Agents encoding CLDN6 and CD3 binding elements for treating CLDN6-positive cancers

Abstract

The present invention generally relates to binding agents that are bispecific for binding to at least CD3 and CLDN6, i.e., capable of binding to at least CD3 and CLDN6. In particular, the present invention relates to RNA encoding these binding agents that can be used to treat or prevent cancer in a subject.

Description

[Background technology]

[0001] Cancer is the second leading cause of death worldwide and was estimated to be responsible for 10 million deaths in 2020. In general, once solid tumors metastasize, with a few exceptions such as germ cell and some carcinoid tumors, the 5-year survival rate rarely exceeds 25%.

[0002] Recent advances in immunotherapy, as well as refinements in traditional treatments such as chemotherapy, radiation therapy, surgery, and targeted therapy, have improved outcomes for patients with advanced solid tumors. In the last few years, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved several immune checkpoint inhibitors for the treatment of patients with multiple cancer types, primarily solid tumors. These approvals have dramatically changed the landscape of cancer treatment.

[0003] The poor prognosis in metastatic or locally advanced cancers highlights the need for additional therapeutic approaches. One such approach is that of targeted therapy, a constantly evolving field with promising modalities.

[0004] CLDN6 belongs to the PMP-22 / EMP / MP20 / claudin superfamily of tetraspanin membrane proteins (Pfam database ID: PF00822) that are involved in the formation of apical tight junction complexes in epithelial and endothelial cell sheets and play a key role in maintaining cell polarity (Krause G.et al.,Biochim Biophys Acta.2008;1778(3):631-645). Importantly, expression of claudin proteins is restricted to the tight junctions of cells and is only available for ion transport under standard physiological conditions (Krause G.et al.,Biochim Biophys Acta.2008;1778(3):631-645). Otherwise, little else is known about the in vivo functions of CLDN6.

[0005] CLDN6 has four transmembrane helices whose N- and C-termini extend into the cytoplasm. The short N-terminal sequence of CLDN6 is followed by a large extracellular loop (EL1), a short intracellular loop, a second extracellular loop (EL2), and a C-terminal cytoplasmic tail (Colegio OR et al., Am J Physiol Cell Physiol. 2002; 283(1):C142-C147).

[0006] No isoforms of CLDN6 have been identified so far (Lal-Nag M.et al.,Genome Biol.2009;10(8):235). Claudin family members CLDN3, CLDN4 and CLDN9 share sequence homology with CLDN6. CLDN3 and CLDN4 are generally expressed in normal epithelial cells of the lung, liver, breast, pancreas, kidney and intestine (Kwon M.et al.,Int J Mol Sci.2013;14(9):18148-18180). CLDN9 expression is absent in most normal tissues; however, CLDN9 expression has been reported in the cochlea and vestibule of the mouse inner ear (Kitajiri SI et al., Hear Res. 2004; 187(1-2):25-34; Nakano Y. et al., PLoS Genet. 2009; 5(8):e1000610), as has been reported in humans where CLDN9 gene truncation is associated with hearing impairment (Sineni C. et al., Human genetics 2019; 138(10):1071-1075).

[0007] The oncofetal protein CLDN6 is almost exclusively expressed in embryonic stem cells, then rapidly downregulated during differentiation into neural or cardiac lineages, and is not expressed in normal adult tissues except placenta (Assou S. et al., Stem Cells. 2007;25(4):961-973; Ben-David U. et al., Nat Commun. 2013;4:1992; Reinhard K. et al., Science. 2020;367(6476):446-453). CLDN6 is expressed in a variety of human cancer types, including testicular, ovarian, endometrial and lung cancer. Representative studies showed that approximately 93% of testicular cancers of all histological subtypes stain highly positive for CLDN6, defined by staining intensity ≥2+. Furthermore, 56% of ovarian cancers stained positive for CLDN6, of which 20–25% showed high (≥2+) cell membrane staining in >50% of tumor cells. Compared with primary ovarian cancers, the frequency of CLDN6-positive samples was significantly increased in metastatic lesions (72%; data not shown), correlating CLDN6 expression with disease progression. 23% of endometrial and 11% of lung cancers stained positive for CLDN6, of which 10–15% and 2–5%, respectively, showed staining intensity of ≥2+. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Krause G. et al., Biochim Biophys Acta.2008;1778(3):631-645 [Non-Patent Document 2] Colegio ORet al.,Am J Physiol Cell Physiol.2002;283(1):C142-C147 [Non-Patent Document 3] Lal-Nag M. et al.,Genome Biol.2009;10(8):235 [Non-Patent Document 4] Kwon M. et al.,Int J Mol Sci.2013;14(9):18148-18180 [Non-Patent Document 5] Sineni C.et al.,Human genetics 2019;138(10):1071-1075 [Non-Patent Document 6] Kitajiri SIet al.,Hear Res.2004;187(1-2):25-34 [Non-Patent Document 7] Nakano Y. et al.,PLoS Genet.2009;5(8):e1000610 [Non-Patent Document 8] Assou S. et al.,Stem Cells.2007;25(4):961-973 [Non-Patent Document 9] Ben-David U.et al.,Nat Commun.2013;4:1992 [Non-Patent Document 10] Reinhard K.et al.,Science.2020;367(6476):446-453 Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention was to provide novel agents and methods for the treatment of CLDN6-positive cancer diseases.

[0010] In some embodiments, the solution to the problem underlying the present invention is based on the concept of administering RNA expressed by cells of a patient to express a polypeptide chain that forms a binder comprising two binding domains specific for CLDN6 expressed by the cancer cells and a binding domain specific for CD3 expressed by the T cells, thus making it possible to target the cytotoxic effect of the T cells against the cancer cells. [Means for solving the problem]

[0011] The present invention generally relates to a binding agent that is bispecific for binding to at least CD3 and CLDN6, i.e., can bind to at least CD3 and CLDN6.Specifically, the present invention relates to RNA that codes for these binding agents that can be used to treat or prevent cancer in a subject.In particular, RNA that codes for the binding agents disclosed herein can be administered to provide the binding agent (after expression of the RNA by appropriate target cells) for targeting CD3 and CLDN6.

[0012] Thus, the pharmaceutical composition described herein may comprise as active ingredient a single-stranded RNA that can be translated into the respective encoded polypeptide when entering the recipient's cell. In addition to the sequence encoding the binding agent, the RNA may comprise one or more structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) that are optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency. In some embodiments, the RNA comprises all of these elements.

[0013] The RNA described herein can be complexed with protein and / or lipid, preferably lipid, to produce RNA particles for administration.Different RNAs can be complexed together, or can be complexed separately with protein and / or lipid to produce RNA particles for administration.

[0014] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a first RNA encoding a first polypeptide chain comprising a variable region (VH) of a heavy chain derived from an immunoglobulin having specificity for CD3 (VH(CD3)), a variable region (VH) of a heavy chain derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region (VL) of a light chain derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)); and (ii) a second RNA encoding a second polypeptide chain comprising a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CD3 (VL(CD3)), a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)). The present invention provides a composition or pharmaceutical formulation comprising:

[0015] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form a binding domain with specificity for CD3 and two binding domains with specificity for CLDN6.

[0016] In some embodiments, the VH(CD3) of the first polypeptide chain and the VL(CD3) of the second polypeptide chain interact to form a binding domain specific for CD3; VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact with each other to form a binding domain specific for CLDN6, VH(CLDN6) and VL(CLDN6) of the second polypeptide chain interact with each other to form a binding domain having specificity for CLDN6.

[0017] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a first RNA encoding a first polypeptide chain comprising a variable region VH(CD3), a variable region VH(CLDN6) and a variable region VL(CLDN6); and (ii) a second RNA encoding a second polypeptide chain comprising a variable region VL(CD3), a variable region VH(CLDN6) and a variable region VL(CLDN6); wherein the VH(CD3) of the first polypeptide chain interacts with the VL(CD3) of the second polypeptide chain to form a binding domain specific for CD3; VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact with each other to form a binding domain specific for CLDN6, VH(CLDN6) and VL(CLDN6) of the second polypeptide chain interact with each other to form a binding domain specific for CLDN6. A composition or pharmaceutical formulation is provided.

[0018] In some embodiments, the first and second polypeptide chains comprise a heavy chain constant region 1 (CH1) derived from an immunoglobulin or functional variant thereof and a light chain constant region (CL) derived from an immunoglobulin or functional variant thereof.

[0019] In some embodiments, the immunoglobulin is IgG1.

[0020] In some embodiments, the IgG1 is human IgG1.

[0021] In some embodiments, the VH, VL, and CH1 on the first polypeptide chain are VH(CD3)-CH1-VH(CLDN6)-VL(CLDN6), or VH(CD3)-CH1-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0022] In some embodiments, CH1 is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence SGPGGGRS(G4S)2 or a functional variant thereof.

[0023] In some embodiments, the VH, VL, and CL on the second polypeptide chain are VL(CD3)-CL-VH(CLDN6)-VL(CLDN6), or VL(CD3)-CL-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0024] In some embodiments, CL is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence DVPGGS or a functional variant thereof.

[0025] In some embodiments, VH(CLDN6) and VL(CLDN6) are connected to each other by a peptide linker. In some embodiments, the peptide linker is the amino acid sequence (G4S) x or a functional variant thereof, wherein x is 2, 3, 4, 5 or 6. In one embodiment, the peptide linker comprises the amino acid sequence (G4S)4 or a functional variant thereof.

[0026] In some embodiments, a CH1 on a first polypeptide chain interacts with a CL on a second polypeptide chain.

[0027] In some embodiments, VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO:4.

[0028] In some embodiments, the VH(CD3) comprises a CDR1 comprising the amino acid sequence GYTFTRYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYSLDY or a functional variant thereof.

[0029] In some embodiments, the VH(CD3) comprises a CDR1 comprising the amino acid sequence GYTFTRYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYCLDY or a functional variant thereof.

[0030] In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, or a functional variant thereof.

[0031] In some embodiments, the VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO:6.

[0032] In some embodiments, the VL(CD3) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence DTS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQWSSNPLT or a functional variant thereof.

[0033] In some embodiments, the VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, or a functional variant thereof.

[0034] In some embodiments, VH(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO:4.

[0035] In some embodiments, VH(CLDN6) comprises a CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY or a functional variant thereof.

[0036] In some embodiments, VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, or a functional variant thereof.

[0037] In some embodiments, VL(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4.

[0038] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO:4).

[0039] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO:4).

[0040] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at a position corresponding to position 449 of SEQ ID NO:4.

[0041] In some embodiments, VL(CLDN6) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence STS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof.

[0042] In some embodiments, VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4, or a functional variant thereof.

[0043] In some embodiments, VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, VH(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, and VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4, and preferably a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO: 4) and / or a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO: 4).

[0044] In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO:4 or a functional variant thereof; VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO:6 or a functional variant thereof; VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4 or a functional variant thereof; and / or VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4 or a functional variant thereof.

[0045] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof.

[0046] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:6, or a functional variant thereof.

[0047] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

[0048] In some embodiments, at least one of the first and second polypeptides is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content has been increased compared to a wild-type coding sequence, wherein the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0049] In some embodiments, each of the first and second polypeptides is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to a wild-type coding sequence, and the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0050] In some embodiments, the RNA includes modified nucleosides in place of uridines. In such cases, preferably, a modified nucleoside is present in place of every or essentially every uridine in the RNA.

[0051] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0052] In some embodiments, at least one RNA is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG.

[0053] In some embodiments, each RNA is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG.

[0054] In some embodiments, at least one RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0055] In some embodiments, each RNA comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0056] In some embodiments, at least one RNA comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0057] In some embodiments, each RNA comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0058] In some embodiments, at least one RNA comprises a polyA sequence.

[0059] In some embodiments, each RNA comprises a polyA sequence.

[0060] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0061] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:10.

[0062] In some embodiments, (i) the first RNA and the second RNA are in a (w / w) ratio of about 1.75:1 to about 1.25:1, or about 1.5:1 to about 1.25:1, or preferably about 1.5:1; and / or (ii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine; and / or (iii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine, the modified nucleoside being independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U); and / or (iv) the first RNA and the second RNA are 5' capped m2 7,3’-O Gppp(m1 2’-O ) ApG; and / or (v) the first RNA and the second RNA comprise a 5'UTR comprising a nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and / or (vi) the first RNA and the second RNA comprise a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9; and / or (vii) the first RNA and the second RNA comprise a polyA tail comprising the nucleotide sequence of SEQ ID NO:10.

[0063] In some embodiments, (i) the first RNA and the second RNA are in a (w / w) ratio of about 1.75:1 to about 1.25:1, or about 1.5:1 to about 1.25:1, or preferably about 1.5:1; (ii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine, the modified nucleoside being N1-methylpseudouridine (m1ψ); (iii) the first RNA and the second RNA are 5' capped m2 7,3’-O Gppp(m1 2’-O ) including ApG; (iv) the first RNA and the second RNA comprise a 5' UTR comprising the nucleotide sequence of SEQ ID NO:8; (v) the first RNA and the second RNA comprise a 3'UTR comprising the nucleotide sequence of SEQ ID NO:9; and (vi) the first RNA and the second RNA comprise a polyA tail comprising the nucleotide sequence of SEQ ID NO:10.

[0064] In some embodiments, (i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an amino acid sequence of SEQ ID NO:4; and / or (ii) the first RNA comprises a nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0065] In some embodiments, (i) the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an amino acid sequence of SEQ ID NO:6; and / or (ii) the second RNA comprises the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0066] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a first RNA encoding a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4; and (ii) a second RNA encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6. The present invention provides a composition or pharmaceutical formulation comprising:

[0067] In some embodiments of all aspects, the first RNA comprises a nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0068] In some embodiments of all aspects, the second RNA comprises a nucleotide sequence of SEQ ID NO:7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0069] In one aspect, the invention provides a composition or pharmaceutical formulation comprising a first RNA comprising the nucleotide sequence of SEQ ID NO:35, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:35, and / or a second RNA comprising the nucleotide sequence of SEQ ID NO:36, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:36.

[0070] In one aspect, the invention provides a composition or pharmaceutical formulation comprising a first RNA comprising the nucleotide sequence of SEQ ID NO:5, and a second RNA comprising the nucleotide sequence of SEQ ID NO:7, wherein the first RNA and the second RNA are present in a first RNA to second RNA (w / w) ratio of about 1.5:1.

[0071] In some embodiments of all aspects, the RNA is mRNA.

[0072] In some embodiments of all aspects, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0073] In some embodiments of all aspects, the RNA is or is to be formulated for injection.

[0074] In some embodiments of all aspects, the RNA is formulated or is to be formulated for intravenous administration.

[0075] In some embodiments of all aspects, the RNA is or is to be formulated as a particle.

[0076] In some embodiments, the particle is a lipid nanoparticle (LNP).

[0077] In some embodiments, the LNP particles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0078] In some embodiments of all aspects, the composition or pharmaceutical formulation is a pharmaceutical composition.

[0079] In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents and / or excipients.

[0080] In some embodiments of all aspects, the composition or pharmaceutical formulation is a kit.

[0081] In some embodiments, the RNA and optionally the particle-forming components are in separate vials.

[0082] In some embodiments, the composition or pharmaceutical preparation further comprises instructions for using the composition or pharmaceutical preparation to treat or prevent cancer.

[0083] In one aspect, the invention provides a composition or pharmaceutical formulation as described herein for pharmaceutical use.

[0084] In some embodiments, medical uses include therapeutic or prophylactic treatment of a disease or disorder.

[0085] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder includes treating or preventing cancer.

[0086] In some embodiments, the therapeutic or prophylactic treatment of a disease or disorder further comprises administering an additional therapy.

[0087] In some embodiments, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, resect, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy.

[0088] In some embodiments, the additional therapy comprises administering an additional therapeutic agent.

[0089] In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent.

[0090] In some embodiments, the compositions or pharmaceutical formulations described herein are for administration to humans.

[0091] In one aspect, the invention provides a method of treating cancer in a subject, comprising: (i) a first RNA encoding a first polypeptide chain comprising a variable region (VH) of a heavy chain derived from an immunoglobulin having specificity for CD3 (VH(CD3)), a variable region (VH) of a heavy chain derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region (VL) of a light chain derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)); and (ii) a second RNA encoding a second polypeptide chain comprising a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CD3 (VL(CD3)), a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)). The method includes administering to a subject

[0092] In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form a binding domain with specificity for CD3 and two binding domains with specificity for CLDN6.

[0093] In some embodiments, the VH(CD3) of the first polypeptide chain and the VL(CD3) of the second polypeptide chain interact to form a binding domain specific for CD3; VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact with each other to form a binding domain specific for CLDN6, VH(CLDN6) and VL(CLDN6) of the second polypeptide chain interact with each other to form a binding domain having specificity for CLDN6.

[0094] In one aspect, the invention provides a method of treating cancer in a subject, comprising: (i) a first RNA encoding a first polypeptide chain comprising a variable region VH(CD3), a variable region VH(CLDN6) and a variable region VL(CLDN6); and (ii) a second RNA encoding a second polypeptide chain comprising a variable region VL(CD3), a variable region VH(CLDN6) and a variable region VL(CLDN6); administering to a subject, wherein the VH(CD3) of the first polypeptide chain interacts with the VL(CD3) of the second polypeptide chain to form a binding domain specific for CD3; VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact with each other to form a binding domain specific for CLDN6, The method includes: a) administering to the subject a polypeptide chain comprising: a first polypeptide chain and a second polypeptide chain; a second polypeptide chain and a second polypeptide chain;

[0095] In some embodiments, the first and second polypeptide chains comprise a heavy chain constant region 1 (CH1) derived from an immunoglobulin or functional variant thereof and a light chain constant region (CL) derived from an immunoglobulin or functional variant thereof.

[0096] In some embodiments, the immunoglobulin is IgG1.

[0097] In some embodiments, the IgG1 is human IgG1.

[0098] In some embodiments, the VH, VL, and CH1 on the first polypeptide chain are VH(CD3)-CH1-VH(CLDN6)-VL(CLDN6), or VH(CD3)-CH1-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0099] In some embodiments, CH1 is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence SGPGGGRS(G4S)2 or a functional variant thereof.

[0100] In some embodiments, the VH, VL, and CL on the second polypeptide chain are VL(CD3)-CL-VH(CLDN6)-VL(CLDN6), or VL(CD3)-CL-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0101] In some embodiments, CL is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence DVPGGS or a functional variant thereof.

[0102] In some embodiments, VH(CLDN6) and VL(CLDN6) are connected to each other by a peptide linker. In some embodiments, the peptide linker is the amino acid sequence (G4S) x or a functional variant thereof, and x is 2, 3, 4, 5 or 6. In some embodiments, the peptide linker comprises the amino acid sequence (G4S)4 or a functional variant thereof.

