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EP4598954A1 - Compositions d'arn ciblant la claudine 18.2 - Google Patents

Compositions d'arn ciblant la claudine 18.2

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Publication number
EP4598954A1
EP4598954A1 EP23785777.6A EP23785777A EP4598954A1 EP 4598954 A1 EP4598954 A1 EP 4598954A1 EP 23785777 A EP23785777 A EP 23785777A EP 4598954 A1 EP4598954 A1 EP 4598954A1
Authority
EP
European Patent Office
Prior art keywords
nucleotide sequence
rna
seq
composition
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23785777.6A
Other languages
German (de)
English (en)
Inventor
Ugur Sahin
Hayat BÄHR-MAHMUD
Ursula ELLINGHAUS
Christiane STADLER
Gábor BOROS
Jonas REINHOLZ
Sergey BESSONOV
Katalin Karikó
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biontech SE
Original Assignee
Biontech SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/EP2022/077839 external-priority patent/WO2024074211A1/fr
Application filed by Biontech SE filed Critical Biontech SE
Publication of EP4598954A1 publication Critical patent/EP4598954A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • Cancer is the second leading cause of death globally and is expected to be responsible for an estimated 9.6 million deaths in 2018 (Bray et al. 2018). In general, once a solid tumor has metastasized, with a few exceptions such as germ cell and some carcinoid tumors, 5-year survival rarely exceeds 25%.
  • Zolbetuximab (development code IMAB362), which is a monoclonal antibody that targets isoform 2 of Claudin- 18, has been under investigation for the treatment of gastrointestinal adenocarcinomas and pancreatic tumors (Tureci et al. 2019).
  • the present disclosure further provides an insight that, in some embodiments, therapy targeting CLDN-18.2, as described herein, may usefully involve administration of RNA (e.g., ssRNA such as mRNA) encoding an antibody agent that targets CLDN-18.2. Still further, the present disclosure provides a particular insight that delivery of RNA via lipid nanoparticles targeting liver cells may be a particularly beneficial strategy for delivering such an antibody agent.
  • RNA e.g., ssRNA such as mRNA
  • RNA e.g., ssRNA such as mRNA
  • a CLDN-18.2-targeting agent e.g., ssRNA such as mRNA
  • IMAB362 e.g., IMAB362
  • the present disclosure proposes that such delivering modality may achieve one or more improvements such as effective administration with reduced incidence (e.g., frequency and/or severity) of TEAEs, and/or with improved relationship between efficacy level and TEAE level (e.g., improved therapeutic window) relative to those observed when a corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered.
  • a corresponding (e.g., encoded) protein e.g., antibody
  • the present disclosure teaches that such improvements in particular may be achieved by delivering IMAB362 via administration of RNA(s) (e.g., ssRNA(s) such as mRNA(s)) encoding it.
  • the present disclosure provides insights that mRNA(s) encoding an antibody agent (e.g., IMAB362) or a functional portion thereof that is/or formulated with lipid nanoparticles (LNP) for intravenous (IV) administration can be taken up by target cells (e.g., liver cells) for efficient production of the encoded antibody agent (e.g., IMAB362) at therapeutically relevant plasma concentrations, for example, as illustrated in Figure 14 for the described RiboMab targeting CLDN-18.2.
  • target cells e.g., liver cells
  • the present disclosure utilizes RiboMabs as CLDN-18.2- targeting agents.
  • RiboMabs are antibody agents encoded by mRNA, e.g., engineered for minimal immunogenicity, and/or formulated in lipid nanoparticles (LNPs).
  • the present disclosure provides an insight that the capability of a CLDN-18.2-targeted antibody agent as described herein to induce antibodydependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while leveraging immune system of recipient subjects can augment cytotoxic effect(s) of chemotherapy and/or other anti-cancer therapy.
  • ADCC antibodydependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • target cells e.g., tumor cells
  • such a combination therapy may prolong progression-free and/or overall survival, e.g., relative to the individual therapies administered alone and/or to another appropriate reference.
  • chemotherapeutic agents for example such as gemcitabine, oxaliplatin, and 5-fluorouracil were shown to upregulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines; moreover, these agents were not observed to increase de novo expression in CLDN-18.2-negative cell lines. See, for example, Tiireci et al. (2019) “Characterization of zolbetuximab in pancreatic cancer models” In Oncoimmimology 8 (1), pp. el523096.
  • CLDN-18.2-targeted therapy e.g., administration of RNA and, more particularly an mRNA encoding a CLDN-18.2-targeting antibody agent
  • CLDN-18.2-targeted therapy may provide synergistic therapeutic when administered in combination with (e.g., to a subject who has received and/or is receiving or has otherwise been exposed to) one or more CDLN18.2- enhancing agents (e.g., one or more certain chemotherapeutic agents).
  • CLDN-18.2-targeted therapy as described herein can be useful in combination with other anti-cancer agents that are expected to and/or have been demonstrated to up-regulate CLDN-18.2 expression in tumor cells.
  • compositions targeting CLDN-18.2. comprises: (a) at least one RNA (e.g., ssRNA) comprising one or more coding regions that encode an antibody agent that binds to a Claudin-18.2 (CLDN-18.2) polypeptide, e.g., binds preferentially to a Claudin- 18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 (CLDN18.1) polypeptide (“CLDN- 18.2-targeting antibody agent”); and (b) lipid nanoparticles; wherein the at least one RNA is encapsulated within at least one of the lipid nanoparticles.
  • RNA e.g., ssRNA
  • such a pharmaceutical composition can comprise and/or deliver one or more RNAs encoding an antibody that binds to CLDN-18.2 polypeptide, e.g., binds preferentially to CLDN-18.2 polypeptide relative to a CLND18.1 polypeptide.
  • such a pharmaceutical composition can comprise and/or deliver one or more RNAs encoding an antigen binding fragment that binds to CLDN-18.2 polypeptide, e.g., binds preferentially to CLDN-18.2 polypeptide relative to a CLND18.1 polypeptide.
  • an antibody agent that targets CLDN-18.2 (and may be encoded by an RNA such as an ssRNA, e.g., an mRNA as described herein) specifically binds to a first extracellular domain (ECD1) of a CLDN-18.2 polypeptide.
  • ECD1 extracellular domain
  • an antibody agent specifically binds to an epitope of ECD 1 that is exposed in cancer cells.
  • at least one RNA e.g., ssRNA such as mRNA
  • VH variable heavy chain
  • CLDN-18.2-targeting antibody agent and a variable light chain (VL) domain of the antibody agent.
  • VH domain(s) and VL domain(s) of a CLDN-18.2-targeting antibody agent may be encoded by a single RNA construct; alternatively in some embodiments they may be encoded separately by at least two individual RNA constructs.
  • an RNA as utilized herein comprises two or more coding regions, which comprises a heavy chain-coding region that encodes at least a VH domain of the antibody agent; and a light chain-coding region that encodes at least a VL domain of the antibody agent.
  • a pharmaceutical composition may comprise: (i) a first RNA comprising a heavy chain-coding region that encodes at least a VH domain of the antibody agent; and (ii) a second RNA comprising a light chain-coding region that encodes at least a VL domain of the antibody agent.
  • RNA(s) that encode a CLDN-18.2-targeting antibody agent may comprise a secretion signal-encoding region.
  • a secretion signal-encoding region allows a CLDN-18.2-targeting antibody agent encoded by one or more RNAs to be secreted upon translation by cells, e.g., present in a subject to be treated, thus yielding a plasma concentration of a biologically active CLDN- 18.2 -targeting antibody agent.
  • RNA(s) each independently comprise, in a 5’ to 3’ direction: (a) a 5’UTR; (b) a secretion signal-coding region; (c) the antibody chain-coding region; (d) a 3’ UTR; and (e) a polyA tail.
  • a polyA tail included in an RNA is or comprises a modified polyA sequence.
  • RNA(s) that encode a CLDN-18.2-targeting antibody agent may comprise a 5’ cap.
  • such a first RNA and a second RNA may be present in a weight ratio of about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4: 1 , about 1.3:1, or about 1.2:1.
  • lipid nanoparticles provided in pharmaceutical compositions described herein are liver-targeting lipid nanoparticles. In some embodiments, lipid nanoparticles provided in pharmaceutical compositions described herein are cationic lipid nanoparticles. In some embodiments, lipid particles provided in pharmaceutical compositions described herein may have an average size of about 50-150 nm.
  • lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid; a cationic lipid; and a neutral lipid.
  • a polymer-conjugated lipid is be present in about 1-2.5 mol% of the total lipids; a cationic lipid is present in 35-65 mol% of the total lipids; and a neutral lipid is present in 35-65 mol% of the total lipids.
  • lipids including, e.g., polymer-conjugated lipids, cationic lipids, and neutral lipids
  • lipid nanoparticles e.g., lipid nanoparticles targeting a specific cell type (e.g., liver cells).
  • a polymer- conjugated lipid included in pharmaceutical compositions described herein may be a PEG- conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof).
  • a cationic lipid included in pharmaceutical compositions described herein may be ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate) or a derivative thereof.
  • neutral lipid included in pharmaceutical compositions described herein may be or comprise a phospholipid or derivative thereof (e.g., l,2-distearoyI-sn-glycero-3-phosphocholine (DPSC)) and/or cholesterol.
  • DPSC l,2-distearoyI-sn-glycero-3-phosphocholine
  • a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions.
  • a pharmaceutical composition may further comprise a cryoprotectant (e.g, sucrose) and/or an aqueous buffered solution, which may in some embodiments include one or more salts (e.g., sodium salts).
  • a pharmaceutical composition described herein may further comprises one or more active agents other than RNA (e.g., an ssRNA such as an mRNA) encoding a CLDN-18.2-targeting agent (e.g., antibody agent).
  • active agents other than RNA e.g., an ssRNA such as an mRNA
  • such other active agent may be or comprise a chemotherapeutic agent.
  • An exemplary chemotherapeutic agent may be or comprise a chemotherapeutic agent indicated for treatment of pancreatic cancer.
  • pharmaceutical compositions described herein can be taken up by target cells for production of an encoded CLDN-18.2-targeting antibody agent at therapeutically relevant plasma concentrations.
  • such pharmaceutical compositions described herein can induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • one aspect of the present disclosure relates to methods of using pharmaceutical compositions described herein.
  • a method comprising administering a provided pharmaceutical composition to a subject suffering from a CLDN- 18.2-positive solid tumor.
  • CLDN- 18.2-positive solid tumor are but are not limited to a biliary tract tumor, a gastric tumor, a gastro-esophageal tumor, an ovarian tumor, a pancreatic tumor, and a tumor that expresses or exhibits a certain level of a CLDN-18.2 polypeptide.
  • compositions and methods described herein may be applicable to a subject of any age suffering from a CLDN-18.2 positive solid tumor.
  • a subject suffering from a CLDN-18.2 positive solid tumor is an adult subject.
  • a method of producing a CLDN-18.2- targeting antibody agent comprises administering to cells a composition comprising at least one RNA (e.g., ones as described herein) comprising one or more coding regions that encode a CLDN-18.2-targeting antibody agent so that such cells express and secrete a CLDN-18.2- targeting antibody agent encoded by such RNA(s).
  • cells to be administered or targeted are or comprise liver cells.
  • cells are present in a cell culture.
  • cells are present in a subject.
  • a pharmaceutical composition described herein may be administered to a subject in need thereof.
  • the present disclosure also provides methods of characterizing one or more features of an RNA or composition thereof, which RNA encodes part or all of an antibody agent.
  • a method comprising a step of: determining one or more features of an antibody agent expressed from at least one mRNA introduced into cells, wherein such at least one mRNA comprises one or more of features of at least one or more RNA comprising a coding region that encodes an antibody agent that binds to a Claudin-18.2 (CLDN-18.2) polypeptide, e.g., binds preferentially to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 polypeptide, wherein such one or more features comprises: (i) protein expression level of an antibody agent; (ii) binding specificity of an antibody agent to CLDN- 18.2; (iii) efficacy of an antibody agent to mediate target cell death through ADCC; and (iv) efficacy of an antibody agent to
  • a method of characterizing a pharmaceutical composition targeting CLDN-18.2. Such a method comprises steps of: (a) contacting cells with at least one composition or pharmaceutical composition described herein (which encodes part or all of a CLDN-18.2-targeting antibody agent); and detecting an antibody agent produced by the cells.
  • the cells may be or comprise liver cells.
  • such a method may further comprise determining one or more features of an antibody agent expressed from one or more RNAs described herein, wherein such one or more features comprises: (i) protein expression level of the antibody agent; (ii) binding specificity of the antibody agent to a CLDN-18.2 polypeptide; (iii) efficacy of the antibody agent to mediate target cell death through ADCC; and (iv) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).
  • a step of determining one or more features of an antibody agent expressed from one or more RNAs described herein may comprise comparing such features of the CLDN-18.2- targeting antibody agent with that of a reference CLDN-18.2-targeting antibody.
  • a step of determining one or more features of an antibody agent expressed from one or more RNAs described herein may comprise assessing the protein expression level of the antibody agent above a threshold level.
  • a threshold level corresponds to a therapeutically relevant plasma concentration.
  • a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise characterizing an antibody agent expressed from one or more RNAs described herein as a CLDN-18.2-targeting antibody agent if the antibody agent comprises the following features: (a) protein level of the antibody agent expressed by the cells above a threshold level; (b) preferential binding of the antibody agent to CLDN-18.2 relative to CLDN18.1 ; and (c) killing of at least 50% target cells (e.g., cancer cells) mediated by ADCC and/or CDC.
  • target cells e.g., cancer cells
  • a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise characterizing an antibody agent expressed from one or more RNAs described herein as a Zolbetuximab or Claudiximab-equivalent antibody if tested features of the antibody are at least comparable to that of Zolbetuximab or Claudiximab.
