Novel combinations and immune therapy using the same

Abstract

The present invention relates to combinations of host cells expression antigen binding proteins that specifically bind to a tumor expressed Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide in a complex with MHC, and mRNAs encoding PRAME antigenic peptides. The invention further relates to combined TCR-T / mRNA therapy employing said mRNAs and host cells. In particular, a combined TCR-T / mRNA therapy is provided for use in cancer treatment.

Description

[0001] P296PC00 / 3000181 -001977 NOVEL COMBINATIONS AND IMMUNE THERAPY USING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to combinations and combination treatments of host cells comprising antigen binding proteins that specifically bind to target cells expressing an Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide and nucleic acids encoding such PRAME antigenic peptides. In embodiments, TCR-T cell therapy in combination with mRNA treatment is provided for use in cancer treatment.

[0004] BACKGROUND OF THE INVENTION TCR-based immunotherapy targets peptide epitopes derived from tumor-associated or tumorspecific proteins, which are presented by molecules of the major histocompatibility complex (MHC). These tumor associated antigens (TAAs) can be peptides derived from all protein classes, such as enzymes, receptors, transcription factors, etc., which are specifically expressed by cancer cells, and / or upregulated in cancer cells. Unlike chimeric antigen receptor (CAR)-T cell therapy and current antibody-based approaches, which can only target cell surface proteins, TCR-based immunotherapy enables the targeting of otherwise inaccessible intracellular proteins and thus significantly increases the number and diversity of targets derived from tumor-associated or tumor-specific proteins.

[0005] Preferentially Expressed Antigen of Melanoma (PRAME) is encoded by the PRAME gene, which is expressed at a high level in a large proportion of cancerous tumors, including melanomas, non-small-cell lung carcinomas, ovarian carcinoma renal cell carcinoma (RCC), breast carcinoma, cervix carcinoma, colon carcinoma, sarcoma, neuroblastoma, as well as several types of leukemia. PRAME is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer / testis antigen (CTA) that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells.

[0006] CTAs are attractive targets for cancer immunotherapy due to their restricted expression in germ cells and aberrant reactivation in various cancers, and their immunogenic properties. Many of these cancers still have a high unmet medical need, with patients needing improved, effective, and specific therapeutics. Accordingly, there exists a need to develop new anti-cancer therapies that specifically target intracellular proteins highly specific to cancer cells. P296PC00 / 3000181 -001977

[0007] SUMMARY OF THE INVENTION

[0008] In one aspect, the invention provides a method for inducing or enhancing an immune response in a subject in need thereof, comprising administering to said subject: a) a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b) an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0009] In a further aspect, the invention provides a method for treating a proliferative disease in a subject in need thereof, comprising administering to said subject: a) a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b) an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0010] In a further aspect, the invention provides a combination comprising: a) a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b) an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0011] In a further aspect, the invention provides a kit comprising a) a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b) an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail; c) optionally packaging material; and d) optionally a label or packaging insert contained within said packaging material indicating that said combination is effective for a method of treating cancer or for use in the treatment of cancer.

[0012] In a further aspect, the invention provides, a combination comprising: a) at least one nucleic acid(s) and / or vector(s) encoding an antigen binding protein capable of specifically binding to a P296PC00 / 3000181 -001977 Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b) an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0013] In a further aspect, the invention provides a kit comprising a) at least one nucleic acid(s) and / or vector(s) encoding an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ); and b) an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail; c) optionally packaging material; and d) optionally a label or packaging insert contained within said packaging material indicating that said combination is effective for a method of treating cancer or for use in the treatment of cancer.

[0014] In a further aspect, the invention provides a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1) for use in a method of treating a proliferative disease, wherein treatment comprises administering to a subject in need thereof a) said host cell, and b) an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0015] In a further aspect, the invention provides an mRNA encoding a PRAME epitope concatemeric polypeptide for use in a method of treating a proliferative disease, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail, for use in a method of treating a proliferative disease, wherein said treatment comprises administering to a subject in need thereof a) said mRNA, and b)a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1) to a subject in need thereof.

[0016] In some embodiments, the polypeptide comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 repeats, about 5-15 repeats, about 7-14 repeats, about 8-13 repeats, or about 9-12 repeats of the PRAME epitope. In some embodiments, the polypeptide comprises no alanine linker residues. In some embodiments, the polypeptide comprises an alanine linker linking adjacent repeats of the PRAME epitope. In some embodiments, P296PC00 / 3000181 -001977 the alanine linker comprises a single alanine reside. In some embodiments, the alanine linker comprises at least two alanine residues. In some embodiments, the alanine linker comprises at least three alanine residues. In some embodiments, the polypeptide comprises 12 repeats of the PRAME epitope. In some embodiments, the polypeptide consists of 12 repeats of the PRAME epitope. In some embodiments, the polypeptide comprises the amino acid sequence of MSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLL QHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGL (SEQ ID NO: 2). In some embodiments, the polypeptide comprises 9 repeats of the PRAME epitope, wherein adjacent repeats are linked by two alanine residues. In some embodiments, the polypeptide consists of the 9 repeats of the PRAME epitope and the alanine residues linkin the adjacent repeats. In some embodiments, the polypeptide comprises the amino acid sequence of MSLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIG LAASLLQHLIGLAASLLQHLIGL (SEQ ID NO: 3). In some embodiments, the polypeptide comprises 9 repeats of the PRAME epitope, wherein adjacent repeats are linked by three alanine residues. In some embodiments, the polypeptide consists of the 9 repeats of the PRAME epitope and the alanine residues linking the adjacent repeats. In some embodiments, the polypeptide comprises the amino acid sequence of MSLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASL LQHLIGLAAASLLQHLIGLAAASLLQHLIGL (SEQ ID NO: 4). In some embodiments, the polypeptide comprises the amino acid sequence of MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLT HISALQSLLQHLIGLSNLTH (SEQ ID NO: 5). In some embodiments, the polypeptide comprises the amino acid sequence of MTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPL ESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVP LESY (SEQ ID NO: 6). In some embodiments, the polypeptide comprises the amino acid sequence of MSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISA LQSLLQHLIGLSNLTHVLYPVPLESYSLSHCSQLTTLSFYGNSISISALQSLLQHLIGL (SEQ ID NO: 7). In some embodiments, the polypeptide comprises the amino acid sequence of MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLT HGNSISISALQSLLQHLIGL (SEQ ID NO: 8). In some embodiments, the polypeptide is about 50-150 amino acids in length, or about 80-120 amino acids in length, or about 90-110 amino acids in length. In some embodiments, the mRNA comprises one or more of the following: a 5'-UTR comprising the sequence of any one of SEQ ID NOs: 19-49 and 118-120, and a 3' UTR comprising the sequence of any one of SEQ ID NOs: 50-58 and 121. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the mRNA comprises the P296PC00 / 3000181 -001977 sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

[0017] In some embodiments, the antigen binding protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a CDRal comprising or consisting of an amino acid sequence accordingto SEQ ID NO: 122, and a CDRa3 comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 124, and wherein the second polypeptide comprises a CDRbl comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 125, and a CDRb3 comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 127, optionally wherein the CDRal, CDRa3, CDRbl and / or CDRb3 sequence(s) may comprise one, two or three amino acid mutations. In some embodiments of the previously mentioned aspects of the invention the first polypeptide further comprises a CDRa2 comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 123 or 134. In some embodiments of the previously mentioned aspects of the invention, said first polypeptide comprises or consists of a variable domain V, which may comprise or consist of a TCR a-chain variable domain (Va). Said TCR a-chain variable domain (Va) may comprise or consist of an amino acid sequence according to SEQ ID NO: 128, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence accordingto SEQ ID NO: 128. In embodiments of the previously mentioned aspects of the invention, the second polypeptide further comprises a CDRb2 comprising or consisting of the amino acid sequence according to SEQ ID NO: 126. In some embodiments of the previously mentioned aspects of the invention, said second polypeptide comprises or consists of a variable domain VB, which may be a TCR p-chain variable domain (VP). Said TCR p-chain variable domain (VP) may comprise or consist of an amino acid sequence accordingto SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO: 130. In some embodiments of the previously mentioned aspects of the invention, said antigen binding protein may be a TCR or a fragment or derivative thereof. In some embodiments of the previously mentioned aspects of the invention, said host cell may be a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

[0018] In some embodiments, the host cell and / or the mRNA is provided in the form of a pharmaceutical composition.

[0019] In some embodiments, the mRNA pharmaceutical composition further comprises a lipid nanoparticle (LNP). In some embodiments, the LNP comprises an ionizable lipid. In some embodiments, the ionizable lipid has a chemical structure selected from any one of (a)-(p):

[0020] (a) P296PC00 / 3000181 -001977

[0021] R

[0022]

[0023] 1

[0024] (I)

[0025] or a salt thereof, wherein:

[0026] R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”); RNis H or Cve alkyl;

[0027] RNis H or Cve alkyl;

[0028] RN” is H or Ci-6alkyl;

[0029] o is 1, 2, 3, or 4;

[0030] n is 4, 5, 6, 7, or 8;

[0031] m is 4, 5, 6, 7, or 8;

[0032] M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;

[0033] M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;

[0034] R2aR2b

[0035] | I p2c

[0036]

[0037] R2is or-(Ci-6alkylene)-(C3.8cycloalkyl)-Ci.6alkyl;

[0038] R2ais -H or C1-10 alkyl;

[0039] R2bis -H or C1-10 alkyl;

[0040] R2cis Ci-8alkyl or C2.8alkenyl;

[0041] □3a p3b

[0042] R

[0043]

[0044] 3isK K

[0045] R3ais H or C1-10 alkyl;

[0046] R3bis H or Ci-8alkyl; and

[0047] R3cis C1-10 alkyl or C2.8alkenyl,

[0048] or

[0049]

[0050] (II)

[0051] or a salt thereof, wherein: P296PC00 / 3000181 -001977

[0052] R1is -OH;

[0053] o is 2, 3, or4;

[0054] n is 4, 5, 6, 7, or 8;

[0055] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0056] m is 6, 7, or 8;

[0057] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0058] R2cis C4-8 alkyl;

[0059] R3ais C7-10 alkyl; and

[0060] R3cis C3-5 alkyl,

[0061] or

[0062] (c)

[0063] R2C

[0064]

[0065] R3a

[0066] (HI)

[0067] or a salt thereof, wherein:

[0068] R1is NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”); RNis H;

[0069] RN’ is CT-2 alkyl;

[0070] RN” is H;

[0071] o is 2, 3, or4;

[0072] n is 6, 7, or 8;

[0073] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0074] m is 6, 7, or 8;

[0075] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0076] R2ais C7-10 alkyl;

[0077] R2cis C4-6 alkyl;

[0078] R3ais C-i-3 alkyl; and

[0079] R3cis C4.6alkyl,

[0080] or

[0081] (d) P296PC00 / 3000181 -001977

[0082]

[0083] (IV)

[0084] or a salt thereof, wherein:

[0085] R1is OH;

[0086] o is 2, 3, or4;

[0087] n is 6, 7, or 8;

[0088] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0089] m is 6, 7, or 8;

[0090] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0091] R2bis C3.5alkyl;

[0092] R2cis C2-4 alkyl;

[0093] R3ais C7-10 alkyl; and

[0094] R3cis C4-6 alkyl,

[0095] (e)

[0096] R2c

[0097]

[0098] (V)

[0099] or a salt thereof, wherein:

[0100] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula I; and R3ais C1-8 alkyl.

[0101] (f)

[0102]

[0103] (VI)

[0104] or a salt thereof, wherein:

[0105] o, M, M’, R2cand R3care as defined for Formula (I); and

[0106] R3ais C1-8alkyl. P296PC00 / 3000181 -001977

[0107]

[0108] (VII)

[0109] or a salt thereof, wherein:

[0110] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula I; and R3ais Ci-8alkyl.

[0111] (

[0112]

[0113] VIII)

[0114] or a salt thereof, wherein:

[0115] o, M, M’, R2cand R3care as defined for Formula (I); and

[0116] R3ais C1-8alkyl.

[0117] (i)

[0118] R2C

[0119]

[0120] R3a

[0121] (IX)

[0122] or a salt thereof, wherein:

[0123] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula (I); R2ais a Ci-8alkyl; and

[0124] R3ais Ci-8alkyl,

[0125]

[0126] P296PC00 / 3000181 -001977

[0127] (X)

[0128] or a salt thereof, wherein:

[0129] R1, o, m, n, M, M’, R2c, and R3care as defined forformula I;

[0130] R2bis a Cva alkyl; and

[0131] R3ais C1-8alkyl,

[0132] or

[0133] (k)

[0134]

[0135] (XI)

[0136] or a salt thereof, wherein:

[0137] R1, o, M, M’, R2c, and R3care as defined forformula I;

[0138] R2ais a Ci-8alkyl; and

[0139] R3ais Ci-8alkyl,

[0140] or

[0141] (I)

[0142] R2a

[0143]

[0144] (XII)

[0145] or a salt thereof, wherein:

[0146] R1, o, M, M’, R2c, and R3care as defined forformula I;

[0147] R2ais a Ci-8alkyl; and

[0148] R3ais Ci-8alkyl,

[0149] or

[0150] (m)

[0151]

[0152] P296PC00 / 3000181 -001977

[0153] (XIII)

[0154] or a salt thereof, wherein:

[0155] R1, o, M, M’, R2c, and R3care as defined forformula I;

[0156] R2ais a Cva alkyl; and

[0157] R3ais C1-8alkyl,

[0158] or

[0159] (n)

[0160] R3a

[0161]

[0162] (XIV)

[0163] or a salt thereof, wherein:

[0164] R1, o, m, n, M, M’, R2c, and R3care as defined for formula I; R2bis a Ci-8alkyl; and

[0165] R3ais Ci-8alkyl,

[0166] or

[0167] (

[0168]

[0169] XV)

[0170] or a salt thereof, wherein:

[0171] R1, o, m, n, M, M’, R2c, and R3care as defined for formula I; R2bis a Ci-8alkyl; and

[0172] R3ais Ci-8alkyl,

[0173] or

[0174]

[0175] P296PC00 / 3000181 -001977 (XV)

[0176] or a salt thereof, wherein:

[0177] o, M, M’, R2c, and R3care as defined for formula I;

[0178] R2ais a Cva alkyl; and

[0179] R3ais C1-8alkyl,

[0180] or N-oxides, salts, or isomers of each thereof. In some embodiments, the ionizable lipid is a compound selected from the group consisting of:

[0181]

[0182] or N-oxides, salts, or isomers thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the mRNA comprises a 5' terminal cap, optionally wherein the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof. In some embodiments, the mRNA comprises a poly-A region, optionally wherein the poly-A region 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 nucleotides in length, or at least about 100 nucleotides in length, or optionally wherein the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in P296PC00 / 3000181 -001977

[0183] length. In some embodiments, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. In some embodiments, the mRNA comprises at least one chemically modified nucleobase, wherein the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof, optionally wherein at least about 25%, 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%, at least about 95%, at least about 99%, or 100% of the uracils are N1 methylpseudouracils. In some embodiments, the mRNA comprises at least one chemically modified nucleobase, wherein the at least one chemically modified nucleobase is N1-methylpseudouracil (m1ψ), optionally wherein at least about 25%, 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%, at least about 95%, at least about 99%, or 100% of the uracils are N1 methylpseudouracils. In some embodiments, the mRNA comprises a 5 ’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length (SEQ ID NO: 92), and wherein all uracils of the polynucleotide are N1 -methylpseudouracils.

[0184] In some embodiments, the immune response is a T cell response. In some embodiments, the proliferative disease is cancer. In some embodiments, said cancer is selected from cutaneous melanoma (CM) and synovial sarcoma (SS). In some embodiments, the cancer is a PRAME-positive cancer, wherein the PRAME-positive solid tumor may express the PRAME peptide SLLQHLIGL (SEQ ID NO: 1) in a complex with a major histocompatibility complex (MHC) protein on cell surface.. In some embodiments, the cancer is an unresectable and / or metastatic cancer. In some embodiments, the subject is a HLA-A*02 positive subject. In some embodiments, the mRNA and the host cell are administered separately from one another. In some embodiments, the mRNA and the host cell are administered simultaneously or sequentially. In some embodiments, the mRNA or the pharmaceutical composition comprising the same is administered to the subject via intravenous (IV), subcutaneous, intramuscular (IM), or intradermal administration. In some embodiments, the host cell or the pharmaceutical composition comprising the same is administered to the subject via intravenous (IV) administration. In some embodiments, the treatment further comprises administering IL-2 to said subject. In some embodiments, IL-2 is administered after administration of said host cell, optionally at low doses, preferably at 1 million IU or less. In some embodiments, IL-2 and is administered daily or twice daily. In some embodiments, IL-2 is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embdiments, IL-2 is administered daily on days 1 -5 after administering said host cell, and / or twice daily on days 6-10, preferably at a dose of 1 million IU. In some embodiments, said host cell is administered to a total dose of 1 -10x109host cells, preferably of 1 -5x109host cells, such as up to 5 x 109host cells per patient. In some embodiments, said mRNA is administered after administering said host cell and P296PC00 / 3000181 -001977 optionally IL-2. In some embodiments, said mRNA is administered 2-4 weeks after administering said host cell or optionally IL-2. In some embodiments, said mRNA is administered every 2-4 weeks for up to 52 weeks or less. In some embodiments, said treatment comprises: a) administering said host cell(s) on Day 1, b) administering IL-2 on Days 2-11; and c) administering said mRNA on Day 15 or later.