[0103] In some embodiments, a CH1 on a first polypeptide chain interacts with a CL on a second polypeptide chain.

[0104] In some embodiments, VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO:4.

[0105] In some embodiments, the VH(CD3) comprises a CDR1 comprising the amino acid sequence GYTFTRYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYSLDY or a functional variant thereof.

[0106] In some embodiments, the VH(CD3) comprises a CDR1 comprising the amino acid sequence GYTFTRYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYCLDY or a functional variant thereof.

[0107] In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, or a functional variant thereof.

[0108] In some embodiments, the VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO:6.

[0109] In some embodiments, the VL(CD3) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence DTS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQWSSNPLT or a functional variant thereof.

[0110] In some embodiments, the VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, or a functional variant thereof.

[0111] In some embodiments, VH(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO:4.

[0112] In some embodiments, VH(CLDN6) comprises a CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARDYGFVLDY or a functional variant thereof.

[0113] In some embodiments, VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, or a functional variant thereof.

[0114] In some embodiments, VL(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4.

[0115] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO:4).

[0116] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO:4).

[0117] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 404 to 510 of SEQ ID NO:4, and a serine residue at a position corresponding to position 449 of SEQ ID NO:4.

[0118] In some embodiments, VL(CLDN6) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence STS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof.

[0119] In some embodiments, VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4, or a functional variant thereof.

[0120] In some embodiments, VH(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, VL(CD3) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, VH(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, and VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4, and preferably a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO: 4) and / or a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO: 4).

[0121] In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO:4 or a functional variant thereof; VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO:6 or a functional variant thereof; VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4 or a functional variant thereof; and / or VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4 or a functional variant thereof.

[0122] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof.

[0123] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:6, or a functional variant thereof.

[0124] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

[0125] In some embodiments, at least one of the first and second polypeptides is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content has been increased compared to a wild-type coding sequence, wherein the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0126] In some embodiments, each of the first and second polypeptides is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to a wild-type coding sequence, and the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0127] In some embodiments, the RNA includes modified nucleosides in place of uridines. In such cases, preferably, a modified nucleoside is present in place of every or essentially every uridine in the RNA.

[0128] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0129] In some embodiments, at least one RNA is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG.

[0130] In some embodiments, each RNA is 5' capped m2 7,3’-O Gppp(m1 2’-O )Contains ApG.

[0131] In some embodiments, at least one RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0132] In some embodiments, each RNA comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0133] In some embodiments, at least one RNA comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0134] In some embodiments, each RNA comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0135] In some embodiments, at least one RNA comprises a polyA sequence.

[0136] In some embodiments, each RNA comprises a polyA sequence.

[0137] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0138] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:10.

[0139] In some embodiments, (i) the first RNA and the second RNA are in a (w / w) ratio of about 1.75:1 to about 1.25:1, or about 1.5:1 to about 1.25:1, or preferably about 1.5:1; and / or (ii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine; and / or (iii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine, the modified nucleoside being independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U); and / or (iv) the first RNA and the second RNA are 5' capped m2 7,3’-O Gppp(m1 2’-O ) ApG; and / or (v) the first RNA and the second RNA comprise a 5'UTR comprising a nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8; and / or (vi) the first RNA and the second RNA comprise a 3'UTR comprising a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9; and / or (vii) the first RNA and the second RNA comprise a polyA tail comprising the nucleotide sequence of SEQ ID NO:10.

[0140] In some embodiments, (i) the first RNA and the second RNA are in a (w / w) ratio of about 1.75:1 to about 1.25:1, or about 1.5:1 to about 1.25:1, or preferably about 1.5:1; (ii) the first RNA and the second RNA comprise a modified nucleoside in place of each uridine, the modified nucleoside being N1-methylpseudouridine (m1ψ); (iii) the first RNA and the second RNA are 5' capped m2 7,3’-O Gppp(m1 2’-O ) including ApG; (iv) the first RNA and the second RNA comprise a 5' UTR comprising the nucleotide sequence of SEQ ID NO:8; (v) the first RNA and the second RNA comprise a 3'UTR comprising the nucleotide sequence of SEQ ID NO:9; and (vi) the first RNA and the second RNA comprise a polyA tail comprising the nucleotide sequence of SEQ ID NO:10.

[0141] In some embodiments, (i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an amino acid sequence of SEQ ID NO:4; and / or (ii) the first RNA comprises a nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0142] In some embodiments, (i) the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an amino acid sequence of SEQ ID NO:6; and / or (ii) the second RNA comprises the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0143] In one aspect, the invention provides a method of treating cancer in a subject, comprising: (i) a first RNA encoding a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4; and (ii) a second RNA encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6. The method includes administering to a subject

[0144] In some embodiments of all aspects, the first RNA comprises a nucleotide sequence of SEQ ID NO:5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5.

[0145] In some embodiments of all aspects, the second RNA comprises a nucleotide sequence of SEQ ID NO:7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0146] In one aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a first RNA comprising the nucleotide sequence of SEQ ID NO:35, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:35, and / or a second RNA comprising the nucleotide sequence of SEQ ID NO:36, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:36.

[0147] In one aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject a first RNA comprising the nucleotide sequence of SEQ ID NO:5 and a second RNA comprising the nucleotide sequence of SEQ ID NO:7, wherein the first RNA and second RNA are administered in a (w / w) ratio of about 1.5:1 of the first RNA to the second RNA.

[0148] In some embodiments of all aspects, the RNA is mRNA.

[0149] In some embodiments of all aspects, the RNA is formulated as a liquid, a solid, or a combination thereof.

[0150] In some embodiments of all aspects, the RNA is administered by injection.

[0151] In some embodiments of all aspects, the RNA is administered once a week.

[0152] In some embodiments of all aspects, the RNA is administered by intravenous administration.

[0153] In some embodiments of all aspects, the RNA is formulated as a particle.

[0154] In some embodiments, the particle is a lipid nanoparticle (LNP).

[0155] In some embodiments, the LNP particles comprise ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0156] In some embodiments of all aspects, the RNA is formulated in a pharmaceutical composition.

[0157] In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents and / or excipients.

[0158] In some embodiments of all aspects, the methods described herein further comprise administering an additional therapy.

[0159] In some embodiments, the additional therapy comprises one or more selected from the group consisting of: (i) surgery to remove, resect, or debulk the tumor; (ii) radiation therapy; and (iii) chemotherapy.

[0160] In some embodiments, the additional therapy comprises administering an additional therapeutic agent.

[0161] In some embodiments, the additional therapeutic agent comprises an anti-cancer therapeutic agent.

[0162] In some embodiments of all aspects, the subject is a human.

[0163] In some embodiments of all aspects, the cancer is a CLDN6 positive cancer.

[0164] In one aspect, the invention provides a composition or pharmaceutical formulation as described herein for use in the methods described herein.

[0165] In one aspect, the invention relates to an agent or composition as described herein for use in the methods described herein. [Brief description of the drawings]

[0166] [Figure 1] General Structure of RNA Drug Substance BNT142 Schematic diagram of the general structure of an RNA drug substance with a 5' cap (herein "Cap 1"), a 5'-UTR and a 3'-UTR. CH = constant heavy domain; CL = constant light domain; DS = drug substance; L = linker; m = messenger; Sec = secretory signal peptide sequence; Poly(A) = polyadenine tail; RNA = ribonucleic acid; UTR = untranslated region; VH = variable heavy domain; VL = variable light domain. [Diagram 2]RiboMab02.1 specifically binds human CD3 and CLDN6 and does not show off-target binding to the closely related CLDN3, 4 and 9 or non-human primate CD3. Target binding of RiboMab02.1 was determined by flow cytometry binding assays using an APC-labeled goat anti-mouse IgG (heavy and light chain [H+L]) secondary antibody. Cells were first gated on singlets and subsequently gated on viable lymphocytes (PBMCs) or viable HEK-293T-17 cells. RiboMab02.1 in HEK-293T-17 supernatant at a concentration of 10 μg / mL was used. Cynomolgus or human PBMCs and HEK-293T-17 transductants stably expressing luciferase (HEK-293T-17_mock), CLDN3, 4, 6 or 9 served as target cells, as indicated. The vertical dotted line indicates the peak position of the unstained population. APC = allophycocyanin; CLDN = claudin; HEK = human embryonic kidney; PBMC = peripheral blood mononuclear cells. [Diagram 3] RiboMab02.1 expressed in mice mediates dose-dependent and target-specific tumor cell lysis in vitro for multiple PBMC donors Human PBMCs from eight healthy donors were co-cultured with luciferase-expressing tumor cells. CLDN6-positive PA-1 (A) or OV-90 (B) cell lines were used as target cells, and the CLDN6-negative MDA-MB-231 (A, B) cell line was used to control for target specificity. The assay was performed in a 384-well plate format with an effector-to-target cell ratio of 20:1. Bioluminescence of viable tumor cells was measured as readout. Specific lysis percentages of tumor cell killing are shown. RiboMab02.1-containing mouse serum was serially diluted (10-fold, 10 points; range: 5.0 × 10-7 to 500 ng / mL) and added to the co-cultures, followed by incubation with (A) PA-1 cells for 24 h or (B) OV-90 cells for 48 h. Each line represents tumor cell lysis by an individual donor's PBMC sample. Error bars indicate standard deviation (SD) of the mean (technical triplicates). CDLN = claudins; EC50 = half maximal effective concentration, PBMC = peripheral blood mononuclear cells. [Figure 4] RiboMab02.1 induces dose-dependent and target-dependent T cell proliferation CFSE-labeled human PBMCs from three healthy donors were co-cultured with CLDN6-positive PA-1 and OV-90 target cells, or with CLDN6-negative but control TAA-positive NUGC-4 and target-negative MDA-MB-231 control cells in a 12-well culture plate format. An effector-to-target cell ratio of 10:1 was applied. In addition, separate (-) PBMCs without target cells were included. HEK-293T-17 supernatants containing 100 and 1 ng / mL RiboMab02.1 (first and second columns of each block of five columns) or control RiboMab (third and fourth columns of each block of five columns) were added to the co-cultures as indicated. OKT3 antibody (anti-human CD3; black bars) served as a positive control for target-independent CD3-driven T cell proliferation. After 72 h of incubation, the percentage of proliferating T cells was analyzed by flow cytometry. Error bars indicate the standard deviation (SD) of the mean of all three donors. CFSE = carboxyfluorescein succinimidyl ester; PBMC = peripheral blood mononuclear cells; w / o = without. [Diagram 5]RiboMab02.1 mediates dose-dependent T cell activation at high concentrations with low target-independent effects Human PBMCs (effector cells) from three healthy donors were cultured in the presence and absence of CLDN6-positive PA-1 cells (target cells) at an effector-to-target cell ratio of 10:1. RiboMab02.1-containing mouse sera were serially diluted (10-fold, 10 points; range: 4.0 × 10-6 to 4,000 ng / mL) before use in the assay. After 48 h of co-incubation, cells were stained with anti-CD5, anti-CD69 and anti-CD25 antibodies for flow cytometric analysis of T cells. Total T cell activation normalized to samples incubated with mock serum from Luc_RNA-LNP-treated mice is shown here. The percentage of activated T cells is shown as the average (right) for each individual donor (left) and for all three donors. Filled symbols represent values ​​with target cells, open symbols represent values ​​without target cells. Error bars indicate standard deviation (SD) of the mean (technical triplicates [per donor, left panel] or biological replicates [across donors, right panel]). EC50 = half maximal effective concentration; w / o = without. [Figure 6A] (Figure 6) BNT142 treatment eliminates advanced xenograft tumors in PBMC-humanized NSG mice by T cell redirection to tumors. NSG mice bearing advanced SC tumor xenografts of OV-90 cells (mean tumor volume at the start of treatment = 100 mm3) were implanted with human PBMCs as effector cells by IP injection. Tumor volumes were measured twice weekly with a digital caliper. Mice were treated once weekly with 5 IV bolus injections of 0.1 or 1 μg BNT142, or 1 μg RNA-LNPs encoding a target-irrelevant RiboMab tribody (negative control for target specificity), 1 μg Luc_RNA-LNPs, 100 μg recombinant purified CD3x(CLDN6)2 tribody reference protein or DPBS (physiological saline) as vehicle control. (Figure 6A) Treatment schedule. [Figure 6B] Tumor volumes of individual mice at the indicated time points. [Figure 6C]Summary of median tumor volumes per group, each containing 13-14 mice at study initiation and a minimum of 5 mice at the last data point. Vertical dotted lines represent time points of IV administration of test / control articles. Four mice from all groups (5 mice from the 0.1 μg BNT142 group) were euthanized on day 38 to obtain samples for ex vivo assays. [Figure 6D] Number of CD3 positive cells per mm2 of xenograft tissue determined by immunohistochemistry (IHC) staining using anti-human CD3 antibody. Tumor xenografts were dissected 72 hours after the third treatment (day 38). Horizontal lines represent the mean (n=4-5). [Figure 6E] Percentage of CLDN6 positive cells in tumor xenografts in mice from each test and control group, as determined by IHC staining using anti-human CLDN6 antibody. Tumor xenografts were dissected at different time points over the course of the study. Horizontal lines represent the mean values ​​(n=8-10). [Figure 6F] Representative IHC photographs of human CD3 (upper panel) and CLDN6 (lower panel) staining in OV-90 tumor xenografts from BNT142-treated and control mice euthanized 72 hours (day 38) after the third treatment. Red-brown staining (dark in black and white) indicates positive IHC signal, whereas blue-purple areas indicate the absence of positive staining (negative IHC signal). Scale bar lengths are as indicated in the panels (BNT142 and reference protein group: 1,000 μm; negative control / Luc_RNA-LNP and saline group: 2,000 μm). CD = cluster of differentiation; CLDN = claudin; ctrl = control; IP = intraperitoneal; IV = intravenous; LNP = lipid nanoparticle; Luc = luciferase encoding; neg = negative; NSG = NOD.Cg-Prkdscid IL2rgtm1Wjl / SzJ; PBMC = peripheral blood mononuclear cells; RNA = ribonucleic acid; SC = subcutaneous. [Figure 7]RiboMab02.1 induces human cytokines in a dose- and target-dependent manner Cell culture supernatants from T cell activation assays (see Figure 5 above) were used to determine human cytokine (IFN-γ, TNF-α, IL-6, IL-2, IL-10 and IL-1β) production driven by different concentrations of RiboMab02.1 using a custom-made multiplex ELISA kit. Cytokine concentration values ​​(mean of technical triplicates) for each donor are shown. Filled symbols represent values ​​with target cells and open symbols represent values ​​without target cells. IFN=interferon; IL=interleukin; TNF=tumor necrosis factor; w / o=without. [Figure 8] BNT142 treatment does not induce human cytokine release in PBMC-humanized NSG mice Serum from NSG / PBMC mice bearing subcutaneous xenograft tumors (see Figure 6 above) was further evaluated 6 and 72 hours after the third injection with 0.1 or 1 μg BNT142, 1 μg RNA-LNP encoding a target-irrelevant RiboMab tribody (negative control) to control for target specificity, 1 μg Luc_RNA-LNP control or 100 μg CD3x(CLDN6)2 tribody reference protein. An additional non-tumor-bearing (tumor-free) group administered 1 μg BNT142 was included. (A) Concentrations of human cytokines (IFN-γ, IL-6, IL-2, IL-10, TNF-α and IL-1β) in mouse serum determined by multiplex ELISA at 6 hours (n=8) and 72 hours (n=4). Data were normalized to saline-treated animals at the respective time points. Horizontal lines indicate median values. Unpaired and paired samples were compared using the Mann-Whitney U test or Wilcoxon signed-rank test, respectively. (B) Serum concentrations of the encoded therapeutic antibody RiboMab02.1. Horizontal lines indicate mean values. ***, p=0.0002; ctrl=control; h=time; IFN=interferon; IL=interleukin; LNP=lipid nanoparticles; Luc=encoded luciferase; neg.=negative; ns=not significant; RNA=ribonucleic acid; TNF=tumor necrosis factor; w / o=none. [Figure 9] Liver targeting of LNP-formulated mRNA in vivo Balb / cJRj mice received a single IV injection of LNP-formulated firefly luciferase mRNA. Bioluminescence was observed 6, 24, 48, 72 and 144 hours after administration. (A) Images taken 6 hours after administration are shown for (left) individual mice in prone position (n=5) and (right) single organs of animals No. 1 and 2. (B) Quantification of luciferase signal (photons / sec) is shown for all analyzed time points (n=5 or 3, average). IV=intravenous; LN=lymph node; LNP=lipid nanoparticles. [Figure 10] RiboMab02.1 encoded by BNT142 is efficiently expressed in vivo Female Balb / cJRj mice (n=3) received an IV bolus injection of 30 μg BNT142 per mouse. Serum was collected 2 and 6 h after administration. (A) Quantification of RiboMab02.1 concentration in serum by ELISA. Horizontal lines represent the mean values. (B) Western blot analysis of RiboMab02.1-containing serum and reference protein (monomer, HMW) in buffer or spiked-in Balb / cJRj serum analyzed under non-reducing conditions. In total, 60 ng of protein was loaded per lane after the serum protein purification step. Serum from untreated mice was used as control. Western blots were performed using horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fd antibody. Ab = antibody; ctrl = control; Fd = detectable fragment; HMW = high molecular weight; HPI = time post injection; ID = identification number; IgG = immunoglobulin gamma; kDa = kilodaltons; MW = molecular weight. [Figure 11]Sustainable RiboMab02.1 exposure and dose-dependent anti-drug antibody responses with repeated BNT142 administration in mice. Female Balb / cJRj mice (n=4) were IV injected with 10 or 30 μg BNT142 as control or 30 μg Luc_RNA-LNP once a week for a total of five administrations at the time points indicated by the horizontal dotted lines. Blood was collected from mice at baseline (0 hours), 6 hours (6, 174, 342, 510 and 678 hours) and 24 hours (144, 312, 480 and 648 hours) after each BNT142 or saline administration, respectively. Final blood collection was performed 816 hours after the first BNT142 / saline administration. Serum RiboMab02.1 concentrations were determined by ELISA. Error bars indicate standard deviation (SD) of the mean. LNP=lipid nanoparticles; Luc=luciferase; RNA=ribonucleic acid. [Figure 12] RiboMab02.1 exposure in cynomolgus monkeys following IV injection of BNT142. The BNT142-treated cohort and saline control group each contained three cynomolgus monkeys from which blood was collected to prepare serum for assessment of RiboMab02.1 concentrations by ELISA. Error bars indicate standard deviation (SD) of the mean (technical triplicates). [Figure 13]RiboMab02.1 is highly monomeric and induces a lower ADA response in mice than the alternative lead structure candidate RiboMab_712 / 711 C53W Female Balb / cJRj were IV injected with 30 μg of RNA-LNPs encoding RiboMab02.1 or RiboMab_712 / 711 or luciferase (control). (A) Serum was sampled 6 h after injection. 50 ng of purified protein references (monomeric and HMW references) were spiked into mouse serum. 5 μL serum from untreated (mock), luciferase RNA-injected (control) or RiboMab RNA-injected mice and spiked references were subjected to Melon G purification and resolved on 4–15% Criterion gels under non-reducing conditions. Western blot analysis was performed using HRP-conjugated goat anti-human IgG Fd antibody. Samples from one representative mouse per group are shown. (B) Serum was sampled at the indicated time points for ADA analysis. Serum samples were analyzed for anti-RiboMab ADA content by sandwich ELISA assay. ADA response (black line) is plotted against RiboMab protein concentration (grey dotted line) over time. RiboMab_712 / 711 C53W mutant (top) and RiboMab02.1 (bottom) are shown. Error bars indicate standard deviation of the mean (n=4). Ab=antibody; ADA=anti-drug antibody; C53S / W=cysteine ​​to serine / tryptophan substitution at position 53 of anti-CLDN6 VL moiety; Fd=fragment difficult; HMW=high molecular weight; IgG=immunoglobulin G; kDa=kilodaltons; MW=molecular weight; RU=relative units. [Figure 14]The HC:LC weight ratio of RiboMab02.1-encoding drug substance intermediates affects the expression efficiency and monomer content of RiboMab02.1. HEK-293T-17 cells were electroporated with the indicated weight (w / w) ratios of two RiboMab02.1-encoding drug substance intermediates (RNAs), encoding the RiboMab02.1 heavy chain (HC) and light chain (LC), respectively. Cell culture supernatants (SNs) were harvested 48 h after transfection. (A) Western blot analysis of RiboMab02.1-containing SNs and reference proteins (monomers, HMW) under non-reducing conditions. SNs from untransfected cells were used as negative controls (mock SNs). Western blots were performed using a combination of HRP-conjugated goat anti-human kappa light chain (1:500) and IgG Fd (1:2,000) antibodies for detection. (B) Mean RiboMab02.1 concentrations in technical duplicate SN samples from two independent experiments were analyzed by ELISA. Error bars indicate standard deviation (SD) of the mean. Ab = antibody; Fd = detectable fragment; HC = heavy chain coding RNA; HMW = high molecular weight; IgG = immunoglobulin gamma; kDa = kilodaltons; MW = molecular weight; LC = light chain coding RNA; LMW = low molecular weight; RNA = ribonucleic acid; SN = supernatant.