  • such a step may comprise determining one or more of the following features:
  • RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and
  • RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2)
  • the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16
  • the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17.
  • the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3
  • the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.
  • RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), wherein the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition or medical preparation comprising:
  • RNA comprising a coding region that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2), and
  • RNA comprising a coding region that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to Claudin-18.2 (CLDN-18.2)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the coding region under (i) comprises the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 1422 of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 79 to 738 of SEQ ID NO: 17, and/or
  • the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 474 of SEQ ID NO: 3
  • the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity to the amino acid sequence of amino acids 27 to 246 of SEQ ID NO: 4.
  • the coding region under (i) comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 16, and the coding region under (ii) comprises the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 17.
  • At least 90% is at least 95%, 96%, 97%, 98%, 99%.
  • the antibody agent binds preferentially to CLDN-18.2 relative to Claudin-
  • the antibody agent binds to a first extracellular domain (ECD1) of CLDN-18.2.
  • the antibody agent binds to an epitope of ECD 1 of CLDN-18.2 that is exposed in cancer cells.
  • the antibody agent is IgGl .
  • the first polypeptide chain interacts with the second polypeptide chain to form a binding domain that binds to CLDN-18.2.
  • the VH(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH(CLDN-18.2) comprises CDR1 , CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 5, 6, and 7, respectively.
  • the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)).
  • VL variable domain of a light chain of an antibody agent that binds to CLDN-18.2
  • the VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 15.
  • the VL(CLDN-18.2) comprises CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 8, 9, and 10, respectively.
  • the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising CDR1 , CDR2 and CDR3 of the amino acid sequence SEQ ID NO: 14, and the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising CDR1 , CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 15.
  • VH heavy chain
  • CLDN-18.2 VH(CLDN-18.2)
  • VL light chain
  • the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 5, 6, and 7, respectively
  • the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising CDR1, CDR2 and CDR3 comprising the sequences as set forth in SEQ ID NO: 8, 9, and 10, respectively.
  • the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)) comprising the amino acid sequence SEQ ID NO: 14, and the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)) comprising the amino acid sequence of SEQ ID NO: 15.
  • VH heavy chain
  • VL light chain
  • the first polypeptide chain comprises a variable domain of a heavy chain (VH) of an antibody agent that binds to CLDN-18.2 (VH(CLDN-18.2)), and the second polypeptide chain comprises a variable domain of a light chain (VL) of an antibody agent that binds to CLDN-18.2 (VL(CLDN-18.2)), wherein the VH(CLDN-18.2) and the VL(CLDN-18.2) interact to form a binding domain that binds to Claudin-18.2 (CLDN-18.2).
  • VH heavy chain
  • VL light chain
  • the VH(CLDN-18.2), CHI, CH2 and CH3 are present in the first polypeptide chain in an immunoglobulin G (IgG) form.
  • IgG immunoglobulin G
  • 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.
  • 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.
  • RNA comprising the nucleotide sequence of SEQ ID NO: 27.
  • composition or medical preparation comprising:
  • RNA comprising the nucleotide sequence of SEQ ID NO: 24 or 26, and
  • RNA comprising the nucleotide sequence of SEQ ID NO: 25 or 27.
  • composition or medical preparation comprising:
  • composition or medical preparation comprising:
  • RNA comprising the nucleotide sequence of SEQ ID NO: 27.
  • the RNA e.g., each RNA, comprises a modified nucleoside in place of uridine.
  • the RNA e.g., each RNA, comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is pseudouridine (y) and/or N1 -methylpseudouridine (mly).
  • the modified nucleoside is N1 -methyl -pseudouridine (mly).
  • the RNA e.g., each RNA, comprises a 5’ cap.
  • the RNA e.g., each RNA, comprises the 5’ cap m2 7 3 _ °Gppp( r ’ 0 )ApG.
  • the RNA e.g., each RNA, is single- stranded RNA.
  • the RNA e.g., each RNA
  • the RNA is mRNA.
  • the RNA, e.g., each RNA is formulated in lipid nanoparticles (LNP), e.g., each RNA is co-formulated in lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • lipids that form the lipid nanoparticles comprise a cationic lipid, a polymer-conjugated lipid; and a neutral lipid.
  • a. the cationic lipid is present in 35-65 mol% of the total lipids; b. the polymer-conjugated lipid is present in about 1-2.5 mol% of the total lipids; and c. the neutral lipid is present in 35-65 mol% of the total lipids.
  • the cationic lipid is ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate).
  • the polymer-conjugated lipid is a PEG-conjugated lipid (e.g., 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).
  • the neutral lipid comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and/or cholesterol.
  • DPSC l,2-distearoyl-sn-glycero-3-phosphocholine
  • the lipid nanoparticles have an average size of about 50-150 nm.
  • the lipid nanoparticles comprise ((3- hydroxypropyl)azanediyl)bis(nonane-9, 1 -diyl)bis(2 -butyloctanoate), 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide, l,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the medical preparation is a kit.
  • the RNA e.g., each RNA, and optionally the particle forming components are in separate vials.
  • the medical preparation further comprises instructions for use of the composition or medical preparation for treating or preventing cancer.
  • the present disclosure also provides the composition or medical preparation described herein for pharmaceutical use.
  • the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.
  • the therapeutic or prophylactic treatment of a disease or disorder comprises treating or preventing cancer.
  • the cancer comprises a CLDN-18.2-positive solid tumor.
  • the cancer comprises a CLDN-18.2-positive pancreatic cancer.
  • the cancer comprises a CLDN-18.2-positive gastric cancer.
  • the cancer comprises a CLDN-18.2-positive biliary tract tumor.
  • the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) radiotherapy, and (iii) chemotherapy.
  • the present disclosure also provides a method of treating cancer in a subject comprising administering to the subject the composition described herein.
  • the cancer comprises a CLDN-18.2-positive cancer.
  • the cancer comprises a CLDN-18.2-positive solid tumor.
  • the cancer comprises a CLDN-18.2-positive pancreatic cancer.
  • the cancer comprises a CLDN-18.2-positive gastric cancer.
  • the composition is administered intravenously.
  • the polypeptide chains of the antibody agent that binds to Claudin-18.2 (CLDN-18.2) are secreted into the bloodstream as fully assembled antibodies and/or as functional antibodies.
  • a fully assembled antibody is a tetramer composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain of an antibody agent that binds to Claudin- 18.2 (CLDN- 18.2).
  • a functional antibody is an antibody that has the expected biological activity of the antibody, such as binding to its target and/or recruitment and/or stimulation of the immune system, for example ADCC, e.g., to the same or a similar level as a corresponding antibody expressed in vitro.
  • composition or medical preparation described herein is for introducing the RNA into liver cells and expressing the polypeptide chains encoded by the RNA in liver cells.
  • composition or medical preparation described herein is for systemic delivery of the polypeptide chains following expression of the polypeptide chains in liver cells.
  • present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising:
  • the present disclosure also provides a method for expressing an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising: (a) administering a composition described herein such that the RNA is introduced into liver cells; and
  • the present disclosure also provides a method for systemic delivery of an antibody agent that binds to Claudin-18.2 (CLDN-18.2) in a subject, said method comprising:
  • administration is parenteral administration.
  • a pharmaceutical composition comprising: a. at least one single- stranded RNA comprising one or more coding regions that encode an antibody agent that binds preferentially to a Claudin-18.2 (CLDN-18.2) polypeptide relative to a Claudin-18.1 polypeptide; and b. lipid nanoparticles; wherein the at least one single-stranded RNA is encapsulated within at least one of the lipid nanoparticles.
  • any one of items 1-4 wherein the at least one singlestranded RNA encodes both of: a variable heavy chain (VH) domain of the antibody agent; and a variable light chain (VL) domain of the antibody agent.
  • the pharmaceutical composition further comprises a second singlestranded RNA comprising a light chain-coding region that encodes at least a VL domain of the antibody agent.
  • IgG immunoglobulin G
  • the pharmaceutical composition of item 8 wherein the IgG is IgGl.
  • the pharmaceutical composition of item 13 wherein the at least one non-coding sequence element comprises a 3’ untranslated region (UTR), a 5’ UTR, a cap structure for co- transcriptional capping of mRNA, and/or a poly adenine (polyA) tail.
  • DPSC ,2-Distearoyl-sn-glycero-3 -phosphocholine
  • a method comprising administering a pharmaceutical composition of any one of item 1-36 to a subject suffering from a CLDN-18.2-positive solid tumor.
  • the method of item 37, wherein the CLDN- 18.2-positive tumor is a pancreatic tumor.
  • the method of item 37, wherein the CLDN- 18.2-positive tumor is a gastric tumor.
  • the method of item 37, wherein the CLDN- 18.2-positive tumor is a biliary tract tumor.
  • the method of any one of items 37-40, wherein the CLDN-18.2-positive solid tumor is locally advanced, unresectable, or metastatic.
  • the method of item 45 further comprising administering to the subject the chemotherapeutic agent such that the subject is receiving the combination therapy.
  • the method of item 47 wherein the chemotherapeutic agent is administered at least four hours after the administration of the pharmaceutical composition.
  • the chemotherapeutic agent is or comprises gemcitabine and/or paclitaxel (e.g., nab-paclitaxel) for a subject suffering from a CLDN-
  • a method of producing a CLDN-18.2-targeting antibody comprising administering to cells the pharmaceutical composition of any one of items 1-35 so that the cells express and secrete the CLDN-18.2-targeting antibody encoded by the at least one single-stranded RNA of the pharmaceutical composition.
  • the method of item 76 further characterizing the antibody agent as a Zolbetuximab or Claudiximab-equivalent antibody if the features of the antibody are at least comparable to that of Zolbetuximab or Claudiximab.
  • the method of any one of items 65 and 66-79, wherein the step of determining comprises determining one or more of the following features:
  • any one of items 66-82 further comprising: administering the pharmaceutical composition to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor to determine anti-tumor activity if the pharmaceutical composition is characterized as CLDN-18.2-targeting.
  • a method of manufacture the method comprising steps of:
  • ssRNA single stranded RNA
  • A determining one or more features of a single stranded RNA (ssRNA) or composition thereof, which ssRNA encodes part or all of an antibody agent, which one or more features are selected from the group consisting of:
  • CLDN-18.2-targeting the method comprising steps of:
  • the present disclosure further provides an insight that the 3 ’end region of mRNA is a very sensitive and exceptional area in terms of translational capacity as well as functionality of mRNA. Both in vitro and in vivo results suggest that a single nucleotide substitution upstream of the poly(A) tail has an impact on translational capacity and functionality of mRNA.
  • the nucleotide sequence linking the 3’ UTR sequence and the poly-A sequence comprises the sequence CUCGAGCUAGC.
  • the RNA comprises in the 5' — > 3' direction the coding sequence that encodes a polypeptide, the 3’ UTR sequence, the nucleotide sequence linking the 3’ UTR sequence and the poly-A sequence, and the poly-A sequence.
  • 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.
  • 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.
  • 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.
  • the poly-A sequence is an interrupted sequence of A nucleotides.
  • the poly-A sequence comprises at least 100 nucleotides. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence A x - L-A y , wherein A x is a sequence of at least 20 A nucleotides, A y is a sequence of at least 60 A nucleotides and L is a linker of 1 to 20 nucleotides which may include nucleotides other than A. In some embodiments, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 23.
  • 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.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20 which is preceded by a sequence comprising the nucleotide sequence AGX1AX3AAACUAGU, wherein XI is any nucleotide, preferably A or C, and X3 is any nucleotide, preferably C or U.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20 which is preceded by a sequence comprising the nucleotide sequence AGAAUAAACUAGU.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 14 to 53 of SEQ ID NO: 20 which is preceded by a sequence comprising the nucleotide sequence AGCACAAACUAGU.
  • 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.
  • 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.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 38, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 38 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 38, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 38 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 36, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 36.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of nucleotides 7 to 53 of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 298 of SEQ ID NO: 36, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of nucleotides 1 to 295 of SEQ ID NO: 37, and a poly-A sequence.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 20 and, downstream of the coding sequence that encodes a polypeptide, a sequence comprising the nucleotide sequence of SEQ ID NO: 37.
  • the RNA comprises two or more coding sequences encoding two or more polypeptides.
  • the RNA does not encode one or more polypeptide chains of a binding agent which binds to Claudin-18.2 (CLDN-18.2).
  • the RNA comprises:
  • RNA comprising a coding sequence that encodes a first polypeptide chain comprising a heavy chain of an antibody agent
  • the antibody agent that binds to CLDN-18.2 is as described herein.
  • the coding sequence that encodes a first polypeptide chain comprising a heavy chain of an antibody agent that binds to CLDN-18.2, and the coding sequence that encodes a second polypeptide chain comprising a light chain of an antibody agent that binds to CLDN-18.2 are as described herein.
  • the first polypeptide chain comprising a heavy chain of an antibody agent that binds to CLDN-18.2 and the second polypeptide chain comprising a light chain of an antibody agent that binds to CLDN-18.2 are as described herein.
  • the RNA e.g., each RNA, comprises a modified nucleoside in place of uridine.
  • the RNA e.g., each RNA
  • LNP lipid nanoparticles
  • each RNA is co-formulated in lipid nanoparticles (LNP).
  • the neutral lipid comprises l ,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and/or cholesterol.
  • DPSC l ,2-distearoyl-sn-glycero-3-phosphocholine
  • the lipid nanoparticles have an average size of about 50-150 nm.
  • the lipid nanoparticles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the medical preparation is a kit.
  • the RNA e.g., each RNA, and optionally the particle forming components are in separate vials.
  • the composition or medical preparation is for introducing the RNA into liver cells and expressing the polypeptide encoded by the RNA in liver cells.
  • composition or medical preparation is for systemic delivery of the polypeptide. In some embodiments, the composition or medical preparation is for systemic delivery of the polypeptide following expression of the polypeptide in liver cells.