[0185] BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1B are graphs illustrating a dose-dependent in vitro experiment. LNPs were titrated in a 2.5fold - dilution series ranging from 10ng - 977ng. Immature DCs were transfected with the respective LNP atthe indicated concentration. Following o / n incubation, PRAME-specificT cells and their NTD counterparts were co-cultured with the LNP-transfected DCs for 24h. FIG. 1A is a graph showing the mean fluorescent intensity (MFI) of CD25, gated on CD8+ T cells as measured by flow cytometry. FIG. 1 B is a graph showing IFNy release in the supernatant as measured by ELISA. The dotted line indicates the maximum detection limit of the plate reader.

[0186] FIGS. 2A-2C are graphs showing the results of a screening experiment directly comparing MRNA-PRAME LNP-01 to LNP-16. The experimental set-up was the same as in the experiment described by FIG. 1. FIG. 2A is a graph showing the mean fluorescent intensity (MFI) of CD25 (left), CD69 (middle) and CD137 (right), gated on CD8+ T cells as measured by flow cytometry. FIG. 2B is a graph showing the same data as FIG. 1 A, subdivided by construct type for clarity. FIG. 2C is a graph showing IFNy release in the supernatant as measured by ELISA. The dotted line indicates the maximum detection limit of the plate reader.

[0187] FIGS. 3A-3B are graphs showing the results of validation experiments to confirm screening results for the lead mRNA-LNPs for immune response induction. The experimental set-up was the same as in the screening experiment described by FIG. 2. FIG. 3A is a graph showing the mean fluorescent intensity (MFI) of CD25 gated on CD8+ T cells in two validation experiments. FIG. 3B is a graph showing IFNy release in the supernatant as measured by ELISA.

[0188] FIGS. 4A-4G are graphs showing that MRNA-PRAME LNPs can induce proliferation of PRAME-specific T cells. The experimental set-up was the same as in the screening experiment described by FIG. 2, but co-culture was conducted for 4 days in the presence of 0.5U / ml IL-2. FIG. 4A shows exemplary microscopy images of PRAME-specific T cells co-cultured with non-transfected dendritic cells (DCs) (left), LNP-04 (middle) or LNP-12 (right) -transfected DCs, respectively. FIG.4B shows an image of supernatant collected after 4 days of co-culture. FIG. 4C is a panel of graphs showing the absolute cell counts (gated on live CD8+ T cells) measured by flow cytometry. FIG. 4D is a panel of graphs showing IFNy response measured on supernatant collected after 4 days of co-culture. FIG. P296PC00 / 3000181 -001977 4E is a panel of graphs showing comparison proliferation experiment 1 vs. proliferation experiment 2. The comparative data from proliferation experiment 1 is the same as in C. FIG. 4F is a panel of graphs showing IFNy response measured on supernatant collected after 4 days of co-culture, comparison of proliferation experiment 1 vs. proliferation experiment 2.

[0189] FIG. 5 shows CD25, CD137 expression and IFNy response in an experimental set-up as described by FIG. 2.

[0190] FIG. 6 shows PRAME-specific T cell responses to co-culture with mRNA-LNP transfected antigen presenting dendritic cells (DCs) for 24h-72h at a T cell: DC ratio of 3:1 -4:1. FIGs. 6 A, B and C show CD25, CD69 and CD137 expression in PRAME-specific T cells, respectively, as measured by by flow cytometry (24h), gated on CD8+ cells. FIG. 6 D shows absolute counts of CD8+ T cells after 72h co-culture. FIG. 6E shows Interferon-y (IFNy) secretion, measured by ELISA (24h).

[0191] FIG. 7 shows an exemplary clinical trial protocol for the combined treatment with mRNA-LNPs and PRAME-specific T cells. Briefly, patients receive PRAME-specific T cells on Day 0 and subsequently receive at least one low dose IL-2 treatment. PRAME mRNA-LNPs are administered starting on Day 15 after T cell infusion at the earliest, and are administered for about 12 cycles (cycle length: 28 days). During Cycle 1, PRAME mRNA-LNPs are administered on Day 1 and Day 15, and in Cycle 2-12, PRAME mRNA-LNPs are administered on Day 1.

[0192] DETAILED DESCRIPTION OF THE INVENTION

[0193] In order for the present invention to be readily understood, several definitions of terms used in the course of the invention are set forth below.

[0194] Definitions

[0195] As used herein, the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Accordingly, the articles “a” and “an” preceding an element or component are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore, “a” or “an” is to be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0196] In the context of the present specification, the term “about” or "approximately" when referring to a specific value is meant to indicate that the value may deviate by ±20%, ±15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%. It also includes the concrete value, e.g., "about 50" includes the value "50".

[0197] As used herein, the term “adoptive cell therapy” (“ACT”) is a type of immunotherapy in which T cells are administered to a patient to treat a disease, in this case cancer. In autologous ACT, T cells P296PC00 / 3000181 -001977

[0198] that have been extracted from a patient are cultured in vitro and are typically genetically modified to improve TCR function and are then returned to the same patient for therapy. Comparatively, allogeneic ACT involves T cells isolated and expanded from a donor or donors different from the patient receiving the T cells for therapy.

[0199] As used herein, the term “administration” of an agent (e.g., host cells or polynucleotides of the disclosure or compositions or formulations comprising such host cells or polynucleotides) to a subject (e.g., a subject in need thereof) includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route as described herein. Administration includes self-administration and the administration by another. Administration of an active agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected bythe treating physician or in the case of animals, by the treating veterinarian. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Embodiments relating to suitable means, dosages and treatment regimen are disclosed in the specification.

[0200] As used herein, the term “administered in combination” or “combined administration” or “combination therapy” means that two or more agents are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient. Embodiments relating to combination administration are disclosed in the specification.

[0201] “Affinity” is defined, in the context of the present disclosure by the equilibrium binding between the antigen binding protein and its antigen, namely the PRAME peptide in a complex with a MHC protein. Affinity is usually expressed as equilibrium dissociation constant (KD).

[0202] As used herein, the term “amino acid” refers to one of the 20 naturally occurring amino acids or any non-natural analogues. Preferably, the term “amino acid” refers to one of the 20 naturally occurring amino acids. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0203] “Amino acid mutations” may be deletions, insertions or substitutions. P296PC00 / 3000181 -001977

[0204] As used herein, the term “amino acid substitution” refers to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. An amino acid can be substituted in a parent or reference sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. In the context of the present disclosure, substitutions (even when they referred to as amino acid substitution) are typically conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid. Accordingly, a reference to a “substitution at position X” refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position X, and Y and Z are alternative substituting amino acid residue.

[0205] Amino acid substitutions may be conservative or non-conservative. For instance, and specifically in the context of antigen binding proteins, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. An amino acid substitution may also be a post-translational modification of the antigen binding protein and is herein also encompassed.

[0206] A conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class, for example, (non-polar amino acids substituted by other nonpolar amino acids.

[0207] A conservative amino acid substitution may be made in accordance with Table 1. Methods for predicting tolerance to protein modification may be found in, for example, Guo et aL, Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0208] Conservative Amino Acid substitutions

[0209] Amino acid Substitutions (others are known in the art)

[0210] Ala Ser, Gly, Cys

[0211] Arg Lys, Gin, His

[0212] Asn Gin, His, Glu, Asp

[0213] Asp Glu, Asn, Gin

[0214] Cys Ser, Met, Thr

[0215] Gin Asn, Lys, Glu, Asp, Arg

[0216] Glu Asp, Asn, Gin P296PC00 / 3000181 -001977

[0217] Gly Pro, Ala, Ser

[0218] His Asn, Gin, Lys

[0219] lie Leu, Vai, Met, Ala

[0220] Leu lie, Vai, Met, Ala

[0221] Lys Arg, Gin, His

[0222] Met Leu, lie, Vai, Ala, Phe

[0223] Phe Met, Leu, Tyr, Trp, His

[0224] Ser Thr, Cys, Ala

[0225] Thr Ser, Vai, Ala

[0226] Trp Tyr, Phe

[0227] Tyr Trp, Phe, His

[0228] Vai lie, Leu, Met, Ala, Thr

[0229] The antigen binding proteins of the present disclosure can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and may include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, |3-phenylserine p-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N'. N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a,y-diaminobutyric acid, a,|3-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0230] The antigen binding protein or the nucleic acid(s) encoding the antigen binding protein of the present disclosure can be recombinant, isolated, engineered and / or purified.

[0231] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0232] Throughout the instant application, the term “and / or” is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. Furthermore, “and / or” where used herein is to betaken as specific disclosure of each ofthe two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). P296PC00 / 3000181 -001977 The term “antibody” as used herein is meant to include native and engineered antibodies. The term “engineered antibody includes functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

[0233] A “native antibody” comprises two heavy and two light chains, wherein the heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct domains (also referred to as regions). The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four or five domains depending on the antibody isotype; a variable domain (VH) and three or four constant domains (CHi, CH2 and CH3, and optionally CH4, collectively referred to as CH). The variable domains of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0234] The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are made up of residues that are primarily from the “antibody complementarity determining regions” (CDRs) or hypervariable regions. Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the binding site. CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native antibody binding site. The light and heavy chains of an antibody each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. An antibody antigen binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. “Antibody framework regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of antibody light and heavy chain variable regions that are relatively conserved among different antibodies in a single species. The light and heavy chains of an antibody each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain may be described as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain may be described as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody. In the context of the disclosure, CDR / FR in a TCR is determined based on IMGT definition (Lefranc et aL, Dev. Comp. ImmunoL, 2003, 27(1 ):55-77; www.imgt.org). Accordingly, amino acid sequences of the CDR1, CDR2 P296PC00 / 3000181 -001977 and CDR3 of a given variable chain and the amino acid sequences of the framework regions (e.g. FR1, FR2, FR3, and FR4) are indicated accordingto said IMGT definition in the herein provided disclosure.

[0235] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule or complex that is capable of being bound by an antigen binding site, wherein said antigen binding site is present in an antigen binding protein, preferably an antigen binding protein of the present invention. A target antigen may generally be a protein or antigenic peptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. The antigen in the context of the present invention is a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide, more particularly a PRAME antigenic peptide that comprises or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO:1), in a complex with a MHC protein, such as an HLA protein, for instance HLA-A*02. Any reference to “PRAME” in the present disclosure preferably refers to the PRAME-004 peptide having the amino acid sequence of SLLQHLIGL (SEQ ID NO: 1), unless denoted otherwise. The peptide SLLQHLIGL (SEQ ID NO: 1) is derived from the amino acid sequence of the known PRAME protein. In instances where the PRAME antigenic peptide comprises further amino acids in addition to the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), it is preferred that the overall length of the PRAME antigenic peptide does not exceed 30 or 20 amino acids, more preferably does not exceed 15 amino acids, even more preferably does not exceed 12 amino acids. In instances where the PRAME antigenic peptide comprises further amino acids in addition to SEQ ID NO: 1, the amino acids of SEQ ID NO: 1 are preferably located within the peptide binding groove of the MHC protein when the antigenic peptide is in a complex with an MHC protein. The person skilled in the art is aware that antigenic peptides presented on MHC I are usually no longer than 12 amino acids.

[0236] The term “antigen binding protein” herein (occasionally abbreviated to “ABP”) refers to a polypeptide or a complex of two or more polypeptides comprising an antigen binding site that specifically binds to a PRAME antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein, wherein the PRAME antigenic peptide comprises or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), and that polypeptide or the two or more polypeptides comprise(s) the CDRs as herein provided, such as CDRal, CDRa3, and optionally CDRa2, and CDRbl, CDRb3, and optionally CDRb2. The two or more polypeptides of the antigen binding protein may be covalently or non-covalently linked together. As used in the context of the present specification, the term antigen binding protein includes antigen binding proteins that comprise fragment(s) of the herein provided TCRs. The herein provided antigen binding proteins may be used in different formats as also described below, such as membrane bound antigen binding proteins, fusion proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins. P296PC00 / 3000181 -001977 The term includes antigen binding proteins having the overall structure of a TCR, an antibody and / or a chimeric antigen receptor (CAR). The antigen binding protein can comprise TCR-derived CDRs, in particular a variable domain VAcomprising TCR-derived CDRal, CDRa3, and optionally CDRa2, and a variable domain VBcomprising TCR-derived CDRbl, CDRb3, and optionally CDRb2. Antigen binding proteins can comprise a variable domain VAcomprising complementarity determining regions (CDRs) CDRal, CDRa2, and CDRa3, e.g. on a first polypeptide, and a variable domain VBcomprising CDRbl, CDRb2, and CDRb3, e.g. on a second polypeptide, wherein CDRal, CDRa2, CDRa3, CDRbl, CDRb2 and CDRb3 form an “antigen binding domain A”. “Antigen binding domain A” denotes a binding domain that binds to the antigenic peptide (PRAME antigenic peptide in the context of the present invention) that is in a complex with a major histocompatibility complex (MHC) protein. For instance, the entire VAdomain and / or the entire VBdomain may be TCR-derived and thus be TCR alpha, beta, gamma or delta variable domains (Va, vp, Vy or V6). Preferably, the antigen binding protein may be a TCR or functional fragment(s) thereof, e.g. the variable domains VAand VBof theTCR. In some instances, the antigen binding protein can comprise CDRs and optionally the VAand VBas herein provided, and can comprise further (an) additional domain(s) fused directly or indirectly to VAor VB. The further domains may form (an) additional binding domain(s) or (a) binding site(s). For example and in particular instances, the additional binding domains may form antigen binding domain B. Further binding domains maybe comprised that, e.g., form further antigen binding domains, e.g. antigen binding domain C, etc. The additional / further domains comprised in the antigen binding protein may also be a further protein.

[0237] The antigen binding protein thus also includes fusion proteins wherein fragment(s) of the herein provided TCRs further comprise other binding domains. Examples of additional domains comprised in an antigen binding protein of the invention that is a fusion protein are listed below.

[0238] “Antigenic peptide in a complex with an MHC protein”, herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. In particular, the antigenic peptide is located to a “peptide-binding groove” formed by the MHC molecule. A complex of an MHC molecule and an antigenic peptide is herein also referred to as “peptide-MHC complex” or “pMHC complex”. In the case of the PRAME antigenic peptide, the complex is also referred to as “PRAME antigenic peptide-MHC complex” or “PRAME: MHC complex”.

[0239] As used herein, the term “at least one” herein refers to one or more of the specified objects such as 1, 2, 3, 4, 5 or 6 or more of the specified objects. For example, at least one binding site herein refers to 1, 2, 3, 4, 5 or 6 or more binding sites.

[0240] As used herein, the term “approximately.” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain instances, the term “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) P296PC00 / 3000181 -001977

[0241] of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0242] As used herein with respect to a disease, the term “associated with” means that the symptom, measurement, characteristic, or status in question is linked to the diagnosis, development, presence, or progression of that disease. As association can, but need not, be causatively linked to the disease. For example, symptoms, sequelae, or any effects causing a decrease in the quality of life of a patient having cancer are considered associated with the cancer and in some instances of the present invention can be treated, ameliorated, or prevented by administering the polynucleotides of the present invention to a subject in need thereof.

[0243] As used herein, the term “binding affinity” refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay).

[0244] As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.

[0245] As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.

[0246] As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and / or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. For instance, a polynucleotide (e.g. mRNA) or antigen binding protein can be considered biologically active if even a only a portion of the polynucleotide or antigen binding protein retains or mimics the desired biological effect.

[0247] As used herein, the term “(pharmaceutically acceptable) carrier” refers to a type of excipient used to deliver a therapeutic agent to a specific target site in the patient’s body. It acts as a vehicle or medium to transport the therapeutic agent, and it can help to enhance the solubility, stability, and P296PC00 / 3000181 -001977 bioavailability of the therapeutic agent. Carriers are usually inert substances that do not interfere with the therapeutic action of the therapeutic agent.

[0248] The carrier typically serves as a vehicle or medium to transport the therapeutic agent to the target site and ensure its proper distribution and absorption in the body. Examples of pharmaceutically acceptable carriers include solvents, diluents, binders, and lubricants, among others. These carriers are typically selected based on their compatibility with the therapeutic agent and other components of the composition or formulation, as well as their safety and effectiveness in delivering the therapeutic agent to the patient. The use of a pharmaceutically acceptable carrier can help to ensure the stability, efficacy, and safety of the final product. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. In the context of polynucleotides, e.g. mRNAs, pharmaceutically acceptable carriers include nanoparticles, such as an polymeric nanoparticle carrier or an lipid nanoparticle (LNP).

[0249] As used herein, the terms “coding region” and “region encoding” and grammatical variants thereof, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.

[0250] As used herein, the terms “codon substitution” or “codon replacement” in the context of sequence optimization refer to replacing a codon present in a reference nucleic acid sequence with another codon. A codon can be substituted in a reference nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a "substitution" or "replacement" at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon.

[0251] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in P296PC00 / 3000181 -001977 two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some instances, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned antiparallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0252] As used herein, the term “comprising” is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term “comprising” also discloses the embodiment wherein no features other than the specifically mentioned features are present ( / .e. “consisting of”). Accordingly, both meanings are specifically intended, and hence individually disclosed, embodiments according to the present invention.

[0253] The term “consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0254] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a compound means that the mammalian cell and compound are made to share a physical connection. Methods of contacting cells with entities both in vivo and ex vivo are known in the biological arts. For example, contacting a compound and a mammalian cell disposed within a mammal can be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve varied amounts of compound. Moreover, more than one mammalian cell can be contacted by a compound.

[0255] “Copy number” herein refers to the number of PRAME / MHC complexes as defined in the context of the present disclosure that are present on the cell surface of a cell, such as a PRAME / MHC P296PC00 / 3000181 -001977

[0256] presenting cell, for example a cancer cell, or a healthy cell. Copy numbers of a protein can be determined by a variety of art known methods including FACS analysis of diseased cells with fluorescently labelled antigen binding proteins.

