的可变结构域(VL)(VL(CLDN-18.2))和抗体剂的轻链的恒定结构域(CL)。39. The composition or pharmaceutical formulation of any one of claims 1 to 38, wherein the second polypeptide chain comprises the variable domain (VL) of the light chain of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) and the constant domain (CL) of the light chain of the antibody agent.40.权利要求39所述的组合物或药物制剂，其中所述VL(CLDN-18.2)和CL以IgG形式存在于所述第二多肽链中。40. The composition or pharmaceutical formulation of claim 39, wherein the VL (CLDN-18.2) and CL are present in the second polypeptide chain in the form of IgG.41.权利要求39或40所述的组合物或药物制剂，其中所述第一多肽链上的CH1与所述第二多肽链上的CL相互作用。41. The composition or pharmaceutical formulation of claim 39 or 40, wherein CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain.42.权利要求1至41中任一项所述的组合物或药物制剂，其中所述第一多肽链和所述第二多肽链各自独立地包含分泌信号，其中所述分泌信号优选地位于所述第一多肽链和所述第二多肽链的N端。42. The composition or pharmaceutical formulation of any one of claims 1 to 41, wherein the first polypeptide chain and the second polypeptide chain each independently comprise a secretion signal, wherein the secretion signal is preferably located at the N-terminus of the first polypeptide chain and the second polypeptide chain.43.权利要求42所述的组合物或药物制剂，其中所述第一多肽链和/或所述第二多肽链的分泌信号包含SEQ ID NO:13的氨基酸序列。43. The composition or pharmaceutical 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.权利要求1至43中任一项所述的组合物或药物制剂，其中44. The composition or pharmaceutical preparation of any one of claims 1 to 43, wherein(i)中所述的编码区包含SEQ ID NO:16的核苷酸序列，并且The coding region described in (i) comprises the nucleotide sequence of SEQ ID NO: 16, and(ii)中所述的编码区包含SEQ ID NO:17的核苷酸序列。The coding region described in (ii) comprises the nucleotide sequence of SEQ ID NO:17.45.权利要求1至44中任一项所述的组合物或药物制剂，其中45. The composition or pharmaceutical preparation of any one of claims 1 to 44, wherein所述第一多肽链包含SEQ ID NO:3的氨基酸序列，并且The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3, and所述第二多肽链包含SEQ ID NO:4的氨基酸序列。The second polypeptide chain comprises the amino acid sequence of SEQ ID NO:4.46.权利要求1至45中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含poly-A序列。46. The composition or pharmaceutical formulation of any one of claims 1 to 45, wherein the RNA, e.g. each RNA, comprises a poly-A sequence.47.权利要求46所述的组合物或药物制剂，其中所述poly-A序列是A核苷酸的间断序列。47. The composition or pharmaceutical formulation of claim 46, wherein the poly-A sequence is an interrupted sequence of A nucleotides.48.权利要求46或47所述的组合物或药物制剂，其中所述poly-A序列包含至少100个核苷酸。48. The composition or pharmaceutical formulation of claim 46 or 47, wherein the poly-A sequence comprises at least 100 nucleotides.49.权利要求46至48中任一项所述的组合物或药物制剂，其中所述poly-A序列包含核苷酸序列A_x-L-A_y或由核苷酸序列A_x-L-A_y组成，其中A_x是至少20个A核苷酸的序列，A_y是至少60个A核苷酸的序列并且L是可包含除A之外的核苷酸的1至20个核苷酸的接头。49. The composition or pharmaceutical formulation of any one of claims 46 to 48, wherein the poly-A sequence comprises or consists of the nucleotide sequence_Ax-LAy , wherein_Ax is a sequence of at least 20 A nucleotides,_Ay is_a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides which may contain nucleotides other than A.50.权利要求46至49中任一项所述的组合物或药物制剂，其中所述poly-A序列包含SEQID NO:23的核苷酸序列或由SEQ ID NO:23的核苷酸序列组成。50. The composition or pharmaceutical formulation 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.权利要求1至50中任一项所述的组合物或药物制剂，其含有：51. The composition or pharmaceutical preparation of any one of claims 1 to 50, comprising:(i)包含SEQ ID NO:24或26的核苷酸序列或者与SEQ ID NO:24或26的核苷酸序列具有至少90％同一性的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 24 or 26, and(ii)包含SEQ ID NO:25或27的核苷酸序列或者与SEQ ID NO:25或27的核苷酸序列具有至少90％同一性的核苷酸序列的RNA。(ii) an RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27, or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 25 or 27.52.权利要求51所述的组合物或药物制剂，其含有：52. The composition or pharmaceutical preparation of claim 51, comprising:(i)包含SEQ ID NO:24的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and(ii)包含SEQ ID NO:25的核苷酸序列的RNA。(ii) RNA comprising the nucleotide sequence of SEQ ID NO:25.53.权利要求51所述的组合物或药物制剂，其含有：53. The composition or pharmaceutical preparation of claim 51, comprising:(i)包含SEQ ID NO:26的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and(ii)包含SEQ ID NO:27的核苷酸序列的RNA。(ii) RNA comprising the nucleotide sequence of SEQ ID NO:27.54.组合物或药物制剂，其含有：54. A composition or pharmaceutical preparation comprising:(i)包含SEQ ID NO:24或26的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and(ii)包含SEQ ID NO:25或27的核苷酸序列的RNA。(ii) RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27.55.组合物或药物制剂，其含有：55. A composition or pharmaceutical preparation comprising:(i)包含SEQ ID NO:24的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 24, and(ii)包含SEQ ID NO:25的核苷酸序列的RNA。(ii) RNA comprising the nucleotide sequence of SEQ ID NO:25.56.组合物或药物制剂，其含有：56. A composition or pharmaceutical preparation comprising:(i)包含SEQ ID NO:26的核苷酸序列的RNA，和(i) an RNA comprising the nucleotide sequence of SEQ ID NO: 26, and(ii)包含SEQ ID NO:27的核苷酸序列的RNA。