  • the present disclosure also provides a method for expressing a polypeptide in a subject, said method comprising:
  • the present disclosure also provides a method for systemic delivery of a polypeptide in a subject, said method comprising:
  • FIG. 1 shows that a CLDN-18.2-targeting antibody (RiboMabOl) encoded by two RNAs encoding a heavy chain and a light chain, respectively, of a CLDN-18.2-targeting antibody is expressed in primary human hepatocytes and CHO-K1 cells.
  • RatoMabOl a CLDN-18.2-targeting antibody encoded by two RNAs encoding a heavy chain and a light chain, respectively, of a CLDN-18.2-targeting antibody
  • RB RMAB01 a CLDN-18.2-targeting antibody
  • FIG. 2 shows that RiboMabOl binds target specific to CLDN-18.2.
  • Targeted binding of RiboMabOl to CLDN-18.2 was determined by flow cytometric binding assays visualized using a fluorescently labeled antibody directed against the F(ab')2 fragment of human IgG (H+L).
  • a dilution row of RiboMabOl -containing CHO-K1 cell culture supernatant (Panels A and B, left) or IMAB362 reference protein (Panels A and B, right) was incubated with 5 x 10 5 (Panel A) CLDN-18.2+ or (Panel B) CLDN18.1+ HEK293 transfectants.
  • FIG 3 shows high target specific cell cytotoxicity mediated by in vitro expressed RiboMabOl.
  • RiboMabOl -containing cell culture supernatant from CHO-K1 cells lipofected with RB RMABOl was subjected to (Panel A) ADCC and (Panel B) CDC assays.
  • Human PBMCs of three different healthy donors were utilized as effector cells (E:T ratio 30:1).
  • Target or control and effector cells were incubated for 48 hours with the indicated RiboMabOl and IMAB362 reference protein concentrations. Specific cell lysis as determined in a luciferase-based assay is shown.
  • Solid lines CLDN- 18.2+ CH0-K1 transfectants (solid lines) served as target cells and CLDN- 18.2-negative CHO-K1 (dotted lines) as control cells.
  • Target and control cells were incubated with human serum and RiboMabOl concentrations as indicated for 2 hours.
  • FIG. 4 shows specific tumor cell lysis mediated by RiboMabOl generated in mice.
  • Plasma of mice dosed with five repetitive injections of either 1 pg (-0.04 mg/kg), 3 pg (-0.10 mg/kg), 10 pg (-0.40 mg/kg) and 30 pg (-1.20 mg/kg) RB_RMAB01 or 80 pg (-3.20 mg/kg) of IMAB362 was sampled 24 hours post 5th injection and directed to luciferasebased ex vivo ADCC assays.
  • Plasma of untreated mice spiked with IMAB362 served as assay reference.
  • CLDN- 18.2+ NUG-C4 transfectants served as target and human PBMCs as effector cells.
  • CLDN-18.2+ NUG-C4 transfectants (solid lines) served as target cells, CLDN- 18.2-negative MDA-MB-231 cells (dotted lines) as control cells.
  • Human PBMCs of a healthy donor served as effector cells.
  • FIG. 6 shows that systemic availability of RiboMabOl mediates tumor growth inhibition in vivo.
  • FIG. 7 shows concentration-time profile of RiboMabOl in mouse serum after single dosing.
  • Balb/cJRj mice received a single IV injection of 1 gg (-0.040 mg/kg), 3 gg (-0.10 mg/kg), 10 gg (-0.40 mg/kg) or 30 gg (-1.20 mg/kg) RB_RMAB01 drug product and 40 gg (-1.60 mg/kg) IMAB362 reference protein.
  • Plasma was sampled 6, 24, 96, 168, 264, 336 and 504 hours post administration.
  • FIG. 8 shows concentration-time profile of RiboMabOl in rat serum after single dosing.
  • mol % is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.
  • mol % of the total lipid is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100.
  • total lipid includes lipids and lipid-like material.
  • patient refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
  • a patient is a human.
  • a patient is suffering from or susceptible to one or more diseases or disorders or conditions.
  • a patient displays one or more symptoms of a disease or disorder or condition.
  • polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).
  • compositions comprising a peptide or polypeptide that can be used in the treatment of an individual where the expression of the peptide or polypeptide would be of benefit, e.g., in ameliorating the symptoms of a disease.
  • a pharmaceutically active peptide or polypeptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease.
  • a pharmaceutically active peptide or polypeptide has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount.
  • pharmaceutically active peptides and polypeptides include, but are not limited to, immunostimulants, e.g., cytokines, honnones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.
  • immunostimulants e.g., cytokines, honnones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.
  • the pharmaceutically active peptide and polypeptide includes a replacement protein.
  • an “immunostimulant” is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells.
  • the immunostimulant may be pro-inflammatory (e.g., when treating infections or cancer), or anti-inflammatory (e.g., when treating autoimmune diseases).
  • the immunostimulant is a cytokine or a variant thereof.
  • cytokines include interferons, such as interferon-alpha (IFN-a) or interferon- amma (IFN- ⁇ ), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor.
  • the immunostimulant includes an adjuvant-type immunostimulatory agent such as APC Toll-like Receptor agonists or costimulatory/cell adhesion membrane proteins.
  • Toll-like Receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.
  • Cytokines differ from hormones in that (i) they usually act at much more variable concentrations than honnones and (ii) generally are made by a broad range of cells (nearly all nucleated cells can produce cytokines).
  • cytokines include erythropoietin (EPO), colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alfa (IFNa), interferon beta (IFNP), interferon gamma (INF ⁇ ), 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
  • a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof.
  • a cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal.
  • One particularly preferred cytokine is interferon-a.
  • interferons Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon (the type I interferons present in humans are IFN ⁇ , IFN ⁇ , IFN ⁇ , IFNK and IFNco), type II interferon (IFN ⁇ in humans), and type III interferon.
  • type I interferon the type I interferons present in humans are IFN ⁇ , IFN ⁇ , IFN ⁇ , IFNK and IFNco
  • type II interferon IFN ⁇ in humans
  • type III interferon type III interferon
  • a type I interferon is preferably IFNa or IFN , more preferably IFN ⁇ .
  • an interferon may be a naturally occurring interferon or a functional fragment or variant thereof.
  • An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.
  • Interleukins are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.
  • an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof.
  • An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.
  • Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant).
  • the immunostimulant may be fused to an extended- pharmacokinetic (PK) group, which increases circulation half-life.
  • PK pharmacokinetic
  • Non-limiting examples of extended-PK groups are serum albumin or fragments thereof or variants of the serum albumin or fragments thereof (e.g., HSA or fragments or variants thereof), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).
  • Other exemplary extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul; 16(7):903- 15 which is herein incorporated by reference in its entirety.
  • a pharmaceutically active peptide or polypeptide comprises a replacement protein.
  • the present disclosure provides a method for treatment of a subject having a disorder requiring protein replacement (e.g., protein deficiency disorders) comprising administering to the subject RNA (in particular, mRNA) as described herein encoding a replacement protein.
  • RNA in particular, mRNA
  • protein replacement refers to the introduction of a protein (including functional variants thereof) into a subject having a deficiency in such protein.
  • the term also refers to the introduction of a protein into a subject otherwise requiring or benefiting from providing a protein, e.g., suffering from protein insufficiency.
  • hormones relates to a class of signaling molecules produced by glands, wherein signaling usually includes the following steps: (i) synthesis of a hormone in a particular tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) relay and amplification of the signal; and (vi) breakdown of the hormone.
  • Hormones differ from cytokines in that (1) hormones usually act in less variable concentrations and (2) generally are made by specific kinds of cells.
  • Adhesion molecules relates to proteins which are located on the surface of a cell and which are involved in binding of the cell with other cells or with the extracellular matrix (ECM).
  • Adhesion molecules are typically transmembrane receptors and can be classified as calcium- independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins).
  • Particular examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.
  • Integrins are also involved in signal transduction.
  • integrins modulate cell signaling pathways, e.g., pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTK).
  • RTK receptor tyrosine kinases
  • integrins include: o ⁇ 1, ⁇ 2 ⁇ 1 , ⁇ 3 ⁇ 1, ⁇ 4 ⁇ 1 , ⁇ 5 ⁇ 1, ⁇ 6 ⁇ 1 , ⁇ 7 ⁇ 1 , ⁇ L ⁇ 2, ⁇ L ⁇ 2, ⁇ IIb ⁇ 3, ⁇ v ⁇ 1, ⁇ v ⁇ 3, ⁇ v ⁇ 5 , ⁇ v ⁇ 6 , ⁇ v ⁇ 8, and ⁇ 6 ⁇ 4.
  • immunoglobulins or “immunoglobulin superfamily” refers to molecules which are involved in the recognition, binding, and/or adhesion processes of cells. Molecules belonging to this superfamily share the feature that they contain a region known as immunoglobulin domain or fold.
  • immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3 ⁇ , CD3- ⁇ , CD-3 ⁇ , CD79a, CD79b), co- stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and other.
  • antibodies e.g., IgG
  • T cell receptors T cell receptors
  • MHC major histocompatibility complex
  • co-receptors e.g., CD4, CD8, CD19
  • antigen receptor accessory molecules e.g., CD-3 ⁇ , CD3- ⁇ , CD-3 ⁇ , CD79a, CD79b
  • co- stimulatory or inhibitory molecules e.g., CD28, CD80, CD86
  • immunologically active compound relates to any compound altering an immune response, e.g., by inducing and/or suppressing maturation of immune cells, inducing and/or suppressing cytokine biosynthesis, and/or altering humoral immunity by stimulating antibody production by B cells.
  • Immunologically active compounds possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2 mediated diseases.
  • Immunologically active compounds can be useful as vaccine adjuvants.
  • immunologically active compounds include interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, in particular tumor-associated antigens, pathogen- associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.
  • An immunologically active compound may be a vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response.
  • the "peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject” is also designated herein as "vaccine antigen", “peptide and protein antigen” or simply "antigen”.
  • the RNA encoding the vaccine antigen is expressed in cells , e.g., muscle cells or antigen-presenting cells (APCs), of the subject to provide the vaccine antigen.
  • expression of the vaccine antigen is at the cell surface.
  • the vaccine antigen is presented in the context of MHC.
  • the RNA encoding the vaccine antigen is administered systemically, e.g., intravenously. In some embodiments, after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in spleen occurs.
  • RNA encoding the vaccine antigen after systemic administration of the RNA encoding the vaccine antigen, expression of the RNA encoding the vaccine antigen in antigen presenting cells, preferably professional antigen presenting cells occurs.
  • antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells.
  • the RNA encoding the vaccine antigen is administered intramuscularly.
  • the vaccine antigen comprises an epitope for inducing an immune response against an antigen in a subject.
  • the vaccine antigen comprises an antigenic sequence for inducing an immune response against an antigen in a subject.
  • Such antigenic sequence may correspond to a target antigen or disease-associated antigen, e.g., a protein of an infectious agent (e.g., viral or bacterial antigen) or tumor antigen, or may correspond to an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or the immunogenic variant thereof.
  • the antigenic sequence may comprise at least an epitope of a target antigen or disease-associated antigen or an immunogenic variant thereof.
  • the antigenic sequence or a procession product thereof may bind to the antigen receptor such as TCR or CAR carried by immune effector cells.
  • the antigenic sequence is selected from the group consisting of the antigen expressed by a target cell to which the immune effector cells are targeted or a fragment thereof, or a variant of the antigenic sequence or the fragment.
  • the RNA encoding the vaccine antigen is expressed in cells of a subject to provide the antigen or a procession product thereof for binding by the antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and/or expansion of the immune effector cells.
  • an “antigen” covers any substance that will elicit an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response and/or humoral response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules.
  • an “antigen” relates to any substance, such as a peptide or polypeptide, that reacts specifically with antibodies or T-lymphocytes (T-cells).
  • the term "antigen" may comprise a molecule that comprises at least one epitope, such as a T cell epitope.
  • an antigen is a molecule which, optionally after processing, induces an immune reaction, which may be specific for the antigen (including cells expressing the antigen).
  • an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.
  • autoantigen or "self-antigen” refers to an antigen which originates from within the body of a subject (z.e., the autoantigen can also be called “autologous antigen") and which produces an abnormally vigorous immune response against this normal part of the body. Such vigorous immune reactions against autoantigens maybe the cause of "autoimmune diseases”.
  • any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may comprise a humoral or cellular immune response, or both.
  • the antigen is presented by a cell, such as by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen.
  • An antigen may be a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen.
  • an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.
  • disease-associated antigen is used in its broadest sense to refer to any antigen associated with a disease.
  • a disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and/or a humoral antibody response against the disease.
  • Disease-associated antigens include pathogen- associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.
  • the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells.
  • the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein.
  • the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, such as CD4+ and CD8+ lymphocytes, in particular via the modulation of the activity of a T-cell receptor.
  • epitope refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies, T cells or B cells, in particular when presented in the context of MHC molecules.
  • An epitope of a protein may comprises a continuous or discontinuous portion of said protein and, e.g., may be between about 5 and about 100, between about 5 and about 50, between about 8 and about 30, or about 10 and about 25 amino acids in length.
  • T cell epitope refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules.
  • major histocompatibility complex and the abbreviation "MHC” includes MHC class 1 and MHC class II molecules and relates to a complex of genes which is present in all vertebrates.
  • an amino acid sequence enhancing antigen processing and/or presentation and/or an amino acid sequence which breaks immunological tolerance is fused, either directly or through a linker, to an antigenic peptide or polypeptide (antigenic sequence).
  • immune response and “immune reaction” are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and may refer to a cellular immune response, a humoral immune response, or both.
  • the term "immune response to” or “immune response against” with respect to an agent such as an antigen, cell or tissue relates to an immune response such as a cellular response directed against the agent.