[0257] As used herein, a " CpG site" refers to a region of DNA in which cytosine nucleotides follow a guanine nucleotide in a linear order of bases in the 5 'to 3' direction. CpG sites occur at high frequency in regions of the genome called CpG islands (or CG islands). Cytosine in CpG dinucleotides can be methylated to form 5-methylcytosine. In mammals, 70% to 80% of CpG cytosines are methylated. Methylation of cytosine within a gene can alter its expression.

[0258] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various antitumor or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxoid, vincas, taxanes, a maytansinoid or maytansinoid analog such as DM1 or DM4, a small drug, a tomaymycin or pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or dolastatin analog, a prodrug, topoisomerase II inhibitors, a DNA alkylating agent, an anti-tubulin agent, a CC-1065 or CC-1065 analog.

[0259] As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a polynucleotide or host cell to a subject can involve administering a composition including the polynucleotide or host cell to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route).

[0260] A “dextramer staining” involves contacting cells expressing an antigen binding protein with fluorescently labelled multimers comprising ten PRAME: MHC complexes.

[0261] The term "disease" or "disorder" refers to any condition that would benefit from treatment with the therapeutic agent(s) of the disclosure. The term may include chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question.

[0262] The term “diagnostic agent” as used herein refers to a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other labels known in the art that provide (either directly or indirectly) a signal.

[0263] As used herein, a “domain” may be any region of a protein, generally defined on the basis of sequence homologies and often having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0264] The term "dosage" or "unit dose" or refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the P296PC00 / 3000181 -001977 desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0265] As used herein, a "dosing regimen" or a "dosing regimen" is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.

[0266] As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of a therapeutic agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the polynucleotide(s) administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. The terms “effective amount” or “therapeutically effective amount,” can be used interchangeably with “effective dose” or “therapeutically effective dose.”

[0267] As used herein, the term “enhanced delivery” in the context of polynucleotides such as mRNAs means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to the level of delivery of a polynucleotide by a control nanoparticle to a target tissue of interest (e.g., MC3, KC2, or DLinDMA). The level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue. It will be understood that the enhanced delivery of a nanoparticle to a P296PC00 / 3000181 -001977 target tissue need not be determined in a subject being treated, it can be determined in a surrogate such as an animal model (e.g., a mouse model).

[0268] As used herein, the term “(therapeutic) efficacy” refers to as a parameter that describes the capability of a therapeutic agent to exert its desired therapeutic effect, e.g. for a host cell expressing an antigen binding protein to kill cancer cells. The efficacy can be determined in a functional assay, for example a live-cell monitoring cytotoxicity assay as described below.

[0269] As used herein, the term “encapsulate” means to enclose, surround, or encase.

[0270] As used herein, “encapsulation efficiency” in the context of polynucleotides, such as mRNAs, refers to the amount of said polynucleotide that becomes part of a nanoparticle composition, relative to the initial total amount of therapeutic and / or prophylactic used in the preparation of an LNP. For example, if 97 mg of polynucleotide are encapsulated in an LNP out of a total 100 mg of polynucleotide initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0271] The term “E: T ratio” refers to the ratio of effector cells (i.e. immune cells, in particular T cells, expressing the antigen binding protein, in particular the TCR) to target cells. In some instances, the E: T ratio corresponds to the seeding ratio, i.e. the ratio of the total number of immune cells, in particular T cells, to target cells. In some instances, the E: T ratio is lower than the seeding ratio. This applies to cases where not all immune cells express the antigen binding protein, i.e. not all immune cells are effector cells, for example due to a low electroporation efficiency. In some instances, the seeding ratio is used as approximation of the E: T ratio. In some instances, the E: T ratio is determined by adjusting the seeding ratio taking into account the electroporation efficiency.

[0272] In an example of a lactate dehydrogenase (LDH)-release assay, the effector cells are immune cells. These effector cells are co-cultured with tumor cells endogenously expressing and presenting the PRAME antigenic peptide and optionally additionally loaded with the PRAME antigenic peptide. In some instances, the tumor cells are SKMEL-5 cells, RPMI7951 cells or SCC25 cells. In some instances of the LDH-release assay, the seeding ratio of total immune cells and target cells is 10:1. The efficacy of an effector cell or its antigen binding protein is considered high if in a LDH-release assay as defined above, killing of tumor cells (as determined LDH release) is observed at an E: T ratio of 10:1. Alternatively, the efficacy of an effector cell or its antigen binding protein is considered high if in a cytotoxicity assay, preferably a LDH-release assay as defined above, the cytotoxic activity of the effector cells against the target cells at the highest concentration of the antigen binding protein tested is at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of the cytotoxic activity of a control toxic reagent. The skilled in the art is aware that the cytotoxic activity can be higher than 100%. This is due to the fact that 100% cytotoxic activity is defined by a “maximum lysis control”, which refers to incubation of the target cells with P296PC00 / 3000181 -001977

[0273] the toxic reagent. In some instances, the toxic reagent is a detergent, e.g. Triton-X100, Tween-20, Tween-80 or NP-40, that effects lysis of the target cells. In some specific examples, the maximum lysis control comprises adding a 0.9% Triton-X100 solution to the target cell culture. The cytotoxic activity of the toxic reagent, i.e. the number of target cells killed by the toxic reagent is defined as 100%. Since the target cells can still proliferate during the co-culture, the effector cells may eventually kill an even higher number of target cells during the cytotoxicity assay than the toxic reagent killed during the maximum lysis control. In such instances, the calculated cytotoxic activity will be higher than 100%.

[0274] In an example of a cytokine production assay and cytokine release assay, the effector cells are immune cells expressing the antigen binding protein as host cells. These effector cells are cocultured with cells (either target cells, e.g. tumor / cancer cells, or peptide loaded cells, e.g. T2 cells) with different expression of the antigenic peptide in complex with MHC, such as an HLA protein, for instance HLA-A*02. Preferably, the effector cells and the target cells are seeded, e.g. at a ratio between 10:1 and 1:1. For the cytokine release assay, after a defined time of co-culture, e.g. 24-48 hours, preferably about 48 hours, the supernatants of the co-culture (effector cells + target cells) are collected and subjected to a cytokine release ELISA assay, for example IFN-gamma, TNF alpha, IL-2 and Granzyme B, to determine the amount of cytokine released by the effector cells. In order to determine the efficacy, a cytokine production assay can be applied using target cells, e.g. tumor or cancer cells. Alternatively, the killing of a target cell / cancer cell / tumor cell can be determined in e.g. an LDH assay or a live cell imaging assay. The efficacy of an antigen binding protein may be considered high if the antigen is capable of activating effector cells in a cytokine production assay, in particular if the amount of produced cytokines upon co-culture with target cells is at least about 100 pg / ml, at least about 300 pg / ml, preferably at least about 500 pg / ml, more preferably at least about 1000 pg / mL

[0275] An “engineered” molecule, e.g. a polynucleotide or antigen binding protein, refers to a molecule that is not naturally occurring or that has been modified by biotechnological methods, in particular by introducing amino acid mutations / substitutions into the native protein sequence. Such biotechnological methods are well known to the skilled in the art.

[0276] The term “epitope”, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system. As used herein, the term epitope comprises the terms “structural epitope” and “functional epitope”. The term “structural epitope” refers those amino acids of the antigen, e.g. peptide-MHC complex, that are covered by the antigen binding protein when bound to the antigen. Typically, all amino acids of the antigen are considered covered that are within 5 A of any atom of an amino acid of the antigen binding protein. The structural epitope of an antigen may be determined by art known methods including X-ray crystallography or NMR analysis. The structural epitope of an antibody typically comprises 20 to 30 amino acids. The structural epitope of a TCR P296PC00 / 3000181 -001977

[0277] typically comprises 20 to 30 amino acids. A “functional epitope” as herein defined is a subset of those amino acids forming the structural epitope and comprises the amino acids of the antigen that are critical for formation of the interface with the antigen binding protein, either by directly forming non-covalent interactions such as H-bonds, salt bridges, aromatic stacking or hydrophobic interactions or by indirectly stabilizing the binding conformation of the antigen and is, for instance, determined by mutational scanning. In the context of the present disclosure, the functional epitope is also referred to as “binding motif”. Typically, the functional epitope of an antigen bound by an antibody comprises between 4 and 6 amino acids. Typically, the functional epitope of a peptide-MHC complex comprises between 2 to 6 or 7 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since MHC I presented peptides typically have a length between 8 to 10 amino acids only a subset of amino acids of each given peptide is part of the functional epitope of a peptide-MHC complex. The epitope, in particular the functional epitope bound by the antigen binding protein comprises or consists of the amino acids of the antigen that are required for formation of the binding interface.

[0278] As used herein, the term “excipient” refers to a substance that is added to a therapeutic agent to facilitate its manufacturing, administration, stability or therapeutic efficacy. Excipients are typically inactive substances that are used, inter alia, as carriers, solvents, fillers, binders, disintegrants, lubricants, or flavoring agents.

[0279] The term "expression system" means a host cell and compatible expression vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[0280] As used herein, an “expression vector” includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0281] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment. P296PC00 / 3000181 -001977 “Fluorescent molecules” are known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or uv laser (e.g. Pacific blue, pacific orange).

[0282] As used herein, a “format” in relation to an antigen binding protein relates to a defined spatial arrangement of domains, in particular of variable and optionally constant domains. Characteristics of such antigen binding protein formats are the number of polypeptide chains (single chain, double chain or multiple chains), the type and length of linkers connecting different domains, the number of variable domains (and thus the number of valences), the number of different variable domains (and thus the number of specificities for different antigens, e.g. bispecific, or multispecific), and the order and orientation of variable domains (e.g. cross-over, parallel).

[0283] As used herein, the term "formulation" refers to a specific therapeutic product that has been developed with a particular set of ingredients, dosage form, and delivery method to meet specific therapeutic needs. It is a final product that has typically undergone several steps of formulation development, such as selecting the appropriate ingredients, optimizing the drug delivery system, and ensuring its safety and efficacy. Different formulations of the same therapeutic agents may have different pharmacokinetic and pharmacodynamic properties, and may be used to treat different conditions or patient populations.

[0284] A "fragment." as used herein, refers to a portion. For example, fragments of proteins can comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. A fragment may be a subsequence of a full-length protein wherein N-terminal, and / or C-terminal, and / or internal subsequences have been deleted. Preferably, fragments of a protein are functional fragments (i.e., exhibit or mimic the protein’s desired biological activity), or exhibit a novel desired biological activity.

[0285] The term “free of”, as used herein, means not comprising the referenced component. For example, when a composition, solution, or formulation is described as being “free of X”, the composition, solution, or formulation does not comprise X.

[0286] In a “functional assay”, an antigen binding protein is, for example, expressed in an “effector cell ”, and the effector cell is co-cultured with “target cells (T)”. i.e. with antigen presenting cells presenting a peptide: MHC complex. Functional assays can thus also be described as “co-culture assays”. For all cell culture assays described herein, the cell culture temperature preferably is at about 37°C. Preferably, the effector cell is a T cell. The target cells may be cells that are artificially loaded with the antigenic peptide (e.g. T2 cells) or may be cells that endogenously present the target antigenic peptide on their surface (e.g. cancer cells expressing PRAME). Binding of the antigen binding protein to the peptide: MHC complex leads to activation of the effector cell. Depending of the type of functional assay, there are different readouts for measuring the degree of activation. In a cytokine production assay or cytokine release assay, such as an ELISA, the production of cytokines P296PC00 / 3000181 -001977 (e.g. TNF-a, IFN-y, CD107a+, IL-2 and / or Granzyme B) by the effector cells is determined. In a cytotoxicity assay, the killing of target cells by the effector cells is determined, e.g. by measuring a decline in proliferation of target cells, in particular cancer cells or by measuring the release of intracellular proteins from the target cells. Suitable intracellular proteins to be measured in a cytotoxicity assay can be endogenous proteins, e.g. LDH release assay.

[0287] “Functional avidity” is defined, in the context of the present disclosure, as a parameter that describes the capability of an antigen binding protein to activate an effector cell, preferably a T cell, upon binding to its target antigenic peptide in a complex with MHC. The activation of the effector cell, preferably T cell, can be measured in a functional assay, e.g., a cytokine production assay or a cytotoxicity assay as described below. In some embodiments, the functional avidity of an antigen binding protein is considered high if the EC50determined in a functional assay is low, such as less than about 50 nM, less than about 20 nM, or less than about 5 nM in a cytotoxicity assay as described below, and / or the activity determined in a functional assay is high, such as at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of a maximal activity defined in the respective functional assay. Depending on the functional assay, the maximal activity may be the activity of a reference protein with known high functional avidity or the activity of a “maximum lysis control” as described below.

[0288] The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0289] A "growth inhibitory agent", or “anti-proliferative agent”, which can be used indifferently, refers to a compound or composition which inhibits growth of a cell, especially a tumor cell, either in vitro or in vivo.

[0290] “Half maximal effective concentration” also called “ECgn”. typically refers to the concentration of a molecule, which induces a response halfway between the baseline and maximum after a specified exposure time. EC50and affinity are inversely related, the lower the EC50value the higher the affinity of the molecule.

[0291] As used herein, the term “helper lipid” refers to a compound or molecule that includes a lipidic moiety (for insertion into a lipid layer, e.g., lipid bilayer) and a polar moiety (for interaction with physiologic solution at the surface of the lipid layer). In some instances, the helper lipid is a phospholipid. A function of the helper lipid is to “complement” the amino lipid and increase the P296PC00 / 3000181 -001977 fusogenicity of the bilayer and / or to help facilitate endosomal escape, e.g., of nucleic acid delivered to cells. Helper lipids are also believed to be a key structural component to the surface of the LNP.

[0292] The term “HLA-A*02” signifies a specific H LA allele, wherein the letter A signifies the allele and “*02” indicates the A2 serotype.

[0293] As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g., between polynucleotides (e.g., DNA molecules and / or RNA molecules) and / or between (poly-)peptides. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity. As used herein, polymeric molecules may be considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0294] A “host cell” is a cell that can be used to express a (heterologous) nucleic acid. The host cell may be transfected, infected or transduced or transformed, in particular with nucleic acid(s) and / or a vector(s) as described herein.

[0295] The term “humanized” in the context of an antigen binding protein or antibody refers to an antibody which is completely or partially of non-human origin and which has been modified by replacing certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are mainly human CHand CLdomains. Numerous methods for humanization of an antibody sequence are known in the art. For example, a “humanized” antibody can be made by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations, see, e.g., Teng et aL, Proc. Natl. Acad. Sci. U. S. A., 80: 7308-7312, 1983; Kozbor et aL, Immunology Today, 4: 7279, 1983; Olsson et aL, Meth. EnzymoL, 92: 3-16, 1982, and the review byAlmagro& Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR grafting, or antibody reshaping, which involves grafting of the CDR sequences of a donor antibody, generally a mouse antibody, into the framework scaffold of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues. Another P296PC00 / 3000181 -001977 alternative technique is known as “guided selection” (Jespers et aL (1994) Biotechnology 12, 899) and can be used to derive from for example a murine or rat antibody a fully human antibody conserving the epitope and binding characteristics of the parental antibody. A further method of humanization is the so-called 4D humanization. The 4D humanization protocol is described in the patent application US20110027266 A1 (the content of which is incorporated by reference in its entirety) (W02009032661A1) and is exemplified in the following applying the 4D humanization to humanize the rat antibody variable light (VL) and heavy (VH) domains.

[0296] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Knowing the amino acid sequence of the CDRs an antigen binding protein of the disclosure, e.g., a TCR, one skilled in the art can easily determine the framework regions, such as the TCR framework regions. In cases where the CDRs are not indicated, the skilled in the art can first determine the CDR amino acid sequences based on the IMGT definition for TCRs and then determine the amino acid sequences of the framework regions.

[0297] The term “immunoglobulin (Ig) domain” refers to a protein domain that consists of a 2-layer sandwich of 7-9 antiparallel p-strands arranged in two p-sheets with a Greek key topology. Proteins containing Ig domains are subsumed into the immunoglobulin superfamily, including e.g. antibodies, T cell receptors (TCRs) and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

[0298] As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0299] As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0300] As used herein, the term “isolated” refers to the separation of a specific molecule or entity from its natural environment or context. For nucleic acids, isolation can refer to the separation of a specific DNAor RNAsequencefrom the rest of the naturally occurringgenomic material. This can be done through techniques such as PCR, gel electrophoresis, or hybridization. An “isolated” nucleic acid can be present in solution, or in a host cell. For polypeptides, isolation can refer to the purification of a specific protein from a complex mixture of proteins or cell culture components. This can be done using techniques such as chromatography, electrophoresis, or immunoaffinity purification. For host cells, isolation can refer to the removal of a specific cell type from a mixed population of cells. This can be done using techniques such as fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or microfluidic cell sorting.

[0301] “Kc” is the equilibrium dissociation constant, a ratio of koff / kon, between an antigen binding protein and its antigen. KDand affinity are inversely related. The KDvalue relates to the concentration of an antigen binding protein and the lower the KDvalue, the higher the affinity of an antigen binding P296PC00 / 3000181 -001977 protein. The KDvalue can be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates with surface plasmon resonance (SPR) or bio-layer interferometry (BLI). For example, an antigen binding protein can be produced as soluble molecule, for instance, by removing the transmembrane domain and introducing an artificial disulfide bridge (Boulter et aL 2003; Stable, soluble T-cell receptor molecules for crystallization and therapeutics; Protein Engineering vol. 16 no. 9 pp. 707-711 ) or dimerization via a leucin zipper domain. (Willcox et al. 1999; TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface; Immunity, Vol. 10, 357-365). Binding interactions can be measured at a broad range of settings, including, but not limited to, a temperature range of 25°C to 37°C and a shake speed range of 500 rpm to 1500 rpm using a suitable buffer that minimizes nonspecific binding and maintains protein stability. Examples of such buffers are phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS), or other physiological buffers, with or without additives such as Tween, BSA, DMSO, or EDTA. The analyte can be immobilized on various sensors at a concentration range, including, but not limited to, 1 pg / ml to 100 pg / ml for a duration range of 30 s to 300 s. KDdetermination can be measured at various molarities of the analyte sample for detecting potential off-target reactivities with high sensitivity. Exemplary determination of the KD is herein provided in the Examples. For example the KD may be determined by the following bio-layer interferometry (BLI) method: using a 384-well tilted bottom microplate, black and loading with 50 pg / ml pMHC (100 pl / well). The antigen binding protein can then be added, e.g. as soluble TCR in 7 concentrations as well as a reference well (only HEPES / Tween): 50 pM; 25 pM; 12,5 pM; 6,25 pM; 3,1 pM; 1,6 pM; 0,8 pM with 60 pl / well. The association of off-target TCRs can be against NYESO1-001 at 25pM TCR concentration. Then, the dissociation of soluble TCR can be 100 pl / well HEPES / Tween. The measurements can be conducted by 16 streptavidin sensors: duplicates measured with different sensors (sensor offset: 3).