(ii) RNA comprising the nucleotide sequence of SEQ ID NO:27.57.权利要求1至56中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含替代尿苷的经修饰核苷。57. The composition or pharmaceutical formulation of any one of claims 1 to 56, wherein the RNA, e.g., each RNA, comprises a modified nucleoside replacing uridine.58.权利要求1至57中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含替代每个尿苷的经修饰核苷。58. The composition or pharmaceutical formulation of any one of claims 1 to 57, wherein the RNA, e.g., each RNA, comprises a modified nucleoside replacing each uridine.59.权利要求57或58所述的组合物或药物制剂，其中所述经修饰核苷是假尿苷(ψ)和/或N1-甲基-假尿苷(m1ψ)。59. The composition or pharmaceutical formulation of claim 57 or 58, wherein the modified nucleoside is pseudouridine (ψ) and/or N1-methyl-pseudouridine (m1ψ).60.权利要求57至59中任一项所述的组合物或药物制剂，其中所述经修饰核苷是N1-甲基-假尿苷(m1ψ)。60. The composition or pharmaceutical formulation of any one of claims 57 to 59, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).61.权利要求1至60中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’帽。61. The composition or pharmaceutical formulation of any one of claims 1 to 60, wherein the RNA, e.g., each RNA, comprises a 5' cap.62.权利要求1至61中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’帽m₂^7，3’-OGpp(m₁^2’-O)ApG。62. The composition or pharmaceutical formulation of any one of claims 1 to 61, wherein the RNA, such as each RNA, comprises a 5' cap_m27,3'^-O Gpp(_m12'^-O )ApG.63.权利要求1至62中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，是单链RNA。63. The composition or pharmaceutical formulation of any one of claims 1 to 62, wherein the RNA, e.g., each RNA, is single-stranded RNA.64.权利要求1至63中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，是mRNA。64. The composition or pharmaceutical formulation of any one of claims 1 to 63, wherein the RNA, e.g., each RNA, is mRNA.65.权利要求1至64中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，配制在脂质纳米粒(LNP)中，例如每一种RNA共配制在脂质纳米粒(LNP)中。65. The composition or pharmaceutical formulation of any one of claims 1 to 64, wherein the RNA, e.g., each RNA, is formulated in a lipid nanoparticle (LNP), e.g., each RNA is co-formulated in a lipid nanoparticle (LNP).66.权利要求65所述的组合物或药物制剂，其中形成所述脂质纳米粒的脂质包含阳离子脂质、聚合物缀合的脂质；和中性脂质。66. The composition or pharmaceutical formulation of claim 65, wherein the lipids forming the lipid nanoparticles comprise cationic lipids, polymer-conjugated lipids; and neutral lipids.67.权利要求66所述的组合物或药物制剂，其中：67. The composition or pharmaceutical formulation of claim 66, wherein:a.所述阳离子脂质以总脂质的35mol％至65mol％存在；a. The cationic lipid is present in 35 mol% to 65 mol% of the total lipids;b.所述聚合物缀合的脂质以总脂质的约1mol％至2.5mol％存在；并且b. the polymer-conjugated lipid is present at about 1 mol % to 2.5 mol % of the total lipid; andc.所述中性脂质以总脂质的35mol％至65mol％存在。c. The neutral lipid is present in an amount of 35 mol% to 65 mol% of the total lipids.68.权利要求66或67所述的组合物或药物制剂，其中所述阳离子脂质是((3-羟基丙基)氮烷二基)双(壬烷-9,1-二基)双(2-丁基辛酸酯)。68. The composition or pharmaceutical formulation of claim 66 or 67, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate).69.权利要求66至68中任一项所述的组合物或药物制剂，其中所述聚合物缀合的脂质是PEG缀合的脂质(例如2-[(聚乙二醇)-2000]-N,N-双十四烷基乙酰胺)。69. The composition or pharmaceutical formulation of any one of claims 66 to 68, wherein the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).70.权利要求66至69中任一项所述的组合物或药物制剂，其中所述中性脂质包含1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DPSC)和/或胆固醇。70. The composition or pharmaceutical formulation of any one of claims 66 to 69, wherein the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and/or cholesterol.71.权利要求65至70中任一项所述的组合物或药物制剂，其中所述脂质纳米粒的平均尺寸为约50至150nm。71. The composition or pharmaceutical formulation of any one of claims 65 to 70, wherein the average size of the lipid nanoparticles is about 50 to 150 nm.72.权利要求65至71中任一项所述的组合物或药物制剂，其中所述脂质纳米粒包含((3-羟基丙基)氮烷二基)双(壬烷-9,1-二基)双(2-丁基辛酸酯)、2-[(聚乙二醇)-2000]-N,N-双十四烷基乙酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱和胆固醇。72. The composition or pharmaceutical formulation of any one of claims 65 to 71, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.73.权利要求1至72中任一项所述的组合物，其是药物组合物。73. The composition of any one of claims 1 to 72, which is a pharmaceutical composition.74.权利要求73所述的组合物，其中所述药物组合物还包含一种或更多种可药用载体、稀释剂和/或赋形剂。74. The composition of claim 73, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.75.权利要求1至72中任一项所述的药物制剂，其是药盒。75. The pharmaceutical formulation of any one of claims 1 to 72, which is a kit.76.权利要求75所述的药物制剂，其中所述RNA，例如每种RNA，以及任选地颗粒形成组分在单独的小瓶中。76. The pharmaceutical formulation of claim 75, wherein the RNA, e.g. each RNA, and optionally the particle-forming components are in separate vials.77.权利要求75或76所述的药物制剂，其还包含所述组合物或药物制剂用于治疗或预防癌症的使用说明。77. The pharmaceutical formulation of claim 75 or 76, further comprising instructions for use of the composition or pharmaceutical formulation for treating or preventing cancer.78.