[0167] Array Description The following table provides a list of the specific sequences referenced herein.

[0168] [Table 1]

[0169] [Table 2]

[0170] [Table 3]

[0171] [Table 4]

[0172] [Table 5]

[0173] [Table 6]

[0174] [Table 7]

[0175] [Table 8] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0176] The present disclosure will be described in detail below, but it should be understood that the disclosure is not limited to the specific methodology, protocol and reagent described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0177] Preferably, the terms used herein are defined as set forth in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0178] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0179] In the following, elements of the present disclosure are described. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be interpreted as limiting the present disclosure to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments in which the explicitly described embodiments are combined with any number of the disclosed elements. Moreover, any permutation and combination of all described elements should be considered to be disclosed by this description, unless otherwise indicated by the context.

[0180] As used herein, the term "approximately" or "about" applied to one or more values ​​of interest refers to a value similar to the stated reference value. In general, a person skilled in the art familiar with the context will understand the relevant degree of dispersion encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated value.

[0181] The terms "a" and "the" and similar references used in the context of describing this disclosure (especially in the context of the claims) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not impose limitations on the claims. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0182] Unless otherwise indicated, the term "comprising" is used in the context of this specification to indicate that in addition to the members of the list introduced by "comprising", further members may optionally be present. However, it is contemplated as a specific embodiment of the present disclosure that the term "comprising" encompasses the possibility that further members are not present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of" or "consisting essentially of".

[0183] Several documents are cited throughout the text of this specification. Each document cited herein, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

[0184] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0185] As used herein, terms such as "reduce", "diminish", "inhibit" or "impair" relate to the ability to cause an overall decrease or overall reduction, preferably at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or even more, in level. These terms include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0186] Terms such as "increase," "enhance," or "exceed" preferably relate to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

[0187] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance that contains about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150 consecutive amino acids linked together by peptide bonds. The term "protein" or "polypeptide" refers to larger peptides, particularly peptides having at least about 150 amino acids, although the terms "peptide", "protein" and "polypeptide" are generally used synonymously herein.

[0188] With respect to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of the amino acid sequence, i.e. a sequence that is an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

[0189] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence can be a natural or wild-type (WT) amino acid sequence, or can be a modified form of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0190] By "wild-type" or "WT" or "native" herein is meant an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0191] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and / or an amino acid substitution variant. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformational variants, isoform variants, allelic variants, species variants and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0192] Amino acid insertion variants include the insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting products is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of the protein are also called N-terminus and / or C-terminus truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include the substitution of one of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, Glutamine; Serine, Threonine; Lysine, arginine; and Phenylalanine, tyrosine.

[0193] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant (e.g., functional variant) of said given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example using Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0194] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0195] The term "% identical", "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) expectation threshold set to 10; (ii) word size set to 28; (iii) maximum match within the query range set to 0; (iv) match / mismatch score set to 1, -2; (v) gap cost set to linear; and (vi) filter for low complexity regions used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10; (ii) word size set to 3; (iii) maximum match within the query range set to 0; (iv) matrix set to BLOSUM62; (v) gap cost set to presence: 11, extension: 1; and (vi) conditional composition score matrix adjustment.

[0196] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0197] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference sequence.For example, if the reference nucleic acid sequence is 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0198] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% of the amino acid residues.

[0199] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.

[0200] In some embodiments, the fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or a "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e. is functionally equivalent. In the context of a binding agent sequence, one particular function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence that comprises an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that still performs one or more of the functions of the parent molecule or sequence, e.g., can bind to a target molecule. In some embodiments, the modification of the amino acid sequence of the parent molecule or sequence does not significantly affect or change the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or variant may be reduced but still significantly present, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the binding of the functional fragment or variant may be enhanced compared to the parent molecule or sequence.

[0201] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that sequences suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0202] For example, the amino acid sequences of the VH, VL, CH1 and CL domains of the polypeptide chains of the binding agents of the invention may be derived from the amino acid sequences of the VH, VL, CH1 and CL domains of an immunoglobulin, but may be altered compared to the domains from which they are derived. For example, according to the invention, a VH or VL derived from an immunoglobulin may comprise an amino acid sequence that may be identical to the amino acid sequence of the respective VH or VL from which it is derived, or may differ at one or more amino acid positions compared to the sequence of the respective parent VH or VL. For example, the VH domain of the binding agent of the invention may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions and / or substitutions compared to the amino acid sequence of the VH domain from which it is derived. For example, the VL domain of the binding agent of the invention may comprise an amino acid sequence that comprises one or more amino acid insertions, additions, deletions and / or substitutions compared to the amino acid sequence of the VL domain from which it is derived. Preferably, a VH or VL having an amino acid sequence that is a functional variant of the amino acid sequence of a parent VH or VL provides the same or essentially the same function as the amino acid sequence of the parent VH or VL, e.g., in terms of binding specificity, binding strength, etc. However, as the skilled artisan will recognize, in some embodiments, it may be preferable to provide a functional variant of, e.g., an amino acid sequence of a VH or VL, that has altered properties compared to the amino acid sequence of the parent molecule. The same considerations apply, e.g., to the amino acid sequences of the CDRs, as well as to other amino acid sequences, e.g., the amino acid sequences of the CH1 and / or CL domains. In some embodiments, the variants of the CH1 and CL sequences described herein have the ability to interact, e.g., to bind to each other.

[0203] As used herein, "instructional material" or "instructions" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be affixed to a container containing the composition of the invention or shipped together with a container containing the composition. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the composition are used in conjunction by the recipient.

[0204] "Isolated" means modified or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0205] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant" such as a recombinant nucleic acid in the context of the present invention is not naturally occurring.

[0206] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in a laboratory, is naturally occurring.

[0207] As used herein, "physiological pH" refers to a pH of about 7.35 to about 7.45, with an average of about 7.40.

[0208] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" relates to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of the present invention, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, the cell being present in a subject, e.g. a patient. Thus, according to the present invention, the cells for transfection of a nucleic acid as described herein can be present in vitro or in vivo, e.g. the cells can form part of an organ, tissue and / or organism of a patient. According to the present invention, the transfection can be transient or stable. In some applications of transfection, it is sufficient that the transfected genetic material is only expressed transiently. RNA can be transfected into cells to transiently express its encoded protein. The nucleic acid introduced in the transfection process is not usually integrated into the nuclear genome, so that the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection. In general, the nucleic acid encoding the antigen is transiently transfected into the cell. RNA can be transfected into the cell to transiently express its encoded protein.

[0209] Claudin 6 (CLDN6) Claudins are a family of proteins that are the most important components of tight junctions, which establish a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that cross the membrane four times, with both their N- and C-termini located in the cytoplasm. The first extracellular loop, called EL1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, called EL2 or ECL2, consists of approximately 24 amino acids.

[0210] The term "claudin 6" or "CLDN6" preferably relates to human CLDN6, in particular to a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80 or 29 to 81, more preferably amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. The first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0211] CLDN6 is expressed in tumors of various origins, and the only adult normal tissue that expresses CLDN6 is the placenta.

[0212] CLDN6 has been found to be expressed in, for example, ovarian cancer, lung cancer, testicular cancer, endometrial cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, bile duct cancer, renal cell carcinoma, and bladder cancer. CLDN6 is expressed in ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, fallopian tube cancer and peritoneal cancer, lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma or non-squamous type non-small cell lung cancer (NSCLC), stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, cancer of the bladder, particularly transitional type. It is a particularly preferred target for the prevention and / or treatment of epithelial and papillary carcinomas, renal cancer, particularly renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer including cancer of the ileum, particularly small intestine adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and testicular embryonal carcinoma, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly testicular germ cell tumors, and metastatic forms thereof. In some embodiments, the cancer disease associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, the ovarian cancer is a carcinoma or adenocarcinoma. Preferably, the lung cancer is a carcinoma or adenocarcinoma, preferably a bronchiolar carcinoma such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0213] As used herein, the term "CLDN6 positive cancer" relates to cancers involving cancer cells which express CLDN6, preferably on the surface of said cancer cells.

[0214] According to the present invention, CLDN6 is not substantially expressed in cells when its expression level is low compared to its expression in placental cells or tissues.Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% or even lower than its expression in placental cells or tissues.Preferably, CLDN6 is not substantially expressed in cells when its expression level exceeds its expression level in non-cancerous tissues other than the placenta by no more than 2-fold, preferably no more than 1.5-fold, preferably does not exceed its expression level in said non-cancerous tissues.Preferably, CLDN6 is not substantially expressed in cells when its expression level is below detection limits and / or when its expression level is too low to allow binding by a CLDN6-specific antibody added to the cells.

[0215] According to the present invention, CLDN6 is expressed in cells when the expression level is preferably more than 2-fold, preferably more than 10-fold, more than 100-fold, more than 1,000-fold, or more than 10,000-fold higher than the expression level in non-cancerous tissues other than placenta.Preferably, CLDN6 is expressed in cells when the expression level is above the detection limit and / or when the expression level is high enough to allow binding by a CLDN6-specific antibody added to the cells.Preferably, the CLDN6 expressed in cells is expressed or exposed on the surface of the cells.

[0216] Cluster of differentiation 3 (CD3) A second target molecule of the binding agents described herein is CD3 (cluster of differentiation 3).

[0217] The CD3 complex is a T cell specific antigen. A T cell specific antigen is an antigen on the surface of a T cell.

[0218] The CD3 complex represents an antigen expressed on a subset of mature human T cells, thymocytes and natural killer cells as part of the multi-molecular T cell receptor (TCR) complex. The T cell coreceptor is a protein complex, composed of four different chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule together constitute the TCR complex.

[0219] Human CD3 epsilon is set forth in GenBank Accession No. NM_000733 and comprises SEQ ID NO: 3. Human CD3 gamma is set forth in GenBank Accession No. NM_000073. Human CD3 delta is set forth in GenBank Accession No. NM_000732. CD3 is responsible for TCR signaling. As described in Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding of MHC-presented specific antigen epitopes results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases, resulting in the activation of Ca. 2+ For example, clustering of CD3 on T cells by immobilized anti-CD3 antibodies results in T cell activation similar to T cell receptor engagement, but independent of the typical specificity of that clone.

[0220] As used herein, "CD3" includes human CD3 and refers to an antigen expressed on human T cells as part of the multimolecular T cell receptor complex.

[0221] In some embodiments, the binding agents described herein recognize the epsilon chain of CD3, and in particular recognize an epitope corresponding to the first 27 N-terminal amino acids of CD3 epsilon or a functional fragment of this stretch of 27 amino acids.

[0222] Binder The present disclosure describes a binding agent, such as a bispecific trivalent binding agent, that can bind at least an epitope of CD3 and an epitope of CLDN6. The binding agent comprises at least three binding domains, a first binding domain can bind to CD3, a second and a third binding domain can bind to CLDN6, and the second and the third binding domains bind to the same or different epitopes of CLDN6. In some embodiments, the second and the third binding domains of the binding agent described herein bind to the same epitope of CLDN6. In some embodiments, the sequences of the second and the third binding domains are identical or essentially identical.

[0223] In some embodiments, the binding agents described herein are recombinant molecules.

[0224] The term "epitope" refers to a portion or fragment of a molecule or antigen, such as CD3 and / or CLDN6, that is recognized by a binding agent. For example, an epitope can be recognized by an antibody or any other binding protein. An epitope can include a continuous or discontinuous portion of an antigen and can be about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length, for example, an epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes structural epitopes.

[0225] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region (herein referred to as C H The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically contains a light chain variable region (herein referred to as V L or VL) and a light chain constant region (herein abbreviated as C Lor CL). The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions where the sequence and / or the shape of structurally defined loops can be hypervariable), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise indicated or contradicted by the context, references to amino acid positions of constant regions in the present invention are according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1): 78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). Generally, the CDRs described herein are as defined by Kabat. In some embodiments, the immunoglobulin is an antibody.

[0226] Throughout this specification, reference to a heavy chain (HC) or a light chain (LC) does not necessarily mean the presence of the entire heavy chain (HC) or light chain (LC), but is used as a shorthand to indicate the presence of at least a relevant or characteristic portion of a heavy chain (HC) or a light chain (LC). For example, if a (Fab)-(scFv)2-based bispecific antibody has two chains, one of which contains a heavy chain variable region (VH) and an scFv derived from a parent immunoglobulin, and the other chain of the light chain variable region (VL) and an scFv derived from a parent immunoglobulin, the two chains can be referred to as a heavy chain (HC) and a light chain (LC), respectively. This can also be true when neither chain actually contains a heavy chain or a light chain, but both chains contain scFv, meaning that both contain elements derived from the parent heavy chain and the parent light chain.

[0227] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, that has the ability to bind, preferably specifically bind, to an antigen. In some embodiments, the binding occurs under typical physiological conditions with a half-life of a significant period, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant functionally defined period (e.g., a time sufficient to induce, promote, enhance and / or modulate a physiological response associated with antibody binding to an antigen). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with an antigen. As used herein, the terms "antigen-binding region", "binding region" or "binding domain" refer to a region or domain that interacts with an antigen and typically includes both the VH and VL regions. The term antibody as used herein includes not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As mentioned above, the term antibody as used herein includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to antigens, and antibody derivatives, i.e., constructs derived from antibodies, unless otherwise indicated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting essentially of the VH domain, also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(11):484-90) (Ward et al., Nature 341,544-546(1989)); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;. 5(1):111-24) and (vii) isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be made using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise stated or clearly indicated by the context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention and exhibit different biological properties and usefulness. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. The term antibody, unless otherwise specified, should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0228] The phrase "single-chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy and light chains (VH and VL) of a traditional two-chain antibody are combined to form a single chain. Optionally, a linker (usually a peptide) is inserted between the two chains to allow proper folding and creation of an active binding site.

[0229] An antibody can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibody of the present invention can be a sequence variant of a naturally occurring IgG1 antibody, including mutations in the constant region.

[0230] In various embodiments, the antibody is an IgG1 antibody, more specifically an IgG1 kappa or IgG1 lambda isotype (i.e. IgG1κ, IgG1λ), an IgG2a antibody (e.g. IgG2aκ, IgG2aλ), an IgG2b antibody (e.g. IgG2bκ, IgG2bλ), an IgG3 antibody (e.g. IgG3κ, IgG3λ) or an IgG4 antibody (e.g. IgG4κ, IgG4λ).

[0231] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas containing B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, whose genome includes human heavy and light chain transgenes fused to an immortalized cell.

[0232] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic use have been developed to reduce antibody immunogenicity. The term "variable region" or "variable domain" as used in the context of a chimeric antibody refers to the region that includes the CDR and framework regions of both the heavy and light chains of an immunoglobulin. Chimeric antibodies can be produced by using standard DNA techniques such as those described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. It is within the knowledge of one skilled in the art to produce chimeric antibodies, and therefore the production of chimeric antibodies according to the present invention can be performed by methods other than those described herein.

[0233] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site into a homologous human acceptor framework region (FR) (see WO 92 / 22653 and EP 0629240). Substitution (back mutation) of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help to identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid back mutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, may be applied to obtain a humanized antibody with favorable characteristics such as affinity and biochemical properties.

[0234] The term "human antibody" as used herein refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are generally preferred, other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g. mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be made using, for example, transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Thus, in some embodiments, human antibodies are obtained from transgenic animals, such as mice or rats, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences.In such embodiments, the antibody is derived from human germline immunoglobulin sequences introduced into the animal, with the final antibody sequence being the result of further modification of said human germline immunoglobulin sequences by somatic hypermutation and affinity maturation by the endogenous animal antibody machinery, see e.g. Mendez et al. 1997 Nat Genet. 15(2):146-56.

[0235] The term "full length" when used in reference to an antibody indicates that the antibody is not a fragment, but includes all of the domains of a particular isotype that are normally found for that isotype in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains of an IgG1 antibody.

[0236] As used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least the hinge region, CH2 domain, and CH3 domain.