[0302] A “linker”, as long as it is not specified otherwise in the respective context, can be from at least 1 to 30 amino acids in length. For instance, a linker can be 2-25, 2-20, or 3-18 amino acids long. In some instances, a linker can be a peptide of a length of no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In other instances, a linker can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids long. In other instances, a linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In a particular instance, a linker can be less than 24, less than 20, less than 16, is less than 12, less than 10, for example from 5 to 24, 10 to 24 or 5-10 amino acid residues in length. In some instances, said linker is equalto 1 or more amino acid residues in length, such as more than 1, more than 2, more than 5, more than 10, more than 20 amino acid residues in length, more than 22 amino acid residues in length. In preferred instances, the linker is an alanine linker, i.e. a linkerconsistingof or essentially consisting of one or more alanine residues. In some instances, the linker is a single alanine linker. In some instances, a linker P296PC00 / 3000181 -001977 comprises or consists of 2 alanine residues. In some instances, a linker comprises or consists of 3 alanine residues.

[0303] As used herein, the term “lipid nanoparticle” or “LNP” in the context of polynucleotides, e.g. mRNAs, refers to a nanoparticle comprising one or more lipids. In some instances, the LNP has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, or about 100 nm or less. In some instances, the LNP has a size ranging from about 1 nm to about 100 nm.

[0304] As used herein, the term “lipop lex” refers to a nucleic acid-liposome complex, where nucleic acid can be DNA, siRNA or mRNA.

[0305] As used herein, the term “liposome” refers to a composite having at least one lipid bilayer. In some instances, a liposome has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, or about 100 nm or less. In some instances, the liposome has a size ranging from about 1 nm to about 100 nm.

[0306] As used herein, the term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells.

[0307] The “Major Histocompatibility Complex” (MHC) is a set of cell surface proteins essentialforthe acquired immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or just HLA). Thus, the terms “MHC” and “HLA” can be used interchangeably. The MHC gene family is divided into three subgroups: class I, class II, and class HL Complexes of peptide and MHC class I molecules (MHC I) are usually recognized by CD8-positive T cells (CD8+ T cells) bearing the appropriate T cell receptor (TCR), whereas complexes of peptide and MHC class II molecules (MHC II) are usually recognized by CD4-positive helper-T cells (CD4+ T cells) bearing the appropriate TCR. CD4 and CD8 usually function as co-receptors of a TCR in binding to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptide and MHC I are recognized by CD8-negative (in particular CD8-negative, CD4-positive) T cells (Soto et aL, 2013, Cancer Immunol Immunother. 2013 Feb; 62(2): 359-369). Since the responses of CD8-positive and CD4-positive T cells contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens and correspondingT cell receptors is important in the development of cancer immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger, a-chain, constituent of HLA-A. Variation of HLA-A a-chain is key to HLA function. P296PC00 / 3000181 -001977 This variation promotes genetic diversity in the population. Since each H LA has a different affinity for peptides of certain structures, greater variety of HLAs means greater variety of antigens to be 'presented' on the cell surface. The MHC class I HLA protein in the context of the present disclosure may be an HLA-A, HLA-Bor HLA-C protein, suitably HLA-A protein, for example HLA-A*02. In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized byT cells bearing specific T cell receptors (TCR).

[0308] As used herein, the term “messenger RNA” or “mRNA” refers to any polynucleotide (a ribonucleic acid) which encodes a (poly-)peptideof interest and which is capable of being translated to produce the encoded (poly-)peptide of interest in vitro, in vivo, in situ, or ex vivo. Traditionally, the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail.

[0309] As used herein, the term “modified messenger RNA” or “modified mRNA” refers to mRNA polynucleotides that include naturally occurring and / or non-naturally occurring modifications, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, orto the phosphodiester backbone). Non-natural modified nucleotides may be introduced during synthesis of post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleoside linkage, purine or pyrimidine base, or sugar. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0310] As used herein, the terms “polypeptide” or “protein” mean a macromolecule composed of one or more sequence(s) of amino acids. A protein can be a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it can be produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence.

[0311] A “PRAME: MHC complex presenting cell” herein refers to a cell that presents on its surface the PRAME antigenic peptide in a complex with an MHC molecule. Specifically, the PRAME: MHC complex presenting cell may be a tumor cell, wherein the tumor is preferably a cancer as defined herein below in the section ‘Therapeutic Methods and Uses’. In the context of the present disclosure, the PRAME: MHC complex is over-presented on the cell surface of a PRAME: MHC complex presenting cell, compared to levels of said complex on the surface of cells in normal (healthy) tissue (also referred to as “healthy cells”) or on the surface of control cells loaded with a different antigen presenting peptide or no peptide. By "over-presented" is meant that the PRAME: MHC complex is P296PC00 / 3000181 -001977

[0312] present at a level at least 2-fold, preferably between 5-fold to 10-fold of the level present in healthy tissue or control cells.

[0313] As used herein, a “nanoparticle composition” in the context of polynucleotides, e.g. mRNAs, is a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0314] As used herein, the “N: P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g., in an LNP including a lipid component and an RNA.

[0315] As used herein, the term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5′to 3′orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

[0316] As used herein, the term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a (poly-)peptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0317] As used herein, “operably linked” with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a (poly-)peptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encodinga first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce P296PC00 / 3000181 -001977

[0318] only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0319] Herein a phrase of the form "optionally substituted X" is intended to be equivalent to " X, wherein X is optionally substituted". It is not intended to mean that the feature " X" per se is optional.

[0320] As used herein, a "part" or "region" of a polynucleotide or polypeptide is defined as any portion of the polynucleotide or polypeptide that is less than the entire length of the polynucleotide or polypeptide, respectively.

[0321] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0322] The terms "pharmaceutical composition" as used herein refers to a mixture or formulation of one or more therapeutic agent(s), typically along with one or more excipients, which are added to the formulation to aid in its manufacture, stability, or administration. The pharmaceutical composition is typically designed to deliver the therapeutic agent(s) in a safe, effective, and convenient manner for the treatment or prevention of a particular disease or medical condition. Pharmaceutical compositions can take various forms, such as tablets, capsules, injections, creams, ointments, or inhalers, and can be administered by various routes, including oral, topical, intravenous, intramuscular, or inhalation. The composition may need to meet regulatory requirements for safety, efficacy, and quality before it can be approved for marketing and distribution to patients.

[0323] " Pharmaceutically acceptable" refers to the suitability of a therapeutic agent for use in a pharmaceutical formulation, which typically means that the substance is safe, effective, and compatible with the other ingredients in the formulation. A therapeutic agent that is considered "pharmaceutically acceptable" may need to meet certain criteria, including purity, stability, and absence of harmful impurities or contaminants, and it must not cause any adverse effects on the patient's health. In addition, the substance may need to be able to perform its intended function within the formulation and be compatible with the manufacturing process, packaging, and storage conditions.

[0324] As used herein, “phosphomimic substitution” or “phosphomimetic substitution” refers to an amino acid substitution that mimic a phosphorylated amino acid. In some instances, a phosphomimic substitution comprises a glutamic acid (E) substitution. In some instances, a phosphomimic substitution comprises an aspartic acid (D) substitution. In some instances, a phosphomimic substitution mimics a phosphorylated tyrosine (Y) residue, a phosphorylated serine (S) residue, a phosphorylated threonine (T) residue, a phosphorylated arginine (R) residue, a phosphorylated histidine (H) residue, ora phosphorylated cysteine (C) residue.

[0325] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or P296PC00 / 3000181 -001977

[0326] ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides encoding antigen binding proteins are typically provided as part of vectors. In some instances, nucleic acids may be isolated nucleic acids. In some instances, nucleic acids may be a recombinant or heterologous nucleic acids. In some instances, nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form.

[0327] As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some instances, the polymeric lipid is a lipid comprising a polymer component. In some instances, the polymeric lipid is a PEG lipid. In some instances, the polymeric lipid is not a PEG lipid. In some instances, the polymeric lipid is Brij or OH-PEG-stearate.

[0328] As used herein, the terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic P296PC00 / 3000181 -001977

[0329] polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0330] As used herein, the term "prevent" refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0331] As used herein, "prophylactic" refers to a therapeutic or course of action used to prevent the spread of disease.

[0332] As used herein, a "prophylaxis" refers to a measure taken to maintain health and prevent the spread of disease. An "immune prophylaxis" refers to a measure to produce active or passive immunity to prevent the spread of disease.

[0333] The term “proliferative diseases refers to a type of disease characterized by an uncontrolled or abnormal growth of cells, which can lead to the formation of tumors or other forms of abnormal tissue growth. This growth can occur in various parts of the body. Examples of proliferative diseases include cancer, benign tumors, and conditions such as hyperplasia and dysplasia. The causes of proliferative diseases can vary, but they may be related to genetic mutations, exposure to toxins or radiation, or other environmental factors.

[0334] A cancer is considered to be a “PRAME-expressing cancer” (also referred to as PRAME “positive” cancer), if the related peptide, such as, for example the PRAME antigenic peptide of SEQ ID NO: 1, is over-presented in patient cancer cells as defined herein. In all other indications named here a biopsy can be performed as it is standard in the treatment of these cancers and the peptide can be identified according to the XPresident’ and related methods (according to WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, US 7,811,828, US 9,791,444, and US 2016 / 0187351, the contents of each are hereby incorporated by reference in their entirety).

[0335] As used herein, pseudouridine (i ) refers to the C-glycoside isomer of the nucleoside uridine. A "pseudouridine analog" is any modification, variant, isoform or derivative of pseudouridine. For example, pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1 -taurinomethyl-pseudouridine, 1 -taurinomethyl-4-thio-pseudouridine, 1 - P296PC00 / 3000181 -001977 methylpseudouridine (m1i ) (also known as N1-methyl-pseudouridine), 1-methyl-4-thio-pseudouridine (m1s4i ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3i ), 2-thio-1 -methyl-pseudouridine, 1 -methyl-1 -deaza-pseudouridine, 2-thio-1 -methyl-1 -deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 i ), and 2'-O-methyl-pseudouridine (i m).

[0336] The term "purified" in the context of nucleic acids, polypeptides, and host cells, refers to the separation of a specific molecule or entity from its (natural) environment or context. Purification is a process of removing impurities, contaminants, or unwanted molecules to obtain a more homogeneous or highly concentrated form of the molecule or entity of interest. “Purified” thus denotes a degree of separation that is higher than isolation. Isolation can be the first step in the purification process, but it does not necessarily imply that the molecule or entity has been completely purified. A “purified” or “biologically pure” nucleic acid, polypeptide, or host cell is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the nucleic acid, polypeptide, or host cell. That is, a nucleic acid, polypeptide or host cell is purified if it is substantially free of cellular material, viral material, culture medium or other supplements and additives.

[0337] For nucleic acids, purification can involve removing proteins, lipids, and other cellular debris to obtain a highly concentrated and pure DNA or RNA sample. This can be done using techniques such as column chromatography, gel electrophoresis, or spin column purification.

[0338] For polypeptides, purification can involve removing other proteins, nucleic acids, or contaminants to obtain a highly purified form of the polypeptide of interest. This can be done using techniques such as affinity chromatography, size exclusion chromatography, or ion exchange chromatography.

[0339] For host cells, purification can involve removing dead cells, debris, and other unwanted materials to obtain a highly purified population of cells. This can be done using techniques such as density gradient centrifugation, magnetic separation, or fluorescence-activated cell sorting (FACS).

[0340] Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0341] The term “radioactive isotope” or “radioactive molecule” is intended to include radioactive isotopes suitable for treating cancer, such as At211, Bi212, Er169, I131, I125, Y90, In111, P32, Re186, Re188, Sm153, Sr89, and radioactive isotopes of Lu. Such radioisotopes generally emit mainly beta-radiation. P296PC00 / 3000181 -001977

[0342] For instance, the radioactive isotope can be an alpha-emitter isotope, more precisely Thorium 227 which emits alpha-radiation. The term also includes spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0343] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means. Recombinant molecules do not exist in nature. Accordingly, “recombinant” refers to a polynucleotide or polypeptide which is made using recombinant techniques, i.e., which is not naturally occurring. Methods and techniques for the production of recombinant nucleic acids and polypeptides are well known in the art.

[0344] The term "reference sequence" refers to a starting nucleic acid or amino acid sequence that can be sequence optimized. A reference nucleic acid sequence may be a wild-type nucleic acid sequence, a fragment or a variant thereof, or a previously sequence optimized nucleic acid sequence.

[0345] As used herein, “regulatory sequence” of a nucleic acid molecule means a cis- acting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0346] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 51of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNAto synthesize precursors of mRNA, and most shRNAand microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EF1 -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. For instance, the promoter can be a constitutive promoter. As used herein, the term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus P296PC00 / 3000181 -001977 40 (SV40) promoter, a polyubiquitin C (UBC) promoter, an EF1 -alpha promoter, a PGK promoter and a CAG promoter. Alternatively, the promoter can be a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. The conditional promoter may be an immune cell specific promoter, which allows for continual transcription of the coding sequence or gene in an immune cell. Non-limiting examples of the immune cell specific promoters include a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-p gene promoter, a WASP gene promoter, a T-cell receptor p-chain gene promoter, a V9 y (TRGV9) gene promoter, a V2 6 (TRDV2) gene promoter, and the like.

[0347] Enhancers are known to influence gene expression when positioned 51or 31of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0348] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0349] The term “safety profile” herein refers to the capacity to distinguish tumor cells from healthy tissue cells and this is often determined by determining the safety window.

[0350] The term “safety window” or “therapeutic window” herein refers to a factor that compares the half maximal concentration of a compound that is required for inducing 100% cytotoxicity in a tumor cell line in comparison to the half maximal concentration of a compound that is required for inducing 100% cytotoxicity healthy tissue cells. If for an antigen binding protein of interest the EC5o determined for a tumor cell line is 1 pM and the EC5o value determined for, for instance, primary cells is 1000 pM then the safety window is 1000 since the EC50 for the tumor cell line is 1000 times smaller than the EC5O for the primary cells.

[0351] As used herein, the term "sample" or "biological sample" refers to a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further can include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, P296PC00 / 3000181 -001977

[0352] tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which can contain cellular components, such as proteins or nucleic acid molecule.

[0353] As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and in some cases capable of formulation into an efficient therapeutic agent.

[0354] As used herein, the term "stabilize." "stabilized." "stabilized region" means to make or become stable.

[0355] As used herein, the term “sequence optimization” refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties, e.g., improved protein expression or decreased immunogenicity of the nucleic acid itself.

[0356] In general, the goal in sequence optimization is to produce a synonymous nucleotide sequence than encodes the same polypeptide sequence encoded by the reference nucleotide sequence. Thus, there are no amino acid substitutions (as a result of codon optimization) in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the reference nucleotide sequence. In some instances, codon optimization is achieved by modification of a coding sequence according to at least one of the following; (i) replacing the naturally occurring codon sequence with an alternative codon that retains the amino acid sequence encoding the protein but alters the composition and / or structure of the encoding RNA; (ii) adjusting the guanosine cytosine content of the coding sequence relative to the naturally occurring guanosine cytosine content of the coding sequence; (iii) adjusting the number of CpG sites of the coding sequence relative to the number of CpG sites in the naturally occurring coding sequence; (iv) substituting the naturally occurring codon sequence with an alternative codon relative to (ii) guanosine cytosine content and / or (iii) number of CpG sites. Codon optimization may include adjusting codons in the context of tRNA expression in a particular tissue and / or may include methods for evading the effects of natural, tissue-specific shRNAs or miRNAs.

[0357] As used herein, the term “sequential” therapeutic use refers to administration of at least two active agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the therapeutic agents before administration of the other or others commences. It is thus possible to administer one of the therapeutic agents over several minutes, hours, or days before administering the other therapeutic agent or ingredients. There is no simultaneous treatment in this case.

[0358] As used herein, the phrases "signal sequence." "signal peptide." and "transit peptide" are used interchangeably and refer to a sequence that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence polypeptide and the nucleic acid sequence encoding the signal sequence. Thus, P296PC00 / 3000181 -001977 references to a signal sequence in the context of a nucleic acid refer in fact to the nucleic acid sequence encoding the signal sequence polypeptide.

[0359] The term “sequence identity” refers to a measure of the similarity between two biological sequences, such as polynucleotide or polypeptide sequences. It is expressed as a percentage of the number of identical positions in the two sequences divided by the total number of positions. In other words, sequence identity indicates how much two sequences match each other in terms of the order and composition of their building blocks, such as nucleotides or amino acids. If the two sequences to be compared are not of equal length, they can be aligned to give the best possible fit, allowing the insertion of gaps or alternatively, truncation at the ends of the nucleic acid sequences or amino acid sequences. The skilled person will acknowledge that various means for comparing sequence identity are available (see below). A higher sequence identity may indicate a closer evolutionary relationship between the two sequences, or a higher degree of functional similarity or homology. Sequence identity is commonly used in bioinformatics and molecular biology to compare and analyze the structure and function of biological molecules, such as proteins, and to infer their evolutionary history and relationships.