权利要求1至77中任一项所述的组合物或药物制剂，其用于药物用途。78. A composition or pharmaceutical formulation as claimed in any one of claims 1 to 77 for use as a medicine.79.权利要求78所述的组合物或药物制剂，其中所述药物用途包含疾病或病症的治疗性或预防性治疗。79. The composition or pharmaceutical formulation of claim 78, wherein the pharmaceutical use comprises therapeutic or prophylactic treatment of a disease or condition.80.权利要求79所述的组合物或药物制剂，其中所述疾病或病症的治疗性或预防性治疗包含治疗或预防癌症。80. The composition or pharmaceutical formulation of claim 79, wherein the therapeutic or prophylactic treatment of the disease or condition comprises treating or preventing cancer.81.权利要求80所述的组合物或药物制剂，其中所述癌症包含CLDN-18.2阳性癌症。81. The composition or pharmaceutical formulation of claim 80, wherein the cancer comprises a CLDN-18.2 positive cancer.82.权利要求80或81所述的组合物或药物制剂，其中所述癌症包含CLDN-18.2阳性实体瘤。82. The composition or pharmaceutical formulation of claim 80 or 81, wherein the cancer comprises a CLDN-18.2-positive solid tumor.83.权利要求80至82中任一项所述的组合物或药物制剂，其中所述癌症包括CLDN-18.2阳性胰腺癌。83. The composition or pharmaceutical formulation of any one of claims 80 to 82, wherein the cancer comprises CLDN-18.2-positive pancreatic cancer.84.权利要求80至83中任一项所述的组合物或药物制剂，其中所述癌症包括CLDN-18.2阳性胃癌。84. The composition or pharmaceutical formulation of any one of claims 80 to 83, wherein the cancer comprises CLDN-18.2-positive gastric cancer.85.权利要求80至84中任一项所述的组合物或药物制剂，其中所述癌症包括CLDN-18.2阳性胆道肿瘤。85. The composition or pharmaceutical formulation of any one of claims 80 to 84, wherein the cancer comprises a CLDN-18.2 positive biliary tract tumor.86.权利要求80至85中任一项所述的组合物或药物制剂，其中所述癌症包括CLDN-18.2阳性的局部晚期、不可切除或转移性癌症。86. The composition or pharmaceutical formulation of any one of claims 80 to 85, wherein the cancer comprises a CLDN-18.2-positive locally advanced, unresectable or metastatic cancer.87.权利要求79至86中任一项所述的组合物或药物制剂，其中所述疾病或病症的治疗性或预防性治疗还包括施用另外的治疗。87. The composition or pharmaceutical formulation of any one of claims 79 to 86, wherein the therapeutic or prophylactic treatment of the disease or condition further comprises the administration of an additional treatment.88.权利要求87所述的组合物或药物制剂，其中所述另外的治疗包括选自以下中的一种或更多种：(i)手术以切去、切除肿瘤或使其减积，(ii)放射治疗，和(iii)化学治疗。88. The composition or pharmaceutical formulation of claim 87, wherein the additional treatment comprises one or more selected from the group consisting of: (i) surgery to resect, excise or debulk the tumor, (ii) radiation therapy, and (iii) chemotherapy.89.权利要求87或88所述的组合物或药物制剂，其中所述另外的治疗包括施用另外的治疗剂。89. The composition or pharmaceutical formulation of claim 87 or 88, wherein the additional treatment comprises administration of an additional therapeutic agent.90.权利要求89所述的组合物或药物制剂，其中所述另外的治疗剂包含抗癌治疗剂。90. The composition or pharmaceutical formulation of claim 89, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.91.权利要求1至90中任一项所述的组合物或药物制剂，其用于向人施用。91. The composition or pharmaceutical formulation of any one of claims 1 to 90 for administration to a human.92.权利要求1至91中任一项所述的组合物或药物制剂，其用于静脉内施用。92. The composition or pharmaceutical formulation of any one of claims 1 to 91 for intravenous administration.93.用于在对象中治疗癌症的方法，其包括向所述对象施用权利要求1至74中任一项所述的组合物。93. A method for treating cancer in a subject, comprising administering to the subject the composition of any one of claims 1 to 74.94.权利要求93中所述的方法，其中所述癌症包括CLDN-18.2阳性癌症。94. The method of claim 93, wherein the cancer comprises a CLDN-18.2 positive cancer.95.权利要求93或94中所述的方法，其中所述癌症包括CLDN-18.2阳性实体瘤。95. The method of claim 93 or 94, wherein the cancer comprises a CLDN-18.2-positive solid tumor.96.权利要求93至95中任一项所述的方法，其中所述癌症包括CLDN-18.2阳性胰腺癌。96. The method of any one of claims 93 to 95, wherein the cancer comprises CLDN-18.2-positive pancreatic cancer.97.权利要求93至96中任一项所述的方法，其中所述癌症包括CLDN-18.2阳性胃癌。97. The method of any one of claims 93 to 96, wherein the cancer comprises CLDN-18.2-positive gastric cancer.98.权利要求93至97中任一项所述的方法，其中所述癌症包括CLDN-18.2阳性胆道肿瘤。98. The method of any one of claims 93 to 97, wherein the cancer comprises a CLDN-18.2-positive biliary tract tumor.99.权利要求93至98中任一项所述的方法，其中所述癌症包括CLDN-18.2阳性的局部晚期、不可切除或转移性癌症。99. The method of any one of claims 93 to 98, wherein the cancer comprises a locally advanced, unresectable, or metastatic cancer that is positive for CLDN-18.2.100.权利要求93至99中任一项所述的方法，其还包括施用另外的治疗。100. The method of any one of claims 93-99, further comprising administering an additional therapy.101.权利要求100中所述的方法，其中所述另外的治疗包括选自以下中的一种或更多种：(i)手术以切去、切除肿瘤或使其减积，(ii)放射治疗，和(iii)化学治疗。101. The method of claim 100, wherein the additional treatment comprises one or more selected from the group consisting of: (i) surgery to remove, excise, or debulk the tumor, (ii) radiation therapy, and (iii) chemotherapy.102.权利要求100或101中所述的方法，其中所述另外的治疗包括施用另外的治疗剂。102. The method of claim 100 or 101, wherein the additional treatment comprises administering an additional therapeutic agent.103.权利要求102中所述的方法，其中所述另外的治疗剂包含抗癌治疗剂。