[0237] As used herein, the terms "bind" or "capable of binding" in reference to the binding of a binding agent, e.g., an antibody, to a given antigen or epitope typically refer to a binding affinity of about 10% or more, as determined, for example, using biolayer interferometry (BLI), as determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the binding agent as the analyte, or as determined using a quartz crystal microbalance system using target (CLDN6)-expressing cells as the "ligand." -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or lower K DIn some embodiments, a binding agent has a K that is at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1,000 times lower, e.g., at least 10,000 times lower, e.g., at least 100,000 times lower, e.g., at least 100,000 times lower, than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The amount of binding agent with a lower affinity is the K D Since it depends on the K of the binder, D is very low (i.e., the binder is highly specific), the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.

[0238] As used herein, "k d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular binding agent-antigen interaction. off Also called value.

[0239] As used herein, "K D The term "" (M) refers to the dissociation equilibrium constant of a particular binding agent-antigen interaction.

[0240] The present invention also contemplates binding agents comprising functional variants of the VL regions, VH regions, or one or more CDRs described herein. A functional variant of a VL, VH, or CDR used in connection with a binding agent still allows the binding agent to retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the "reference" or "parent" binding agent, and in some cases, such binding agents may be associated with higher affinity, selectivity, and / or specificity than the parent binding agent.

[0241] Such functional variants typically retain significant sequence identity to the parent sequence.

[0242] Exemplary variants include those which differ from the VH and / or VL and / or CDR regions of a parent sequence primarily by conservative substitutions; for example, up to 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[0243] Functional variants of the sequences described herein, such as the VL or VH regions, or sequences having a degree of homology or identity to the sequences described herein, such as the VL or VH regions, preferably include modifications or mutations of the non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0244] A binding agent that comprises a variant of the heavy chain and / or light chain variable region sequences described herein, e.g., a binding agent that includes modifications and / or a degree of identity in the CDRs described herein, may compete with another binding agent, e.g., a binding agent that includes a heavy chain variable region and a light chain variable region described herein, for binding to an antigen, e.g., CD3 and / or CLDN6, or may have the specificity for an antigen of another binding agent, e.g., a binding agent that includes a heavy chain variable region and a light chain variable region described herein.

[0245] The term "specificity", as used herein, unless contradicted by context, is intended to have the following meaning: Two binding agents have the "same specificity" if they bind to the same antigen and the same epitope.

[0246] The terms "compete" and "competition" can refer to the competition between a first binding agent and a second binding agent for the same antigen. Methods for testing the competition of binding agents such as antibodies for binding to target antigens are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be carried out, for example, as ELISA or by flow cytometry. Alternatively, competition can be determined using biolayer interferometry.

[0247] Binders that compete for binding to a target antigen can bind to different epitopes on the antigen, and the epitopes are so close to each other that a first binder that binds to one epitope prevents a second binder from binding to the other epitope. However, in other situations, two different binders can bind to the same epitope on an antigen and compete for binding in a competitive binding assay. Such binders that bind to the same epitope are considered to have the same specificity herein. Thus, in some embodiments, binders that bind to the same epitope are considered to bind to the same amino acid on the target molecule. The binding of a binder to the same epitope on a target antigen can be determined by standard alanine scanning experiments or antibody-antigen crystallization experiments known to those skilled in the art. Preferably, binders or binding domains that bind to different epitopes do not compete with each other for binding to their respective epitopes.

[0248] As mentioned above, various types of antibodies are described in the art. The binding agent of the present invention can in principle comprise the sequence of an antibody of any isotype. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either human light chain constant region kappa or lambda may be used. In some embodiments, the sequences of the binding agents described herein, such as CH1 and CL, are derived from an antibody of IgG1 isotype, for example an IgG1, κ antibody.

[0249] Preferably, each of the antigen-binding regions or domains comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of said variable regions comprising three CDR sequences, CDR1, CDR2 and CDR3 respectively, and four framework sequences, FR1, FR2, FR3 and FR4 respectively.Furthermore, preferably, the binding agents described herein comprise a heavy chain constant region (CH) and a light chain constant region (CL).

[0250] The term "binding agent" in the context of the present invention refers to any agent capable of binding to one or more desired antigens, such as CD3 and CLDN6. The term "binding agent" includes antibodies, antibody fragments, or any other binding proteins, or any combination thereof. In some embodiments, the binding proteins include antibody fragments, such as Fab and scFv.

[0251] Naturally occurring antibodies are generally monospecific, i.e., they bind to a single antigen. The present invention provides binding agents that bind to cytotoxic cells such as T cells (by engaging the CD3 receptor) and target cells such as cancer cells (by engaging CLDN6). Such binding agents are at least bispecific or multispecific, e.g., trispecific, tetraspecific, etc. In some embodiments, the binding agents described herein are artificial proteins composed of fragments of two different antibodies (said fragments of two different antibodies form three binding domains).

[0252] According to the present invention, a bispecific binding agent, particularly a bispecific protein, is a molecule that has two different binding specificities and can therefore bind to two epitopes. In particular, the term "bispecific binding agent" as used herein includes antibody-derived molecules that contain three antigen-binding sites, i.e., a first binding site that has affinity for a first epitope, and a second and a third binding site that have binding affinity for a second epitope that is different from the first epitope.

[0253] The term "bispecific" in the context of the present invention refers to an agent that comprises two different antigen-binding regions that bind to different epitopes, in particular different epitopes on different antigens, such as CD3 and CLDN6.

[0254] A "multispecific binding agent" is a molecule that has three or more different binding specificities.

[0255] In some embodiments, the binding agents described herein that bind to CD3 and CLDN6 are at least trivalent. As used herein, "valent", "valency", "valency" or other grammatical variations thereof refer to the number of antigen binding sites or binding domains in a binding agent. In some embodiments, the binding agents described herein have at least one antigen binding site or binding domain for CD3 and at least two antigen binding sites or binding domains for CLDN6. Antigen binding sites that bind to the same antigen may recognize the same epitope or different epitopes.

[0256] In some embodiments, the binding agent described herein is in the format of a Fab-scFv2 construct, i.e. a Fab fragment specific for CD3 is provided with two scFv fragments specific for CLDN6 at the C-terminus of the constant region of the Fab fragment. In some embodiments, the binding agent is a dimer, preferably composed of two polypeptide chains linked together by a disulfide bridge, the first polypeptide comprising an scFv linked to a further VH domain via a CH1 polypeptide chain, and the second polypeptide comprising an scFv linked to a further VL domain via a CL polypeptide chain. The disulfide bridge is preferably formed between a Cys residue in CH1 and a Cys residue in CL, such that the further VH of the first polypeptide associates with a further VL of the second polypeptide in an antigen-binding configuration, such that the binding agent comprises overall three antigen-binding domains. Thus, in some embodiments, the binding agent comprises a heavy chain (Fd fragment) and a light chain (L) of a Fab fragment capable of heterodimerizing, in which the scFv binding domain is incorporated (preferably at the C-terminus of Fd / L). In some embodiments, the VH and VL domains of the scFv portion are connected by a peptide linker and / or the Fab chain and the scFv are connected by a peptide linker. In some embodiments, the VH and VL domains of the scFv portion have the amino acid sequence (G4S): xand x is 2, 3, 4, 5 or 6. In some embodiments, the Fab chain and the scFv are connected by a peptide linker comprising the amino acid sequence SGPG3RS(G4S)2 or DVPG2S. In some embodiments, a linker comprising the amino acid sequence SGPG3RS(G4S)2 connects the scFv binding domain to the Fd fragment and a linker comprising the amino acid sequence DVPG2S connects the scFv binding domain to the L fragment. In some embodiments, the scFv portion binds to CLDN6 and the Fab portion binds to CD3.

[0257] The term "linker" refers to any means that serves to link two different functional units (e.g., antigen-binding moieties). Types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. In some embodiments, the polypeptide linker is preferably non-immunogenic and flexible, for example, containing serine and glycine sequences. Depending on the particular construct, the linker may be long or short.

[0258] In some embodiments, the linker connecting the VH and VL domains to form the VH-VL or VL-VH scFv domains preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In some embodiments, the linker comprises the amino acid sequence (G4S) x and x is 2, 3, 4, 5 or 6. In some embodiments, for scFv domains comprising a VH domain and a VL domain in a VH-VL orientation, the linker comprises the amino acid sequence (G4S)4. In some embodiments, for scFv domains comprising a VH domain and a VL domain in a VL-VH orientation, the linker comprises the amino acid sequence (G4S)5.

[0259] In some embodiments, the linker connecting the scFv domain and the Fd domain comprises the amino acid sequence DVPG2S or SGPG3RS(G4S)2, preferably SGPG3RS(G4S)2, preferably at the C-terminus of CH1. In some embodiments, the linker connecting the scFv domain and the L domain comprises the amino acid sequence DVPG2S or SGPG3RS(G4S)2, preferably DVPG2S, preferably at the C-terminus of CL.

[0260] A binding agent may also contain an amino acid sequence to facilitate secretion of the molecule, such as an N-terminal secretion signal, and / or one or more epitope tags to facilitate binding, purification or detection of the molecule.

[0261] According to some embodiments, each of the polypeptide chains of the binding agents described herein comprises a signal peptide.

[0262] Such signal peptides are sequences that typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of a polypeptide chain, but are not limited thereto. A signal peptide as defined herein preferably enables the transport of one or more polypeptide chains, for example encoded by an RNA, to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or an endosomal-lysosomal compartment.

[0263] Signal peptide sequences as defined herein include, but are not limited to, immunoglobulin signal peptide sequences, such as the signal peptide sequence of an immunoglobulin heavy chain variable region or the signal peptide sequence of an immunoglobulin light chain variable region, and the immunoglobulin may be a human immunoglobulin. In some embodiments, the signal peptide sequence is a signal peptide sequence of an MHC molecule, such as an MHC class I molecule, and the MHC molecule may be a human MHC molecule (HLA molecule).

[0264] In some embodiments, the secretion signal is a signal sequence (e.g., an amino acid sequence comprising amino acids 1-26 of SEQ ID NO:4) that allows for sufficient passage through the secretory pathway and / or secretion of the binding agent or its polypeptide chain into the extracellular environment. In some embodiments, the secretion signal sequence is cleavable and removed from the mature binding agent. In some embodiments, the secretion signal sequence is selected with respect to the cell or organism in which the binding agent is produced.

[0265] In further embodiments, the binding agents described herein are linked or conjugated to one or more therapeutic moieties, such as cytokines, immunosuppressants, and / or immunostimulatory molecules.

[0266] In some embodiments, the binding agents described herein comprise a Fab antibody fragment comprising a first binding domain. In some embodiments, the binding agents described herein comprise two scFv antibody fragments comprising second and third binding domains covalently linked to a Fab antibody fragment comprising a first binding domain. In some embodiments, the binding agents comprise scFv antibody fragments covalently linked to the C-terminus of each chain of the Fab antibody fragment.

[0267] The CH1 and CL sequences of the binding agents described herein may each be of any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4, and may contain one or more mutations or modifications. In some embodiments, each of the CH1 and CL sequences is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications.

[0268] In some embodiments of the present invention, the binding agent described herein does not comprise a full-length antibody. In some embodiments of the present invention, the binding agent described herein does not comprise the CH2 domain and the CH3 domain of an antibody. In some embodiments of the present invention, the binding agent described herein does not comprise an Fc region. In some embodiments of the present invention, the binding agent described herein does not comprise an Fc sequence that can exert effector function.

[0269] The term "effector function" in the context of the present invention includes any function mediated by components of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor seeding and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such functions include ADCC, ADCP, or CDC.

[0270] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the killing of antibody-coated target cells by cytotoxic effector cells through a non-phagocytic process characterized by the release of the contents of cytotoxic granules or the expression of cell death-inducing molecules. ADCC is independent of the immune complement system, which also lyses targets but does not require other cells. ADCC is triggered by the interaction of target-bound antibodies (belonging to the IgG or IgA or IgE classes) with specific Fc receptors (FcRs), glycoproteins present on the effector cell surface that bind to the Fc region of immunoglobulins (Ig). Effector cells that mediate ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells. ADCC is a rapid effector mechanism whose effectiveness depends on many parameters (density and stability of antigens on the surface of the target cells; antibody affinity and FcR binding affinity). ADCC involving human IgG1, the most used IgG subclass for therapeutic antibodies, is highly dependent on the glycosylation profile of its Fc portion and on the polymorphism of Fcγ receptors.

[0271] Antibody-dependent cellular phagocytosis (ADCP) is one of the key mechanisms of action of many antibody therapeutics. It is defined as a highly regulated process in which an antibody eliminates bound targets by connecting its Fc domain to specific receptors on phagocytes and inducing phagocytosis. Unlike ADCC, ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells via FcγRIIa, FcγRI and FcγRIIIa, of which FcγRIIa (CD32a) on macrophages is the major pathway.

[0272] Complement-dependent cytotoxicity (CDC) is another cell killing method that can be induced by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both very effective in inducing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex is followed by the C-terminal end of the participating antibody molecule, such as an IgG molecule. H IgG-binding sites in close proximity on the 2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low affinity C1q-IgG interaction to a high avidity one, triggering a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of effector cell chemotactic / activating agents C3a and C5a. Preferably, the complement cascade ends with the formation of the membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0273] In some embodiments, the binding agent comprises two polypeptide chains forming a binding domain with specificity for CD3 and two binding domains with specificity for CLDN6. In some embodiments, the two polypeptide chains are encoded by two RNA molecules. In some embodiments, the binding agent is a dimer composed of two polypeptide chains, a first polypeptide comprises a scFv specific for CLDN6 linked to a further VH domain via the constant region 1 (CH1) of the immunoglobulin heavy chain, and a second polypeptide comprises a scFv specific for CLDN6 linked to a further VL domain via the constant region (CL) of the immunoglobulin light chain. In some embodiments, the two polypeptide chains are linked together by a disulfide bridge. The disulfide bridge is preferably formed between a Cys residue in the CH1 domain and a Cys residue in the CL domain, such that the further VH domain of the first polypeptide associates with the further VL domain of the second polypeptide in a CD3-binding configuration, such that the binding agent comprises overall three antigen-binding domains. In some embodiments, the binding domain specific for CD3 is comprised in a Fab fragment and the binding domain specific for CLDN6 is comprised in a respective scFv. In some embodiments, each chain of the Fab fragment is linked to one scFv, and the scFv is preferably linked to the C-terminus of the Fab fragment. According to the invention, the VH and VL domains of the scFv portion are preferably connected by a peptide linker, such as a peptide linker comprising the amino acid sequence (G4S)4, and the Fab chain and the scFv are preferably connected by a peptide linker, such as a peptide linker comprising the amino acid sequence SGPG3RS(G4S)2 or DVPG2S.

[0274] In some embodiments, the binding agent comprises (i) a first polypeptide chain comprising a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CD3 (VH(CD3)), a VH derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)); and (ii) a second polypeptide chain comprising a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CD3 (VL(CD3)), a VH derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)). In some embodiments, the first polypeptide chain interacts with the second polypeptide chain to form the binding agent. In some embodiments, the VH(CD3) of the first polypeptide chain and the VL(CD3) of the second polypeptide chain interact to form a binding domain having specificity for CD3. In some embodiments, the VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact to form a binding domain with specificity for CLDN6. In some embodiments, the VH(CLDN6) and VL(CLDN6) of the second polypeptide chain interact to form a binding domain with specificity for CLDN6. In some embodiments, the first and second polypeptide chains comprise a heavy chain constant region 1 (CH1) derived from an immunoglobulin or functional variant thereof, and a light chain constant region (CL) derived from an immunoglobulin or functional variant thereof. In some embodiments, the immunoglobulin is an IgG1. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the VH, VL, and CH1 on the first polypeptide chain are VH(CD3)-CH1-VH(CLDN6)-VL(CLDN6), or VH(CD3)-CH1-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0275] In some embodiments, CH1 is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence SGPGGGRS(G4S)2 or a functional variant thereof.

[0276] In some embodiments, the VH, VL, and CL on the second polypeptide chain are VL(CD3)-CL-VH(CLDN6)-VL(CLDN6), or VL(CD3)-CL-VL(CLDN6)-VH(CLDN6) They are arranged in the following order from N-terminus to C-terminus.

[0277] In some embodiments, CL is connected to VH(CLDN6) or VL(CLDN6) by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence DVPGGS or a functional variant thereof.

[0278] In some embodiments, VH(CLDN6) and VL(CLDN6) are connected to each other by a peptide linker. In some embodiments, the peptide linker is the amino acid sequence (G4S) x or a functional variant thereof, and x is 2, 3, 4, 5 or 6. In some embodiments, the peptide linker comprises the amino acid sequence (G4S)4 or a functional variant thereof.

[0279] In some embodiments, a CH1 on a first polypeptide chain interacts with a CL on a second polypeptide chain.

[0280] In some embodiments, CH1 comprises the amino acid sequence of amino acids 146 to 248 of SEQ ID NO: 4, or a functional variant thereof.

[0281] In some embodiments, the CL comprises the amino acid sequence of amino acids 133 to 239 of SEQ ID NO: 6, or a functional variant thereof.

[0282] In some embodiments, VH(CD3) comprises a CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27-145 of SEQ ID NO:4 (SEQ ID NOs:18, 19 and 20, respectively). In some embodiments, VH(CD3) comprises a CDR1 comprising the amino acid sequence GYTFTRYT or a functional variant thereof, a CDR2 comprising the amino acid sequence INPSRGYT or a functional variant thereof, and a CDR3 comprising the amino acid sequence ARYYDDHYSLDY or ARYYDDHYCLDY or a functional variant thereof. In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27-145 of SEQ ID NO:4 or a functional variant thereof.

[0283] In some embodiments, the VL(CD3) comprises a CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 27-132 of SEQ ID NO:6 (SEQ ID NOs:22, 23 and 24, respectively). In some embodiments, the VL(CD3) comprises a CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, a CDR2 comprising the amino acid sequence DTS or a functional variant thereof, and a CDR3 comprising the amino acid sequence QQWSSNPLT or a functional variant thereof. In some embodiments, the VL(CD3) comprises the amino acid sequence of amino acids 27-132 of SEQ ID NO:6 or a functional variant thereof.

[0284] In some embodiments, VH(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 267-383 of SEQ ID NO:4 (SEQ ID NOs:25, 26 and 27, respectively). In some embodiments, VH(CLDN6) comprises CDR1 comprising the amino acid sequence GYSFTGYT or a functional variant thereof, CDR2 comprising the amino acid sequence INPYNGGT or a functional variant thereof, and CDR3 comprising the amino acid sequence ARDYGFVLDY or a functional variant thereof. In some embodiments, VH(CLDN6) comprises the amino acid sequence of amino acids 267-383 of SEQ ID NO:4 or a functional variant thereof.