  • An immune response may comprise one or more reactions selected from the group consisting of developing antibodies against one or more antigens and expansion of antigen-specific T-lymphocytes, such as CD4 + and CD8 + T-lymphocytes, e.g. CD8 + T-lymphocytes, which maybe detected in various proliferation or cytokine production tests in vitro.
  • vaccination and “immunization” describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s).
  • RNA especially mRNA
  • allergen refers to a kind of antigen which originates from outside the body of a subject (i.e., the allergen can also be called “heterologous antigen”) and which produces an abnormally vigorous immune response in which the immune system of the subject fights off a perceived threat that would otherwise be harmless to the subject.
  • allergen usually is an antigen which is able to stimulate a type-I hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE) responses.
  • IgE immunoglobulin E
  • allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phi p 5), and proteins of dust mites (e.g., Der p 2).
  • peanut proteins e.g., Ara h 2.02
  • ovalbumin e.g., ovalbumin
  • grass pollen proteins e.g., Phi p 5
  • proteins of dust mites e.g., Der p 2
  • growth factors refers to molecules which are able to stimulate cellular growth, proliferation, healing, and/or cellular differentiation. Typically, growth factors act as signaling molecules between cells.
  • growth factors include particular cytokines and hormones which bind to specific receptors on the surface of their target cells.
  • growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, ephrins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain- derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P)), and tumor necrosis factor-alpha (TNF-a).
  • BMPs bone morphogenetic proteins
  • protease inhibitors refers to molecules, in particular peptides or polypeptides, which inhibit the function of proteases.
  • Protease inhibitors can be classified by the protease which is inhibited (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serpins).
  • protease inhibitors include serpins, such as alpha 1 -antitrypsin, aprotinin, and bestatin.
  • enzymes refers to macromolecular biological catalysts which accelerate chemical reactions. Like any catalyst, enzymes are not consumed in the reaction they catalyze and do not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are much more specific. In some embodiments, an enzyme is essential for homeostasis of a subject, e.g., any malfunction (in particular, decreased activity which may be caused by any of mutation, deletion or decreased production) of the enzyme results in a disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.
  • HSV1-TK herpes simplex virus type 1 thymidine kinase
  • hexosaminidase hexosaminidase
  • phenylalanine hydroxylase phenylalanine hydroxylase
  • pseudocholinesterase pseudocholineste
  • receptors refers to protein molecules which receive signals (in particular chemical signals called ligands) from outside a cell.
  • signals in particular chemical signals called ligands
  • the binding of a signal (e.g., ligand) to a receptor causes some kind of response of the cell, e.g., the intracellular activation of a kinase.
  • Receptors include transmembrane receptors (such as ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors).
  • receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1 ).
  • growth factor receptors refers to receptors which bind to growth factors.
  • apoptosis regulators refers to molecules, in particular peptides or polypeptides, which modulate apoptosis, i.e., which either activate or inhibit apoptosis. Apoptosis regulators can be grouped into two broad classes: those which modulate mitochondrial function and those which regulate caspases.
  • the first class includes proteins (e.g., BCL-2, BCL-xL) which act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Also to this first class belong proapoptotic proteins (e.g., BAX, BAK, BIM) which promote release of cytochrome C.
  • the second class includes proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP which block the activation of caspases.
  • transcription factors relates to proteins which regulate the rate of transcription of genetic information from DNA to messenger RNA, in particular by binding to a specific DNA sequence. Transcription factors may regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and/or in response to signals from outside the cell, such as a hormone. Transcription factors contain at least one DNA-binding domain which binds to a specific DNA sequence, usually adjacent to the genes which are regulated by the transcription factors. Particular examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.
  • tumor suppressor proteins relates to molecules, in particular peptides or polypeptides, which protect a cell from one step on the path to cancer.
  • Tumor-suppressor proteins (usually encoded by corresponding tumor-suppressor genes) exhibit a weakening or repressive effect on the regulation of the cell cycle and/or promote apoptosis.
  • Their functions may be one or more of the following: repression of genes essential for the continuing of the cell cycle; coupling the cell cycle to DNA damage (as long as damaged DNA is present in a cell, no cell division should take place); initiation of apoptosis, if the damaged DNA cannot be repaired; metastasis suppression (e.g., preventing tumor cells from dispersing, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair.
  • tumor-suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI/SNF (SWItch/Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (STI 4), and Yippee-like 3 (YPEL3).
  • the term "structural proteins” refers to proteins which confer stiffness and rigidity to otherwisefluid biological components.
  • reprogramming factors or "reprogramming transcription factors” relates to molecules, in particular peptides or polypeptides, which, when expressed in somatic cells optionally together with further agents such as further reprogramming factors, lead to reprogramming or dedifferentiation of said somatic cells to cells having stem cell characteristics, in particular pluripotency.
  • reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.
  • genomic engineering proteins relates to proteins which are able to insert, delete or replace DNA in the genome of a subject.
  • genomic engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9).
  • blood proteins relates to peptides or polypeptides which are present in blood plasma of a subject, in particular blood plasma of a healthy subject.
  • Blood proteins have diverse functions such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., alpha 1 -antitrypsin), etc.
  • 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.
  • the pharmaceutically active peptide or polypeptide is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2), and interleukin 10 (IL-11), more preferably EPO; (ii) an adhesion molecule, in particular an integrin; (iii) an immunoglobulin, in particular an antibody; (iv) an immunologically active compound, in particular an antigen, such as a viral or bacterial antigen, e.g., an antigen of SARS-CoV-2, e.g., a spike (S) protein of SARS-CoV-2 or a variant thereof; (v) a hormone, in particular vasopressin, insulin or growth hormone; (vi) a growth factor, in particular VEGFA; (vii) a protease inhibitor, in particular alpha 1 -antitrypsin; (viii) an enzyme, preferably selected from the group consisting of EPO, interleuk
  • a pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response against the one or more antigens or one or more epitopes in a subject which may be therapeutic or partially or fully protective.
  • 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.
  • the target antigen is a tumor antigen and the antigenic sequence (e.g., an epitope) is derived from the tumor antigen.
  • the tumor antigen may be a "standard” antigen, which is generally known to be expressed in various cancers.
  • the tumor antigen may also be a "neoantigen", which is specific to an individual’s tumor and has not been previously recognized by the immune system.
  • a neo-antigen or neo-epitope may result from one or more cancer-specific mutations in the genome of cancer cells resulting in amino acid changes.
  • the vaccine antigen preferably comprises an epitope or a fragment of said neo-antigen comprising one or more amino acid changes.
  • the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.
  • the RNA e.g., mRNA, used in the present disclosure encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus protein or the immunogenic variant thereof.
  • Recombinant means “made through genetic engineering”. In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.
  • RNA Ribonucleic acid
  • an RNA refers to a polymer of ribonucleotides.
  • an RNA is single stranded.
  • an RNA is double stranded.
  • an RNA comprises both single and double stranded portions.
  • an RNA can comprise a backbone structure as described in the definition of “ Nucleic acid / Polynucleotide” above.
  • An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA).
  • mRNA messenger RNA
  • an RNA is a mRNA.
  • secretion signal refers to an amino acid sequence present in a polypeptide that can target the polypeptide towards the secretory pathway. Typically, the secretion signal is cleaved after translocation into the endoplasmic reticulum following translation of an RNA. Typically, a secretion signal is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptide. A secretion signal may be present at the N-terminus of a polypeptide.
  • a CLDN-18.2-targeting antibody agent binds with higher on-rate, lower off- rate, increased affinity, decreased dissociation, and/or increased stability to CLDN-18.2 polypeptide as compared with its competing alternative target(s), including, e.g., CLDN18.1 polypeptide.
  • a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Susceptible to An individual who is “susceptible to” a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition.
  • a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of individuals suffering from the disease, disorder, or condition; carrier of a genetic marker or other biomarker associated with the disease, disorder or condition, etc.).
  • a population-based risk e.g., family members of individuals suffering from the disease, disorder, or condition; carrier of a genetic marker or other biomarker associated with the disease, disorder or condition, etc.
  • Three prime untranslated region ' refers to the sequence of an mRNA molecule that begins following the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after the stop codon of the coding region of an open reading frame sequence. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence
  • Threshold level refers to a level that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay.
  • a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria).
  • a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population.
  • a threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.
  • Transfection relates to the introduction of nucleic acids, in particular RNA, into a cell.
  • the term “transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient.
  • a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or the body of a patient.
  • transfection can be transient or stable.
  • RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution.
  • RNA can be transfected into cells to transiently express its coded protein.
  • Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition.
  • Unresectable tumor typically refers to a tumor characterized by one or more features that, in accordance with sound medical judgement, are considered to indicate that the tumor cannot safely (e.g., without undue harm to the subject) be removed by surgery, and/or with respect to which a competent medical profession has determined that risk to the subject of tumor removal outweighs benefits associated with such removal.
  • an unresectable tumor refers to a tumor that involves and/or has grown into an essential organ or tissue (including blood vessels that may not be reconstructable) and/or that is otherwise in a location that cannot readily be surgically accessed without unreasonable risk of damage to one or more other critical or essential organs and/or tissues (including blood vessels).
  • “unresectability” of a tumor refers to the likelihood of achieving a margin-negative (RO) resection.
  • a tumor such as superior mesenteric artery (SMA) or celiac axis, portal vein occlusion, and the presence of celiac or para-aortic lymphadenopathy are generally acknowledged as findings that preclude RO surgery.
  • SMA superior mesenteric artery
  • celiac axis portal vein occlusion
  • para-aortic lymphadenopathy are generally acknowledged as findings that preclude RO surgery.
  • SOC Standard of Care
  • Treatment options typically include further palliative chemotherapy, which might be less tolerated after previous repeated exposure to cytotoxic compounds, or best supportive care, and investigational treatments without proven benefit. Therapy in this population is not curative, with an expected overall survival of a few months.
  • Immunotherapy has emerged as an effective treatment option in some cancers with high unmet medical need. Specifically, immune checkpoint inhibitors are approved for treatment across various cancer indications and act by invigorating pre-existent anti-tumor-specific T cells. The medical need is still high for various cancer types.
  • the present disclosure provides insights and technologies for treating cancer (e.g., pancreatic cancer and/or biliary cancer) with a therapy targeting Claudin-18.2 (CLDN-18.2).
  • the present disclosure provides RNA technologies to deliver a monoclonal antibody targeting CLDN-18.2 that combines both potent anti-tumoral features and an excellent safety profile, skipping the hurdle of slow and cumbersome antibody manufacturing process.
  • RNA delivering modality may achieve one or more improvements such as effective administration with reduced incidence e.g. , frequency and/or severity) of treatment emergent adverse events (“TEAEs”), and/or with improved relationship between efficacy level and TEAE level (e.g., improved therapeutic window) relative to those observed when a corresponding (e.g., encoded) protein (e.g., antibody) agent itself is administered.
  • TEAEs treatment emergent adverse events
  • the present disclosure teaches that such improvements in particular may be achieved by delivering IMAB362 via administration of RNA(s) (e.g., ssRNA(s) such as mRNA(s))) encoding it.
  • antibody agents are expressed from mRNA, e.g., engineered for minimal immunogenicity, and/or formulated in lipid nanoparticles (LNPs).
  • mRNA that encodes an antibody agent may comprise modified nucleotides (e.g., but not limited to pseudouridine and/or 1-methyl-pseudouridine).
  • the present disclosure provides an insight that the capability of a CLDN-18.2-targeting antibody agent delivered as described herein can induce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against target cells (e.g., tumor cells) while leveraging immune system of recipient subjects can augment cytotoxic effect(s) of chemotherapy and/or other anti-cancer therapy.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • target cells e.g., tumor cells
  • such a combination therapy may prolong progression- free and/or overall survival, e.g., relative to the individual therapies administered alone and/or to another appropriate reference.
  • chemotherapeutic agents for example such as gemcitabine, oxaliplatin, and 5-fluorouracil were shown to upregulate existing CLDN-18.2 expression levels in pancreatic cancer cell lines; moreover, these agents were not observed to increase de novo expression in CLDN-18.2 -negative cell lines. See, for example, Tureci et al., (2019) “Characterization of Zolbetuximab in pancreatic cancer models.” In Oncoimmunology 8 (1), pp. el 523096.
  • the present disclosure provides an insight that CLDN-18.2- targeted therapy as described herein may be particularly useful and/or effective when administered to tumor(s) (e.g., tumor cells, subjects in whom such tumor(s) and/or tumor cell(s) are suspected and/or have been detected, etc.) characterized by (e.g., that have been determined to display and/or that are expected or predicted to display) elevated expression and/or activity of CLDN-18.2 expression in tumor cells (e.g., as may result or have resulted from exposure to one or more chemotherapeutic agents).
  • tumor(s) e.g., tumor cells, subjects in whom such tumor(s) and/or tumor cell(s) are suspected and/or have been detected, etc.
  • elevated expression and/or activity of CLDN-18.2 expression in tumor cells e.g., as may result or have resulted from exposure to one or more chemotherapeutic agents.
  • CLDN-18.2-targeted therapy e.g., administration of RNA and, more particularly an mRNA encoding a CLDN-18.2-targeting antibody agent
  • CLDN-18.2-targeted therapy as described herein can be useful in combination with other anti-cancer agents that are expected to and/or have been demonstrated to up-regulate CLDN-18.2 expression and/or activity in tumor cells.
  • provided technologies are effective for treatment of pancreatic cancers.
  • provided technologies are effective for treatment of gastric or gastro-esophageal cancers.
  • provided technologies are effective for treatment of biliary cancers.
  • provided technologies are effective for treatment of ovarian cancers.
  • provided technologies are effective when applied to locally advanced tumors.
  • provided technologies are effective when applied to unresectable tumors.
  • provided technologies are effective when applied to metastatic tumors.
  • Claudin-18.2 (CLDN-18.2) is a cancer-associated splice variant of Claudin-18.