103. The method of claim 102, wherein the additional therapeutic agent comprises an anti-cancer therapeutic agent.104.权利要求93至103中任一项所述的方法，其中所述对象是人。104. The method of any one of claims 93-103, wherein the subject is a human.105.权利要求93至104中任一项所述的方法，其中所述组合物静脉内施用。105. The method of any one of claims 93-104, wherein the composition is administered intravenously.106.权利要求1至74中任一项所述的组合物，其用于权利要求93至105中任一项所述的方法中。106. The composition of any one of claims 1 to 74, for use in the method of any one of claims 93 to 105.107.权利要求1至92中任一项所述的组合物或药物制剂，其用于将所述RNA引入到肝细胞中并在肝细胞中表达由所述RNA编码的多肽链。107. The composition or pharmaceutical preparation of any one of claims 1 to 92, which is used to introduce the RNA into hepatocytes and express the polypeptide chain encoded by the RNA in the hepatocytes.108.权利要求1至92中任一项所述的组合物或药物制剂，其用于所述多肽链的全身递送。108. The composition or pharmaceutical formulation of any one of claims 1 to 92, for systemic delivery of the polypeptide chain.109.权利要求1至92中任一项所述的组合物或药物制剂，其用于所述多肽链在肝细胞中表达之后所述多肽链的全身递送。109. The composition or pharmaceutical formulation of any one of claims 1 to 92, for systemic delivery of the polypeptide chain following expression of the polypeptide chain in hepatocytes.110.用于在对象中表达与密蛋白-18.2(CLDN-18.2)结合的抗体剂的方法，所述方法包括：110. A method for expressing an antibody agent that binds to claudin-18.2 (CLDN-18.2) in a subject, the method comprising:(a)施用权利要求1至74中任一项所述的组合物以使得RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 1 to 74 so that the RNA is introduced into hepatocytes; and(b)在所述肝细胞中表达由所述RNA编码的多肽链。(b) expressing the polypeptide chain encoded by the RNA in the hepatocyte.111.用于在对象中表达与密蛋白-18.2(CLDN-18.2)结合的抗体剂的方法，所述方法包括：111. A method for expressing an antibody agent that binds to claudin-18.2 (CLDN-18.2) in a subject, the method comprising:(a)施用权利要求1至74中任一项所述的组合物以使得RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 1 to 74 so that the RNA is introduced into hepatocytes; and(b)在所述肝细胞中表达由所述RNA编码的多肽链，(b) expressing the polypeptide chain encoded by the RNA in the hepatocyte,其中，在表达之后，所述多肽链被分泌到血流中。Therein, following expression, the polypeptide chain is secreted into the blood stream.112.用于在对象中全身递送与密蛋白-18.2(CLDN-18.2)结合的抗体剂的方法，所述方法包括：112. A method for systemically delivering an antibody agent that binds to claudin-18.2 (CLDN-18.2) in a subject, the method comprising:(a)施用权利要求1至74中任一项所述的组合物以使得RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 1 to 74 so that the RNA is introduced into hepatocytes; and(b)在所述肝细胞中表达由所述RNA编码的多肽链，(b) expressing the polypeptide chain encoded by the RNA in the hepatocyte,其中，在表达之后，所述多肽链被分泌到血流中。Therein, following expression, the polypeptide chain is secreted into the blood stream.113.权利要求110至112中任一项所述的方法，其中施用是肠胃外施用。113. The method of any one of claims 110 to 112, wherein the administration is parenteral administration.114.权利要求110至113中任一项所述的方法，其中施用是静脉内施用。114. The method of any one of claims 110 to 113, wherein the administration is intravenous.115.组合物或药物制剂，其包含RNA，115. A composition or pharmaceutical preparation comprising RNA,其中所述RNA包含：Wherein the RNA comprises:(i)编码多肽的编码序列，(i) a coding sequence encoding a polypeptide,(ii)3’UTR序列，(ii) 3'UTR sequence,(iii)poly-A序列，和(iii) poly-A sequence, and(iv)连接所述3’UTR序列和所述poly-A序列的核苷酸序列，其包含序列CUXGAGCUAGC，其中X是C、A或U。(iv) a nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, which comprises the sequence CUXGAGCUAGC, wherein X is C, A or U.116.权利要求115所述的组合物或药物制剂，其中连接所述3’UTR序列和所述poly-A序列的核苷酸序列包含序列CUXGAGCUAGC。116. The composition or pharmaceutical preparation of claim 115, wherein the nucleotide sequence linking the 3'UTR sequence and the poly-A sequence comprises the sequence CUXGAGCUAGC.117.权利要求115或116所述的组合物或药物制剂，其中所述RNA以5’至3’方向包含所述编码多肽的编码序列、所述3’UTR序列、连接所述3’UTR序列和所述poly-A序列的核苷酸序列以及所述poly-A序列。117. The composition or pharmaceutical preparation of claim 115 or 116, wherein the RNA comprises, in 5' to 3' direction, the coding sequence of the encoded polypeptide, the 3'UTR sequence, a nucleotide sequence connecting the 3'UTR sequence and the poly-A sequence, and the poly-A sequence.118.权利要求115至117中任一项所述的组合物或药物制剂，其中所述3’UTR序列包含SEQ ID NO:22的核苷酸序列或者与SEQ ID NO:22的核苷酸序列具有至少90％同一性的核苷酸序列。118. The composition or pharmaceutical preparation of any one of claims 115 to 117, wherein the 3'UTR sequence comprises the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:22.119.权利要求115至118中任一项所述的组合物或药物制剂，其中所述RNA包含3’UTR，所述3’UTR包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列或者与SEQ ID NO:36的第1至298位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。119. The composition or pharmaceutical preparation of any one of claims 115 to 118, wherein the RNA comprises a 3'UTR comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36.120.权利要求115至118中任一项所述的组合物或药物制剂，其中所述RNA包含3’UTR，所述3’UTR包含SEQ ID NO:37的第1至295位核苷酸的核苷酸序列或者与SEQ ID NO:37的第1至295位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。