[0360] For example, in the context of the present disclosure, a sequence that is “at least 85% identical to a reference sequence” may be a sequence having, over its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of a reference sequence (e.g., a variable domain disclosed herein). Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% Identical” to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence.

[0361] In the context of the present disclosure, the sequence identity can be calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle” program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol.48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp.276-277). The percentage of identity between two polypeptides, in accordance with the disclosure, is calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

[0362] In the context of the present disclosure “similar peptides” herein refers to potential off-target peptides, i.e. peptides that may potentially be bound by the antigen binding proteins of the disclosure based on their biochemical / biophysical characteristics, including but not limited to a homologous P296PC00 / 3000181 -001977

[0363] sequence or a similar motif. Similar peptides comprise typically 8 to 12 amino acids in length. The similar peptides in the context of the present disclosure are typically MHC, in particular MHC I, presented. Furthermore, similar peptides in the context of the present disclosure include peptides that comprise or consist of an amino acid sequence that is similar to the amino acid sequence of the PRAME antigenic peptide, more particular, peptides that, in comparison to the epitope of the PRAME antigenic peptide, comprise an epitope wherein some or all amino acids have identical and / or similar biochemical / biophysical characteristics as the amino acids that constitute the epitope of the PRAME antigenic peptide. In some examples, similar peptides investigated in the context of the present disclosure were selected from a database of tumor and normal tissue-presented HLA-A*02 bound peptides (XPRESIDENT* database) using a similarity scoring within the binding-relevant positions of PRAME and the requirement of at least one detection on normal tissues. Binding of an antigen binding protein to a similar peptide presented by an MHC protein may lead to adverse reactions. Such adverse reactions may be “off-tumor” side effects, such as cross-reactivity of a specific TCR with a similar peptide in healthy tissues as reported in Lowdell et al., Cytotherapy, published on December 4, 2018).

[0364] In particular, the “similar” peptides disclosed in WO2018172533 A1 are similar peptides in the context of the present disclosure.

[0365] The skilled person is aware that among the similar peptides, there are some that are not bound by the antigen binding proteins of the disclosure to a detectable degree, e.g. peptides for which no binding signal during affinity determination or no response in a functional assay beyond the background level is detectable. “Background level” in this context refers to a response in a functional assay observed for the co-culture of target cells and effector cells at the respective E: T ratio without the addition of bispecific TCR-antibody fusion protein.

[0366] For other similar peptides, a low, but non-significant binding may be detectable. These latter similar peptides may also be described as "potentially relevant" similar peptides. An antigen binding protein is considered to not significantly bind to a similar peptide and to be specific for its target antigenic peptide if at least one of the following applies when binding to the similar peptide and the target antigenic peptide is compared under similar, preferably identical experimental conditions:

[0367] The functional avidity in response to the similar peptide, determined in a functional assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the functional avidity in response to the target antigenic peptide PRAME.

[0368] The cytotoxic activity in response to the similar peptide, determined in a cytotoxicity assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the cytotoxic activity in response to the target antigenic peptide PRAME. P296PC00 / 3000181 -001977 The EC5O of the similar peptide, determined in a functional assay, preferably a cytotoxicity assay, as described above, is increased by a factor of at least 50, at least 100, at least 200 or at least 500, compared to the EC5o of the target antigenic peptide PRAME.

[0369] The KDfor the similar peptide is increased by a factor at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100, compared to the KDfor the target antigenic peptide PRAME.

[0370] The relative response signal for the similar peptide is not higher than 30%, not higher than 25%, not higher than 20%, or not higher than 15%, compared to the response signalto the target antigenic peptide.

[0371] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two therapeutic agents by the same or different routes and at the same time or at substantially the same time.

[0372] As used herein, a "single unit dose" is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0373] The term “specificity” or “specific binding” or “specifically binds” or “specifically targets” in the context of the present invention denotes the capacity of an antigen binding protein to discriminate its target peptide from peptides having a different amino acid sequence, e.g. similar peptides as defined below. An antigen binding protein is considered specific for a target peptide, if bindingto the target peptide presented in an MHC molecule occurs with a significantly higher affinity and / or higher functional avidity than the binding to similar peptides. The specificity of the antigen binding protein is determined by the amino acid sequences CDRal, CDRa3, CDRbl and CDRb3. The amino acid sequences of CDRa2 and CDRb2 contact the MHC molecule and may in some instances not be required for antigen specificity.

[0374] Therefore, as used herein, the term “specifically binds” or “specifically binds to” or “specifically target” can be exhibited, for example, by a molecule having a Kd for the molecule to which it binds to of about 10“4M, 10“5M, 10“6M, 10’7M, 10“8M, 10’9M, 10’10M, 10“11M, or 10“12M.

[0375] As used herein, the term “specific delivery.” “specifically deliver.” or “specifically delivering”, in particular in the context of polynucleotides (e.g., mRNAs) means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen). The level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue. For example, for renovascular targeting, a polynucleotide is specifically provided to a P296PC00 / 3000181 -001977

[0376] mammalian kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more polynucleotide per 1 g of tissue is delivered to a kidney compared to that delivered to the liver or spleen following systemic administration of the polynucleotide. It will be understood that the ability of a nanoparticle to specifically deliver to a target tissue need not be determined in a subject beingtreated, it can be determined in a surrogate such as an animal model (e.g., a rat model).

[0377] As used herein, the term “start codon”, used interchangeably with the term “initiation codon”, refers to the first codon of an open readingframethat is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases. The initiation codon is depicted by the first letter codes of adenine (A), uracil (U), and guanine (G) and is often written simply as “AUG”. Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as “alternative initiation codons”, the initiation codons of polynucleotides described herein use the AUG codon. During the process of translation initiation, the sequence comprising the initiation codon is recognized via complementary base-pairing to the anticodon of an initiator tRNA (Met-tRNAiMet) bound by the ribosome. Open reading frames may contain more than one AUG initiation codon, which are referred to herein as “alternate initiation codons”.

[0378] As used herein, by "subject" or "individual" or "animal" or "patient" or "mammal." is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, prophylaxis or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferably, the subject is a human subject.

[0379] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness in many biological and chemical characteristics.

[0380] As used herein, the term “substantially equal” as it relates to time differences between doses, the term means plus / minus 2%.

[0381] As used herein, the term “sub-therapeutic dose” refers to a dose of an agent that does not achieve a particular therapeutic effect (e.g., wherein the particular therapeutic effect is achieved using a therapeutically effective amount). Typically, a sub-therapeutic dose of an agent is an amount of a therapeutic dose that is less than a therapeutically effective amount of the agent. P296PC00 / 3000181 -001977

[0382] As used herein, the term “suffering from” refers to a subject who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0383] A subject who is "susceptible to" a disease, disorder, and / or condition has not been diagnosed with and / or cannot exhibit symptoms of the disease, disorder, and / or condition but harbors a propensity to develop a disease or its symptoms. In some instances, a subject who is susceptible to a disease, disorder, and / or condition (for example, cancer) can be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microbe associated with development of the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0384] In the context of the present disclosure, the term “T2 cell” refers to a cell that expresses an MHCI molecule (HLA-A2) that lacks TAP function. T2 cells can be easily artificially loaded with different concentrations of exogenous antigenic peptides. T2 cell are described e.g. in (Hosken and Bevan, Science 1990 Apr 20;248(4953):367-70). T2 cells are commercially available, e.g. from ATCC (American Type Culture Collection). Loading of T2 cells can be achieved under standard cell culture conditions known to the skilled in the art by incubating the T2 cells for about 2 hours with a desired concentration of antigenic peptide. In the context of the present disclosure, T2 cells that are incubated with a certain concentration of antigenic peptide, such as 1 pM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, are referred to as T2 cells loaded with said concentration of antigenic peptide, e.g. T2 cells incubated with 10 pM of antigenic peptide are referred to as T2 cells loaded with 10 pM of antigenic peptide.

[0385] The term “TCR” as used herein includes both native and engineered TCRs.

[0386] A “native” TCR refers to a wildtype TCR that can be isolated from nature, whereas an “engineered” TCR may be a protein resembling a native TCR, but comprising further modifications e.g. in the variable and / or constant domains compared to the naturally occurring sequence, e.g. a humanized TCR or a TCR with altered characteristics (e.g. altered binding, heterodimerization or expression level).

[0387] “Native TCRs” are heterodimeric cell surface proteins of the immunoglobulin super-family, which are associated with invariant proteins of the CD3 complex involved in mediating signal P296PC00 / 3000181 -001977 transduction. Native heterodimeric TCRs exist in ap and y6 forms, which are structurally similar but have distinct locations and probably functions. The terms “a / p TCR” or a ”y / 6 TCR” thus refer to a TCR comprising an a-chain and a p-chain as described above, or a y-chain and a 6-chain, respectively. Such TCRs may also be described as “full length TCRs” or “conventional TCRs”. An a / p TCR or a y / 6 TCR may be a native TCR or may be an engineered TCR that retains the structure of a native TCR, i.e. an engineered TCR comprising minor modifications in the variable and / or constant domains as described above, such as a humanized TCR.

[0388] Native, full-length ap heterodimeric TCRs consist of an a-chain and a p-chain. The a-chain comprises a variable region (V region) encoded by a TRAV gene, a joining region (J region) encoded by a TRAJ gene, and a constant region (C region) encoded by a TRAC gene. The p-chain comprises a variable region (V region) encoded by a TRBV gene, a joining region (J region) encoded by a TRBJ gene and a constant region (C region) encoded by a TRBC gene, and usually a short diversity region (D region) encoded by a TRBD gene between the V and J regions, although this D region is often considered as part of the J region (Lefranc, (2001 ), Curr Protoc Immunol Appendix 1: Appendix 10). The genes encoding different a-chain and p-chain variable, joining and constant regions are referred to in IMGT nomenclature by unique numbers (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scavinerand Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFrancand LeFranc, (2001), " T cell Receptor Factsbook", Academic Press). Further information on TCR genes can be found in the international ImMunoGeneTics information system®, Lefranc M-P et aL, (Nucleic Acids Res. 2015 Jan;43(Database issue): D413-22; and http: / / www.imgt.org / ).

[0389] The alpha chain TRAC constant domain sequence and the beta chain TRBC1 orTRBC2 constant domain are in the following, also referred to as TCR constant domain sequences. The TCR constant domain sequences may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human. The TCR constant domain sequences may be modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. Also, further stabilizing mutations as known from the state of the art (e.g. WO 2018 / 104407, PCT / EP2018 / 069151, WO 2011 / 044186, WO 2014 / 018863) may be introduced, such as replacement of unfavorable amino acids in the variable regions and / or the introduction of a disulfide bridge between the TCR C domains and the removal of unpaired cysteine. Thus, the disclosure also relates to proteins that may comprise the antigen binding domains (e.g. the CDRs as provided herein) and further comprise domains / amino acid sequences that are not found in the naturally occurringTCR.

[0390] On the protein level, TCR a-, p-, y- and 6-chains comprise two immunoglobulin domains, the variable domain and the constant domain. The variable domain corresponds to the V(D) J region. The constant domain corresponds to the C region. The constant domain is the membrane-proximal domain and in the context of the present disclosure also includes the transmembrane (TM) domain P296PC00 / 3000181 -001977 and a short cytoplasmic tail. Each of the constant and variable domains include an intra-chain disulfide bond. The variable domains (VQand Vpin a(3 TCRs and VYand V5in y6 TCRs) contain highly polymorphic loops comprising the complementarity determining regions (CDRs).

[0391] Each TCR variable domain comprises three “TCR complementarity determining regions embedded in a framework sequence, one being the hypervariable region named CDR3. In the context of the present disclosure, CDRal, CDRa2 and CDRa3 denote a-chain CDRs, and CDRbl, CDRb2 and CDRb3 denote p-chain CDRs. The sequences encoding CDRal and CDRa2 are comprised inTRAV, the sequences encoding CDRa3 are comprised inTRAVandTRAJ, the sequences encoding CDRbl and CDRb2 are comprised in TRBV, and the sequences encoding CDRb3 are comprised in TRBV, TRBD and TRBJ. In TCRs, the CDR1 and CDR3 amino acid residues make contact with the antigenic peptide, while the CDR2 amino acid residues mainly contact the HLA molecule (Stadinski et aL, J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et aL, J Biol Chem. 2014 Jan 10;289(2):628-38). The antigen specificity of a TCR is thus defined by the CDR3 and CDR1 sequences. The CDR2 sequences are not required for the determination of antigen specificity, but may play a role in the overall affinity of a TCR towards a peptide: MHC complex.

[0392] “TCR framework regions” (FRs) refer to amino acid sequences interposed between the CDRs, i.e. to those portions of the variable domains that are to some extent conserved among different TCRs. The a-, (B-, y- and 6-chain variable domains each have four FRs, herein designated FR1 -a, FR2-a, FR3-a, FR4-a (for an a- or y-chain), and FR1-b, FR2-b, FR3-b, FR4-b (for a |3- or 6-chain), respectively. Accordingly, an a-chain or y-chain variable domain may be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a) and a |3- or 6-chain variable domain may be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In the context of the present disclosure, the CDR / FR sequences in an a-, p, y- or 6-chain variable domain is determined based on IMGT definition (Lefranc et aL, Dev. Comp. ImmunoL, 2003, 27(1 ):55-77; www.imgt.org). Accordingly, CDR / FR amino acid positions when related to TCR or TCR-derived domains are indicated according to said IMGT definition. Preferably, the IMGT position of the CDR / FR amino acid positions of the variable domain Va is given in analogy to the IMGT numbering of TRAV24*01 and / or the IMGT position of the CDR / FR amino acid positions of the variable domain vp is given in analogy to the IMGT numbering of TRBV12-3*01.

[0393] “Functional fragment of a TCR” refers to a fragment of a TCR that retains or substantially retains the affinity, functional avidity and / or specificity of the parental TCR from which it is derived for a target antigen. “Parental TCR” in this context refers to a full length TCR from which a functional fragment may be derived.

[0394] As binding to the target antigenic peptide is defined by the CDR1 and CDR3 sequences, and binding to the target antigenic peptide MHC complex is defined by CDR1, CDR2 and CDR3, antigen binding proteins comprising the CDR1 and CDR3 and optionally CDR2 sequences of a TCR retain the P296PC00 / 3000181 -001977 affinity, functional avidity and / or specificity of the parental TCR for a target antigen. The person skilled in the art is aware that the CDRs have to be interspersed with framework regions (FRs), however the specific amino acid sequences of the framework regions are not directly involved in target antigen specificity. Examples of functional TCR fragments include single variable domains, such as TCR alpha, beta, gamma or delta variable domains, or fragments of the a, p, 6 or y chain, such as an a, p, 6 or y chain without transmembrane domain and short cytoplasmic tail. The term “fragment” as used herein refers to naturally occurring fragments (e.g. splice variants or peptide fragments) as well as artificially constructed fragments, in particular to those obtained by gene-technological means.

[0395] A functional fragment of a TCR is considered to have retained or substantially retained the affinity for a target antigen, if, for example, the KDfor binding to the target antigen measured as outlined below is identical to the KDof the TCR or is increased or reduced, preferably reduced, no more than 10x, 5x, 3x, or 2x.

[0396] A functional fragment of a TCR may have retained or substantially retained the functional avidity for a target antigen, if, for example, the functional avidity for the target antigen is identical to that of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1%. In particular, a functional fragment of a TCR is considered to have retained or substantially retained the functional avidity for a target antigen, if, for example, its cytotoxic activity in response to the target of the parent protein measured in a cytotoxicity assay is identical to the cytotoxic activity of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1%, preferably 10%, 8%, 5%, 3%, 2% or 1%.

[0397] A functional fragment of a TCR is considered to have retained or substantially retained the specificity for a target antigen (i.e. the ability to specifically bind to a target antigen), if it does not significantly bind to peptides other than the target antigenic peptide of the TCR.

[0398] “Does not significantly bind” in the context of antigenic peptide variants and in the context of antigen binding proteins of the disclosure, denotes, typically, a functional avidity was determined for the antigen binding protein binding to an antigenic peptide variant that is not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, preferably not higher than 20% of the functional avidity obtained for binding to the PRAME peptide consisting of the amino acid sequence of SEQ ID NO: 1, preferably in the same experimental conditions. For instance, the functional avidity obtained for the antigen binding protein binding to a similar peptide is not higher than 30% of the signal obtained in the same experimental conditions for the antigen binding protein binding to the PRAME peptide consisting of the amino acid sequence of SEQ ID NO: 1 (see WO2018172533 A1 ). The skilled person knows howto determine whether an antigen binding protein does not significantly bind P296PC00 / 3000181 -001977 to an antigenic peptide. An exemplary method is herein disclosed below and exemplified in the appended examples.

[0399] “Single chain TCR (scTCR)” as used herein denotes a TCR in which the variable domains of the TCR are located on a single polypeptide. Typically, the variable domains in scTCRs are separated by a linker, wherein said linker typically comprises 10 to 30 amino acids, such as 25 amino acids.

[0400] The term "therapeutic agent" or “active agent” refers to an agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. For example, a host cell expressing an antigen binding protein, or an mRNA encoding an antigenic peptide can be a therapeutic agent.

[0401] By a "therapeutically effective amount" of a therapeutic agent (e.g., a host cell or mRNA) or pharmaceutical composition thereof is meant a sufficient amount of the therapeutic agent to treat said proliferative disease, at a reasonable benefit / risk ratio applicable to any medical treatment. The total daily / monthly / yearly dose may be determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the therapeutic agent; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion, exhaustion or degradation of the therapeutic agent; the duration of the treatment; drugs used in combination or coincidental with the specific therapeutic agent employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of a therapeutic agent at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0402] As used herein, the term "therapeutically effective outcome" means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0403] As used herein, the term "transcription" refers to methods to produce mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence).