[0285] In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404-510 of SEQ ID NO:4 (SEQ ID NO:28, 29 and 30, respectively). In some embodiments, VL(CLDN6) comprises CDR1 comprising the amino acid sequence SSVSY or a functional variant thereof, CDR2 comprising the amino acid sequence STS or a functional variant thereof, and CDR3 comprising the amino acid sequence QQRSNYPPWT or a functional variant thereof. In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404-510 of SEQ ID NO:4, and a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO:4). In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404-510 of SEQ ID NO:4, and a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO:4). In some embodiments, VL(CLDN6) comprises CDR1, CDR2 and CDR3 of the amino acid sequence of amino acids 404-510 of SEQ ID NO: 4, a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 404-510 of SEQ ID NO: 4, and a serine residue at a position corresponding to position 449 of SEQ ID NO: 4. In some embodiments, VL(CLDN6) comprises the amino acid sequence of amino acids 404-510 of SEQ ID NO: 4, or a functional variant thereof.

[0286] A serine residue at position +15 relative to CDR1 means that the 15th amino acid position counting from the end of CDR1 is a serine residue. A serine residue at position -3 relative to CDR2 means that the third amino acid before the start of CDR2 is a serine. These include, for example, the following (from N to C): XXXXX-Y 14 -S and S-Y2-ZZZ, respectively, where X represents a CDR1 amino acid, Y represents an intervening amino acid between the CDRs, S represents a serine residue, and Z represents a CDR2 amino acid.

[0287] In some embodiments, VH(CD3) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, VL(CD3) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, VH(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, and VL(CLDN6) comprises CDR1, CDR2, and CDR3 of the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4, and preferably VL(CLDN6) comprises a serine residue at position +15 relative to CDR1 (corresponding to position 449 of SEQ ID NO: 4) and / or a serine residue at position -3 relative to CDR2 (corresponding to position 449 of SEQ ID NO: 4).

[0288] In some embodiments, VH(CD3) comprises the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4 or a functional variant thereof, VL(CD3) comprises the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6 or a functional variant thereof, VH(CLDN6) comprises the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4 or a functional variant thereof, and / or VL(CLDN6) comprises the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4 or a functional variant thereof.

[0289] In some embodiments, the first polypeptide chain comprises the amino acid sequence of amino acids 27-510 of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27-510 of SEQ ID NO:4, or a functional fragment of the amino acid sequence of amino acids 27-510 of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27-510 of SEQ ID NO:4. In some embodiments, the first polypeptide chain comprises the amino acid sequence of amino acids 27-510 of SEQ ID NO:4.

[0290] In these and other embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5, or a functional fragment of the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5.

[0291] In some embodiments, the second polypeptide chain comprises the amino acid sequence of amino acids 27-489 of SEQ ID NO:6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27-489 of SEQ ID NO:6, or a functional fragment of the amino acid sequence of amino acids 27-489 of SEQ ID NO:6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27-489 of SEQ ID NO:6. In some embodiments, the second polypeptide chain comprises the amino acid sequence of amino acids 27-489 of SEQ ID NO:6.

[0292] In these and other embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7, or a functional fragment of the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7. In some embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7.

[0293] In some embodiments, (i) the first polypeptide chain comprises an amino acid sequence of amino acids 27 to 510 of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27 to 510 of SEQ ID NO:4, or a functional fragment of an amino acid sequence of amino acids 27 to 510 of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27 to 510 of SEQ ID NO:4. and (ii) the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 489 of SEQ ID NO: 6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27 to 489 of SEQ ID NO: 6, or a functional fragment of the amino acid sequence of amino acids 27 to 489 of SEQ ID NO: 6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 27 to 489 of SEQ ID NO: 6. In some embodiments, the first polypeptide chain comprises the amino acid sequence of amino acids 27 to 510 of SEQ ID NO: 4 and the second polypeptide chain comprises the amino acid sequence of amino acids 27 to 489 of SEQ ID NO: 6.

[0294] In these and other embodiments, (i) the RNA encoding the first polypeptide chain is a functional polypeptide comprising the nucleotide sequence of nucleotides 132 to 1583 of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132 to 1583 of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132 to 1583 of SEQ ID NO:5 or the nucleotide sequence of nucleotides 132 to 1583 of SEQ ID NO:5. and (ii) the RNA encoding the second polypeptide chain comprises a nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7, or a functional fragment of the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of nucleotides 132-1583 of SEQ ID NO:5 and the RNA encoding the second polypeptide chain comprises the nucleotide sequence of nucleotides 132-1520 of SEQ ID NO:7.

[0295] According to some embodiments, a signal peptide is fused, either directly or via a linker, to a polypeptide chain described herein. Thus, in some embodiments, a signal peptide is fused to the amino acid sequence described above.

[0296] In some embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-26 of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-26 of SEQ ID NO:4, or a functional fragment of the amino acid sequence of amino acids 1-26 of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-26 of SEQ ID NO:4. In some embodiments, the signal sequence comprises the amino acid sequence of amino acids 1-26 of SEQ ID NO:4.

[0297] In these and other embodiments, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 54-131 of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-131 of SEQ ID NO:5, or a functional fragment of the nucleotide sequence of nucleotides 54-131 of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-131 of SEQ ID NO:5. In some embodiments, the RNA encoding the signal sequence comprises the nucleotide sequence of nucleotides 54-131 of SEQ ID NO:5.

[0298] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:4.

[0299] In these and other embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of nucleotides 54-1583 of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-1583 of SEQ ID NO:5, or a functional fragment of the nucleotide sequence of nucleotides 54-1583 of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-1583 of SEQ ID NO:5. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of nucleotides 54-1583 of SEQ ID NO:5.

[0300] In further embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a functional fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of SEQ ID NO:5.

[0301] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6, or a functional fragment of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:6.

[0302] In these and other embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of nucleotides 54-1520 of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-1520 of SEQ ID NO:7, or a functional fragment of the nucleotide sequence of nucleotides 54-1520 of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-1520 of SEQ ID NO:7. In some embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of nucleotides 54-1520 of SEQ ID NO:7.

[0303] In further embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a functional fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the RNA encoding the second polypeptide chain comprises the nucleotide sequence of SEQ ID NO:7.

[0304] In some embodiments, (i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, or a functional fragment of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:4, and (ii) the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6, or a functional fragment of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:4 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:6.

[0305] In these and other embodiments, (i) the RNA encoding the first polypeptide chain is a functional polypeptide comprising the nucleotide sequence of nucleotides 54 to 1583 of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 1583 of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 1583 of SEQ ID NO:5 or the nucleotide sequence of nucleotides 54 to 1583 of SEQ ID NO:5. and (ii) the RNA encoding the second polypeptide chain comprises a nucleotide sequence from nucleotides 54 to 1520 of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence from nucleotides 54 to 1520 of SEQ ID NO:7, or a functional fragment of the nucleotide sequence from nucleotides 54 to 1520 of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence from nucleotides 54 to 1520 of SEQ ID NO:7. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence from nucleotides 54 to 1583 of SEQ ID NO:5 and the RNA encoding the second polypeptide chain comprises the nucleotide sequence from nucleotides 54 to 1520 of SEQ ID NO:7.

[0306] In a further embodiment, (i) the RNA encoding the first polypeptide chain comprises the nucleotide sequence of SEQ ID NO:5, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5, or a functional fragment of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:5. and (ii) the RNA encoding the second polypeptide chain comprises the nucleotide sequence of SEQ ID NO:7, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7, or a functional fragment of the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the RNA encoding the first polypeptide chain comprises the nucleotide sequence of SEQ ID NO:5 and the RNA encoding the second polypeptide chain comprises the nucleotide sequence of SEQ ID NO:7.

[0307] In some embodiments, the binding agent binds to the extracellular domain of CLDN6. In some embodiments, the binding agent binds to a native epitope of CLDN6 present on the surface of a living cell. In some embodiments, the binding agent binds to the first extracellular loop of CLDN6.

[0308] The binding agents described herein are specific for CLDN6, i.e., have the ability to bind to CLDN6, i.e., to an epitope present on CLDN6, preferably located within the extracellular domain of CLDN6, in particular the first extracellular loop, preferably amino acid positions 28-76 or 29-81 of CLDN6, or the second extracellular loop, preferably amino acid positions 141-159 of CLDN6. In some embodiments, the agent capable of binding to CLDN6 binds to an epitope on CLDN6 that is not present on CLDN9. In some embodiments, the agent capable of binding to CLDN6 binds to an epitope on CLDN6 that is not present on CLDN4 and / or CLDN3. In some embodiments, the agent capable of binding to CLDN6 binds to an epitope on CLDN6 that is not present on claudin proteins other than CLDN6.

[0309] In some embodiments, the agent capable of binding to CLDN6 preferably binds to CLDN6 but not to CLDN9, and preferably not to CLDN4 and / or CLDN3. In some embodiments, the agent capable of binding to CLDN6 binds to CLDN6 expressed on the cell surface. In some embodiments, the agent capable of binding to CLDN6 binds to a native epitope of CLDN6 present on the surface of a living cell.

[0310] The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule, such as an antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of said cell and accessible from the outside of said cell, for example by an antibody located on the outside of said cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that may be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion, or may be a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0311] "Cell surface" or "surface of a cell" is used according to its normal meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible to binding, for example, by an antigen-specific antibody added to the cell.

[0312] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, for example by binding to a molecule, such as an antibody, that is located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0313] nucleic acid The term "polynucleotide" or "nucleic acid" as used herein is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0314] The nucleic acid may be contained in a vector. The term "vector" as used herein includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Said vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors, and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment, and may lack functional sequences required for expression of the desired DNA fragment.

[0315] In some embodiments of all of the aspects of the invention, RNA encoding a binding agent, e.g., a bispecific or multispecific binding agent, as described herein, is expressed in cells (e.g., hepatocytes) of a treated subject to provide the binding agent.

[0316] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0317] In the present disclosure, the term "RNA" refers to a nucleic acid molecule that comprises ribonucleotide residues. In a preferred embodiment, the RNA comprises all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends (either or both) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0318] In some embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to an RNA transcript that codes for a peptide or protein. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that comprises deoxyribonucleotides.

[0319] In some embodiments of the present disclosure, the RNA is a "replicon RNA" or simply a "replicon", in particular a "self-replicating RNA" or a "self-amplifying RNA". In a particularly preferred embodiment, the replicon or self-replicating RNA is derived from or contains elements derived from a ssRNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses is typically in the range of 11,000-12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification and replication and protein modification) and structural proteins (forming the virus particle). Typically, there are two open reading frames (ORFs) in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF that starts near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together by a second ORF that is found downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences that code for the nonstructural proteins are translated from the genomic RNA, while the genetic information that codes for the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-stranded genomic RNA acts directly like a messenger RNA for the translation of the open reading frame that codes for the nonstructural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed to deliver foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated by said replicase in trans (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated by the replicase in trans must contain specific alphavirus sequence elements to allow recognition and RNA synthesis by the alphavirus replicase.

[0320] In some embodiments, the RNA described herein can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridine.

[0321] The term "uracil" as used herein refers to one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: [ka] It is.

[0322] The term "uridine" as used herein refers to one of the nucleosides that can occur in RNA. The structure of uridine is: [ka] It is.

[0323] UTP (uridine 5'-triphosphate) has the following structure: [ka] has.

[0324] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: [ka] has.

[0325] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring through a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0326] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the structure: [ka] has.

[0327] N1-methylpseudoUTP has the following structure: [ka] has.

[0328] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure: [ka] has.

[0329] In some embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0330] In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine, In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0331] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise two or more modified nucleosides, the modified nucleosides being independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U).

[0332] In some embodiments, the modified nucleoside that replaces one or more, e.g., all, of the uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyluridine (cm 5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 Ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3 Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0333] In some embodiments, the RNA comprises other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in some embodiments, cytidine is partially or completely replaced with 5-methylcytidine, preferably completely, in the RNA. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the RNA comprises 5-methylcytidine and N1-methylpseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methylpseudouridine (m1ψ) in place of each uridine.

[0334] In some embodiments, the RNA according to the present disclosure includes a 5' cap. In some embodiments, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In some embodiments, the RNA may be modified by a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide connected to the mRNA via a 5'-5' triphosphate bond. In some embodiments, the guanosine is methylated at position 7. Providing the RNA with a 5' cap or a 5' cap analog may be accomplished by in vitro transcription, where the 5' cap is co-transcriptionally expressed on the RNA strand, or may be attached to the RNA post-transcriptionally using a capping enzyme.

[0335] In some embodiments, the building block cap for RNA is m2 7,3’-O Gppp(m1 2’-O )ApG (sometimes m2 7,3’O G(5')ppp(5')m 2’-O ApG), which has the following structure: [ka] has.

[0336] The following are RNA and m2 7,3’O G(5')ppp(5')m 2’-O An exemplary Cap1 RNA containing ApG is: [ka]

[0337] The following is another exemplary Cap1 RNA (no cap analog): [ka]

[0338] In some embodiments, the RNA has the structure: [ka] Cap analogue anti-reverse cap (ARCA cap (m2 7,3`O G(5')ppp(5')G)) is used to modify the "cap 0" structure.

[0339] The following are RNA and m2 7,3’O An exemplary Cap 0 RNA containing G(5')ppp(5')G: [ka]

[0340] In some embodiments, the “cap 0” structure has the structure: [ka] Cap analogue beta-S-ARCA (m2 7,2’O G(5')ppSp(5')G).

[0341] Below is the Beta-S-ARCA(m2 7,2’O An exemplary Cap 0 RNA includes: [ka]

[0342] The "D1" diastereomer of beta-S-ARCA or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) (see WO 2011 / 015347, incorporated herein by reference).

[0343] A particularly preferred cap is m2 7,3’-O Gppp(m1 2’-O In some embodiments, the RNA sequences described herein (e.g., SEQ ID NOs: 5 and / or 7) are m2 7,3’-O Gppp(m1 2’-O ) ApG. In some embodiments, the ApG of the cap corresponds to the two 5' nucleotides of the RNA sequence described herein.

[0344] In some embodiments, the RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). The 5'-UTR, if present, is located at the 5' end upstream of the start codon of a protein coding region. The 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end downstream of the stop codon of a protein coding region, although the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0345] In some embodiments, the RNA comprises a 5'-UTR comprising a nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0346] In some embodiments, the RNA comprises a 3'-UTR comprising a nucleotide sequence of SEQ ID NO:9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:9.

[0347] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 8. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 9.

[0348] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence.

[0349] As used herein, the term "poly(A) sequence" or "poly(A tail)" refers to a continuous or discontinuous sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3'UTR of the RNA described herein. A continuous poly(A) sequence is characterized by continuous adenylic acid residues. In nature, continuous poly(A) sequences are typical. The RNA disclosed herein may have a poly(A) sequence that is attached to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or a poly(A) sequence that is encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0350] Poly(A) sequences of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of RNA in transfected eukaryotic cells and on the levels of protein translated from open reading frames located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0351] The poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, in particular about 120 A nucleotides. In this context, "consists essentially of" means that most of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly(A) sequence, are A nucleotides, but allow for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that every nucleotide in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence, is an A nucleotide. The term "A nucleotide" or "A" refers to adenylic acid.

[0352] In some embodiments, poly(A) sequences are attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template that contains repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence that encodes the poly(A) sequence (coding strand) is called a poly(A) cassette.

[0353] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides long. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 may be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides, but interrupted by a random sequence in which the four nucleotides (dA, dC, dG, dT) are evenly distributed and have a length of, for example, 5-50 nucleotides, show, at the DNA level, sustained growth of plasmid DNA in Escherichia coli (E. coli) and, at the RNA level, are still associated with beneficial properties with respect to supporting RNA stability and translation efficiency. As a result, in some embodiments, the poly(A) sequences contained in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0354] In some embodiments, no nucleotides other than A nucleotides are adjacent to the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed at its 3' end by nucleotides other than A.

[0355] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0356] In some embodiments, the RNA comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO:10, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:10.

[0357] A particularly preferred poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:10.

[0358] According to the present disclosure, the binding agents are preferably administered as single-stranded 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject to which the RNA is administered. Preferably, the RNA contains structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence) that are optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency.

[0359] In some embodiments, m 7,3’-O Gppp(m12’-O ) ApG is utilized as a specific capping structure at the 5'-end of the RNA. In some embodiments, the 5'-UTR sequence is derived from human alpha globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. In some embodiments, a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term persistence of the mRNA. These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see WO 2017 / 060314, incorporated herein by reference). In some embodiments, a poly(A) sequence measuring 110 nucleotides in length is used, consisting of a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence and another 70 adenosine residues. The poly(A) sequence was designed to increase RNA stability and translation efficiency.

[0360] In some embodiments of all aspects of the invention, the RNA encoding the binding agent is expressed in the cells of the treated subject, such as hepatocytes, to provide the binding agent. In some embodiments of all aspects of the invention, the RNA is transiently expressed in the cells of the subject. In some embodiments of all aspects of the invention, the RNA is in vitro transcribed RNA. In some embodiments of all aspects of the invention, the expression of the binding agent is in the extracellular space, i.e., the binding agent is secreted. In some embodiments of all aspects of the invention, the expression of the binding agent is in the bloodstream.

[0361] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0362] According to the present invention, the term "transcription" includes "in vitro transcription", which relates to a process in which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the generation of the transcript. These cloning vectors are generally called transcription vectors and are included in the term "vector" according to the present invention. According to the present invention, the RNA used in the present invention is preferably an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, the in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of the RNA.

[0363] With respect to RNA, the term "expression" or "translation" refers to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0364] In some embodiments, after administration (e.g., intravenous administration) of the RNA described herein, for example formulated as an RNA-lipid particle, at least a portion of the RNA is delivered to a target cell (e.g., a liver cell). In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein that it codes for. Thus, the present disclosure also relates to a method for delivering RNA to a target cell of a subject, comprising administering to a subject an RNA particle described herein. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein coded by the RNA.

[0365] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0366] In some embodiments, RNA encoding a binding agent administered in accordance with the invention is non-immunogenic.

[0367] The term "non-immunogenic RNA" as used herein refers to an RNA that, for example, when administered to a mammal, does not induce a response by the immune system or induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In a preferred embodiment, the non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that suppress RNA-mediated activation of innate immune receptors and removing double-stranded RNA (dsRNA).

[0368] To render a non-immunogenic RNA non-immunogenic by incorporation of a modified nucleoside, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises the replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is a 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-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 In a particularly preferred embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methylpseudouridine (mψ) or 5-methyluridine (m5U), in particular N1-methylpseudouridine.