120. The composition or pharmaceutical formulation of any one of claims 115 to 118, wherein the RNA comprises a 3'UTR comprising a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37 or a nucleotide sequence that is at least 90% identical to a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37.121.权利要求115至120中任一项所述的组合物或药物制剂，其中所述poly-A序列是A核苷酸的间断序列。121. The composition or pharmaceutical formulation of any one of claims 115 to 120, wherein the poly-A sequence is an interrupted sequence of A nucleotides.122.权利要求115至121中任一项所述的组合物或药物制剂，其中所述poly-A序列包含至少100个核苷酸。122. The composition or pharmaceutical formulation of any one of claims 115 to 121, wherein the poly-A sequence comprises at least 100 nucleotides.123.权利要求115至122中任一项所述的组合物或药物制剂，其中所述poly-A序列包含核苷酸序列A_x-L-A_y或由核苷酸序列A_x-L-A_y组成，其中A_x是至少20个A核苷酸的序列，A_y是至少60个A核苷酸的序列并且L是可包含除A之外的核苷酸的1至20个核苷酸的接头。123. The composition or pharmaceutical formulation of any one of claims 115 to 122, wherein the poly-A sequence comprises or consists of the nucleotide sequence_Ax-LAy , wherein_Ax is a sequence of at least 20 A nucleotides,_Ay is_a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides which may comprise nucleotides other than A.124.权利要求115至123中任一项所述的组合物或药物制剂，其中所述poly-A序列包含SEQ ID NO:23的核苷酸序列或与SEQ ID NO:23的核苷酸序列具有至少90％同一性的核苷酸序列或者由SEQ ID NO:23的核苷酸序列或与SEQ ID NO:23的核苷酸序列具有至少90％同一性的核苷酸序列组成。124. The composition or pharmaceutical formulation 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 that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 23.125.权利要求115至124中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。125. The composition or pharmaceutical formulation of any one of claims 115 to 124, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence that is at least 90% identical to a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20.126.权利要求115至125中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，其之前是包含核苷酸序列AGX1X2X3X4XAACUAGU的序列，其中X1是任意核苷酸，优选A或C，X2是任意核苷酸，优选A或C，X3是任意核苷酸，优选C、U或G，并且X4是A或是缺失的。126. The composition or pharmaceutical formulation of any one of claims 115 to 125, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity thereto, preceded by a sequence comprising the nucleotide sequence AGX1X2X3X4XAACUAGU, wherein X1 is any nucleotide, preferably A or C, X2 is any nucleotide, preferably A or C, X3 is any nucleotide, preferably C, U or G, and X4 is A or is absent.127.权利要求115至126中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，其之前是包含核苷酸序列AGX1AX3AAACUAGU的序列，其中X1是任意核苷酸，优选A或C，并且X3是任意核苷酸，优选C或U。127. The composition or pharmaceutical formulation of any one of claims 115 to 126, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity thereto, and preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, wherein X1 is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.128.权利要求115至127中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，其之前是包含核苷酸序列AGX1AX3AAACUAGU的序列。128. The composition or pharmaceutical formulation of any one of claims 115 to 127, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU.129.权利要求115至127中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第14至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第14至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列，其之前是包含核苷酸序列AGX1AX3AAACUAGU的序列。129. The composition or pharmaceutical formulation of any one of claims 115 to 127, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO:20, which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU.130.权利要求115至129中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第7至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列。130. The composition or pharmaceutical formulation of any one of claims 115 to 129, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence that is at least 90% identical to a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20.131.权利要求115至128和130中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列。131. The composition or pharmaceutical formulation 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 that is at least 90% identical to the nucleotide sequence of SEQ ID NO:20.132.权利要求115至127、129和130中任一项所述的组合物或药物制剂，其中所述RNA包含5’UTR，所述5’UTR包含SEQ ID NO:38的核苷酸序列或者与SEQ ID NO:38的核苷酸序列具有至少90％同一性的核苷酸序列。132. The composition or pharmaceutical formulation 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 that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 38.133.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第7至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列或者与SEQ IDNO:36的第1至298位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；以及poly-A序列。133. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding a polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.134.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列或者与SEQ ID NO:36的第1至298位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；以及poly-A序列。134. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.135.