[0404] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or heterologous) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, e.g. the PRAME antigenic peptide or the antigen-binding protein described herein. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0405] As used herein, the term "treating" or "treatment" or "therapy" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a disease, e.g., P296PC00 / 3000181 -001977

[0406] cancer. For example, "treating" cancer can refer to diminishing symptoms associate with the disease, prolong the lifespan (increase the survival rate) of patients, reducing the severity of the disease, preventing or delaying the onset of the disease, etc. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. Therapeutic effects of treatment include, without limitation, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “treating cancer” is meant that the symptoms associated with the cancer are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0407] As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in anyway. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.

[0408] “Uracil” is one of the four nucleobases in the nucleic acid of RNA, and it is represented by the letter U. Uracil can be attached to a ribose ring, or more specifically, a ribofuranose via a b-N1-glycosidic bond to yield the nucleoside uridine. The nucleoside uridine is also commonly abbreviated according to the one letter code of its nucleobase, i.e., U. Thus, in the context of the present disclosure, when a monomer in a polynucleotide sequence is U, such U is designated interchangeably as a "uracil" or a "uridine."

[0409] The terms "uridine content" or "uracil content" are interchangeable and refer to the amount of uracil or uridine present in a certain nucleic acid sequence. Uridine content or uracil content can be expressed as an absolute value (total number of uridine or uracil in the sequence) or relative (uridine or uracil percentage respect to the total number of nucleobases in the nucleic acid sequence).

[0410] The terms "uridine-modified sequence" refers to a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with a different overall or local uridine content (higher or lower uridine content) or with different uridine patterns (e.g., gradient distribution orclustering) with respect to the uridine content and / or uridine patterns of a candidate nucleic acid sequence. In the content of the present disclosure, the terms "uridine-modified sequence" and "uracil-modified sequence" are considered equivalent and interchangeable.

[0411] A "high uridine codon" is defined as a codon comprising two or three uridines, a "low uridine codon" is defined as a codon comprising one uridine, and a "no uridine codon" is a codon without any uridines. In some instances, a uridine-modified sequence comprises substitutions of high uridine codons with low uridine codons, substitutions of high uridine codons with no uridine codons, P296PC00 / 3000181 -001977 substitutions of low uridine codons with high uridine codons, substitutions of low uridine codons with no uridine codons, substitution of no uridine codons with low uridine codons, substitutions of no uridine codons with high uridine codons, and combinations thereof. In some instances, a high uridine codon can be replaced with another high uridine codon. In some instances, a low uridine codon can be replaced with another low uridine codon. In some instances, a no uridine codon can be replaced with another no uridine codon. A uridine-modified sequence can be uridine enriched or uridine rarefied.

[0412] As used herein, the terms "uridine enriched" and grammatical variants refer to the increase in uridine content (expressed in absolute value or as a percentage value) in a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with respect to the uridine content of the corresponding candidate nucleic acid sequence. Uridine enrichment can be implemented by substituting codons in the candidate nucleic acid sequence with synonymous codons containing less uridine nucleobases. Uridine enrichment can be global (i.e., relative to the entire length of a candidate nucleic acid sequence) or local (i.e., relative to a subsequence or region of a candidate nucleic acid sequence).

[0413] As used herein, the terms "uridine rarefied" and grammatical variants refer to a decrease in uridine content (expressed in absolute value or as a percentage value) in a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with respect to the uridine content of the corresponding candidate nucleic acid sequence. Uridine rarefication can be implemented by substituting codons in the candidate nucleic acid sequence with synonymous codons containing less uridine nucleobases. Uridine rarefication can be global (i.e., relative to the entire length of a candidate nucleic acid sequence) or local (i.e., relative to a subsequence or region of a candidate nucleic acid sequence).

[0414] As used herein, the term “variant” in relation to a nucleic acid or amino acid sequence refers to both natural variants (e.g., polymorphisms, isoforms, etc.) and artificial variants in which at least one nucleic or amino acid residue in a native or starting sequence (e.g., a wild type sequence) has been removed and a different nucleic or amino acid inserted in its place at the same position, respectively. These variants can be described as “substitutional variants.” The substitutions can be single, where only one nucleic or amino acid in the molecule has been substituted, or they can be multiple, where two or more nucleic or amino acids have been substituted in the same molecule. If nucleic or amino acids are inserted or deleted, the resulting variant would be an “insertional variant” or a “deletional variant” respectively.

[0415] “Vu” in the context of the present disclosure refers to a variable domain of a TCR a-chain. “VA” or Vain the context of the present disclosure refers to a TCR variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (VQ), 0-chain (Vp), y-chain (VY) or P296PC00 / 3000181 -001977 6-chain (V5), preferably from a VQ. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, P-chain, y-chain or 6-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR a-chain.

[0416] The CDR and framework sequences of the VAdomain may not necessarily be derived from the same TCR chain. For example, the CDRs derived from one TCR variable domain (of the donor TCR) could also be grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VAencoded by TRAV5 and TRAJ17, and the acceptor TCR may comprise a VAencoded byTRAV14 and TRAJ33.

[0417] ”VP” in the context of the present disclosure refers to a variable domain of a TCR p-chain. “VB” or Vbin the context of the present disclosure refers to a variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (VQ), p-chain (Vp), y-chain (VY) or 6-chain (V5), preferably from a Vp. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, P-chain, y-chain or 6-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR p-chain. In the examples, various framework and CDR mutations / substitutions are shown.

[0418] The CDR and framework sequences of the VBdomain in context of the present disclosure may not necessarily be derived from the same TCR. For example, the CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VBencoded by TRBV2 and TRBJ2-1, and the acceptor TCR may comprise a VBencoded byTRBV27 and TRBJ1-5.

[0419] CDRs may not only be exchanged / grafted between different alpha variable domains or different beta variable domains, but also may be grafted from a TCR alpha to a TCR beta, gamma or delta variable domain, or from a TCR beta to a TCR alpha, gamma or delta variable domain.

[0420] ”Vv” in the context of the present disclosure refers to a variable domain of a TCR y-chain. ’’Vs” in the context of the present disclosure refers to a variable domain of a TCR 6-chain. ”VL” in the context of the present disclosure refers to a variable domain of an antibody light chain.

[0421] “VH” in the context of the present disclosure refers to a variable domain of an antibody heavy chain.

[0422] The terms "vector" includes "cloning vectors" and "expression vectors" and refers to a vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.. P296PC00 / 3000181 -001977

[0423] The term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle and encodes at least an exogenous nucleic acid. The vector and / or particle can be utilized for the purpose of transferring a nucleic acid of interest into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Useful viral vectors include vectors based on retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). Recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replicationdefective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0424] The terms “of the invention” or “according to the invention” as used herein are intended to refer to all aspects and embodiments of the invention disclosed and / or claimed herein. Any aspects, items or embodiments referred to herein as being “disclosed herein” or “described herein” are to be understood as being aspects, items or embodiments “of the invention” or “according to the invention”.

[0425] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0426] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within this disclosure. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having P296PC00 / 3000181 -001977

[0427] a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0428] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0429] The invention will now be described in more details with reference to the following figures and examples. All literature and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the foregoing description, the examples are to be considered illustrative or exemplary and not restrictive.

[0430] Antigen binding proteins

[0431] In a first aspect, the invention provides a combination comprising:

[0432] a. a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and

[0433] b.an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail

[0434] The host cell will typically be transformed to comprise a recombinant or heterologous nucleic acid sequence encoding the antigen binding protein. The host cell will typically express the antigen binding protein on its surface.

[0435] In some embodiments, the antigen binding protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a CDRal comprising or consisting of an amino acid sequence SSNFYA according to SEQ ID NO: 122, and a CDRa3 comprising or consisting of an amino acid sequence CALYNNNDMRF according to SEQ ID NO: 124, and wherein the second polypeptide comprising a CDRbl comprising or consisting of an amino acid sequence SGHNS according to SEQ ID NO: 125, and a CDRb3 comprising or consisting of an amino acid sequence CASSPGSTDTQYF according to SEQ ID NO: 127. In some embodiments, the CDRal, CDRa3, CDRbl and / or CDRb3 sequence(s) may comprise one, two or three amino acid mutations. P296PC00 / 3000181 -001977 In some embodiment, the CDRal, CDRa3, CDRbl and / or CDRb3 sequence(s) may each comprise at most one, at most two or at most three amino acid mutations.

[0436] In some embodiments, said first polypeptide comprises or consists of a variable domain VA. In some embodiments, said VAcomprises or consists of a TCR a-chain variable domain (Va).

[0437] In some embodiments, said second polypeptide comprises or consists of a variable domain VB. In some embodiments, said VBcomprises or consists of a TCR p-chain variable domain (VP).

[0438] In some embodiments, the first polypeptide further comprises a CDRa2 comprising or consisting of the amino acid sequence MTL according to SEQ ID NO: 123 or SEQ ID NO: 134. In some embodiments, a CDRa2 comprising or consisting of the amino acid sequence of SEQ ID NO: 134 is preferred. In some embodiments, the first polypeptide further comprises a CDRb2 comprising or consisting of the amino acid sequence FNNNVP accordingto SEQ ID NO: 126.

[0439] In some embodiments, the Va comprises or consists of an amino acid sequence of SEQ ID NO: 128 as shown in Table 9 or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 128 as shown in Table 9, and comprises the CDRal, CDRa2, and CDRa3 of SEQ ID NOs: 122, 123 or 134, and 124, respectively.

[0440] In some embodiments, the vp comprises or consists of an amino acid sequence of SEQ ID NO: 130 as shown in Table 9 or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 130 as shown in Table 9, and comprises the CDRbl, CDRb2, and CDRb3 of SEQ ID NOs: 125, 126, and 127, respectively.

[0441] In all embodiments, any amino acid mutations within the CDRal, CDRa2, CDRa3, CDRbl, CDRb2 and CDRb3 sequences- if present-are preferably amino acid substitutions, more preferably conservative amino acid substitutions (see Table “Conservative Amino Acid Substitutions”). It is preferred thattheCDR sequences comprise not more than two, preferably not more than one, amino acid mutations. It is further preferred that the amino acid mutations - if present - are at the first or last position of the respective CDR sequence. In most preferred embodiments, the CDR sequences do not comprise any amino acid mutation.

[0442] Introducing a mutation into a known amino acid sequence is standard procedure well-known in the art and routine work for the skilled person. Respective methods are known in the field (e.g. Stratagene’s QuikChange Site Directed Mutagenesis Kit since 2007). The skilled person is thus very well capable of introducing specific mutations such as substitutions into an amino acid sequence in general and into a CDR sequence in particular.

[0443] Screening of variants of a given CDR for binding to its target is also a procedure applied by the skilled person. The present application describes functional assays, including cytokine production assays to determine bindingof an antigen binding protein to the PRAME antigenic peptide. Binding of an antigen binding protein to the PRAME antigenic peptide can also be determined by dextramer staining. P296PC00 / 3000181 -001977 While the outcome of an amino acid mutation in a CDR may not be readily predictable, the skilled person would be well capable of generating and screening multiple mutants without undue burden. The skilled person would thus be able to generate antigen binding proteins carrying one, two or three amino acid mutations within their CDRs and subsequently identify antigen binding proteins having the same binding characteristics as an antigen binding protein comprising the CDR sequences of Table 9.

[0444] In some embodiments, not more than 1 or 2 amino acid mutations, preferably not more than 1 amino acid mutation, more preferably not more than 1 amino acid substitution, most preferably not more than 1 conservative amino acid substitution is comprised within the central 8 amino acids of the CDRa3 and / or CDRb3, i.e. within the CDR3 core. In cases where the CDR3 comprises more than 12 amino acids, it is even more preferred that not more than 1 or 2 amino acid mutations, preferably not more than 1 amino acid mutation, more preferably not more than 1 amino acid substitution, most preferably not more than 1 conservative amino acid substitution is comprised within the central 9- 13 amino acids of the CDRa3 and / or CDRb3, i.e. within the CDR3 core max.

[0445] In some embodiments of the antigen binding protein, the first and the last two positions of the CDR1, CDR2 and / or CDR3 of the VAand / or the VBcomprise a conservative substitution, and preferably wherein the CDRal, CDRa3, CDRbl and / or CDRb3 may comprise one, twoorthree amino acid mutations. In particular embodiments of the antigen binding protein, the first and the last two positions of the CDR3 of the VAand / or the VBdomain comprise a conservative substitution, and preferably wherein the CDRa3 and / orCDRb3 may comprise one, two orthree amino acid mutations.

[0446] In some embodiments, the antigen binding protein maybe engineered, for example, by the introduction of heterologous sequences, e.g. mouse sequences, which may increase expression and stability. Also, further stabilizing mutations as known from the state of the art (e.g. WO2018 / 104407, PCT / EP2018 / 069151, WO2011 / 044186, WO2014 / 018863) may be introduced, such as replacement of amino acids in the variable domains and / or the introduction of a disulfide bonds, e.g. between the constant domains of a TCR and the removal of unpaired cysteine.

[0447] In some embodiments, the antigen binding protein further comprises a TCR constant domain comprising or consisting of an amino acid sequence according to SEQ ID NOs: 129 or 131, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 129 or 131 as shown in Table 9.

[0448] In some embodiments, the antigen binding protein comprises a TCR alpha chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 132, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 132 as shown in Table 9. In some embodiments, the antigen binding protein comprises a TCR beta chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 133, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 133 as shown in Table 9. P296PC00 / 3000181 -001977

[0449] The TCR constant domain sequences may be modified by truncation or substitution to delete the native disulphide bond, e.g. between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and / or beta chain constant domain sequence(s) may also be modified by substitution of cysteine residues for example for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulphide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may additionally include a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. The constant domain may additionally or alternatively contain further mutations, substitutions or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. The term TRAC and TRBC1 / 2 encompasses natural polymophic variants, for example N to K at position 4 of TRAC (Bragado et al Int Immunol. 1994 Feb;6(2):223-30).

[0450] In some embodiments, the antigen binding protein is derived from a TCR, or is a TCR or fragment(s) thereof.

[0451] Preferably, the antigen binding protein is the TCR as disclosed in WO2018 / 172533 A1 (R11 P3D3_KE clone) and / or Wermke, M., Araujo, D. M., Chatterjee, M. et al. Autologous T cell therapy for PRAME+ advanced solid tumors in HLA-A*02+ patients: a phase 1 trial. Nat Med 31, 2365-2374 (2025). https: / / doi.org / 10.1038 / s41591-025-03650-6, which are incorporated by reference in their entirety herein.

[0452] In a preferred embodiment, the TCR is selected from the group consisting of an a / p TCR, a y / 6 TCR, functional fragments of a TCR, and a fusion protein or chimeric protein comprising (a) functional fragment(s) of a TCR.

[0453] In some embodiments, VAand VBare TCR variable domains, in particular TCR alpha, beta, gamma or delta variable domains. In some embodiments, VAis a TCR alpha, gamma or delta variable domain and VBis a TCR beta, gamma or delta variable domain. Preferably, VAis a TCR alpha variable domain and VBis a TCR beta variable domain, or VAis a TCR gamma variable domain and VBis a TCR delta variable domain, or VAis a TCR alpha variable domain and VBis a TCR gamma variable domain, or VAis a TCR delta variable domain and VBis a TCR beta variable domain. In preferred embodiments, VAand VBare TCRa and TCR|3 variable domains, respectively. In some embodiments, VAis a TCR gamma variable domain comprising CDR1 and CDR3 and optionally CDR2 derived from a TCR alpha variable domain, and / or VBis a TCR delta variable domain comprising CDR1 and CDR3 and optionally CDR2 derived from a TCR beta variable domain.

[0454] In some embodiments, the MHC protein is an HLA protein, preferably HLA-A, more preferably HLA-A*02.

[0455] The antigen binding protein has a high specificity for the PRAME antigenic peptide (SEQ ID NO: 1 ), in particular an increased specificity in comparison to a reference protein when measured under P296PC00 / 3000181 -001977

[0456] similar, preferably identical experimental conditions. As demonstrated in WO 2018 / 172533 A1, the antigen binding protein binds the target antigen, i.e. the PRAME antigenic peptide in a complex with a MHO protein, with high specificity, but does not or essentially not bind to off-target peptides that are, for example, similar to the sequence and / or motif of PRAME, and thus have an increased risk of being bound by an antigen binding protein binding to PRAME. In some embodiments, the antigen binding protein thus specifically binds to the PRAME: MHC complex.

[0457] In some embodiments, the MHC protein is an HLA protein, preferably HLA-A, more preferably HLA-A*02.

[0458] In some embodiments, the antigen binding protein further comprises a transmembrane domain, optionally including a cytoplasmic signaling region.

[0459] In some embodiments, the antigen binding protein is membrane-bound, for example, a membrane-bound TCR or a membrane-bound functional fragment of a TCR.

[0460] In some embodiments, the variable domains herein provided or the CDRs as herein provided are comprised / included in antigen binding proteins in various formats.

[0461] In some aspects, the antigen binding protein is in the format of an scFv or scTV. In some aspects, the herein provided antigen binding protein comprise the variable domains comprising the herein provided CDRs and further comprise e.g. linkers, such as a serine-glycine linker. For example, the antigen binding protein comprises a single chain TCR (scTCR). Further antigen binding sites may also be comprised in the antigen binding protein in some embodiments.

[0462] In some embodiments, the amino acid sequenxces of the variable regions V and VBare chimeric, humanized or human.