[0369] In some embodiments, the substitution of one or more uridines with nucleosides comprising modified nucleobases comprises substitution of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0370] During synthesis of mRNA by in vitro transcription (IVT), for example using T7 RNA polymerase, significant amounts of abnormal products, including double-stranded RNA (dsRNA), are produced due to unusual activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, for example by ion-pair reversed-phase HPLC using non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrices. Alternatively, an enzyme-based method can be used using E. coli RNase III, which specifically hydrolyzes dsRNA but not ssRNA, thereby removing dsRNA contaminants from the IVT RNA preparation. Additionally, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions that allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.

[0371] "Removing" or "removal", as the term is used herein, refers to a characteristic of a population of a first substance, such as non-immunogenic RNA, that is separated from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, the population of the first substance that is characterized by the removal of the population of the second substance has a measurably lower content of the second substance compared to an unseparated mixture of the first substance and the second substance.

[0372] In some embodiments, removing dsRNA from non-immunogenic RNA comprises removing dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, or less than 0.01% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, a purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0373] In some embodiments, the non-immunogenic RNA is translated in cells more efficiently than a standard RNA having the same sequence. In some embodiments, translation is enhanced 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1,000-fold. In some embodiments, translation is enhanced 2,000-fold. In some embodiments, the factor is between 10-1,000-fold. In some embodiments, the fold is 10-100 fold. In some embodiments, the fold is 10-200 fold. In some embodiments, the fold is 10-300 fold. In some embodiments, the fold is 10-500 fold. In some embodiments, the fold is 20-1,000 fold. In some embodiments, the fold is 30-1,000 fold. In some embodiments, the fold is 50-1,000 fold. In some embodiments, the fold is 100-1,000 fold. In some embodiments, the fold is 200-1,000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0374] In some embodiments, the non-immunogenic RNA exhibits significantly less natural immunogenicity than standard RNA having the same sequence. In some embodiments, the non-immunogenic RNA exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced 3-fold. In some embodiments, the natural immunogenicity is reduced 4-fold. In some embodiments, the natural immunogenicity is reduced 5-fold. In some embodiments, the natural immunogenicity is reduced 6-fold. In some embodiments, the natural immunogenicity is reduced 7-fold. In some embodiments, the natural immunogenicity is reduced 8-fold. In some embodiments, the natural immunogenicity is reduced 9-fold. In some embodiments, the natural immunogenicity is reduced 10-fold. In some embodiments, the natural immunogenicity is reduced 15-fold. In some embodiments, the natural immunogenicity is reduced 20-fold. In some embodiments, the natural immunogenicity is reduced 50-fold. In some embodiments, the natural immunogenicity is reduced 100-fold. In some embodiments, the natural immunogenicity is reduced 200-fold. In some embodiments, the natural immunogenicity is reduced 500-fold. In some embodiments, the natural immunogenicity is reduced by 1,000-fold. In some embodiments, the natural immunogenicity is reduced by 2,000-fold.

[0375] The term "exhibits significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, the term refers to a reduction such that an effective amount of the non-immunogenic RNA can be administered without eliciting a detectable natural immune response. In some embodiments, the term refers to a reduction such that the non-immunogenic RNA can be repeatedly administered without eliciting a sufficient natural immune response to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA can be repeatedly administered without eliciting a sufficient natural immune response to eliminate the detectable production of the protein encoded by the non-immunogenic RNA.

[0376] "Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively non-specific, immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0377] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue or system.

[0378] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0379] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0380] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the association of two or more elements or components or domains.

[0381] Codon optimization / increased G / C content In some embodiments, the amino acid sequence of the binding agent described herein is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0382] The term "codon-optimized" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, the coding region is preferably codon-optimized for optimal expression in a subject treated with the RNA molecule described herein. Codon optimization is based on the observation that translation efficiency is also determined by the different frequencies of occurrence of tRNAs in a cell. Thus, the sequence of an RNA can be modified such that a codon for which a frequently occurring tRNA is available is inserted in place of the "codon". Rare

[0383] In some embodiments of the invention, the guanosine / cytosine (G / C) content of the coding region of the RNA described herein is increased compared to the G / C content of the corresponding coding sequence of the wild-type RNA, and the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of its mRNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. With regard to the fact that several codons code for exactly the same amino acid (the so-called degeneracy of the genetic code), it is possible to determine the most favorable codons for stability (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for the modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that code for the same amino acid but do not contain A and / or U or contain a lower content of A and / or U nucleotides.

[0384] In various embodiments, the G / C content of the coding region of the RNA 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.

[0385] Nucleic acid containing particles The nucleic acids described herein, such as RNA encoding a binding agent, can be formulated and administered as particles.

[0386] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecular complexes.

[0387] In some embodiments, the particles described herein are nanoparticles. The term "nanoparticle" refers to nano-sized particles, in which all three external dimensions of the particle are nanoscale, i.e., at least about 1 nm and less than about 1,000 nm. Preferably, the size of a particle is its diameter.

[0388] In some embodiments, a nucleic acid particle comprises two or more types of nucleic acid molecules, the molecular parameters of which may be similar or different from each other, such as with regard to molar mass or basic structural elements, e.g., molecular structure, capping, coding regions or other features.

[0389] In a particle formulation comprising RNA, for example a first RNA and a second RNA, it is possible to formulate each RNA species separately as an individual particle formulation. In that case, each individual particle formulation comprises one RNA species. The individual particle formulations may be present as separate entities, for example in separate containers. Such a formulation may be obtained by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Each particle comprises only the specific RNA species provided when the particle is formed (individual particle formulation). In some embodiments, a composition, such as a pharmaceutical composition, comprises a plurality of individual particle formulations. Each pharmaceutical composition is referred to as a mixed particle formulation. A mixed particle formulation according to the present invention may be obtained by forming the individual particle formulations separately, followed by mixing the individual particle formulations. The mixing step may result in a formulation comprising a mixed population of RNA-containing particles. The individual particle populations may be present together in one container comprising a mixed population of the individual particle formulations. Alternatively, it is possible to formulate all RNA species of the pharmaceutical composition together as a combined particle formulation. Such a formulation can be obtained by providing a combined formulation (typically a combined solution) of all RNA species together with a particle forming agent, thereby allowing the formation of particles.In contrast to a mixed particle formulation, a combined particle formulation typically comprises particles that contain multiple RNA species.In a combined particle composition, different RNA species typically exist together in a single particle.

[0390] "Nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from nucleic acid and at least one particle-forming component, such as at least one cationic or cationically ionizable lipid or lipid-like substance, at least one cationic polymer, such as protamine, or a mixture thereof. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. Without intending to be bound by any theory, it is believed that cationic or cationically ionizable lipid or lipid-like substances and / or cationic polymers form aggregates together with nucleic acids, and this aggregation results in colloidally stable particles. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0391] The term "colloid" as used herein refers to a type of homogenous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have a particle size between 1 and 1,000 nanometers. The mixture may be called a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the entire suspension.

[0392] In some embodiments, the particles described herein further comprise at least one lipid or lipid-like substance other than a cationic or cationic ionizable lipid or lipid-like substance, at least one polymer other than a cationic polymer, or a mixture thereof.

[0393] The nucleic acid particles described herein, in some embodiments, can have an average diameter in the range of about 30 nm to about 1,000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0394] The term "mean diameter" refers to the so-called Z 平均Z refers to the average hydrodynamic diameter of a particle measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in a polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle refers to this Z 平均 Used synonymously with the value of

[0395] The nucleic acid particles described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or less than about 0.2. By way of example, the nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0396] The "polydispersity index", as mentioned in the definition of "average diameter", is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis. Under certain prerequisites, it can be considered as a measure of the size distribution of the nanoparticle ensemble.

[0397] The N / P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid, e.g., RNA. This correlates with the charge ratio, since the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. If there is a charge balance, the N / P ratio is pH dependent. Since positively charged nanoparticles are thought to favor transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. The RNA is then considered to be fully bound to the nanoparticles.

[0398] Various types of nucleic acid-containing particles have been previously described as suitable for delivery of nucleic acids in particulate form (e.g. Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids can physically protect the nucleic acid from degradation and, depending on the specific chemical properties, aid in cellular uptake and endosomal escape.

[0399] The present disclosure describes a particle that comprises nucleic acid, at least one cationic or ionizable lipid or lipid-like substance, and / or at least one cationic polymer that associates with nucleic acid to form nucleic acid particle, as well as a composition that comprises such particle.Nucleic acid particle can comprise nucleic acid that is complexed to particle in various forms by non-covalent interaction.The particles described herein are not virus particles, particularly infectious virus particles, i.e., they cannot infect cells virally.

[0400] Suitable cationic or cationically ionizable lipids or lipid-like substances and cationic polymers form nucleic acid particles and are included in the term "particle-forming component" or "particle-forming agent". The term "particle-forming component" or "particle-forming agent" refers to any component that associates with nucleic acid to form a nucleic acid particle. Such components include any component that can be part of a nucleic acid particle.

[0401] Cationic Polymer Polymers are commonly used materials for nanoparticle-based delivery, given their high degree of chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change state of protonation in the pH range of 5.5-7.5, which is thought to lead to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamines, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0402] As used herein, "polymer" is given its usual meaning, i.e., a molecular structure that includes one or more repeat units (monomers) that are linked by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties as described herein, may also be present in the polymer.

[0403] When two or more types of repeat units are present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer as used herein can be a copolymer. The repeat units forming the copolymer can be arranged in any manner. For example, the repeat units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., to include one or more regions each including a first repeat unit (e.g., a first block), and one or more regions each including a second repeat unit (e.g., a second block), etc. A block copolymer can have two (diblock copolymer), three (triblock copolymer), or more different blocks.

[0404] In some embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be degraded chemically and / or biologically in a physiological environment, such as within the body.

[0405] In some embodiments, the polymer can be protamine or a polyalkyleneimine, particularly protamine.

[0406] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, soluble in water, do not coagulate with heat, and produce primarily arginine upon hydrolysis. In purified form, they are used in long-acting formulations of insulin to neutralize the anticoagulant effect of heparin.

[0407] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence and fragments thereof obtained or derived from natural or biological sources, and multimeric forms of said amino acid sequence or fragments thereof, as well as man-made, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from natural or biological sources.

[0408] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~10 7 Da, preferably 1,000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0409] According to the present disclosure, linear polyalkyleneimines, such as linear polyethyleneimine (PEI), are preferred.

[0410] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In some embodiments, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is enclosed or encapsulated.

[0411] The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0412] Lipids and lipid-like substances The terms "lipid" and "lipid-like material" are broadly defined herein as molecules that contain one or more hydrophobic moieties or groups, and optionally one or more hydrophilic moieties or groups. Molecules that contain hydrophobic and hydrophilic moieties are also often referred to as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and various phases. One of those phases consists of lipid bilayers when they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0413] As used herein, the term "amphiphilic" refers to a molecule that has both polar and non-polar portions. Often, amphipathic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion may have either a formal positive or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a formal negative charge and may be a zwitterion or an inner salt. For purposes of this disclosure, an amphipathic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0414] The terms "lipid-like substances", "lipid-like compounds" or "lipid-like molecules" refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense, especially amphiphiles. For example, the term includes compounds that can form amphiphilic layers when present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants, or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules that contain hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble those of lipids. Examples of lipid-like compounds capable of spontaneous incorporation into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS), and artificial amphiphilic molecules. Lipids are generally cylindrical in shape. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Lipids have low solubility as monomers and tend to aggregate into water-insoluble planar bilayers. Conventional surfactant monomers are generally conical in shape. Hydrophilic head groups tend to occupy more molecular space than linear alkyl chains. Surfactants tend to aggregate into water-soluble spherical or ellipsoidal micelles. Lipids also have the same general structure as surfactants - a polar hydrophilic head group and a non-polar hydrophobic tail - but lipids differ from surfactants in the shape of the monomer, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" should be construed to encompass both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by context.

[0415] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0416] In some embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. In general, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0417] Fatty acids, or fatty acid residues, are a diverse group of molecules consisting of a hydrocarbon chain that terminates in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a non-polar, hydrophobic end that is insoluble in water. The carbon chain, which is typically 4 to 24 carbons long, may be saturated or unsaturated and may be attached to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, there is the possibility of either cis or trans geometric isomerism, which significantly affects the configuration of the molecule. Cis double bonds cause the fatty acid chain to bend, an effect that combines with more double bonds in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0418] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best known being the fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, each of the three hydroxyl groups of glycerol is typically esterified by a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0419] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached by ester bonds to two fatty acid-derived "tails" and by a phosphate ester bond to a "head" group. Examples of glycerophospholipids, commonly called phospholipids (although sphingomyelins are also classified as phospholipids), are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0420] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The major sphingoid base in mammals is commonly called sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated with chain lengths of 16 to 26 carbon atoms. The major sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues attached to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0421] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids together with glycerophospholipids and sphingomyelins.

[0422] Saccharolipids represent compounds in which fatty acids are directly linked to a sugar backbone, forming structures that are compatible with membrane bilayers. In saccharolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best known saccharolipids are the acylated glucosamine precursors of the lipid A component of the lipopolysaccharides of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine that is derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-lipid A, a hexacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0423] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classical enzymes, as well as by iterative and multi-modular enzymes that share mechanistic features with fatty acid synthases. They include numerous secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0424] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0425] Cationic or cationic ionizable lipids or lipid-like substances The nucleic acid particles described herein can comprise at least one cationic or cationically ionizable lipid or lipid-like substance as particle forming agent.The cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein includes any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acid.In some embodiments, the cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein can be associated with nucleic acid, for example, by forming a complex with nucleic acid or by forming a vesicle in which nucleic acid is enclosed or encapsulated.

[0426] As used herein, "cationic lipid" or "cationic lipid-like substance" refers to lipid or lipid-like substance that has a net positive charge.Cationic lipid or lipid-like substance binds to negatively charged nucleic acid by electrostatic interaction.In general, cationic lipid has a lipophilic portion such as sterol, acyl chain, diacyl or higher acyl chain, and the head group of the lipid typically carries a positive charge.

[0427] In some embodiments, the cationic lipid or lipid-like substance has a net positive charge only at a certain pH, particularly an acidic pH, but preferably has no net positive charge, preferably has no charge, i.e., is neutral at a different, preferably higher, pH, such as physiological pH. This ionizable behavior is believed to enhance efficacy by aiding in endosomal escape and reducing toxicity, compared to particles that remain cationic at physiological pH.

[0428] For the purposes of this disclosure, such "cationically ionizable" lipids or lipid-like substances are included in the term "cationic lipid or lipid-like substance," unless the context indicates differently.

[0429] In some embodiments, the cationic or cationically ionizable lipid or lipid-like substance comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated.

[0430] Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3 -Dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-Dimyristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-Dioleyloxy-3-dimethyl ... dioleoyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanamide trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3- beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-Dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N, N-Dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2 -({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-Di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl- 2,3-Bis(dodecyloxy)propan-1-amine (DLDMA), N,N-Dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

[0431] In some embodiments, the cationic lipid may constitute from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, from about 30 mol% to about 100 mol%, from about 40 mol% to about 100 mol%, or from about 50 mol% to about 100 mol% of the total lipid present in the particle.

[0432] Further lipids or lipid-like substances The particles described herein may also comprise lipids or lipid-like substances other than cationic or cationic ionizable lipids or lipid-like substances, i.e. non-cationic lipids or lipid-like substances (including non-cationic ionizable lipids or lipid-like substances).Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances.In addition to ionizable / cationic lipids or lipid-like substances, the formulation of nucleic acid particles may be optimized by adding other hydrophobic moieties such as cholesterol and lipids to enhance particle stability and nucleic acid delivery effectiveness.

[0433] Additional lipids or lipid-like substances may be incorporated that may or may not affect the overall charge of the nucleic acid particle. In some embodiments, the additional lipids or lipid-like substances are non-cationic lipids or lipid-like substances. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipids include one of the following neutral lipid components, such as neutral lipid components: (1) phospholipids; (2) cholesterol or its derivatives; or (3) a mixture of phospholipids and cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0434] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DPC), diac ... BPC), ditricosanoyl phosphatidylcholine (DTPC), dilignoceroyl phosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with various hydrophobic chains.

[0435] In certain preferred embodiments, the additional lipid is DSPC, or DSPC and cholesterol.

[0436] In some embodiments, the nucleic acid particle comprises both a cationic lipid and an additional lipid.

[0437] Without wishing to be bound by theory, the amount of at least one cationic lipid relative to the amount of at least one additional lipid can affect important nucleic acid particle properties such as charge, particle size, stability, tissue selectivity, and nucleic acid bioactivity.Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0438] In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may constitute from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the particle.

[0439] Polymer-conjugated lipids In some embodiments, particle can comprise at least one polymer-conjugated lipid.Polymer-conjugated lipid is typically a molecule that comprises lipid portion and polymer portion conjugated thereto.In some embodiments, polymer-conjugated lipid is PEG-conjugated lipid, also referred to herein as PEGylated lipid or PEG lipid.

[0440] In some embodiments, the polymer-conjugated lipid is designed to sterically stabilize the lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, the polymer-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.

[0441] A variety of PEG-conjugated lipids are known in the art, including, but not limited to, PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0442] In some embodiments, the particles may include one or more PEG-conjugated or PEGylated lipids as described in WO 2017 / 075531 and WO 2018 / 081480, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.

[0443] Lipoplex particles In some embodiments of the present disclosure, the RNA described herein may be present in an RNA lipoplex particle.

[0444] Lipoplexes (LPXs) are generally electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic RNA. The formed lipoplexes have different internal arrangements of molecules resulting from the conversion of the liposomal structure to a compact RNA-lipoplex. These formulations are generally characterized by poor encapsulation of nucleic acids and incomplete capture of nucleic acids.

[0445] Liposomes are spherical vesicles that contain a single or multilamellar phospholipid bilayer surrounding an aqueous core. They are prepared from materials that have a polar head (hydrophilic) group and a non-polar tail (hydrophobic) group. The interaction between these groups induces the formation of vesicles. The cationic lipids used to formulate liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0446] Positively charged liposomes can generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In some embodiments, the RNA lipoplex particles are nanoparticles.

[0447] In some embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0448] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0449] The RNA lipoplex particles described herein, in some embodiments, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0450] RNA lipoplex particles can be prepared using liposomes, which can be obtained by injecting an ethanol solution of lipids into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase contains acetic acid, for example in an amount of about 5 mM. Liposomes can be used to prepare RNA lipoplex particles by mixing the liposomes with RNA.

[0451] In some embodiments, the liposome and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0452] Lipid Nanoparticles (LNPs) In some embodiments, the RNA described herein is present in the form of a lipid nanoparticle (LNP). LNPs can include any lipid capable of forming a particle to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated.