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的核苷酸序列或者与SEQ ID NO:36核苷酸序列具有至少90％同一性的核苷酸序列。135. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:36.136.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:38的核苷酸序列或者与SEQ ID NO:38的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列或者与SEQ ID NO:36的第1至298位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；以及poly-A序列。136. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.137.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:38的核苷酸序列或者与SEQ ID NO:38的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的核苷酸序列或者与SEQ ID NO:36核苷酸序列具有至少90％同一性的核苷酸序列。137. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:36 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:36.138.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列；以及poly-A序列。138. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.139.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列；以及poly-A序列。139. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.140.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的核苷酸序列。140. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:36.141.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:38的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的第1至298位核苷酸的核苷酸序列；以及poly-A序列。141. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO:36; and a poly-A sequence.142.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:38的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:36的核苷酸序列。142. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:38; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:36.143.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列或者与SEQ ID NO:20的第7至53位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的第1至295位核苷酸的核苷酸序列或者与SEQ IDNO:37的第1至295位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；以及poly-A序列。143. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding a polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity with a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37; and a poly-A sequence.144.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的第1至295位核苷酸的核苷酸序列或者与SEQ ID NO:37的第1至295位核苷酸的核苷酸序列具有至少90％同一性的核苷酸序列；以及poly-A序列。144. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37 or a nucleotide sequence having at least 90% identity with the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37; and a poly-A sequence.145.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列或者与SEQ ID NO:20的核苷酸序列具有至少90％同一性的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的核苷酸序列或者与SEQ ID NO:37核苷酸序列具有至少90％同一性的核苷酸序列。145. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence encoding the polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:37 or a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:37.146.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的第7至53位核苷酸的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的第1至295位核苷酸的核苷酸序列；以及poly-A序列。146. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising a nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising a nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37; and a poly-A sequence.147.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的第1至295位核苷酸的核苷酸序列；以及poly-A序列。147. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO:37; and a poly-A sequence.148.权利要求115至132中任一项所述的组合物或药物制剂，其中所述RNA包含：5’UTR，其包含SEQ ID NO:20的核苷酸序列；和位于所述编码多肽的编码序列下游的序列，该序列包含SEQ ID NO:37的核苷酸序列。148. The composition or pharmaceutical preparation of any one of claims 115 to 132, wherein the RNA comprises: a 5'UTR comprising the nucleotide sequence of SEQ ID NO:20; and a sequence located downstream of the coding sequence for the encoded polypeptide, the sequence comprising the nucleotide sequence of SEQ ID NO:37.149.权利要求118至142中任一项所述的组合物或药物制剂，其中至少90％是至少95％、96％、97％、98％或99％。149. The composition or pharmaceutical formulation of any one of claims 118 to 142, wherein at least 90% is at least 95%, 96%, 97%, 98% or 99%.150.权利要求115至149中任一项所述的组合物或药物制剂，其中所述RNA包含编码两个或更多个多肽的两个或更多个编码序列。150. The composition or pharmaceutical formulation of any one of claims 115 to 149, wherein the RNA comprises two or more coding sequences encoding two or more polypeptides.151.权利要求115至150中任一项所述的组合物或药物制剂，其中所述RNA不编码与密蛋白-6(CLDN-6)和/或CD3结合的多肽。151. The composition or pharmaceutical formulation of any one of claims 115 to 150, wherein the RNA does not encode a polypeptide that binds to claudin-6 (CLDN-6) and/or CD3.152.权利要求115至151中任一项所述的组合物或药物制剂，其中所述RNA不编码与密蛋白-6(CLDN-6)和/或CD3结合的结合剂的一条或更多条多肽链。152. The composition or pharmaceutical formulation of any one of claims 115 to 151, wherein the RNA does not encode one or more polypeptide chains of a binding agent that binds to claudin-6 (CLDN-6) and/or CD3.153.权利要求115至152中任一项所述的组合物或药物制剂，其中所述RNA不编码细胞因子。153. The composition or pharmaceutical formulation of any one of claims 115 to 152, wherein the RNA does not encode a cytokine.154.