[0463] In preferred embodiments, the antigen binding protein induces an immune response, for example, in the host cell expressing the antigen binding protein (if the antigen binding protein is membrane-bound), preferably a lymphocyte, more preferably a T cell or an NK cell, more preferably a T cell. Preferably, the immune response is characterized by an increased production of interferon (IFN) y and / or tumor necrosis factor (TNF) a. The immune response is preferably directed against a tumor cell presenting on its surface a complex of the PRAME antigenic peptide and an MHC protein.

[0464] In some embodiments, the antigen binding protein is capable of activating the host cell upon binding to a PRAME: MHC complex.

[0465] In some embodiments, the antigen binding protein specifically binds to the PRAME antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 and a HLA molecule, preferably HLA-A*02, with a KDwhich is < 200 pM, preferably <100 pM, more preferably < 50 pM, or most preferably < 30 pM. In the context of cell therapy, e.g. adoptive autologous or heterologous cell therapy, higher KD values maybe sufficient for targeting cancer cells. In an embodiment, the antigen binding protein of the disclosure specifically binds to the PRAME antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A*02, P296PC00 / 3000181 -001977 with a KDwhich is between less than about 100 pM and higher than about 1 pM. In case the antigen binding proteins are used as soluble proteins, lower KD values may be suitable for targeting cancer cells. Thus, in an embodiment, the antigen binding protein of the disclosure specifically binds to the PRAME antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A*02, with a KDwhich is < 25 pM, < 1 pM, < 500 nM, < 100 nM, < 50 nM, < 10 nM, < 5 nM. “KD” and “affinity” are as defined herein above. The KD is preferably determined by BLI as disclosed above.

[0466] Preferably, the host cell expresses an antigen binding protein as disclosed in WO 2018 / 172533 A1, which is incorporated by reference in its entirety. Preferably, the antigen binding protein is an a(3 TOR comprising a TOR a-chain variable domain (Va) comprising a CDRal having the sequence of SEQ ID NO: 122, a CDRa2 having the sequence of SEQ ID NO: 123 or 134, and a CDRa3 having a sequence of SEQ ID NO: 124, and a TOR p-chain variable domain (VP) comprising a CDRbl having a sequence of SEQ ID NO: 125, a CDRb2 having a sequence of SEQ ID NO: 126, and a CDRb3 having the sequence of SEQ ID NO: 127.

[0467] In some embodiments, the host cell expressingthe antigen binding protein is capable of killing PRAME-expressing tumor cells in an in vitro cytotoxicity assay wherein the PRAM E-expressing tumor cells have a PRAME copy number per cell of less than 200, preferably less than 100, more preferably less than 50. In a preferred embodiment, the PRAME-expressing tumor cells have a PRAME copy number per cell that is determined by AbsQuant® (e.g. as disclosed in PCT / EP2015 / 079873).

[0468] Nucleic Acids and Vectors encoding antigen binding protein

[0469] The nucleic acid(s) encoding the antigen binding protein can be expressed by the host cell in vivo, ex vivo or in vitro.

[0470] Nucleic acid molecules can be obtained using standard molecular biology techniques, including but not limited to methods of amplification, and reverse transcription of RNA. Once DNA fragments encoding, for example, variable chains are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length chain genes. In these manipulations, a variant-encoding DNA fragment is operatively linked to another DNA molecule, or to a fragment encoding another protein, such as a constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed undercontrolof a desired promoter. The isolated DNA encoding the variable region, e.g. the variable alpha region and / or variable beta region, can be converted to a full-length chain gene by operatively linking the variable-encoding DNA to another DNA molecule encoding constant regions. The sequences of human constant region genes, e.g. forTCRs or antibodies, are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. P296PC00 / 3000181 -001977

[0471] Typically, said nucleic acid comprises one or more DNA or RNA molecules, which may be included in one or more suitable vectors.

[0472] The nucleic acid may also be a DNA or RNA molecule, which may be included in a suitable vector.

[0473] Accordingly, the host cell may comprise an expression vector or a collection of vectors.

[0474] Preferably, the sequence encoding the antigen binding protein is operably linked to a promoter sequence. “Collection of vectors” herein refers to two or more vectors. The first and second polypeptide chains of the antigenic binding peptide can be encoded by one or several nucleic acid(s) or vector(s). If two or more antigen binding protein polypeptide chains are encoded on one vector, the two or more antigen binding protein polypeptide chains can be under the control of the same promoter or under the control of separate promoters.

[0475] Various expression vectors can be employed to express the polynucleotides encoding the antigen binding proteins or functional fragments thereof. Both viral-based and non-viral expression vectors can be used to produce the antigen binding proteins or functional fragments thereof described herein in a mammalian host cell. Non-viral vectors and systems include plasmids, plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0476] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of antibody heavy chain and the like.

[0477] For example, non-viral vectors useful for expression of polynucleotides and polypeptides described herein in mammalian (e.g. human or non-human) cells include all suitable vectors known in the art for expressing proteins Other examples of plasmids and include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.

[0478] The host cell may have been transfected, infected or transformed with a nucleic acid and / or a vector according to the disclosure. The nucleic acid(s) and / or vectors described herein are useful for preparing a recombinant host cell that can be used in therapy (see “Therapeutic Methods and Uses”).

[0479] Host cells

[0480] In some embodiments, the host cells disclosed herein as part of the inventive combination and combination therapies comprise at least one nucleic acid or vector encoding the antigen binding protein described herein. P296PC00 / 3000181 -001977

[0481] In some embodiments, the host cell and mRNA are present as two separate entities in the combination. In some embodiments, the host cell does not, or substantially does not comprise the mRNA of the combination: For example, the host cell and mRNA of the combination may be provided in separate containers.

[0482] In some embodiments, the host cells disclosed herein as part of the inventive combination express the antigen binding protein described herein. Preferably, the host cell is an immune cell. In some embodiments, the host cell or immune cell is a lymphocyte, such as an NK cell, a T cell or T cell progenitor, preferably a CD4 and / or CD8 positive T cell or a y6 T cell, most preferably a CD4 and / or CD8 positive T cell, most preferably a CD4 and / or CD8 positive T cell.

[0483] The host cells disclosed herein as part of the inventive combination are useful for inducing an immune response in a subject in need thereof. Accordingly, the host cell, preferably the T cell, is used as an therapeutic agent of a therapeutic composition. Thus, the the host cells are useful for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO: 1.)

[0484] By "aberrantly expressed" it is meant that the peptide is over-expressed compared to levels of expression in normal (healthy) tissues or that the gene is silent in the tissue from which the tumor is derived but, in the tumor, it is expressed. By "overexpressed" the inventors mean that the peptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferablyat least 2-fold, and more preferably at least 5-fold or 10-fold the level present in normal tissue.

[0485] In an aspect, the TCR-induced immune response or T cell response may refer to the proliferation and activation of effector functions induced by a peptide, such as SLLQHLIGL (SEQ ID NO: 1), in vitro, ex vivo or in vivo. For MHC class I restricted cytotoxic T cells, for example, effector functions may be lysis of peptide-pulsed, peptide-precursor pulsed or naturally peptide-presenting target cells, secretion of cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion of effector molecules, for example, granzymes or perforins induced by peptide, or degranulation.

[0486] T cells can be collected from a subject by apheresis and can be genetically engineered to express the antigen binding protein disclosed herein on their cell surface. The genetically engineered T cells can then be expanded and then re-infused into the subject. In this example, the antigen binding protein is preferably a membrane bound antigen binding protein, more preferably a TCR.

[0487] Accordingly, the host cell has been transfected, infected ortransformed with (a) nucleic acid(s) and / or (a) vector(s) encoding the antigen binding protein according to the disclosure.

[0488] When the host cell is transfected to express the antigen binding protein of the disclosure, preferablythe cell comprises an expression vector capable of expressing the antigen binding protein. The expression vector may (partly) integrate into the host cell’s genome, e.g. in case of lentiviral P296PC00 / 3000181 -001977 expression vectors, or may remain extrachromosomaL The host cell may then be referred to as activated, transformed or engineered host cell.

[0489] Protocols for this so-called adoptive transfer of T cells are well known in the art. Reviews can be found in: Gattioni etal. and Morgan etal. (Gattinoni, L. et aL, Nat. Rev.lmmunoL 6 (2006): 383-393; Morgan, R. A. et aL, Science 314 (2006): 126-129).

[0490] Anumberof other methods maybe used for generating? cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used in the generation of CTL. Piebanski et al. (Piebanski, M. et aL, Eur. J Immunol 25 (1995): 1783-1787) made use of autologous peripheral blood lymphocytes (PLBs) in the preparation of T cells. Also, B cells can be used in the production of autologous T cells. Further, methods forgeneratingT cells are disclosed in Guha, Katz, Methods Cell BioL 2022;167:203-226.

[0491] In some embodiments, T cells may be manufactured according to the methods described in US20190175650, US Application No. 16 / 271,393, and US Application No. 16 / 361,043, the contents of which are hereby incorporated by reference in their entireties.

[0492] Allogeneic cells may also be used in the preparation of T cells and a method is described in detail in US6805861, incorporated herein by reference.

[0493] Host cells expressing the antigen binding protein directed against the peptide SLLQHLIGL (SEQ ID NO: 1 ) are useful in therapy. Activated or transformed host cells may specifically recognize a cell that aberrantly expresses a polypeptide that comprises the peptide SLLQHLIGL (SEQ ID NO: 1).

[0494] In an aspect, the host cell, in particular the T cell, recognizes the cell by interacting through its antigen binding protein, in particular its TCR, with the PRAME antigenic peptide in a complex with a MHC protein on the surface of a target cell, such as a cancer cell. The host cells are useful for killing target cells in a patient whose target cells aberrantly express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO: 1), wherein the patient is administered an effective number of the activated / transformed host cells. The T cells that are administered to the patient may be derived from the patient and activated / transformed as described above (i.e. they are autologous T cells). Alternatively, the T cells are not from the patient but are from another individual or a group of other individuals (i.e. they are heterologous T cells). In such instances, it is preferred that said individual or group of individuals is / are a healthy individual or a group of healthy individuals. By "healthy individual" it is meant that the individual(s) is / are generally in good health, preferably has a competent immune system and, more preferably, is not suffering from any disease that can be readily tested for and detected.

[0495] In vivo, the target cells for the host cells, such as CD8-positiveT cells, according to the present disclosure can be cells of the tumor (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel, J. et aL, Clin Cancer Res 12 (2006): 4163-4170). P296PC00 / 3000181 -001977 According to the above, the host cell comprises the antigen binding protein which is defined herein above, or the nucleic acid(s) encoding the antigen binding protein, or the vector(s) encoding the antigen binding protein, wherein said host cell preferably is a) a lymphocyte, such as a T lymphocyte or T lymphocyte progenitor cell, for example a CD4 or CD8 positive T cell or, as a less preferred alternative b) a cell for recombinant expression, specifically in case of soluble antigen binding proteins.

[0496] Preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a lymphocyte, such as a T cell, a T cell progenitor or a NK cell. NK cells are naturally occurring lymphoid non-T cells that can rapidly kill virally infected cells and tumour cells. NK cells can be engineered to express a tumor-specific TCR for use as a cell therapy product in cancer therapy (Shimasaki et aL, Nat Rev Drug Discov. 2020 Mar;19(3):200-218). In preferred embodiments, the host cell is a T cell, for example a CD4 or CD8 positive T cell. The T cell can be any T cell, such as a cultured T cell, preferably a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., oraT cell obtained from a mammal, preferably a T cell or T cell precursor obtained from a human patient. In some embodiments, the host cell is a primary T cell isolated from a cancer patient and may be used for autologous therapy. In other embodiments, the host cell is a primary T cell isolated from a healthy individual and may be used for allogeneic therapy. Such host cells may require further genetic engineering to be safe and effective in allogeneic settings. In some embodiments, the host cell is thus autologous or allogeneic to the treated patient. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4-positive helper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like.

[0497] The present invention also provides methods of eliciting an immune response and methods of treating proliferative diseases involving host cells and mRNAs as defined herein (see section “Therapeutic Methods and Uses).

[0498] Without wishingto be bound by theory, it is envisaged that “boosting” a patient who has received the host cell(s) with a PRAM E polynucleotide as described herein may stimulate antigen-presenting cells to express the PRAME antigen, and thereby support PRAME-targeting T cell expansion and persistence after infusion. The appended Examples show that the PRAME polynucleotides disclosed herein are preferably capable of enhancing tumor infiltration of PRAME T lymphocytes, promoting in P296PC00 / 3000181 -001977

[0499] vivo expansion of T lymphocytes, increasing persistence of T lymphocytes, delaying and / or reducing exhaustion markers in T lymphocytes, and / or inducing development of memory T cells.

[0500] Concatemeric PRAME polypeptides for immune stimulation

[0501] In some aspects, the invention provides a combination comprising a) a host cell as described herein, and b) a PRAME epitope concatemeric polypeptide.

[0502] In some embodiments, the PRAME epitope concatemeric polypeptide comprises at least one PRAME epitope. In some embodiments, the at least one PRAME epitope comprises the sequence of SLLQHLIGL (SEQ ID NO: 1).

[0503] In some embodiments, the PRAME epitope polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 repeats, about 5-15 repeats, about 7-14 repeats, about 8-13 repeats, or about 9-12 repeats of the PRAME epitope. In some embodiments, the PRAME epitope polypeptide comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 repeats, about 5-15 repeats, about 7-14 repeats, about 8-13 repeats, or about 9-12 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 3 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 4 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 5 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 6 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 7 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 8 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 9 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 10 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 11 repeats of the PRAME epitope. In some embodiments the PRAME epitope polypeptide comprises or consists of 12 repeats of the PRAME epitope.

[0504] In some embodiments, the PRAME epitope polypeptide does not comprise a linker. In some embodiments, the PRAME epitope polypeptide comprises a linker. In some embodiments, the linker comprises or consists of one or more alanine residues. In some embodiments, the linker comprises or consists of a single alanine residue. In some embodiments, the linker comprises or consists of two alanine residues. In some embodiments, the linker comprises or consists of three alanine residues. In some embodiments, the linker comprises or consists of four alanine residues. In some embodiments, the linker comprises or consists of five alanine residues. In some embodiments, the linker links adjacent repeats of the PRAME epitope.

[0505] In another aspect, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or P296PC00 / 3000181 -001977 100% sequence identity to any one of the sequences presented in Table 1. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the PRAME epitope polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 2. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 3. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 4. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 5. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 6. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 7. In some embodiments, the PRAME epitope polypeptide comprises or consists of the sequence of SEQ ID NO: 8.

[0506] Table 1. Exemplary concatemeric PRAME epitope polypeptides

[0507] Name Sequence SEQ ID NO: 9mer-12x MSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQ 2

[0508] HLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLS LLQHLIGL

[0509] 9mer-AA-9x MSLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQH 3

[0510] LIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGL

[0511] 9mer-AAA-9x MSLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAAS 4

[0512] LLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLL QHLIGL

[0513] 19mer-5x MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLI 5

[0514] GLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTH P296PC00 / 3000181 -001977 39mer-4x MTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISI 6

[0515] SALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIG LSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVP LESY

[0516] Mixed1-4x MSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLS 7

[0517] NLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLE SYSLSHCSQLTTLSFYGNSISISALQSLLQHLIGL

[0518] Mixed2-5x MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLI 8

[0519] GLSNLTHISALQSLLQHLIGLSNLTHGNSISISALQSLLQHLIGL

[0520] In some embodiments, the PRAME epitope polypeptide is about 50-150 amino acids in length, or about 80-120 amino acids in length, or about 90-110 amino acids in length. In some embodiments, the PRAME epitope polypeptide is about 100 amino acids in length.

[0521] Engineered polynucleotides encoding concatemeric PRAME epitope polypeptides

[0522] In preferred aspects, the invention provides a combination of a) a host cell as described herein, and b) an engineered, non-naturally-occurring polynucleotide encoding one or more concatemeric PRAME polypeptides.

[0523] In some embodiments, the polynucleotide comprises at least one non-naturally modified nucleotide. In some embodiments, the polynucleotide is codon optimized for expressing in mammalian cells.

[0524] In some embodiments, the polynucleotide is an mRNA.

[0525] The instant invention features combinations comprising host cells and mRNAs as described herein for use in treating cancer. The mRNAs featured for use in embodiments of this disclosure are administered to subjects and encode PRAME epitope polypeptides in vivo. In some embodiments, polynucleotides, e.g., mRNA, comprise an open reading frame of linked nucleosides encoding PRAME epitope polypeptides. Specifically, in some embodiments, sequence-optimized polynucleotides comprise nucleotides encoding one or more polypeptide sequences of PRAME epitope polypeptides, or sequences having high sequence identity with those sequence optimized polynucleotides.

[0526] Exemplary open reading frames (ORFs) of polynucleotides encoding PRAME epitope polypeptides are shown in Table 2.