[0453] LNPs typically contain four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a PEG lipid. LNPs can be prepared by mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0454] In some embodiments, in the RNA LNPs described herein, the mRNA is bound by an ionizable lipid that occupies the central core of the LNP. The PEG lipid forms the surface of the LNP together with the phospholipid. In some embodiments, the surface comprises a bilayer. In some embodiments, the charged and uncharged forms of cholesterol and the ionizable lipid can be distributed throughout the LNP.

[0455] In some embodiments, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and PEGylated lipids.

[0456] In some embodiments, the LNPs comprise cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA encapsulated within or associated with the lipid nanoparticles.

[0457] In some embodiments, the LNP comprises 40-55 mol%, 40-50 mol%, 41-50 mol%, 42-50 mol%, 43-50 mol%, 44-50 mol%, 45-50 mol%, 46-50 mol%, 46-49 mol%, or about 47 or 48 mol% cationic lipid. In some embodiments, the LNPs comprise about 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, or 49 mol% cationic lipid.

[0458] In some embodiments, the neutral lipid is present at a concentration ranging from 5-15 mol%, 7-13 mol%, or 9-11 mol%. In some embodiments, the neutral lipid is present at a concentration of about 10 mol%.

[0459] In some embodiments, the steroid is present in a concentration ranging from 30-50 mol%, 35-45 mol%, or 38-43 mol%, hi some embodiments, the steroid is present in a concentration of about 41 mol%.

[0460] In some embodiments, the LNP comprises 1-10 mol%, 1-5 mol%, or 1-2.5 mol% of a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is present at a concentration of about 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mol%.

[0461] In some embodiments, the LNPs comprise 45-50 mol % cationic lipid; 5-15 mol % neutral lipid; 35-45 mol % steroid; 1-5 mol % polymer-conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

[0462] In some embodiments, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle. In some embodiments, the mole percentage is determined based on the total moles of cationic lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.

[0463] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0464] In some embodiments, the steroid is cholesterol.

[0465] In some embodiments, the polymer-conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure: [ka] or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. 12 and R 13 are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 is each independently a linear saturated alkyl chain containing about 14 carbon atoms and w has an average value of about 45.

[0466] In some embodiments, the pegylated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0467] In some embodiments, the cationic lipid component of the LNP has formula (III): [ka] or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a-OR-NR a C(=O)O-, L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O- or a direct bond; G 1 or G 2 are each independently an unsubstituted C1-C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0468] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] or [ka] where: A is a 3-8 membered cycloalkyl or cycloalkylene ring; R 6 is, at each occurrence, independently H, OH or C-C 24 is alkyl; n is an integer ranging from 1 to 15.

[0469] In some of the foregoing embodiments of formula (III), the lipid has the structure (IIIA), and in other embodiments, the lipid has the structure (IIIB).

[0470] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] or [ka] where y and z are each independently an integer in the range of 1 to 12.

[0471] In any of the foregoing embodiments of formula (III), L 1 or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. In some different embodiments of any of the foregoing, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L2 Each of is -(C=O)O-.

[0472] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] or [ka] It has one of the following.

[0473] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] [ka] [ka] or [ka] has one of the following:

[0474] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, such as from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0475] In some other embodiments of the foregoing embodiments of Formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

[0476] In some of the foregoing embodiments of formula (III), R6 is H. In other embodiments of the foregoing embodiments, R6 is C1-C24 alkyl. In other embodiments, R 6 is OH.

[0477] In some embodiments of formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or straight chain C1-C 24 It is alkenylene.

[0478] In some other aforementioned embodiments of formula (III), R 1 Or R 2 , or both are C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka] where R 7a and R 7b is, in each occurrence, independently H or C 12 is alkyl; and a is an integer from 2 to 12; Here, R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0479] In some of the foregoing embodiments of formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the foregoing embodiments, R 7bAt least one occurrence of is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0480] In different embodiments of formula (III), R 1 Or R 2 or both have the following structure: [ka] It has one of the following.

[0481] In some of the foregoing embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0482] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

[0483] Representative compounds of formula (III). [Table 9]

[0484] [Table 10]

[0485] [Table 11]

[0486] [Table 12]

[0487] [Table 13]

[0488] [Table 14]

[0489] [Table 15]

[0490] [Table 16]

[0491] A variety of lipids (including, for example, cationic lipids, neutral lipids, and polymer-conjugated lipids) are known in the art and can be used herein to form lipid nanoparticles, for example lipid nanoparticles targeted to specific cell types (e.g., hepatocytes). In some embodiments, the cationic lipid included in the pharmaceutical composition described herein can be ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) or a derivative thereof. In some embodiments, the neutral lipid included in the pharmaceutical composition described herein can be or include a phospholipid or a derivative thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol. In some embodiments, the polymer-conjugated lipid included in the pharmaceutical composition described herein can be a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof).

[0492] In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (III) is compound III-45. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159. ALC-0159: [ka]

[0493] In some embodiments, the cationic lipid is present in the LNP in an amount of about 45 to about 50 mol %. In some embodiments, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mol %. In some embodiments, the steroid is present in the LNP in an amount of about 35 to about 45 mol percent. In some embodiments, the pegylated lipid is present in the LNP in an amount of about 1 to about 5 mol %.

[0494] In some embodiments, the LNP comprises compound III-45 in an amount of about 45 to about 50 mol %, DSPC in an amount of about 5 to about 15 mol %, cholesterol in an amount of about 35 to about 45 mol %, and ALC-0159 in an amount of about 1 to about 5 mol %.

[0495] In some embodiments, the LNPs comprise compound III-45 in an amount of about 47 or 48 mol%, DSPC in an amount of about 10 mol%, cholesterol in an amount of about 41 mol%, and ALC-0159 in an amount of about 1.6 or 1.7 mol%.

[0496] 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.

[0497] Embodiments of Administered RNA In some embodiments, the compositions or medical preparations described herein comprise an RNA encoding a first polypeptide chain and an RNA encoding a second polypeptide chain, and the first and second polypeptide chains interact with each other to form the binding agent described herein.Similarly, the methods described herein comprise administering such RNA.In some embodiments, the RNA is an in vitro transcribed RNA.

[0498] In some embodiments, the RNA is a nucleoside-modified mRNA (modRNA). In some embodiments, the active ingredient of a nucleoside-modified messenger RNA (modRNA) drug substance is a single-stranded mRNA that is translated upon entry into a cell, e.g., a hepatocyte. In some embodiments, the modRNA contains common structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) that are optimized for maximum efficacy of the RNA. In some embodiments, the modRNA contains 1-methylpseudouridine instead of uridine. In some embodiments, the 5' cap structure is m2 7,3’-O Gppp(m1 2’-O ) ApG. In some embodiments, the 5'-UTR and 3'-UTR comprise the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9, respectively. In some embodiments, the poly(A) tail comprises the sequence of SEQ ID NO: 10. In some embodiments, a further purification step is applied to the modRNA to reduce dsRNA contaminants generated during the in vitro transcription reaction.

[0499] Several embodiments of the first and second RNAs are described below. Certain terms used in describing the elements have the following meanings: 5'UTR: 5'-UTR sequence of human α-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency. sec:sec corresponds to the secretory signal peptide (sec), which directs the translocation of the nascent polypeptide chain to the endoplasmic reticulum. 3'UTR: The 3'-UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression. PolyA: A poly(A) tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues followed by a linker sequence of 10 nucleotides and another 70 adenosine residues designed to increase RNA stability and translation efficiency in dendritic cells. VH(aCD3): variable region of the heavy chain of an immunoglobulin with specificity for CD3 VL(aCD3) variable region of the immunoglobulin light chain with specificity for CD3 CH1: constant region 1 of the immunoglobulin heavy chain CL: constant region of the immunoglobulin light chain VH(aCLDN6): variable region of the heavy chain of an immunoglobulin with specificity for CLDN6 VL(aCLDN6): variable region of the immunoglobulin light chain with specificity for CLDN6

[0500] RBP022.1 (SEQ ID NO:4; SEQ ID NO:5) - "First RNA" embodiments Cap 1(m2 7,3’-O Gppp(m1 2’-O )ApG)-5'UTR-sec-VH(aCD3)-CH1-VH(aCLDN6)-VL(aCLDN6)-3UTR-PolyA

[0501] RBP021.1 (SEQ ID NO:6; SEQ ID NO:7) - "Second RNA" embodiments Cap 1(m2 7,3’-O Gppp(m1 2’-O )ApG)-5'UTR-sec-VL(aCD3)-CL-VH(aCLDN6)-VL(aCLDN6)-3'UTR-polyA

[0502] Nucleotide sequences of RBP022.1 and RBP021.1 The nucleotide sequence is shown with the individual sequence elements shown in bold, and the sequence of the translated protein is shown in italics below the coding nucleotide sequence (*=stop codon).

[0503]

number

[0504]

number

[0505]

number

[0506]

number

[0507]

number

[0508]

number

[0509]

number

[0510]

number

[0511] In some embodiments, the first RNA described herein comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the second RNA described herein comprises the nucleotide sequence of SEQ ID NO: 7.

[0512] The RNA described herein is preferably formulated into lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA. In some embodiments, the cationic lipids are ALC-0366, the neutral lipids are DSPC, the steroids are cholesterol, and the polymer-conjugated lipids are ALC-0159. The preferred method of administration is intravenous administration.

[0513] In some embodiments, the formulation comprises the following ingredients: [Table 17]

[0514] In some embodiments, the formulation comprises the following ingredients, preferably in the proportions or concentrations shown: [Table 18]

[0515] ALC-0366, ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine; DS = drug substance; Ph.Eur. = European Pharmacopoeia; qs = quantity required (quantity that may be sufficient); USP-NF = United States Pharmacopoeia and National Formulary

[0516] In some embodiments, the ratio of mRNA to total lipids (N / P) is 6.0 to 6.5, for example about 6.0 or about 6.3.

[0517] Pharmaceutical Compositions The agents described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition.

[0518] In some embodiments where a pharmaceutical composition comprises a first RNA and a second RNA as described herein, such first RNA and second RNA may be present in a molar ratio of about 3:1 to about 1:3, or in some embodiments, in a molar ratio of about 2:1 to about 1:2, or in some embodiments, in a molar ratio of about 1.5:1 to about 1:1.5. In some embodiments, such first RNA and second RNA may be present in a molar ratio of about 3:1 to about 1:1, or in some embodiments, in a molar ratio of about 2:1 to about 1:1, or in some embodiments, in a molar ratio of about 1.5:1.

[0519] In some embodiments in which a pharmaceutical composition comprises a first RNA and a second RNA as described herein, such first RNA and second RNA may be present in a weight (w / w) ratio of about 3:1 to about 1:3, or in some embodiments in a (w / w) ratio of about 2:1 to about 1:2, or in some embodiments in a (w / w) ratio of about 1.5:1 to about 1:1.5. In some embodiments, such first and second RNAs may be present in a (w / w) ratio of about 3:1 to about 1:1, or in some embodiments in a (w / w) ratio of about 2:1 to about 1:1, or in some embodiments in a (w / w) ratio of about 1.75:1 to about 1.25:1, or in some embodiments in a (w / w) ratio of about 1.75:1 to about 1.5:1, or in some embodiments in a (w / w) ratio of about 1.5:1 to about 1.25:1, or in some preferred embodiments in a (w / w) ratio of about 1.5:1.

[0520] In some embodiments, the pharmaceutical compositions described herein are compositions for treating cancer in a subject.

[0521] In some embodiments of all aspects of the invention, the components described herein, such as RNA encoding a binding agent, may be administered in a pharmaceutical composition, which may include a pharma- ceutically acceptable carrier, and may optionally include one or more adjuvants, stabilizers, etc. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatment, e.g., for use in treating or preventing cancer.

[0522] The term "pharmaceutical composition" refers to a formulation comprising a therapeutically active agent, preferably together with a pharma-ceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering said pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0523] Pharmaceutical compositions according to the present disclosure are generally applied in "pharmaceutical effective amounts" and "pharmaceutical acceptable preparations."

[0524] The term "pharmaceutical acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of a pharmaceutical composition.

[0525] The term "pharmaceutical effective amount" or "therapeutically effective amount" refers to an amount that alone or together with further doses achieves the desired response or the desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay of the onset or prevention of the onset of said disease or said condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, the individual parameters of the patient, including age, physiological state, size and weight, the duration of the treatment, the type of concomitant treatment (if any), the specific route of administration and similar factors. Thus, the dose administered of the compositions described herein may depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0526] In some embodiments, each dose or cumulative dose of the pharmaceutical compositions described herein may contain RNA encoding a CLDN6-targeted binding agent in an amount of 0.05 μg / kg or more, for example, in an amount ranging from 0.05 μg / kg to 5 mg / kg, or from 0.05 μg / kg to 500 μg / kg, or from 0.5 μg / kg to 500 μg / kg, or from 1 μg / kg to 50 μg / kg, or from 5 μg / kg to 150 μg / kg, or from 15 μg / kg to 150 μg / kg, where kg refers to kg of body weight of the subject to be treated. As will be apparent to one of skill in the art, the binding agents disclosed herein include two polypeptides, each encoded by a separate RNA, and therefore the amounts shown relate to the cumulative amount of RNA encoding the first and second polypeptide chains. Preferably, each dose or cumulative dose comprises a mixture of RNA encoding the first and second polypeptide chains in a total amount of 5 μg / kg to 150 μg / kg, or 15 μg / kg to 150 μg / kg.

[0527] In some embodiments, the pharmaceutical compositions described herein are administered to deliver an RNA (e.g., mRNA) described herein encoding a binding agent to CLDN6 to achieve levels (e.g., plasma and / or tissue levels) of the binding agent of about 0.05 ng / mL or greater.

[0528] In some embodiments, administration may include only a single dose. In some embodiments, administration may include application of a fixed number of doses. In some embodiments, administration may include administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) for at least a selected period of time.

[0529] In some embodiments, the pharmaceutical compositions described herein are administered to a subject suffering from a CLDN6-positive cancer, e.g., a CLDN6-positive solid tumor, in at least one or more (e.g., including at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more) dosing cycles. In some embodiments, each dosing cycle can be a three-week dosing cycle. In some embodiments, the pharmaceutical compositions described herein are administered in at least one administration per dosing cycle. In some embodiments, a dosing cycle includes administration of a set number and / or pattern of administration; in some embodiments, a dosing cycle includes administering a set cumulative dose, e.g., over a specific period of time, and optionally, via multiple administrations, which can be administered, e.g., at set intervals (one or more) and / or according to a set pattern.

[0530] Those skilled in the art will recognize that cancer therapeutics are often administered in dosing cycles. In some embodiments, the pharmaceutical compositions described herein are administered in one or more dosing cycles.

[0531] In some embodiments, one administration cycle is at least 3 days or more (e.g., including at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days).

[0532] In some embodiments, one administration cycle can include multiple administrations, for example, according to a pattern where a dose can be administered every day within the cycle, or where a dose can be administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, etc. within the cycle.

[0533] In some embodiments, multiple cycles may be administered. For example, in some embodiments, at least two cycles (including, for example, at least three cycles, at least four cycles, at least five cycles, at least six cycles, at least seven cycles, at least eight cycles, at least nine cycles, at least ten cycles, or more) may be administered. In some embodiments, at least three to eight cycles of administration may be administered.

[0534] In some embodiments, there may be a "drug holiday" between cycles; in some embodiments, there may not be a drug holiday between cycles. In some embodiments, there may or may not be a drug holiday between cycles.

[0535] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure include one or more pharma- ceutically acceptable carriers, diluents, and / or excipients.

[0536] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0537] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0538] The term "diluent" refers to an agent that dilutes and / or thins. Furthermore, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and / or mixture medium. Examples of suitable diluents include ethanol, glycerol and water.

[0539] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which the active ingredient is combined to facilitate, enhance, or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0540] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0541] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0542] In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, which in some embodiments may enhance the stability of such compositions under certain conditions. For example, in some embodiments, the pharmaceutical compositions may further comprise a cryoprotectant (e.g., sucrose) and / or an aqueous buffer, which in some embodiments may include one or more salts (e.g., sodium salts).

[0543] In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In some embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the digestive tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the digestive tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration, for example, intravenous administration.

[0544] As used herein, the term "co-administration" refers to the process by which different compounds or compositions are administered to the same patient. The different compounds or compositions can be administered simultaneously, essentially simultaneously, or sequentially.

[0545] Efficacy of the Agents and Treatments Described Herein The treatments described herein may result in immune effector functions, such as T cell-mediated effector functions, on target cells, which may result in the killing of target cells. In some embodiments, effector functions in the context of the present invention include the activation of cytotoxic CD4+ and / or CD8+ lymphocytes (CTLs) and the elimination of target cells, i.e., cells characterized by the expression of antigens, i.e., CLDN6, for example, via apoptosis or perforin-mediated apoptosis and cytolysis, the production of cytokines such as IFN-γ and TNF-α, and specific cytotoxicity of antigen-expressing target cells.

[0546] Once activated, cytotoxic lymphocytes can cause the destruction of target cells. For example, cytotoxic T cells can cause the destruction of target cells by either or both of the following means. First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in target cells, and granzymes enter the cells and trigger the cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cells. Second, apoptosis can be induced via Fas-Fas ligand interaction between T cells and target cells.

[0547] In some embodiments, the binding agent described herein, which comprises two binding domains specific for CLDN6 expressed by cancer cells and a binding domain specific for CD3 expressed by T cells, targets the cytotoxic effect of T cells expressing CD3 to cancer cells expressing CLDN6. In some embodiments, binding of the binding agent to CD3 on T cells results in proliferation and / or activation of the T cells. In some embodiments, the T cells release cytotoxic factors, such as perforin and granzymes, initiating cytolysis and apoptosis of the cancer cells. In some embodiments, the binding agent induces T cell-mediated cytotoxicity against cancer cells expressing CLDN6. In some embodiments, the binding agent induces immune effector functions described herein. In some embodiments, the immune effector functions are directed to cells bearing the tumor-associated antigen CLDN6 on their surface.

[0548] In some embodiments, the cancer cell(s) are selected from the group consisting of bladder cancer, ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung carcinoma and adenocarcinoma or non-squamous types of non-small cell lung cancer (NSCLC), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, particularly renal cell carcinoma including renal clear cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including cancer of the ileum, particularly small intestinal adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis, and metastatic forms thereof.

[0549] As used herein, "immune response" refers to the integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. The immune system is divided into the most important innate immune system and the adaptive or adaptive immune system of vertebrates, each of which contains a humoral component and a cellular component.

[0550] "Cell-mediated immunity", "cell-mediated immunity", "cell-mediated immune response", or similar terms are intended to include cellular responses to cells characterized by the expression of antigens, particularly those characterized by the presentation of antigens by class I or class II MHC. The cellular response involves immune effector cells, particularly cells called T cells or T lymphocytes, which act as either "helpers" or "killers". Helper T cells (also called CD4+ T cells) play a central role by regulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells) kill diseased cells, such as virus-infected cells, and prevent the production of further diseased cells.