  • CLDN-18.2 is a member of the Claudin family of more than 20 structurally related proteins that are involved in the formation of tight junctions in epithelia and endothelia.
  • CLDN18 expression in healthy tissues Claudinl 8.2 is a 27.8 kDa protein with four membrane-spanning domains and two small extracellular loops (Niimi et al. 2001).
  • CLDN- 18.2 is a tight junction molecule of the gastric epithelia. Gastric tight junctions are highly specialized on repelling gastric acid, which may injure the gastric lining.
  • CLDN-18.2 is a highly selective gastric lineage antigen (Sahin et al. 2008). Typically, its expression is restricted to short-lived differentiated cells of gastric epithelia in the pit and base regions of gastric glands. The stem cell zone, from which differentiated epithelial cells of the gastric glands are continuously replenished, is CLDN-18.2-negative. Without wishing to be bound by theory, it is commonly believed that no other normal cell type of the human body expresses CLDN-18.2 at transcript level or at protein level.
  • CLDN18 expression in cancer is expressed in various human cancers including gastric, gastroesophageal (GE) and pancreatic cancers (PC) (Karanjawala et al. 2008; Coati et al. 2019) and precancerous lesions (Woll et al. 2014; Tanaka et al. 2011).
  • Tumor- associated expression of CLDN-18.2 has also been detected in ovarian (Sahin et al. 2008), biliary (Shinozaki et al. 2011) and lung cancers (Micke et al. 2014).
  • CLDN-18.2+ About 77% of primary gastric adenocarcinomas (GAC) are CLDN-18.2+ . 56% of GAC display strong CLDN-18.2 expression defined as staining intensity > 2+ by immunohistochemical analysis in at least 60% of tumor cells. CLDN-18.2 expression is more frequent in diffuse than in intestinal gastric cancers. The CLDN-18.2 protein is also frequently detected in lymph node metastases of gastric cancer and in distant metastases into the ovaries (so-called Krukenberg tumors). Moreover, 50% of esophageal adenocarcinomas display significant expression of CLDN-18.2.
  • an antibody agent targeting CLDN-18.2 specifically binds to a CLDN-18.2 polypeptide.
  • an antibody agent targeting CLDN-18.2 specifically binds to a first extracellular domain (ECD1) of a CLDN-18.2 polypeptide.
  • ECD1 extracellular domain
  • such an antibody agent specifically binds to an epitope of ECD 1 that is exposed in cancer cells.
  • such an antibody agent may have a binding affinity (e.g., as measured by a dissociation constant) for a CLDN-18.2 polypeptide, e.g., an epitope of ECD 1 of a CLDN-18.2 polypeptide) of at least about 10 -M, at least about 10 -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.
  • binding affinity e.g., as measured by a dissociation constant
  • binding affinity may be influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces between the two molecules.
  • binding affinity between a ligand and its target molecule may be affected by the presence of other molecules.
  • Those skilled in the art will be familiar with a variety of technologies for measuring binding affinity and/or dissociation constants in accordance with the present disclosure, including, e.g., but not limited to ELISAs, gel-shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), bio-layer interferometry, grating-coupled interferometry, and spectroscopic assays.
  • an antibody targeting CLDN-18.2 may bind specifically to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide. In some embodiments, an antibody targeting CLDN-18.2 does not bind to any other claudin family member including the closely related splice variant 1 of Claudin-18 (CLDN18.1) that is predominantly expressed in tissues, e.g., lung.
  • an antibody agent targeting CLDN-18.2 may be any one of CLDN-18.2-targeting antibodies described in WO 2007/059997, WO2008/145338, and W02013/174510, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.
  • an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues (GYTFTSYW); (ii) CDR2 represented by amino acid residues (IYPSDSYT); and (iii) CDR3 represented by amino acid residues (TRSWRGNSFDY); and/or (b) a variable light chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of (i) CDR1 represented by amino acid residues (QSLLNSGNQKNY); (ii) CDR2 represented by amino acid residues (WAS); and (iii) CDR3 represented by amino acid residues (QNDYSYPFT).
  • CDR including, e.g., 1 CDR, 2 CDRs,
  • an antibody agent targeting CLDN-18.2 has a heavy chain amino acid sequence and a light chain amino acid sequence, that is or includes relevant sequences (e.g., variable region sequences, e.g., CDR and/or framework (FR) sequences) as described in U.S. 9,751,934.
  • relevant sequences e.g., variable region sequences, e.g., CDR and/or framework (FR) sequences
  • an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising an amino acid sequence represented by amino acid residues 20-467 of SEQ ID NO: 1 as set forth below (wherein SEQ ID NO: 1 here corresponds to SEQ ID NO: 118 of U.S.
  • SEQ ID NO: 1 corresponds to a secretion signal sequence
  • a light chain consisting of or comprising an amino acid represented by amino acid residues 21-240 of SEQ ID NO: 2 as set forth below (wherein SEQ ID NO: 2 here corresponds to SEQ ID NO: 125 of U.S. 9,751,934 and the underlined amino acid sequence of SEQ ID NO: 2 corresponds to a secretion signal sequence).
  • MGWS C 11 L FL VATATGVHS QVQLQQPGAELVRPGAS VKL S CKASGYTFTS YW INWVKQRP GQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWR GNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
  • an antibody agent targeting CLDN-18.2 comprises (a) a variable heavy chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues 45- 52 of SEQ ID NO: 1 ; (ii) CDR2 represented by amino acid residues 70-77 of SEQ ID NO: 1; and (iii) CDR3 represented by amino acid residues 116-126 of SEQ ID NO: 1 ; and/or (b) a variable light chain domain having at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of (i) CDR1 represented by amino acid residues 47-58 of SEQ ID NO: 2; (ii) CDR2 represented by amino acid residues 76-78 of SEQ ID NO: 2; and (iii) CDR3 represented by amino acid residues 115
  • an antibody agent targeting CLDN-18.2 comprises a variable heavy chain domain comprising the amino acid sequence SEQ ID NO: 14, and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 15.
  • QVQLQQPGAELVRPGAS VKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATL TVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS ( SEQ ID NO : 14 ) DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTG
  • an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence of SEQ ID NO: 1 and a light chain consisting of or comprising the amino acid sequence of SEQ ID NO: 2.
  • an antibody agent targeting CLDN-18.2 can be engineered to decrease potential immunogenicity and/or improve secretion.
  • a murine secretion signal sequence of an antibody agent targeting CLDN-18.2 can be replaced by a human one.
  • an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising an amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO: 3 as set forth below (wherein the underlined amino acid sequence corresponds to a secretion signal sequence); and a light chain consisting of or comprising an amino acid represented by amino acid residues 27-246 of SEQ ID NO: 4 as set forth below (wherein the underlined amino acid sequence corresponds to a secretion signal sequence).
  • an antibody agent targeting CLDN-18.2 has a heavy chain consisting of or comprising the amino acid sequence of SEQ ID NO: 3 and a light chain consisting of or comprising the amino acid sequence of SEQ ID NO: 4.
  • proteins in accordance with the invention can be generated with or without encoding or having a terminal lysine.
  • sequences with a tenninal lysine such as a constant region sequence having a terminal lysine
  • sequences without a tenninal lysine can also be understood as the corresponding sequences with a terminal lysine.
  • an antibody targeting CLDN-18.2 is IMAB362 (also known as Zolbetuximab, Claudiximab).
  • IMAB362 an antibody targeting CLDN-18.2 is in advanced clinical development (NCT01630083, NCT03816163, NCT03653507, NCT03505320, NCT03504397) and known in the art (see, e.g., Sahin et al. 2018; Sahin et al. 2017; Al-Batran et al. 2017a; Al-Batran et al. 2017b; Tiireci et al. 2019; Trarbach et al. 2014; Morlock et al. 2018a; Schuler et al. 2016; Lordick et al. 2016; Morlock et al. 2018b).
  • Its target CLDN-18.2 is a highly selective tumor-associated surface marker.
  • IMAB362 developed by Ganymed Pharmaceuticals GmbH and acquired by Astellas Pharma Inc., is a full IgGl antibody targeting the tight junction protein CLDN-18.2 and mediates cell death through antibody-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC).
  • IMAB362 recognizes the first extracellular domain (ECD1) of CLDN-18.2 with high affinity and specificity (Sahin et al. 2008; Tiireci et al. 2011).
  • the epitope is not accessible in normal epithelial barriers to the antibody. Disruption of tight junctions and loss of cell polarization are early hallmarks of cancer. In this process, the epitope of IMAB362 is exposed.
  • IMAB362 does not bind to any other claudin family member including the closely related splice variant 1 of Claudin 18 (CLDN18.1) that is predominantly expressed in tissues, e.g., lung.
  • IMAB362 plus epirubicin, oxaliplatin, and capecitabine (EOX) were tested in phase 2 FAST trial (NCT01630083) against EOX in first-line patients with gastric and gastro- esophageal cancer (Morlock et al. 2018a; Schuler et al. 2016; Al-Batran et al. 2016; Lordick et al. 2016; Morlock et al. 2018b).
  • the FAST patient population included patients whose tumors had > 40% of tumor cells expressing CLDN-18.2 with a moderate-to-strong (> 2+) staining intensity.
  • IMAB362 has been tested in various clinical trials as shown in Table 1 below.
  • Table 1 Summary of certain clinical trials involving administration of IMAB362
  • Recombinant protein antibodies are widely used biologies for the treatment of diseases or disorders (e.g., cancer) but show a number of limitations, including, e.g., lengthy manufacturing process development and, for antibody derivatives, short serum half-life.
  • the present disclosure provides technologies that address certain limitations of recombinant antibody technologies, including for example, lengthy manufacturing process development, and for antibody derivatives, short serum half-life, by utilizing RNA technologies as a modality to express antibody agents, called RiboMabs, directly in the patient’s cells as a novel class of antibody-based therapeutics.
  • RiboMab technology can be utilized to deliver various antibody formats.
  • RiboMab technology can be used to express a full immunoglobulin (Ig), including, e.g., but not limited to IgG.
  • Ig immunoglobulin
  • a full immunoglobulin may be encoded by a single RNA comprising a first coding region that encodes a heavy chain of an antibody and a second coding region that encodes a light chain variable domain of the antibody, wherein the single RNA comprises or encodes either an internal ribosome entry sides (IRES) or another internal promoter or peptide sequence such as “selfcleaving” 2A or 2A-like sequences (see, e.g., Szymczak et al. Nat Biotechnol 22:589, May 2004; ePub April 42004) to yield a respective heavy chain and light chain, which can then be processed to form a full IgG.
  • Ig an internal ribosome entry sides
  • a full Ig may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a heavy chain of an antibody; and a second RNA comprising a coding region that encodes a light chain of the antibody. Such first and second RNAs are then translated into respective chains of an antibody and form a full Ig antibody in target cells.
  • RiboMab technology can be used to express a bispecific antibody variant, e.g., as illustrated in Figure 12 (Panel A) or described in Stadler et al. (2016) Oncoimmunology 5(3): el091555; and/or in Stadler et al.
  • a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a scFv for a first target and a coding region that encodes a heavy chain antigen binding fragment (Fab) for a second target; and a second RNA comprising a coding region that encodes a scFv for the same first target and a coding region that encodes a light chain Fab for the same second target.
  • first and second RNAs are then translated into subunits of an antibody and form a bispecific antibody in target cells.
  • RNA agents e.g., ssRNAs described herein
  • RNA/LNP is intravenously (IV) administered and taken up by target cells (e.g., liver cells) for efficient production of the encoded RiboMab antibody at therapeutically relevant plasma concentrations.
  • At least one RNA comprises one or more coding regions that encode an antibody agent as described in the section entitled “Exemplary antibody agents targeting Claudin-18.2 polypeptides” above. In some embodiments, at least one RNA comprises one or more coding regions that encode an antibody agent IMAB362 as described above or exemplified herein.
  • an antibody agent IMAB362 may be particularly useful and/or effective at least in part because it binds specifically to CLDN- 18.2 and, moreover, binds preferentially to CLDN-18.2 relative to CLDN18.1.
  • teachings provided herein may be applicable to other antibody agents specific to CLDN-18.2, and in particular to such antibodies that bind preferentially to CLDN-18.2 even relative to CLDN 18.1.
  • At least one RNA comprises one or more coding regions that encode an antibody agent that binds preferentially to a CLDN-18.2 polypeptide relative to a CLDN18.1 polypeptide.
  • an antibody agent has a binding affinity for a CLDN-18.2 polypeptide higher than that for a CLDN18.1 polypeptide by at least 50% or more including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher.
  • such an antibody agent has a binding affinity for a CLDN-18.2 polypeptide higher than that for a CLDN18.1 polypeptide by at least 1.1-fold or more including, e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50- fold, at least 75-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10,000-fold or higher.
  • such an antibody agent does not detectably bind to any other claudin family member including CLDN 18.1.
  • an antibody agent may be or comprise an antibody.
  • an antibody agent may be or comprise an antigen binding fragment.
  • an antibody agent that targets CLDN-18.2 (and may be encoded by an RNA such as an ssRNA, e.g., an mRNA as described herein) specifically binds to a first extracellular domain (ECD1) of a CLDN- 18.2 polypeptide.
  • an antibody agent specifically binds to an epitope of ECD 1 that is exposed in cancer cells.
  • At least one RNA encodes a variable heavy chain (VH) domain of a CLDN-18.2-targeting antibody agent and a variable light chain (VL) domain of the antibody agent.
  • VH domain(s) and VL domain(s) of a CLDN- 18.2- targeting antibody agent may be encoded by a single RNA construct; alternatively in some embodiments they may be encoded separately by at least two individual RNA constructs.
  • an RNA as utilized herein comprises two or more coding regions, which comprises a heavy chain-coding region that encodes at least a VH domain of a CLDN-18.2-targeting antibody agent; and a light chain-coding region that encodes at least a VL domain of a CLDN-18.2-targeting antibody agent.