权利要求115至153中任一项所述的组合物或药物制剂，其中所述RNA不编码IL2和/或IL7。154. The composition or pharmaceutical formulation of any one of claims 115 to 153, wherein the RNA does not encode IL2 and/or IL7.155.权利要求115至154中任一项所述的组合物或药物制剂，其中所述RNA不编码与HIV结合的多肽。155. The composition or pharmaceutical formulation of any one of claims 115 to 154, wherein the RNA does not encode a polypeptide that binds to HIV.156.权利要求115至155中任一项所述的组合物或药物制剂，其中所述RNA不编码与HIV结合的结合剂的一条或更多条多肽链。156. The composition or pharmaceutical formulation of any one of claims 115 to 155, wherein the RNA does not encode one or more polypeptide chains of a binding agent that binds to HIV.157.权利要求115至156中任一项所述的组合物或药物制剂，其中所述RNA不编码与密蛋白-18.2(CLDN-18.2)结合的多肽。157. The composition or pharmaceutical formulation of any one of claims 115 to 156, wherein the RNA does not encode a polypeptide that binds to claudin-18.2 (CLDN-18.2).158.权利要求115至157中任一项所述的组合物或药物制剂，其中所述RNA不编码与密蛋白-18.2(CLDN-18.2)结合的结合剂的一条或更多条多肽链。158. The composition or pharmaceutical formulation of any one of claims 115 to 157, wherein the RNA does not encode one or more polypeptide chains of a binding agent that binds to claudin-18.2 (CLDN-18.2).159.权利要求115至158中任一项所述的组合物或药物制剂，其中所述RNA编码抗体或抗体样分子。159. The composition or pharmaceutical formulation of any one of claims 115 to 158, wherein the RNA encodes an antibody or antibody-like molecule.160.权利要求115至159中任一项所述的组合物或药物制剂，其中所述RNA包含至少两种，例如两种RNA分子，并且所述RNA分子的至少一种，例如所述RNA分子的全部，包含如其所定义的5’UTR、3’UTR、3’UTR序列、poly-A序列以及/或者连接3’UTR序列和poly-A序列的核苷酸序列。160. The composition or pharmaceutical 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 of the RNA molecules, e.g., all of the RNA molecules, comprises a 5'UTR, a 3'UTR, a 3'UTR sequence, a poly-A sequence, and/or a nucleotide sequence connecting a 3'UTR sequence and a poly-A sequence as defined herein.161.权利要求115至160中任一项所述的组合物或药物制剂，其中所述RNA含有：161. The composition or pharmaceutical formulation of any one of claims 115 to 160, wherein the RNA comprises:(i)包含编码第一多肽链的编码序列的RNA，所述第一多肽链包含抗体剂的重链，和(i) an RNA comprising a coding sequence encoding a first polypeptide chain comprising a heavy chain of an antibody agent, and(ii)包含编码第二多肽链的编码序列的RNA，所述第二多肽链包含抗体剂的轻链。(ii) an RNA comprising a coding sequence encoding a second polypeptide chain comprising the light chain of the antibody agent.162.权利要求161所述的组合物或药物制剂，其中(i)中所述的RNA是第一RNA分子并且(ii)中所述的RNA是第二RNA分子。162. The composition or pharmaceutical formulation of claim 161, wherein the RNA described in (i) is a first RNA molecule and the RNA described in (ii) is a second RNA molecule.163.权利要求161或162所述的组合物或药物制剂，其中所述抗体剂与密蛋白-18.2(CLDN-18.2)结合。163. The composition or pharmaceutical formulation of claim 161 or 162, wherein the antibody agent binds to claudin-18.2 (CLDN-18.2).164.权利要求115至163中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含替代尿苷的经修饰核苷。164. The composition or pharmaceutical formulation of any one of claims 115 to 163, wherein the RNA, e.g., each RNA, comprises a modified nucleoside replacing uridine.165.权利要求115至164中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含替代每个尿苷的经修饰核苷。165. The composition or pharmaceutical formulation of any one of claims 115 to 164, wherein the RNA, e.g., each RNA, comprises a modified nucleoside replacing each uridine.166.权利要求164或165所述的组合物或药物制剂，其中所述经修饰核苷是假尿苷(ψ)和/或N1-甲基-假尿苷(m1ψ)。166. The composition or pharmaceutical formulation of claim 164 or 165, wherein the modified nucleoside is pseudouridine (ψ) and/or N1-methyl-pseudouridine (m1ψ).167.权利要求164至166中任一项所述的组合物或药物制剂，其中所述经修饰核苷是N1-甲基-假尿苷(m1ψ)。167. The composition or pharmaceutical formulation of any one of claims 164 to 166, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).168.权利要求115至167中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’帽。168. The composition or pharmaceutical formulation of any one of claims 115 to 167, wherein the RNA, e.g., each RNA, comprises a 5' cap.169.权利要求115至168中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，包含5’帽m₂^7，3’-OGppp(m₁^2’-O)ApG。169. The composition or pharmaceutical formulation of any one of claims 115 to 168, wherein the RNA, e.g., each RNA, comprises a 5' cap_m27,3'^- OGppp(_m12'^-O )ApG.170.权利要求115至169中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，是单链RNA。170. The composition or pharmaceutical formulation of any one of claims 115 to 169, wherein the RNA, e.g., each RNA, is a single-stranded RNA.171.权利要求115至170中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，是mRNA。171. The composition or pharmaceutical formulation of any one of claims 115 to 170, wherein the RNA, e.g., each RNA, is mRNA.172.权利要求115至171中任一项所述的组合物或药物制剂，其中所述RNA，例如每种RNA，配制在脂质纳米粒(LNP)中，例如每一种RNA共配制在脂质纳米粒(LNP)中。172. The composition or pharmaceutical formulation of any one of claims 115 to 171, wherein the RNA, e.g., each RNA, is formulated in a lipid nanoparticle (LNP), e.g., each RNA is co-formulated in a lipid nanoparticle (LNP).173.