[0551] The term "immune effector cells" or "immunoreactive cells" in the context of the present invention relates to cells that exert effector functions during an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, "immune effector cells" are T cells, preferably CD4+ and / or CD8+ T cells, most preferably CD8+ T cells. According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, in particular T helper cells, or cytolytic T cells) with appropriate stimulation. Immune effector cells include CD34+ hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system when exposed to an antigen.

[0552] The terms "T cells" and "T lymphocytes" are used interchangeably herein and include, but are not limited to, T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs), including cytolytic T cells.

[0553] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor on the surface of these cells, called the T cell receptor (TCR). The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a different function.

[0554] T helper cells assist other white blood cells in immunological processes, including, among other functions, maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in active immune responses.

[0555] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface, and are therefore also known as CD8+ T cells. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0556] The majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The TCR of a T cell can bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade in the T cell, leading to proliferation and differentiation into mature effector T cells. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two separate peptide chains called the α-TCR chain and the β-TCR chain. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is much rarer than αβ T cells (2% of all T cells).

[0557] "Humoral immunity" or "humoral immune response" is the aspect of immunity mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It is in contrast to cell-mediated immunity. The aspect involving antibodies is often referred to as antibody-mediated immunity.

[0558] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically phagocytose and kill foreign pathogens within the macrophage by hydrolytic and oxidative attacks that result in the degradation of the pathogen. Peptides derived from degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, resulting in the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0559] The term "dendritic cells" (DC) refers to another subtype of phagocytes that belong to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitors first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated to become mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small fragments, and upon maturation, present the fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as co-receptors in T cell activation, such as CD80, CD86 and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they act as antigen-presenting cells, activating helper and killer T cells and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce immune responses associated with T cells or B cells. In some embodiments, the dendritic cells are splenic dendritic cells.

[0560] The term "antigen-presenting cell" (APC) is one of a variety of cells that can display, acquire, and / or present at least one antigen or antigen fragment on (or at) their cell surface. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0561] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cells, the antigen-presenting cells produce costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0562] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules, but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0563] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of proteins into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells, such as antigen-presenting cells.

[0564] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also involve the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector functions. The term "activated immune effector cells" refers, among other things, to immune effector cells that undergo cell division.

[0565] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, resulting in differentiation into an effector cell, such as an effector T cell.

[0566] The term "clonal expansion" or "expansion" refers to the process by which a particular entity increases. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated, proliferate, and a particular immune effector cell is amplified. Preferably, clonal expansion results in differentiation of the immune effector cells.

[0567] treatment The present invention provides methods and agents for treating or preventing a disease or disorder described herein, particularly a disease associated with expression of CLDN6, such as CLDN6-positive cancer, in a subject. The methods described herein may comprise administering an effective amount of a composition comprising an RNA encoding a binding agent described herein.

[0568] The term "disease associated with expression" when referring to an antigen refers to any disease related to the antigen, e.g., a disease characterized by the presence of the antigen. The antigen may be a disease-associated antigen, such as a tumor-associated antigen, e.g., CLDN6. In some embodiments, the disease associated with expression of the antigen is preferably a disease involving cells that express the antigen on the cell surface.

[0569] The therapeutic compounds or compositions of the present invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from a disease or disorder or at risk (or susceptible) of developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration is performed prior to the onset of overt clinical symptoms of the disease, such that the disease or disorder is prevented or its progression is delayed. In the context of the medical field, the term "preventing" includes any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can be performed at primary, secondary and tertiary prevention levels. While primary prevention avoids the onset of the disease, secondary and tertiary levels of prevention include activities aimed at preventing the progression and emergence of symptoms of the disease by restoring function and reducing disease-related complications, as well as reducing the adverse effects of an already established disease.

[0570] In some embodiments, administration of an agent or composition of the invention may be performed by a single dose or may be boosted by multiple doses.

[0571] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with certain symptoms and signs. A disease may be caused by factors from an external source, such as an infection, or may be caused by an internal malfunction, such as an autoimmune disease. In humans, "disease" is often used more broadly to refer to a condition that causes pain, disability, distress, social problems, or death to the affected individual, or similar problems to those who come into contact with the individual. In this broader sense, disease may sometimes include damage, disability, disorder, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Diseases usually affect individuals not only physically but also emotionally, as suffering from and living with many diseases can change one's outlook on life and personality.

[0572] As used herein, the term "disease" includes cancer, particularly the forms of cancer described herein. Reference herein to cancer or a particular form of cancer also includes cancer metastasis thereof. In some embodiments, the disease treated according to the present application is associated with cells expressing CLDN6.

[0573] "Diseases associated with cells expressing CLDN6" or similar expressions means, according to the present invention, that CLDN6 is expressed in cells of diseased tissues or organs. In some embodiments, the expression of CLDN6 in cells of diseased tissues or organs is increased compared to the state in healthy tissues or organs. Increase refers to an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1,000%, at least 10,000% or even more. In some embodiments, expression is found only in diseased tissues, while expression in healthy tissues is suppressed. According to the present invention, diseases associated with cells expressing CLDN6 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those in which cancer cells express CLDN6.

[0574] As used herein, "cancer disease" or "cancer" includes diseases characterized by abnormally controlled cell growth, proliferation, differentiation, adhesion, and / or migration. "Tumor cells" refers to abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow after the stimuli that initiated new growth have ceased. Preferably, the "cancer disease" is characterized by cells that express CLDN6, and the cancer cells express CLDN6. The cells that express CLDN6 are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0575] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, digestive cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, nasopharyngeal (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, as well as metastases thereof. Examples thereof are lung carcinoma, breast carcinoma, prostate carcinoma, colon carcinoma, renal cell carcinoma, cervical carcinoma, or metastases of the above cancer types or tumors. The term cancer according to the present invention also includes cancer metastases.

[0576] According to the present invention, "carcinoma" is a malignant tumor derived from epithelial cells. This group is the most common cancer, including the common forms of breast, prostate, lung and colon cancer.

[0577] An "adenocarcinoma" is a cancer that originates from glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line the body cavities and organs of the body. Epithelia are embryologically derived from ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, the cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while those that are not well-differentiated may not. By staining the cells from the biopsy, the pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinoma can occur in many tissues of the body due to the ubiquity of glands in the body. Although each gland may not secrete the same substances, as long as there is an exocrine function to the cells, they are considered glands, and therefore their malignant forms are named adenocarcinomas. Malignant adenocarcinomas often invade other tissues and metastasize if given enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma and endometrioid adenocarcinoma.

[0578] "Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process, which depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter the body cavities and blood vessels, then transported by the blood, and then invasion of the target organ. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, since tumor cells or components may remain and develop metastatic potential. In some embodiments, the term "metastasis" according to the present invention relates to "distant metastasis", which refers to metastasis away from the primary tumor and the regional lymph node system. In some embodiments, the term "metastasis" according to the present invention relates to lymph node metastasis. One particular form of metastasis treatable using the treatment of the present invention is metastasis originating from gastric cancer as a primary site. In a preferred embodiment, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0579] As used herein, the term "unresectable tumor" typically refers to a tumor characterized by one or more features that, according to sound medical judgment, are believed to indicate that the tumor cannot be safely removed by surgery (e.g., without undue harm to the subject), and / or a tumor for which a competent medical professional has determined that the risks to the subject of tumor removal outweigh the benefits associated with such removal. In some embodiments, an unresectable tumor refers to a tumor that involves and / or has grown into an essential organ or tissue (including blood vessels that may not be reconstructable), and / or is in a location that cannot be easily accessed surgically without undue risk of damage to one or more other important or essential organs and / or tissues (including blood vessels). In some embodiments, the "unresectable nature" of a tumor refers to the possibility of achieving a margin-negative (R0) resection. In the context of pancreatic cancer, encasement of major vessels by the tumor, such as the superior mesenteric artery (SMA) or celiac artery, portal vein obstruction, and the presence of celiac or para-aortic lymphadenopathy are commonly recognized findings that preclude R0 surgery. One of ordinary skill in the art would understand the parameters that determine whether a tumor is unresectable.

[0580] "Target cell" means an unwanted cell, such as a cancer cell. In a preferred embodiment, the target cell expresses CLDN6.

[0581] In some embodiments, the CLDN6 positive cancer comprises CLDN6 positive advanced solid tumor. In some embodiments, the CLDN6 positive cancer is selected from the group consisting of advanced / metastatic CLDN6 positive ovarian cancer, non-squamous non-small cell lung cancer (NSCLC), endometrial cancer and testicular cancer, which do not have available standard treatments that are particularly likely to provide clinical benefit. In some embodiments, the CLDN6 positive cancer comprises tumors not otherwise specified (NOS), including rare tumors and cancers of unknown primary. Such cancers can be tested for CLDN6 expression.

[0582] In some embodiments, the CLDN6 positive cancer is selected from the group consisting of bladder cancer, ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung carcinoma and adenocarcinoma or non-squamous type of non-small cell lung cancer (NSCLC), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, The cancer is selected from the group consisting of bile duct cancer, bladder cancer, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, particularly renal cell carcinoma including renal clear cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including cancer of the ileum, particularly small intestinal adenocarcinoma and adenocarcinoma of the ileum, embryonal carcinoma of the testis, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis, and metastatic forms thereof.

[0583] In this context, the term "treatment", "treat" or "therapeutic intervention" refers to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition suffered by a subject, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or relieve symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the aim of combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0584] The term "therapeutic treatment" refers to any treatment that improves the health status and / or extends (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, may halt or delay the onset of the disease in an individual, may inhibit or delay the onset of the disease in an individual, may reduce the frequency or severity of symptoms in an individual, and / or may reduce recurrence in an individual who currently has or has previously had the disease.

[0585] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0586] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder, but may be affected or susceptible to a disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderly people, children, and newborns. In an embodiment of the present disclosure, an "individual" or "subject" is a "patient."

[0587] The term "patient" refers to an individual or subject for treatment, in particular a diseased individual or subject.

[0588] Citation of documents and tests referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to the applicants and do not constitute any admission as to the accuracy of the contents of these documents.

[0589] The following description is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded the scope consistent with the claims. EXAMPLES

[0590] Example 1: Physical, chemical and pharmaceutical properties of BNT142. drug substance The BNT142 drug substance is an RNA mixture encoding the heavy (HC) and light (LC) chains of an antigen-binding fragment (Fab)-(scFv)2-based bispecific antibody-or tribody-against CLDN6 and CD3, hereafter referred to as RiboMab02.1. RiboMab02.1 recognizes conformational epitopes of CLDN6 and CD3 epsilon (ε) chains. The active ingredient of the drug substance is a mixture of two single-stranded 5'-capped and N1-methylpseudouridine modified RNAs that are translated into their respective protein subunits and form a complete bispecific antibody in target cells. Figure 1 shows a schematic diagram of the general structure of protein-coding RNAs, as determined by the respective nucleotide sequences of linearized plasmid deoxyribonucleic acid (DNA) used as a template for in vitro RNA transcription. In addition to sequences encoding the HC and LC, each RNA contains common structural elements (5' cap, 5'-untranslated region [UTR], 3'-UTR, and poly[A] tail) that are optimized for maximum effectiveness with respect to stability and translation efficiency.

[0591] formulation Description of the formulation The formulation is a sterile RNA-LNP dispersion in an aqueous buffer for IV administration. Manufacturing of the formulation involves encapsulation of RNA in nanoscale lipid particles composed of specialized lipid components. When formulated into LNPs, the RNA drug substance is protected from degradation in serum. The LNP formulation allows escape of the RNA from endosomal compartments into the cell cytosol, where the RNA becomes available for translation into functional proteins. The composition of the BNT142 formulation is shown in Table 2. [Table 19]

[0592] ALC-0366, ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine; DS = drug substance; Ph.Eur. = European Pharmaco...

Claims

1. (i)A variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CD3 (VH(CD3)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 18, 19, and 20 or 21; a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 25, 26, and 27; and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 28, 29, and 30, and a first RNA encoding a first polypeptide chain containing VL(CLDN6); and (ii)A variable region of a light chain (VL) derived from an immunoglobulin having specificity for CD3 (VL(CD3)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 22, 23, and 24; a variable region of a heavy chain (VH) derived from an immunoglobulin having specificity for CLDN6 (VH(CLDN6)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 25, 26, and 27; and a variable region of a light chain (VL) derived from an immunoglobulin having specificity for CLDN6 (VL(CLDN6)), which contains CDR1, CDR2, and CDR3 of SEQ ID NO: 28, 29, and 30, and a second RNA encoding a second polypeptide chain containing VL(CLDN6) A composition or pharmaceutical preparation comprising the same.

2. The composition or pharmaceutical preparation according to Claim 1, wherein the ratio of the first RNA to the second RNA is 1.5:1 to 1.25:1 (w / w).

3. (i) The first polypeptide chain interacts with the second polypeptide chain to form a binding domain having specificity for CD3 and two binding domains having specificity for CLDN6; and / or (ii)VH(CD3) of the first polypeptide chain interacts with VL(CD3) of the second polypeptide chain to form a binding domain having specificity for CD3, VH(CLDN6) and VL(CLDN6) of the first polypeptide chain interact to form a binding domain having specificity for CLDN6, and The VH (CLDN6) and VL (CLDN6) of the second polypeptide chain interact with each other to form a binding domain having specificity for CLDN6; and / or (iii) the first and second polypeptide chains include a constant region 1 (CH1) of a heavy chain derived from an immunoglobulin and a constant region (CL) of a light chain derived from an immunoglobulin, and CH1 on the first polypeptide chain interacts with CL on the second polypeptide chain; and / or (iv) The composition or pharmaceutical preparation according to claim 1, wherein the immunoglobulin is IgG1. **Claim 4** (i) The VH, VL, and CH1 on the first polypeptide chain are VH(CD3) - CH1 - VH(CLDN6) - VL(CLDN6), or VH(CD3) - CH1 - VL(CLDN6) - VH(CLDN6) arranged in order from the N-terminus to the C-terminus; and / or, (ii) The CH1 is connected to the VH(CLDN6) or VL(CLDN6) by a peptide linker, and the peptide linker includes the amino acid sequence SGPGGGRSS(G4S)2; and / or (iii) The VH, VL, and CL on the second polypeptide chain are VL(CD3) - CL - VH(CLDN6) - VL(CLDN6), or VL(CD3) - CL - VL(CLDN6) - VH(CLDN6) arranged in order from the N-terminus to the C-terminus; and / or (iv) The CL is connected to the VH(CLDN6) or VL(CLDN6) by a peptide linker, and the peptide linker includes the amino acid sequence DVPGGS; and / or (v) The VH(CLDN6) and the VL(CLDN6) are connected to each other by a peptide linker, and the peptide linker includes the amino acid sequence (G4S)4. The composition or pharmaceutical preparation according to claim 1. **Claim 5** (i) The VH(CD3) includes the amino acid sequence of amino acids 27 to 145 of SEQ ID NO: 4, the VL(CD3) includes the amino acid sequence of amino acids 27 to 132 of SEQ ID NO: 6, the VH(CLDN6) includes the amino acid sequence of amino acids 267 to 383 of SEQ ID NO: 4, and / or the VL(CLDN6) includes the amino acid sequence of amino acids 404 to 510 of SEQ ID NO: 4; and / or (ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 4; and / or (iii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 6; and / or (iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 6; and / or (v) at least one of the first polypeptide and the second polypeptide is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to the wild-type coding sequence, wherein the codon optimization and / or the increase in G / C content do not change the sequence of the encoded amino acid sequence; and / or (vi) the RNA contains a modified nucleoside instead of uridine, and the modified nucleoside is selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U), the composition or pharmaceutical formulation according to claim 1.

6. (i) at least one RNA contains a 5' cap m ２ ７，３’－Ｏ Gppp(m １ ２’－Ｏ )ApG; and / or (ii) at least one RNA comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8; and / or (iii) at least one RNA comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 9; and / or (iv) at least one RNA comprises a polyA sequence, the polyA sequence comprising at least 100 nucleotides, or comprising or consisting of the nucleotide sequence of SEQ ID NO: 10, the composition or pharmaceutical formulation according to claim 1.

7. (i) the first RNA comprises the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 5; and / or (ii) the second RNA comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 7; and / or (iii) the first RNA comprises the nucleotide sequence of SEQ ID NO: 5, and the second RNA comprises the nucleotide sequence of SEQ ID NO: 7, The composition or pharmaceutical formulation according to claim 1.

8. (i) the RNA is mRNA; and / or (ii) the RNA is formulated as a liquid, as a solid, or a combination thereof; and / or (iii) the RNA is formulated or is to be formulated for injection; and / or (iv) the RNA is formulated or is to be formulated for intravenous administration, The composition or pharmaceutical formulation according to claim 1.

9. The RNA is formulated or is to be formulated as particles, and the particles are lipid nanoparticles (LNP), The composition or pharmaceutical formulation according to claim 1.

10. The pharmaceutical composition comprises RNA in a dose of 0.05 μg / kg or more, or 0.05 μg / kg to 5 mg / kg, or 0.05 μg / kg to 500 μg / kg, or 0.5 μg / kg to 500 μg / kg, or 1 μg / kg to 50 μg / kg, or 5 μg / kg to 150 μg / kg, or 15 μg / kg to 150 μg / kg for encoding the first and second polypeptides, where kg refers to the body weight kg of the subject to be treated, and the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients, The composition or pharmaceutical formulation according to claim 1.

11. The pharmaceutical formulation is a kit, (i) the RNA and the particle-forming component are in separate vials; and / or (ii) the kit further comprises instructions for use of the composition or pharmaceutical formulation for treating or preventing cancer, The composition or pharmaceutical formulation according to claim 1. The composition or pharmaceutical preparation according to claim 1, which is for administration to humans.

13. The composition or pharmaceutical preparation according to any one of claims 1 to 12 for pharmaceutical use.

14. The composition or pharmaceutical preparation according to any one of claims 1 to 12 for use in a method for the therapeutic or prophylactic treatment of CLDN6-positive cancer.

15. The therapeutic or prophylactic treatment of CLDN6-positive cancer further comprises administering a further therapy, wherein the further therapy comprises one or more selected from the group consisting of (i) surgery to remove, excise, or debulk the tumor, (ii) radiotherapy, (iii) chemotherapy, and (iv) administering a further therapeutic agent, The composition or pharmaceutical preparation according to claim 14, wherein the further therapeutic agent comprises an anti-cancer therapeutic agent.

16. (i) the RNA is administered once a week by injection; and / or (ii) the RNA is administered by intravenous administration, the composition or pharmaceutical preparation according to claim 15.

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