  • a composition comprises (i) a first RNA comprising a heavy chain-coding region that encodes at least a VH domain of a CLDN-18.2-targeting antibody agent; and (ii) a second RNA comprising a light chain-coding region that encodes at least a VL domain of a CLDN-18.2-targeting antibody agent.
  • a heavy chain-coding region can further encode a constant heavy chain (CH) domain; and/or a light chain-coding region can further encode a constant light chain (C ) domain.
  • a heavy chain-coding region may encode a VH domain, a CHI domain, aCn2 domain, and a CH3 domain of a CLDN-18.2-targeting antibody agent in an immunoglobulin form (e.g., IgG); and/or a light chain-coding region may encode a VL domain and a CL domain of a CLDN-18.2-targeting antibody agent in an Ig form (e.g., IgG).
  • a full immunoglobulin maybe encoded by a single RNA comprising a first coding region that encodes a heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG) and a second coding region that encodes a light chain variable domain of the CLDN-18.2 Ig antibody (e.g. ,IgG), which single RNA requires protein translation to yield a fusion protein comprising a heavy chain and a light chain of the antibody and post-translational cleavage of the fusion protein by a suitable protease into respective heavy chain and light chain, which can then be processed to form a full Ig (e.g., IgG).
  • a full immunoglobulin e.g., IgG
  • a full Ig may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a heavy chain of a CLDN-18.2 Ig antibody (e.g., IgG); and a second RNA comprising a coding region that encodes a light chain of the CLDN-18.2 Ig antibody (e.g., IgG).
  • first and second RNAs are then translated into respective chains of an antibody and form a full Ig antibody (e.g. , IgG) in target cells.
  • an antibody agent encoded by one or more RNAs in an IgG form is IgGl .
  • a heavy chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes at least one CDR (including, e.g., 1 CDR, 2 CDRs, and 3 CDRs) selected from the group consisting of: (i) CDR1 represented by amino acid residues (GYTFTSYW); (ii) CDR2 represented by amino acid residues (IYPSDSYT); and (iii) CDR3 represented by amino acid residues (TRSWRGNSFDY).
  • a light chaincoding region of an RNA consists of or comprises a nucleotide sequence that encodes at least one CDR (including, e.g.
  • CDR1 represented by amino acid residues
  • CDR2 represented by amino acid residues
  • CDR3 represented by amino acid residues
  • a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an amino acid sequence represented by amino acid residues 20-467 of SEQ ID NO: 1.
  • one or more amino acid modifications may be present to one or more non-CDR regions of SEQ ID NO: 1.
  • 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.
  • amino acid modifications including, e.g., amino acid insertions, deletions, and/or substitutions
  • no more than 50 including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less
  • amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 1.
  • a light-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an amino acid sequence represented by amino acid residues 21-240 of SEQ ID NO: 2.
  • one or more amino acid modifications may be present to one or more non-CDR regions of SEQ ID NO: 2.
  • 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.
  • amino acid modifications including, e.g., amino acid insertions, deletions, and/or substitutions
  • no more than 50 including, e.g., no more than 40, no more than 30, no more than 20, no more than 10, or no more 5, or less
  • amino acid modifications may be present in one or more non-CDR regions of SEQ ID NO: 2.
  • a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1.
  • a light-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2.
  • a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes an amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO: 3.
  • one or more amino acid modifications may be present to one or more non-CDR regions of SEQ ID NO: 3.
  • a heavy-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 3.
  • a light-chain coding region of an RNA consists of or comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 4.
  • a heavy chain-coding region of an RNA consists of or comprises a nucleotide sequence that encodes a full-length heavy chain of Zolbetuximab or Claudiximab (e.g., as described and/or exemplified herein).
  • a light chaincoding region of an RNA consists of or comprises a nucleotide sequence that encodes a full- length light chain of Zolbetuximab or Claudiximab.
  • one or more RNAs can be used to encode a bispecific or multispecific antibody agent, which binds to two or more target molecules, e.g., one of which is a CLDN-18.2 polypeptide.
  • Figure 12A illustrates exemplary bispecific antibody encoded by one or more RNAs. See also, e.g., Stadler et al. (2016) Oncoimmunology 5(3): el091555; and/or in Stadler et al. (2017) Nature Medicine 23(7): 815-817.
  • a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a scFv that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a coding region that encodes a heavy chain antigen binding fragment (Fab) for a second target (e.g., in some embodiments which may be a T cell receptor); and a second RNA comprising a coding region that encodes a scFv targeting the CLDN-18.2 polypeptide and a coding region that encodes a light chain Fab for the same second target.
  • a first RNA comprising a coding region that encodes a scFv that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide) and a coding region that encodes a heavy chain antigen binding fragment (Fab) for a second target (e
  • a bivalent antibody agent may be encoded by two separate RNAs: a first RNA comprising a coding region that encodes a scFv for a first target e.g., in some embodiments which may be a T cell receptor) and a coding region that encodes a heavy chain antigen binding fragment (Fab) that preferentially binds to a CLDN-18.2 polypeptide (relative to a CLDN18.1 polypeptide); and a second RNA comprising a coding region that encodes a scFv for the same first target and a coding region that encodes a light chain Fab targeting the CLDN-18.2 polypeptide.
  • first and second RNAs are then translated into subunits of an antibody and form a bispecific antibody in target cells.
  • such a non-human secretion signal may be a murine secretion signal, which may in some embodiments be or comprises the amino acid sequence of MGWSCIILFLVATA GVHS or MESQTQVLMSLLFWVSGTCG.
  • a secretion signal-encoding region included in an RNA consists of or comprises a nucleotide sequence that encodes a human secretion signal, which may in some embodiments be or comprises the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS.
  • a secretion signal-encoding region included in an RNA encoding a light chain domain of a CLDN-18.2-targeting antibody agent may comprise a nucleotide sequence (i) that encodes a murine secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MESQTQVLMSLLFWVSGTCG; or that (ii) encodes a human secretion signal amino acid sequence, which in some embodiments may be or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS .
  • RNA(s) that encode a CLDN-18.2-targeting antibody agent may comprise at least one non-coding sequence element (e.g., to enhance RNA stability and/or translation efficiency).
  • non-coding sequence elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure for co-transcriptional capping of mRNA, a poly adenine (polyA) tail, and any combinations thereof.
  • RNA can comprise a nucleotide sequence that encodes a 5’UTR of interest and/or a 3’ UTR of interest.
  • untranslated regions e.g., 3’ UTR and/or 5’ UTR
  • mRNA stability can contribute to mRNA stability, mRNA localization, and/or translational efficiency.
  • a provided RNA can comprise a 5’ UTR nucleotide sequence and/or a 3’ UTR nucleotide sequence.
  • a 5’ UTR sequence can be operably linked to a 3’ of a coding sequence (e.g., encompassing one or more coding regions).
  • a 3’ UTR sequence can be operably linked to 5’ of a coding sequence e.g., encompassing one or more coding regions).
  • 5' and 3' UTR sequences included in an RNA can consist of or comprise naturally occurring or endogenous 5' and 3' UTR sequences for an open reading frame of a gene of interest.
  • 5’ and/or 3’ UTR sequences included in an RNA are not endogenous to a coding sequence (e.g., encompassing one or more coding regions); in some such embodiments, such 5’ and/or 3’ UTR sequences can be useful for modifying the stability and/or translation efficiency of an RNA sequence transcribed.
  • a skilled artisan will appreciate that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, as will be understood by a skilled artisan, 3’ and/or 5’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • a nucleotide sequence consisting of or comprising a Kozak sequence of an open reading frame sequence of a gene or nucleotide sequence of interest can be selected and used as a nucleotide sequence encoding a 5’ UTR.
  • Kozak sequences are known to increase the efficiency of translation of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation.
  • a provided RNA polynucleotide can comprise a nucleotide sequence that encodes a 5' UTR derived from an RNA virus whose RNA genome is stable in cells.
  • a 5’ UTR included in an RNA may be derived from human a-globin mRNA combined with Kozak region.
  • an RNA may comprise one or more 3 ’UTRs.
  • an RNA may comprise two copies of 3'-UTRs derived from a globin mRNA, such as, e.g., alpha2-globin, alpha 1 -globin, beta-globin (e.g., a human beta-globin) mRNA.
  • two copies of 3’UTR derived from a human beta-globin mRNA may be used, e.g., in some embodiments which may be placed between a coding sequence of an RNA and a poly(A)-tail, to improve protein expression levels and/or prolonged persistence of an RNA.
  • a 3’ UTR included in an RNA may be or comprise one or more (e.g., 1, 2, 3, or more) of the 3’UTR sequences disclosed in WO 2017/060314, the entire content of which is incorporated herein by reference for the purposes described herein.
  • a 3‘-UTR may be a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called 1). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017/060314, herein incorporated by reference).
  • a 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 18 or 20, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, or 90% identity to the nucleotide sequence of SEQ ID NO: 18 or 20.
  • a 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 19 or 21, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, or 90% identity to the nucleotide sequence of SEQ ID NO: 19 or 21.
  • RNA can comprise a nucleotide sequence that encodes a polyA tail.
  • a polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenosine nucleotides.
  • a polyA tail may comprise one or more modified adenosine nucleosides, including, but not limited to, cordiocipin and 8-azaadenosine. In some embodiments, a polyA tail may comprise one or more non-adensoine nucleotides. In some embodiments, a polyA tail may be or comprise a disrupted or modified polyA tail as described in WO 2016/005324, the entire content of which is incorporated herein by reference for the purpose described herein.
  • a polyA tail included in an RNA described herein may be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 20 A consecutive nucleotides, which is 3’ of the linker sequence.
  • a modified polyA sequence may comprise: a linker sequence which is not a polyA sequence comprising at least ten nucleotides (e.g., T, G, and/or C nucleotides); a first sequence of at least 30 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 70 A consecutive nucleotides, which is 3’ of the linker sequence.
  • a linker sequence which is not a polyA sequence comprising at least ten nucleotides (e.g., T, G, and/or C nucleotides); a first sequence of at least 30 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 70 A consecutive nucleotides, which is 3’ of the linker sequence.
  • an RNA described herein may comprise a 5’ cap, which may be incorporated into such an RNA during transcription, or joined to such an RNA post-transcription.
  • an RNA may comprise a 5’ cap structure for co- transcriptional capping of RNA.
  • a cap structure for co-transcriptional capping examples include, e.g., as described in WO 2017/053297, the entire content of which is incorporated herein by reference for the purposes described herein.
  • a 5’ cap included in an RNA described herein is or comprises m7G(5')ppp(5')(2'OMeA)pG.
  • a 5’ cap included in an RNA described herein is or comprises a cap1 structure [e.g., m2 7 ’ 3 ’°Gppp(m1 2 -0)ApG].
  • RNA sequence described herein has a 5' end with the nucleotides 5'-AG, and it is described that the RNA comprises a 5’ cap containing as second and third nucleotides A and G, respectively, [e.g., m2 7 ’ 3 -0Gppp(mi 2 -0)ApG], it is to be understood that in some embodiments, the second and third nucleotides of the cap correspond to the nucleotides 5'-AG of the RNA sequence.
  • RNA(s) that encode a CLDN- 18.2-targeting antibody agent may comprise at least one modified ribonucleotide, for example, in some embodiments to increase the stability of such RNA(s) and/or decrease immunogenicity of such RNA(s) and/or to decrease cytotoxicity of such RNAs.
  • at least one of A, U, C, and G ribonucleotide of RNA(s) may be replaced by a modified ribonucleotide.
  • cytidine residues present in an RNA may be replaced by a modified cytidine, which in some embodiments may be, e.g., 5- methylcytidine.
  • some or all of uridine residues present in an RNA may be replaced by a modified uridine, which in some embodiments may be 3 -methyl -uridine (m3U), 5 -methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5 -hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo- uridineor 5-bromo-uridine), uridine 5-oxyacetic acid
  • some or all of uridine residues present in an RNA may be replaced by a modified uridine selected from the group consisting of pseudouridine ( ⁇
  • some or all of uridine residues present in an RNA may be replaced by pseudouridine or a derivative thereof, e.g., 1 -methylpseudouridine.
  • some or all of uridine residues present in an RNA may be replaced by pseudouridine.
  • some or all of uridine residues present in an RNA may be replaced by 1 - methylpseudouridine.
  • all uridine residues present in an RNA are replaced by pseudouridine.
  • all uridine residues present in an RNA are replaced by 1 -methylpseudouridine.
  • Codon optimization and GC enrichment The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and/or to replace codons which are rare in the cell (or subject) in which a peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject).
  • the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence.
  • this also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence.
  • the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
  • the guanosine/cytosine (G/C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA.
  • RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so- called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence.
  • codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.
  • the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.
  • any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA.
  • Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors.
  • the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a modified uracil.
  • the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m 3 U), 5 -methoxy-uridine (mo 5 U), 5-aza- uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5-iodo-uridine or 5 -bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5- oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxy
  • the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.
  • an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.
  • Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.
  • the non-immunogenic RNA is free or essentially free of dsRNA.
  • the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single- stranded nucleoside modified RNA.
  • the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA).
  • RNA may be taken as a measure for the amount of dsRNA in the sample.
  • a sample containing a known amount of dsRNA may be used as a reference.
  • RN A may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v/v) TWEEN-20) containing 5% (w/v) skim milk powder.
  • the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • dsRNA-specific antibody e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • the non-immunogenic RNA
  • translation is enhanced by a factor of 2- fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3- fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10- fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor.
  • translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1 OOO-fold. In some embodiments, the factor is 30-1000-fold.
  • the factor is 50-1000- fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200- 1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.
  • the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3 -fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor.
  • innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.
  • the term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response.
  • the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA.
  • the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.