权利要求172所述的组合物或药物制剂，其中形成所述脂质纳米粒的脂质包含阳离子脂质、聚合物缀合的脂质；和中性脂质。173. The composition or pharmaceutical formulation of claim 172, wherein the lipids forming the lipid nanoparticles comprise cationic lipids, polymer-conjugated lipids; and neutral lipids.174.权利要求173所述的组合物或药物制剂，其中：174. The composition or pharmaceutical formulation of claim 173, wherein:a.所述阳离子脂质以总脂质的35mol％至65mol％存在；a. The cationic lipid is present in 35 mol% to 65 mol% of the total lipids;b.所述聚合物缀合的脂质以总脂质的约1mol％至2.5mol％存在；并且b. the polymer-conjugated lipid is present at about 1 mol % to 2.5 mol % of the total lipid; andc.所述中性脂质以总脂质的35mol％至65mol％存在。c. The neutral lipid is present in an amount of 35 mol% to 65 mol% of the total lipids.175.权利要求173或174所述的组合物或药物制剂，其中所述阳离子脂质是((3-羟基丙基)氮烷二基)双(壬烷-9,1-二基)双(2-丁基辛酸酯)。175. The composition or pharmaceutical formulation of claim 173 or 174, wherein the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate).176.权利要求173至175中任一项所述的组合物或药物制剂，其中所述聚合物缀合的脂质是PEG缀合的脂质(例如2-[(聚乙二醇)-2000]-N,N-双十四烷基乙酰胺)。176. The composition or pharmaceutical formulation 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.权利要求173至176中任一项所述的组合物或药物制剂，其中所述中性脂质包含1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DPSC)和/或胆固醇。177. The composition or pharmaceutical formulation of any one of claims 173 to 176, wherein the neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and/or cholesterol.178.权利要求172至177中任一项所述的组合物或药物制剂，其中所述脂质纳米粒的平均尺寸为约50至150nm。178. The composition or pharmaceutical formulation of any one of claims 172 to 177, wherein the average size of the lipid nanoparticles is about 50 to 150 nm.179.权利要求172至178中任一项所述的组合物或药物制剂，其中所述脂质纳米粒包含((3-羟基丙基)氮烷二基)双(壬烷-9,1-二基)双(2-丁基辛酸酯)、2-[(聚乙二醇)-2000]-N,N-双十四烷基乙酰胺、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱和胆固醇。179. The composition or pharmaceutical formulation of any one of claims 172 to 178, wherein the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.180.权利要求115至179中任一项所述的组合物，其是药物组合物。180. The composition of any one of claims 115 to 179, which is a pharmaceutical composition.181.权利要求180所述的组合物，其中所述药物组合物还包含一种或更多种可药用载体、稀释剂和/或赋形剂。181. The composition of claim 180, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.182.权利要求115至179中任一项所述的药物制剂，其是药盒。182. The pharmaceutical formulation of any one of claims 115 to 179, which is a kit.183.权利要求182所述的药物制剂，其中所述RNA，例如每种RNA，以及任选地颗粒形成组分在单独的小瓶中。183. The pharmaceutical formulation of claim 182, wherein the RNA, e.g., each RNA, and optionally the particle-forming components are in separate vials.184.权利要求115至183中任一项所述的组合物或药物制剂，其用于静脉内施用。184. The composition or pharmaceutical formulation of any one of claims 115 to 183, for intravenous administration.185.权利要求115至184中任一项所述的组合物或药物制剂，其用于将所述RNA引入到肝细胞中并在肝细胞中表达由所述RNA编码的多肽。185. The composition or pharmaceutical preparation of any one of claims 115 to 184, for introducing the RNA into a hepatocyte and expressing a polypeptide encoded by the RNA in the hepatocyte.186.权利要求115至185中任一项所述的组合物或药物制剂，其用于所述多肽的全身递送。186. The composition or pharmaceutical formulation of any one of claims 115 to 185, for systemic delivery of the polypeptide.187.权利要求115至186中任一项所述的组合物或药物制剂，其用于在肝细胞中表达所述多肽之后所述多肽的全身递送。187. The composition or pharmaceutical formulation of any one of claims 115 to 186, for systemic delivery of the polypeptide following expression of the polypeptide in hepatocytes.188.用于在对象中表达多肽的方法，所述方法包括：188. A method for expressing a polypeptide in a subject, the method comprising:(a)施用权利要求115至181中任一项所述的组合物以使得编码所述多肽的RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 115 to 181 so that RNA encoding the polypeptide is introduced into hepatocytes; and(b)在所述肝细胞中表达所述多肽。(b) expressing the polypeptide in the hepatocytes.189.用于在对象中表达多肽的方法，所述方法包括：189. A method for expressing a polypeptide in a subject, the method comprising:(a)施用权利要求115至181中任一项所述的组合物以使得编码所述多肽的RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 115 to 181 so that RNA encoding the polypeptide is introduced into hepatocytes; and(b)在所述肝细胞中表达所述多肽，(b) expressing the polypeptide in the hepatocytes,其中，在表达之后，所述多肽被分泌到血流中。Therein, following expression, the polypeptide is secreted into the bloodstream.190.用于在对象中全身递送多肽的方法，所述方法包括：190. A method for systemically delivering a polypeptide in a subject, the method comprising:(a)施用权利要求115至181中任一项所述的组合物以使得编码所述多肽的RNA被引入到肝细胞中；以及(a) administering the composition of any one of claims 115 to 181 so that RNA encoding the polypeptide is introduced into hepatocytes; and(b)在所述肝细胞中表达所述多肽，(b) expressing the polypeptide in the hepatocytes,其中，在表达之后，所述多肽被分泌到血流中。Therein, following expression, the polypeptide is secreted into the bloodstream.191.权利要求188至190中任一项所述的方法，其中施用是肠胃外施用。191. The method of any one of claims 188-190, wherein the administration is parenteral.192.权利要求188至191中任一项所述的方法，其中施用是静脉内施用。192. The method of any one of claims 188-191, wherein the administration is intravenous.