  • an RNA encoding a heavy chain of a CLDN-18.2- targeting antibody agent comprises, in a 5’ to 3’ direction: (a) a 5’UTR; (b) a secretion signalcoding region; (c) a heavy chain-coding region; (d) a 3’ UTR; and (e) a polyA tail.
  • a 5’UTR is or comprises a sequence derived from human a-globin mRNA combined with Kozak region.
  • a secretion signalcoding region is or comprises a nucleotide sequence that encodes the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS .
  • a heavy chain-coding region encodes a VH domain, a Cm domain, aCH2 domain, and a CH3 domain of a CLDN-18.2-targeting antibody agent in an IgG form (e.g., ones as described herein, such as IMAB262, or an amino acid sequence represented by amino acid residues 27-474 of SEQ ID NO: 3.
  • a 3’ UTR is or comprises a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I).
  • a polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines).
  • a modified polyA sequence e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines.
  • such an RNA comprises a 5’ cap structure comprising a CAP1 structure, or m? 7 ’ 3 ’ °Gppp(mi 2 ‘°)ApG.
  • such an RNA comprises all uridines replaced by Nl- methylpseudouridine.
  • an RNA encoding a light chain of a CLDN-18.2-targeting antibody agent comprises, in a 5’ to 3’ direction: (a) a 5’UTR; (b) a secretion signal-coding region; (c) a light chain-coding region; (d) a 3’ UTR; and (e) a polyA tail.
  • a 5’UTR is or comprises a sequence derived from human a- globin mRNA combined with Kozak region.
  • a secretion signal-coding region is or comprises a nucleotide sequence that encodes the amino acid sequence of MRVMAPRTLILLLSGALALTETWAGS .
  • a light chain-coding region encodes a VL domain and a CL domain of a CLDN-18.2-targeting antibody agent in an IgG form (e.g., ones as described herein, such as IMAB262, or an amino acid sequence represented by amino acid residues 27-246 of SEQ ID NO: 4.
  • a 3’ UTR is or comprises a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I).
  • a polyA tail is or comprises a modified polyA sequence (e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines).
  • a modified polyA sequence e.g., a polyA sequence of 100 adenosines disrupted by a linker sequence inserted immediately following 30 consecutive adenosines.
  • such an RNA comprises a 5’ cap structure comprising a CAP1 structure, or m2 7 - 3 -0Gppp(m1 2 -0)ApG.
  • such an RNA comprises all uridines replaced by N1 -methylpseudouridine.
  • RNA(s) is or comprises one or more single-stranded RNA(s), e.g., single-stranded mRNAs.
  • a composition comprises a single- stranded mRNA encoding a heavy chain (e.g., open reading frame, ORF) of an antibody agent targeting CLDN- 18.2 (e.g., ones described herein) and a single- stranded mRNA encoding a light chain (e.g., open reading frame, ORF) of an antibody agent targeting CLDN-18.2 (e.g., ones described herein), which upon introduction into target cells, are translated into respective subunits and form a full IgG antibody in target cells.
  • a heavy chain e.g., open reading frame, ORF
  • a light chain e.g., open reading frame, ORF
  • an RNA drug substance is or comprises a combination of two RNAs, respectively, encoding a heavy (HC) and a light chain (LC) of an IgG CLDN-18.2 targeting antibody.
  • each of such two RNAs can be manufactured separately and an RNA drag substance can be prepared by mixing RNAs, respectively, encoding HC and LC of an IgG CLDN-18.2-targeting antibody in an appropriate weight ratio, e.g., a weight ratio such that the resulting molar ratio of HC- and LC-encoding RNAs is about 1.5:1 - 1: 1.5 for proper IgG formation.
  • a first RNA encoding a polypeptide comprising a heavy chain of a CLDN-18.2-targeting antibody agent, and a second RNA encoding a polypeptide comprising a light chain of a CLDN-18.2-targeting antibody agent may be present in a molar ratio of about 1.5:1 to about 1:1.5.
  • such a first RNA and a second RNA may be present in a molar ratio of about 1.30, about 1.29, about 1.28, about 1.27, about 1.26, about 1.25, about 1.24, about 1.23, about 1.22, about 1.21, about 1.20, about 1.19, about 1.18, about 1.17, about 1.16, about 1.15, about 1.14, about 1.13, about 1.12, about 1.11, about 1.10, about 1.09, about 1.08, about 1.07, about 1.06, about 1.05, about 1.04, about 1.03, about 1.02, about 1.01, about 1.00, about 0.99, about 0.98, about 0.97, about 0.96, about 0.95, about 0.94, about 0.93, about 0.92, about 0.91, about 0.90, about 0.89, about 0.88, about 0.87, about 0.86, about 0.85, about 0.84, about 0.83, about 0.82, about 0.81, or about 0.80.
  • such a first RNA and a second RNA may be present in a weight ratio of 3: 1 to 1 : 1. In some embodiments, such a first RNA and a second RNA may be present in a weight ratio of about 2: 1. In some embodiments, such a first RNA and a second RNA may be present in a weight ratio of about 2.2:1, about 2.1 :1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3 : 1 , or about 1.2:1.
  • RNAs encoding the HC and/or LC of a CLDN-18.2- targeting IgG antibody can comprise one or more non-coding sequence elements, for example, to enhance RNA stability and/or translational efficiency.
  • such RNA can comprise a cap structure, for example, a cap structure that can increase the resistance of RNA molecules to degradation by extracellular and intracellular RNases and leads to higher protein expression.
  • an exemplary cap structure is or comprises (nu 73 - °Gppp(mi 2 ’ °))ApG (cap1).
  • RNA can comprise one or more noncoding sequence elements at one or both of 5’ and 3’ untranslated regions (UTRs), for example, a naturally occurring sequence element at 5 ' and 3 ' UTRs that can significantly increase the intracellular half-life and the translational efficiency of the molecule (see, e.g., Holtkamp et al. 2006; Orlandini von Niessen et al. 2019).
  • an exemplary 5’ UTR sequence element is or comprises a characteristic sequence from human a-globin and a Kozak consensus sequence.
  • an RNA polymerase typically traverses at least a portion of a DNA template in the 3'— » 5' direction to produce a complementary RNA in the 5'—* 3' direction.
  • RNA e.g., mRNA
  • a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life.
  • capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g., capping enzymes of vaccinia virus).
  • RNA(s) may be further processed by one or more steps of filtration and/or concentration.
  • RNA(s) may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of RNAs to a desirable RNA concentration and/or to exchange buffer to a drug substance buffer.
  • RNAs e.g., ones described herein
  • compositions thereof may be stored stable at a fridge temperature (e.g., about 4°C to about 10°C) for at least 1 month or longer 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 longer.
  • RNAs (e.g., ones described herein) and/or compositions thereof may be stored stable at a sub-zero temperature (e.g., -20°C or below) for at least 1 month or longer 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 longer.
  • RNAs (e.g., ones described herein) and/or compositions thereof may be stored stable at room temperature (e.g., at about 25°C) for at least 1 month or longer.
  • lipid nanoparticles can be designed to protect RNAs (e.g., mRNA) from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells (e.g., liver cells).
  • RNAs e.g., mRNA
  • target cells e.g., liver cells
  • lipid nanoparticles may be particularly useful to deliver RNAs (e.g., mRNA) when RNAs are intravenously administered to a subject in need thereof.
  • a lipid nanoparticle for delivery of RNA(s) described herein comprises a neutral helper lipid.
  • neutral helper lipids include, but are not limited to phosphatidylcholines such as l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 ,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidylethanolamines such as 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins
  • DOPE 1,2-dio
  • Neutral lipids may be synthetic or naturally derived.
  • Other neutral helper lipids that are known in the art, e.g., as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein, can also be used in lipid nanoparticles described herein.
  • a lipid nanoparticle for delivery of RNA(s) described herein comprises DSPC and/or cholesterol.
  • a lipid nanoparticle for delivery of RNA(s) described herein comprises at least two helper lipids (e.g., ones described herein).
  • a lipid nanoparticle may comprise DSPC and cholesterol.
  • a lipid nanoparticle for delivery of RNA(s) described herein comprises a cationic lipid.
  • a cationic lipid is typically a lipid having a net positive charge.
  • a cationic lipid may comprise one or more amine group(s) which bear a positive charge.
  • a cationic lipid may comprise a cationic, meaning positively charged, headgroup.
  • a cationic lipid may 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.
  • a cationic lipid comprises a polar headgroup, which in some embodiments may comprise one or more amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperizine and amino acid headgroup s such as lysine, arginine, ornithine and/or tryptophan.
  • a polar headgroup of a cationic lipid comprises one or more amine derivatives.
  • a polar headgroup of a cationic lipid comprises a quaternary ammonium.
  • a headgroup of a cationic lipid may comprise multiple cationic charges. In some embodiments, a headgroup of a cationic lipid comprises one cationic charge.
  • monocationic lipids include, but are not limited to l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), 1 ,2-di-O-octadecenyl- 3 -trimethylammonium propane (DOTMA) and/or 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)- dimethylazanium bromide (DMRIE), didodecyl(dimethyl)azanium bromide (DDAB), 1 ,2- dioleyloxypropyl-3 -dimethyl -
  • a positively charged lipid structure described herein may also include one or more other components that may be typically used in the formation of vesicles (e.g. for stabilization).
  • other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or cholesterol esters or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
  • sterols include cholesterol, cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
  • the at least one cationic lipid comprises DMEPC and/or DOTMA.
  • a cationic lipid is ionizable such that it can exist in a positively charged form or neutral form depending on pH. Such ionization of a cationic lipid can affect the surface charge of the lipid particle under different pH conditions, which in some embodiments may influence plasma protein absorption, blood clearance, and/or tissue distribution as well as the ability to form endosomolytic non-bilayer structures. Accordingly, in some embodiments, a cationic lipid may be or comprise a pH responsive lipid. In some embodiments a pH responsive lipid is a fatty acid derivative or other amphiphilic compound which is capable of forming a lyotropic lipid phase, and which has a pKa value between pH 5 and pH 7.5.
  • a pH responsive lipid may be used in addition to or instead of a cationic lipid for example by binding one or more RNAs to a lipid or lipid mixture at low pH.
  • pH responsive lipids include, but are not limited to, 1,2- dioleyloxy-3 - dimethylamino-propane (DODMA).
  • a lipid nanoparticle may comprise one or more cationic lipids as described in WO 2017/075531 (e.g., as presented in Tables 1 and 3 therein) and WO 2018/081480 (e.g., as presented in Tables 1-4 therein), the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • a cationic lipid that may be useful in accordance with the present disclosure is an amino lipid comprising a titratable tertiary amino head group linked via ester bonds to at least two saturated alkyl chains, which ester bonds can be hydrolyzed easily to facilitate fast degradation and/or excretion via renal pathways.
  • an amino lipid has an apparent pKa of about 6.0-6.5 (e.g., in one embodiment with an apparent pKa of approximately 6.25), resulting in an essentially fully positively charged molecule at an acidic pH (e.g., pH 5).
  • such an amino lipid when incorporated in LNP, can confer distinct physicochemical properties that regulate particle formation, cellular uptake, fusogenicity and/or endosomal release of RNA(s).
  • introduction of an aqueous RNA solution to a lipid mixture comprising such an amino lipid at pH 4.0 can lead to an electrostatic interaction between the negatively charged RNA backbone and the positively charged cationic lipid. Without wishing to be bound by any particular theory, such electrostatic interaction leads to particle formation coincident with efficient encapsulation of RNA drug substance.
  • RNA encapsulation After RNA encapsulation, adjustment of the pH of the medium surrounding the resulting LNP to a more neutral pH (e.g., pH 7.4) results in neutralization of the surface charge of the LNP.
  • a more neutral pH e.g., pH 7.4
  • charge-neutral particles display longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are rapidly cleared by the reticuloendothelial system.
  • the low pH of the endosome renders LNP comprising such an amino lipid fusogenic and allows the release of the RNA into the cytosol of the target cell.
  • a cationic lipid that may be useful in accordance with the present disclosure has one of the structures set forth in Table 3 below:
  • a cationic lipid that may be useful in accordance with the present disclosure is or comprises ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2- butyloctanoate) with a chemical structure shown in Example 14.
  • Cationic lipids may 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.
  • neutral lipids e.g., cholesterol and/or neutral phospholipids
  • a polymer-conjugated lipid is a PEG-conjugated lipid.
  • a PEG-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer.
  • a PEG-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.
  • PEG-conjugated lipids include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)- 2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-O-( ⁇ - methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di
  • PEG-conjugated lipids also known as PEGylated lipids
  • PEG-conjugated lipids are known to affect cellular uptake, a prerequisite to endosomal localization and payload delivery.
  • the present disclosure provides an insight that the pharmacology of encapsulated nucleic acid can be controlled in a predictable manner by modulating the alkyl chain length of a PEG-lipid anchor.
  • the present disclosure provides an insight that such PEG-conjugated lipids may be selected for an RNA/LNP drug product formulation to provide optimum delivery of RNAs to the liver.
  • such PEG- conjugated lipids may be designed and/or selected based on reasonable solubility characteristics and/or its molecular weight to effectively perform the function of a steric barrier.
  • a PEGylated lipid does not show appreciable surfactant or permeability enhancing or disturbing effects on biological membranes.
  • PEG in such a PEG-conjugated lipid can be linked to diacyl lipid anchors with a biodegradable amide bond, thereby facilitating fast degradation and/or excretion.
  • a LNP comprising a PEG-conjugated lipid retain a full complement of a PEGylated lipid. In the blood compartment, such a PEGylated lipid dissociates from the particle over time, revealing a more fusogenic particle that is more readily taken up by cells, ultimately leading to release of the RNA payload.
  • a lipid nanoparticle may comprise one or more PEG- conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • a PEG-conjugated lipid that may be useful in accordance with the present disclosure can have a structure as described in WO 2017/075531, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Rg 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 bonds; and w has a mean value ranging from 30 to 60.
  • R8 and R9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • w has a mean value ranging from 43 to 53.
  • a PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N -ditetradecylacetamide with a chemical structure as shown in Example 14.
  • the molar ratio of total cationic lipid to total polymer-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 about 35:1 to about 25: 1.
  • total steroid neutral lipid e.g., cholesterol
  • 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.
  • lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid (e.g., PEG-conjugated lipid); a cationic lipid; and a neutral lipid, wherein the polymer-conjugated lipid is present in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; and the neutral lipid is present in 45- 55 mol% of the total lipids.
  • a polymer-conjugated lipid e.g., PEG-conjugated lipid
  • lipids that form the lipid nanoparticles comprise: 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 in about 1-2 mol% of the total lipids; the cationic lipid is present in 45-48.5 mol% of the total lipids; the non-steroid neutral lipid is present in 9-11 mol% of the total lipids; and the steroid neutral lipid is present in about 36-44 mol% of the total lipids.
  • a polymer-conjugated lipid e.g., PEG-conjugated lipid
  • a cationic lipid e.g., PEG-conjugated lipid
  • a neutral lipid comprising a non-steroid neutral lipid and a steroid neutral lipid
  • a PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide or a derivative thereof.
  • a cationic lipid is or comprises ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2 -butyloctanoate) or a derivative thereof.
  • a neutral lipid comprises DSPC and cholesterol, wherein DSPC is a non-steroid neutral lipid and cholesterol is a steroid neutral lipid.
  • RNA described herein is formulated in a lipid nanoparticle composition comprising Cationic lipid A, a neutral lipid, a steroid, and a pegylated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is PEG-conjugated lipid A.
  • RNA described herein is formulated in a lipid nanoparticle composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in a lipid nanoparticle composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG-conjugated lipid A.
  • RNA described herein is formulated in a lipid nanoparticle composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and PEG-conjugated lipid A.
  • RNA described herein is 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,l-diyl) bis(2- butyloctanoate)
  • the N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 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.
  • cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a predetermined molar ratio (e.g., ones described herein).
  • lipid nanoparticles (LNP) are prepared at a total lipid to RNAs weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such RNAs can be diluted to 0.2 mg/mL in acetate buffer.
  • an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • such therapeutic agents may include one or more chemo therapeutics, such as Taxol derivatives, taxotere, paclitaxel (e.g., nab-paclitaxel), gemcitabin, 5 -Fluoruracil, doxorubicin (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan, Procytox, Neosar), folinic acid, irinotecan, oxaliplatin.
  • chemo therapeutics such as Taxol derivatives, taxotere, paclitaxel (e.g., nab-paclitaxel), gemcitabin, 5 -Fluoruracil, doxorubicin (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan, Procytox, Neosar), folinic acid, irinotecan, oxaliplatin.
  • composition described herein may be administered in conjunction with radiotherapy and/or autologous peripheral stem cell or bone marrow transplantation.
  • compositions described herein may be administered in combination with one or more antibodies selected from anti-CD25 antibodies, anti- EPCAM antibodies, anti-EGFR, anti-Her2/neu, and anti-CD40 antibodies.
  • the present disclosure provides methods of characterizing one or more features of an RNA or composition thereof, which RNA encodes part or all of an antibody agent.
  • RNA integrity assessment of RNA(s) can be performed by adaptation of a capillary gel electrophoresis assay.
  • the proportion of the area of the longer HC- coding RNA is evaluated to describe the integrity of both RNAs encoding different chains of a CLDN-18.2-targeting antibody agent.
  • an RNA composition comprising two or more RNAs can be analyzed by capillary gel electrophoresis, which gives an electropherogram as a result.
  • an RNA composition comprising two different RNAs elutes in two separated peaks, for example, each corresponding to RNA encoding for a distinct chain (e.g., heavy chain or light chain) of an antibody. See, e.g., Figure 15.
  • residual host cell DNA and/or host cell protein may be measured in compositions comprising RNAs.
  • compositions and components thereof can be assessed to determine their efficacy.
  • primary pharmacodynamics and/or pharmacokinetics of pharmaceutical compositions described herein in vitro and/or in vivo can be determined. Examples of useful pharmacokinetics measurements may include one or more parameters:
  • Cmax corresponds to maximum (or peak) plasma/serum concentration that a drug achieves in a specified compartment or test area of the body after the drug has been administered and before the administration of a second dose.
  • the related pharmacokinetics parameter tmax is the time at which the Cmax is observed.
  • Cmin corresponds to minimum plasma/serum concentration that a drug achieves after dosing.
  • Chough corresponds to trough plasma concentration at the end of a dosing interval at steady state (typically taken directly before next administration)
  • RNAs functional assembly of a CLDN-18.2-targeting antibody agent encoded by RNAs can be determined in vitro and in vivo in a dose-dependent manner, e.g., as described in Example 6.
  • binding specificity, mediation of ADCC and CDC, and/or anti-tumor activity of CLDN-18.2-targeting antibody agent encoded by RNA(s) described herein can be determined, e.g., as described in Examples 1-4.
  • the present disclosure provides a method comprising a step of: determining one or more features of an antibody agent expressed from at least one mRNA introduced into cells, wherein such at least one mRNA comprises one or more of features of at least one or more RNA comprising a coding region that encodes an antibody agent that binds to a Claudin-18.2 (CLDN-18.2) polypeptide, e.g., binds preferentially to a Claudin-18.2 (CLDN- 18.2) polypeptide relative to a Claudin-18.1 polypeptide, wherein such one or more features comprises: (i) protein expression level of an antibody agent; (ii) binding specificity of an antibody agent to CLDN-18.2; (iii) efficacy of an antibody agent to mediate target cell death through ADCC; and (iv) efficacy of an antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • a method of characterizing a pharmaceutical composition targeting CLDN-18.2. Such a method comprises steps of: (a) contacting cells with at least one composition or pharmaceutical composition described herein (which encodes part or all of a CLDN-18.2-targeting antibody agent); and detecting an antibody agent produced by the cells.
  • the cells may be or comprise liver cells.
  • such a method may further comprise determining one or more features of an antibody agent expressed from one or more RNAs described herein, wherein such one or more features comprises: (i) protein expression level of the antibody agent; (ii) binding specificity of the antibody agent to a CLDN-18.2 polypeptide; (iii) efficacy of the antibody agent to mediate target cell death through ADCC; and (iv) efficacy of the antibody agent to mediate target cell death through complement dependent cytotoxicity (CDC).
  • a step of determining one or more features of an antibody agent expressed from one or more RNAs described herein may comprise comparing such features of the CLDN-18.2- targeting antibody agent with that of a reference CLDN-18.2-targeting antibody.
  • a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise characterizing an antibody agent expressed from one or more RNAs described herein as a CLDN-18.2-targeting antibody agent if the antibody agent comprises the following features: (a) protein level of the antibody agent expressed by the cells above a threshold level; (b) preferential binding of the antibody agent to CLDN-18.2 relative to CLDN18.1; and (c) killing of at least 50% target cells (e.g., cancer cells) mediated by ADCC and/or CDC.
  • target cells e.g., cancer cells
  • a provided method of characterizing a pharmaceutical composition targeting CLDN-18.2 or components thereof may further comprise characterizing an antibody agent expressed from one or more RNAs described herein as a Zolbetuximab or Claudiximab-equivalent antibody if tested features of the antibody are at least comparable to that of Zolbetuximab or Claudiximab.
  • such a method of characterizing may further comprise administering a composition or pharmaceutical composition described herein to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor to determine anti-tumor activity, if such a composition or pharmaceutical composition is characterized as a CLDN-18.2-targeting antibody agent.
  • RNA or composition thereof which RNA encodes part or all of an antibody agent, which one or more features are selected from the group consisting of:
  • a reference standard can be any quality control standard, including, e.g., a historical reference, a set specification. As will be understood by a skilled artisan, in some embodiments, a direct comparison is not required.
  • a reference standard is an acceptance criterion based on, for example, physical appearance, lipid identity and/or content, LNP size, LNP polydispersity, RNA encapsulation, RNA length, identity (as RNA), integrity, sequence, and/or concentration, pH, osmolality, RNA ratio (e.g., ratio of a HC RNA to a LC RNA), potency, bacterial endotoxins, bioburden, residual organic solvent, osmolality, pH, and combinations thereof.
  • RNA e.g., ones described herein
  • a composition comprising an RNA e.g., ones described herein
  • such a method may further comprise administering the formulation and/or composition to a group of animal subjects each bearing a huma CLDN-18.2 positive xenograft tumor to determine antitumor activity.
  • cells are present in a cell culture.
  • pancreatic adenocarcinoma or metastatic biliary tract cancers still do not yet benefit from existing immunotherapies.
  • This phenomenon is multifactorial, attributed to pancreatic ductal adenocarcinoma (PDAC)’s systemic and aggressive nature, its complex mutational landscape, its desmoplastic stroma, and a potently immunosuppressive tumor microenvironment.
  • PDAC pancreatic ductal adenocarcinoma
  • the poor prognosis of these two cancer types highlights the need for additional treatment approaches.
  • the present disclosure provides an insight that CLDN-18.2 represents a particularly useful tumor-associated antigen against which therapies may be targeted. To date, no therapy targeting CLDN-18.2 has been approved for any cancer indication. Accordingly, in some embodiments, the present disclosures provides an insight that RNA-encoded antibodies targeting CLDN-18.2 can induce ADCC and/or CDC and/or augment cytotoxic effect(s) of chemotherapy and/or other anti-cancer therapy, thus translating into prolonged progression- free and/or overall survival, e.g., relative to the individual therapies administered alone and/or to another appropriate reference.
  • Pancreatic ductal adenocarcinoma is the most prevalent neoplastic disease of the pancreas accounting for more than 90% of all pancreatic malignancies (Kleeff et al. 2016). To date, PDAC is the fourth most frequent cause of cancer-related deaths worldwide with a 5-year overall survival of less than 8% (Siegel et al. 2018). The incidence of PDAC is expected to rise further in the future, and projections indicate a more than 2-fold increase in the number of cases within the next 10 years, both in terms of new diagnoses as well as in terms of PDAC -related deaths in the United States and European countries (Quante et al. 2016; Rahib et al. 2014; Cancer Research UK).
  • the randomized controlled trial comparing erlotinib with placebo showed a 0.4-month median OS benefit and a 0.3-month median PFS benefit.
  • the therapy described herein, targeting the CLDN-18.2+ subpopulation of PDAC, could potentially address a population with significantly high unmet medical need.
  • technologies provided herein can be useful for treatment of diseases or conditions associated with elevated expression and/or activity of CLDN-18.2.
  • technologies provided herein can be useful for treatment of CLDN-18.2 positive solid tumors.
  • CLDN-18.2 positive solid tumors are determined by immunohistochemical analysis with a staining intensity score of 2 or higher in accordance with the practice of skilled pathologists.
  • the present disclosure recognizes that pancreatic cancers and biliary cancers typically have high expression of CLDN-18.2. Accordingly, in some embodiments, technologies provided herein can be useful for treatment of pancreatic cancers.
  • technologies provided herein can be useful for treatment of pancreatic ductal adenocarcinoma (PDAC).
  • technologies provided herein can be useful for treatment of biliary cancers.
  • technologies provided herein can be useful for treatment of gastroesophageal cancer that are determined to be CLDN-18.2 positive, e.g., by immunohistochemical analysis.
  • technologies provided herein can be useful for treatment of non-small cell lung cancer (NSCLC) that are determined to be CLDN- 18.2 positive, e.g., by immunohistochemical analysis.
  • NSCLC non-small cell lung cancer
  • technologies provided herein can be useful for treatment of patients e.g., adult patients) with CLDN-18.2+ solid tumors that are metastatic. In some embodiments, technologies provided herein can be useful for treatment of patients (e.g., adult patients) with CLDN-18.2+ solid tumors that are unresectable, e.g., in some embodiments where surgical resection is likely to result in severe morbidity. In some embodiments, technologies provided herein can be useful for treatment of patients (e.g., adult patients) with CLDN-18.2+ solid tumors that are locally advanced. Additionally or alternatively, in some embodiments, cancer in such patients may have progressed following treatment or such cancer patients may have no satisfactory alternative therapy.
  • such chemotherapy may be expected or predicted to elevate expression and/or activity of CLDN-18.2, or may result or have resulted in expression and/or activity of CLDN-18 by at least 50% or more, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or higher, as compared to expression and/or activity of CLDN-18.2 in the absence of such chemotherapy.
  • one aspect of the present disclosure relates to methods of using pharmaceutical compositions described herein.
  • a method comprising administering a provided pharmaceutical composition to a subject suffering from a CLDN-18.2-positive solid tumor.
  • a provided pharmaceutical composition is administered by intravenous injection or infusion.

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Abstract

La présente divulgation concerne des technologies d'ARN ciblant des polypeptides de la claudine 18.2. Selon certains modes de réalisation, de telles technologies d'ARN peuvent être utiles dans le traitement de maladies associées à l'expression positive de la claudine 18.2. Par exemple, selon certains modes de réalisation, de telles technologies d'ARN peuvent être utiles dans le traitement des cancers exprimant positivement la claudine 18.2, notamment, et de façon non limitative, le cancer de la vésicule biliaire, le cancer des ovaires, le cancer de l'estomac, le cancer gastro-œsophagien et le cancer du pancréas. Selon certains modes de réalisation, de telles technologies d'ARN peuvent être utilisées en polythérapie (par exemple, en association à un agent chimiothérapeutique). La présente invention concerne en outre des squelettes d'ARN contenant des séquences spécifiques en amont et/ou en aval de la séquence codante.
EP23785777.6A 2022-10-06 2023-10-05 Compositions d'arn ciblant la claudine 18.2 Pending EP4598954A1 (fr)

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