[0527] Table 2. Exemplary open reading frames (ORFs) of polynucleotides encoding concatemeric PRAME epitope polypeptides

[0528] Name mRNA Sequence (ORF) SEQ ID NO: 9mer-12x AUGUCUCUCCUACAACACUUGAUUGGGCUGUCACUGCUCCAA 9

[0529] CAUCUGAUAGGCCUUAGCCUACUGCAACAUUUGAUCGGUCUU AGCCUCUUGCAACACCUCAUCGGCCUUUCCCUCUUACAACAC CUGAUUGGCCUGAGCCUUCUGCAGCAUCUGAUCGGCCUCAGC CUGUUACAGCAUCUCAUUGGCUUGUCACUCCUGCAACACUUA AUCGGGCUGAGCCUCCUGCAGCACCUCAUAGGACUGUCCCUG P296PC00 / 3000181 -001977 CUACAGCACCUGAUCGGACUGAGCUUGCUUCAGCACCUAAUC GGCCUGUCUCUGCUUCAACAUCUUAUUGGACUG

[0530] 9mer-AA-9x AUGAGCCUGCUACAGCACUUGAUCGGCUUAGCUGCAAGCCUG 10

[0531] UUGCAGCACCUUAUUGGCCUGGCUGCUAGCUUAUUGCAACAC CUCAUCGGUCUGGCAGCCUCACUGCUCCAGCACCUGAUCGGA UUAGCCGCUUCUCUCCUGCAACACUUAAUUGGCCUAGCUGCC AGCCUACUGCAACAUUUAAUCGGCUUGGCCGCCUCUCUGCUU CAGCAUCUGAUUGGACUCGCCGCCAGCCUCCUGCAGCAUUUG AUUGGCCUCGCCGCUAGCCUUCUGCAGCACUUAAUCGGACUG

[0532] 9mer-AAA-9x AUGAGCUUACUGCAACAUCUGAUCGGGUUGGCAGCUGCAAGC 11

[0533] UUAUUGCAACAUUUGAUUGGCCUAGCCGCCGCUAGCCUACUG CAGCAUCUGAUUGGAUUAGCAGCCGCCUCCCUGCUACAACAC CUGAUUGGGCUGGCAGCCGCAAGCCUGUUACAGCACCUGAUC GGCUUGGCCGCAGCCUCCUUGCUCCAACACUUGAUCGGACUG GCCGCCGCCAGCUUGCUUCAACACCUCAUCGGCCUGGCCGC UGCCAGUCUGUUGCAGCACUUAAUUGGCCUGGCUGCCGCCU CGCUCCUUCAGCACCUCAUUGGACUG

[0534] 19mer-5x AUGAUCAGCGCCCUGCAAUCGCUGUUACAACACCUGAUAGGG 12

[0535] CUUAGCAAUCUGACACAUAUCAGCGCAUUACAGAGCUUACUCC AGCACUUAAUCGGGCUGAGCAACCUCACCCACAUCUCAGCCC UGCAGAGCUUGCUCCAACAUCUGAUUGGCCUGAGUAACUUGA CCCAUAUUAGCGCCCUUCAGAGCCUUCUCCAGCAUUUAAUAG GCCUGUCUAACCUGACUCACAUUAGCGCACUGCAAAGCCUGU UGCAGCACCUGAUCGGCCUUAGCAACCUGACCCAC

[0536] 39mer-4x AUGACUCUGUCGUUCUACGGGAACUCGAUCUCUAUAUCUGCC 13

[0537] CUGCAAUCCCUUCUCCAGCAUCUAAUCGGACUGAGCAAUCUG ACUCACGUGCUCUACCCCGUGCCACUGGAGAGUUAUACCUUA UCAUUCUACGGUAACUCCAUCUCUAUCAGCGCACUUCAAAGC CUUCUGCAGCAUCUGAUCGGCCUGAGCAACCUGACACACGUC CUGUACCCGGUGCCCCUCGAGUCUUACACCCUCAGUUUCUAC GGAAAUUCAAUCUCCAUCAGCGCCCUGCAGAGCUUGCUUCAA CAUCUGAUUGGCCUGUCAAACCUUACACACGUGCUUUACCCC GUUCCCCUGGAAAGCUAUACACUGAGCUUCUACGGCAAUAGC AUUAGUAUUAGCGCACUGCAAAGCCUGCUCCAACACUUGAUAG GCCUGAGUAACCUGACCCACGUGCUGUAUCCCGUGCCGCUG GAGAGCUAC

[0538] Mixed! -4x AUGAGCCUGCUUCAACAUCUGAUCGGGCUGAGCAAUCUGACA 14

[0539] CACGUCCUGUACCCUGUGCCCCUGGAGUCUUACACCUUGAGU UUCUACGGGAACAGCAUCAGUAUCAGUGCCCUGCAGUCGCUG CUGCAACACCUAAUCGGCCUGAGCAACCUGACCCACGUACUG UACCCCGUGCCUCUGGAGAGCUAUACACUGAGCUUCUACGGU AACUCAAUCAGUAUUAGCGCCCUGCAAAGCUUGCUCCAACAUC UCAUCGGCCUCAGCAACCUUACCCACGUGCUGUAUCCCGUGC CCUUAGAGAGCUACAGCCUGAGCCACUGCAGCCAGCUGACCA CCCUGUCAUUCUACGGCAAUAGCAUCUCUAUCAGCGCCCUCC AGAGCCUCCUCCAGCAUCUGAUUGGCCUG

[0540] Mixed2-5x AUGAUCAGCGCACUGCAGUCUCUGCUGCAACAUCUGAUCGGU 15

[0541] CUGUCGAACCUGACACAUAUCAGCGCCCUGCAGAGCCUACUC CAACAUCUAAUCGGCCUGAGCAACUUGACCCAUAUUAGCGCC CUCCAAAGCCUGCUCCAGCACCUGAUCGGCCUUAGCAACCUC ACCCAUAUCAGUGCCCUGCAAUCCCUCCUGCAGCACCUCAUU GGCCUCAGCAAUCUGACCCACGGCAACAGCAUCUCCAUCAGC GCUCUGCAAAGCCUCCUCCAGCAUCUGAUUGGCCUG P296PC00 / 3000181 -001977 In some embodiments, the polynucleotides, e.g., mRNA, comprise an open reading frame of linked nucleosides encoding PRAME epitope polypeptides, variants thereof, functional fragments thereof, and fusion proteins comprising the same. Specifically, in some embodiments, the sequence-optimized polynucleotides comprise nucleotides encoding the polypeptide sequence of a PRAME epitope polypeptide, or sequence having high sequence identity with those sequence optimized polynucleotides.

[0542] In some embodiments, polynucleotides (e.g., a RNA such as an mRNA) comprise a nucleotide sequence (e.g., an ORF) encoding one or more polypeptides. In some embodiments, polynucleotides (e.g., a RNA such as an mRNA) comprise a nucleotide sequence (e.g., an ORF) encoding any of the polypeptides disclosed herein. In some embodiments, the encoded polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0543] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0544] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 99% to 10)%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% sequence identity to the sequence SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0545] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0546] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the P296PC00 / 3000181 -001977 nucleotide sequence is 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% identical to the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0547] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence encodes an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0548] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% sequence identity to the sequence of any of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0549] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0550] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99%, sequence identity to the sequence of any of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0551] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence is between 90% and 100% identical; between 91 % and 99% identical; between 92% and 98% identical; between 93% and 97% identical, or between 94% and 96% identical to the P296PC00 / 3000181 -001977 sequence of any of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0552] Exemplary polynucleotides encoding PRAME epitope polypeptides or full-length PRAME are shown in Table 3.

[0553] Table 3. Exemplary polynucleotides encoding concatemeric PRAME epitope polypeptides or full-length PRAME.

[0554] Name Sequence (5’UTR - ORF - 3’UTR, excluding cap and tail) SEQ ID NO: PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 101 01 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGGAACG GCGGCGUCUGUGGGGCAGCAUCCAGAGCCGGUACAUUAGCAU GAGCGUGUGGACCAGCCCUCGGAGACUGGUGGAGCUGGCAG GCCAAAGCCUCUUAAAGGACGAGGCCCUAGCCAUCGCCGCCC UCGAGCUGCUCCCUCGGGAGCUGUUUCCUCCCCUGUUCAUG GCCGCCUUUGACGGCCGGCACAGCCAGACCCUGAAGGCCAUG GUACAGGCCUGGCCCUUCACCUGCCUGCCCCUGGGUGUGCU GAUGAAAGGCCAGCAUCUGCAUUUGGAGACUUUCAAGGCAGU GUUAGACGGGUUGGACGUGUUGCUGGCCCAGGAAGUACGGCC ACGCCGGUGGAAACUGCAGGUGCUGGACCUGCGUAAGAACAG CCACCAGGAUUUCUGGACCGUGUGGUCGGGCAACCGGGCCAG CCUGUACAGCUUCCCUGAACCCGAAGCCGCCCAGCCCAUGAC CAAGAAGCGGAAGGUGGACGGCCUCAGCACCGAGGCCGAGCA GCCCUUCAUACCCGUGGAGGUCCUGGUUGACCUGUUCCUGAA GGAGGGCGCCUGUGACGAACUGUUUAGCUACCUUAUCGAGAA GGUGAAGAGAAAGAAGAACGUGCUGCGGCUGUGCUGCAAGAAG CUGAAGAUAUUCGCCAUGCCCAUGCAGGACAUCAAGAUGAUCC UGAAGAUGGUGCAGCUGGACAGCAUCGAGGACUUGGAGGUGA CCUGUACCUGGAAGCUGCCCACCCUGGCCAAGUUCAGCCCCU ACCUGGGGCAGAUGAUCAACCUGCGGCGGCUGCUGCUGAGCC ACAUACACGCCAGCAGCUACAUCAGCCCCGAGAAGGAGGAGCA GUACAUCGCCCAGUUCACCAGCCAGUUCCUGAGCCUGCAGUG CCUGCAAGCCCUGUACGUGGACAGCCUGUUCUUUCUGCGGG GCCGGCUGGACCAAUUACUGCGGCACGUGAUGAACCCACUGG AGACGCUGAGCAUCACCAACUGCCGCCUGAGCGAAGGCGACG UGAUGCACCUGAGCCAGAGCCCUAGCGUGAGCCAACUGAGCG UGCUGUCCCUGAGCGGCGUGAUGCUGACUGACGUGAGCCCC GAACCUCUGCAGGCCCUCCUGGAGCGGGCCUCUGCCACCCU GCAGGAUCUGGUGUUCGACGAGUGCGGCAUCACCGACGACCA GCUGCUAGCCCUGCUGCCUAGCCUGAGUCACUGCAGCCAGCU GACCACCCUGAGCUUCUACGGCAACAGCAUCUCCAUCAGCGC CCUGCAAAGCCUACUUCAGCACCUCAUCGGCCUGAGCAACCU CACCCACGUGCUGUACCCUGUGCCUCUGGAGAGCUACGAGGA CAUCCACGGCACCUUGCACUUGGAGCGGCUGGCCUAUCUGCA CGCCCGGCUGCGAGAGCUGCUGUGCGAGCUGGGCAGACCCA GCAUGGUGUGGCUGAGCGCCAACCCCUGCCCACACUGCGGG GACCGGACCUUCUACGACCCCGAGCCCAUCCUGUGCCCCUGC UUCAUGCCCAACUAAAGCUCCCCGGGGGCCUCGGUGGCCUAG CUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU P296PC00 / 3000181 -001977 CCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 102 02 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGUCUCU CCUACAACACUUGAUUGGGCUGUCACUGCUCCAACAUCUGAU AGGCCUUAGCCUACUGCAACAUUUGAUCGGUCUUAGCCUCUU GCAACACCUCAUCGGCCUUUCCCUCUUACAACACCUGAUUGG CCUGAGCCUUCUGCAGCAUCUGAUCGGCCUCAGCCUGUUACA GCAUCUCAUUGGCUUGUCACUCCUGCAACACUUAAUCGGGCU GAGCCUCCUGCAGCACCUCAUAGGACUGUCCCUGCUACAGCA CCUGAUCGGACUGAGCUUGCUUCAGCACCUAAUCGGCCUGUC UCUGCUUCAACAUCUUAUUGGACUGUAAAGCUCCCCGGGGGC CUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGC CCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGA AUAAAGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 103 03 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCCU GCUACAGCACUUGAUCGGCUUAGCUGCAAGCCUGUUGCAGCA CCUUAUUGGCCUGGCUGCUAGCUUAUUGCAACACCUCAUCGG UCUGGCAGCCUCACUGCUCCAGCACCUGAUCGGAUUAGCCGC UUCUCUCCUGCAACACUUAAUUGGCCUAGCUGCCAGCCUACU GCAACAUUUAAUCGGCUUGGCCGCCUCUCUGCUUCAGCAUCU GAUUGGACUCGCCGCCAGCCUCCUGCAGCAUUUGAUUGGCCU CGCCGCUAGCCUUCUGCAGCACUUAAUCGGACUGUAAAGCUC CCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 104 04 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCUU ACUGCAACAUCUGAUCGGGUUGGCAGCUGCAAGCUUAUUGCA ACAUUUGAUUGGCCUAGCCGCCGCUAGCCUACUGCAGCAUCU GAUUGGAUUAGCAGCCGCCUCCCUGCUACAACACCUGAUUGG GCUGGCAGCCGCAAGCCUGUUACAGCACCUGAUCGGCUUGGC CGCAGCCUCCUUGCUCCAACACUUGAUCGGACUGGCCGCCGC CAGCUUGCUUCAACACCUCAUCGGCCUGGCCGCUGCCAGUCU GUUGCAGCACUUAAUUGGCCUGGCUGCCGCCUCGCUCCUUC AGCACCUCAUUGGACUGUAAAGCUCCCCGGGGGCCUCGGUGG CCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 105 05 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAUCAG CGCCCUGCAAUCGCUGUUACAACACCUGAUAGGGCUUAGCAA UC UGACAC AU AUCAGCGCAU U AC AGAGC UU ACUCC AGC AC U UA AUCGGGCUGAGCAACCUCACCCACAUCUCAGCCCUGCAGAGC UUGCUCCAACAUCUGAUUGGCCUGAGUAACUUGACCCAUAUU AGCGCCCUUCAGAGCCUUCUCCAGCAUUUAAUAGGCCUGUCU AACCUGACUCACAUUAGCGCACUGCAAAGCCUGUUGCAGCAC CUGAUCGGCCUUAGCAACCUGACCCACUAAAGCUCCCCGGGG GCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCA GCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUU GAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 106 06 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGACUCU P296PC00 / 3000181 -001977 GUCGUUCUACGGGAACUCGAUCUCUAUAUCUGCCCUGCAAUC CCUUCUCCAGCAUCUAAUCGGACUGAGCAAUCUGACUCACGU GCUCUACCCCGUGCCACUGGAGAGUUAUACCUUAUCAUUCUA CGGUAACUCCAUCUCUAUCAGCGCACUUCAAAGCCUUCUGCA GCAUCUGAUCGGCCUGAGCAACCUGACACACGUCCUGUACCC GGUGCCCCUCGAGUCUUACACCCUCAGUUUCUACGGAAAUUC AAUCUCCAUCAGCGCCCUGCAGAGCUUGCUUCAACAUCUGAU UGGCCUGUCAAACCUUACACACGUGCUUUACCCCGUUCCCCU GGAAAGCUAUACACUGAGCUUCUACGGCAAUAGCAUUAGUAUU AGCGCACUGCAAAGCCUGCUCCAACACUUGAUAGGCCUGAGU AACCUGACCCACGUGCUGUAUCCCGUGCCGCUGGAGAGCUAC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCC UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGU ACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 107 07 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCCU GCUUCAACAUCUGAUCGGGCUGAGCAAUCUGACACACGUCCU GUACCCUGUGCCCCUGGAGUCUUACACCUUGAGUUUCUACGG GAACAGCAUCAGUAUCAGUGCCCUGCAGUCGCUGCUGCAACA CCUAAUCGGCCUGAGCAACCUGACCCACGUACUGUACCCCGU GCCUCUGGAGAGCUAUACACUGAGCUUCUACGGUAACUCAAU CAGUAUUAGCGCCCUGCAAAGCUUGCUCCAACAUCUCAUCGG CCUCAGCAACCUUACCCACGUGCUGUAUCCCGUGCCCUUAGA GAGCUACAGCCUGAGCCACUGCAGCCAGCUGACCACCCUGUC AUUCUACGGCAAUAGCAUCUCUAUCAGCGCCCUCCAGAGCCU CCUCCAGCAUCUGAUUGGCCUGUAAAGCUCCCCGGGGGCCUC GGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCC UCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 108 08 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAUCAG CGCACUGCAGUCUCUGCUGCAACAUCUGAUCGGUCUGUCGAA CCUGACACAUAUCAGCGCCCUGCAGAGCCUACUCCAACAUCU AAUCGGCCUGAGCAACUUGACCCAUAUUAGCGCCCUCCAAAG CCUGCUCCAGCACCUGAUCGGCCUUAGCAACCUCACCCAUAU CAGUGCCCUGCAAUCCCUCCUGCAGCACCUCAUUGGCCUCAG CAAUCUGACCCACGGCAACAGCAUCUCCAUCAGCGCUCUGCA AAGCCUCCUCCAGCAUCUGAUUGGCCUGUAAAGCUCCCCGGG GGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 109 09 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGGAACG GCGGCGUCUGUGGGGCAGCAUCCAGAGCCGGUACAUUAGCAU GAGCGUGUGGACCAGCCCUCGGAGACUGGUGGAGCUGGCAG GCCAAAGCCUCUUAAAGGACGAGGCCCUAGCCAUCGCCGCCC UCGAGCUGCUCCCUCGGGAGCUGUUUCCUCCCCUGUUCAUG GCCGCCUUUGACGGCCGGCACAGCCAGACCCUGAAGGCCAUG GUACAGGCCUGGCCCUUCACCUGCCUGCCCCUGGGUGUGCU GAUGAAAGGCCAGCAUCUGCAUUUGGAGACUUUCAAGGCAGU GUUAGACGGGUUGGACGUGUUGCUGGCCCAGGAAGUACGGCC ACGCCGGUGGAAACUGCAGGUGCUGGACCUGCGUAAGAACAG CCACCAGGAUUUCUGGACCGUGUGGUCGGGCAACCGGGCCAG CCUGUACAGCUUCCCUGAACCCGAAGCCGCCCAGCCCAUGAC P296PC00 / 3000181 -001977 CAAGAAGCGGAAGGUGGACGGCCUCAGCACCGAGGCCGAGCA GCCCUUCAUACCCGUGGAGGUCCUGGUUGACCUGUUCCUGAA GGAGGGCGCCUGUGACGAACUGUUUAGCUACCUUAUCGAGAA GGUGAAGAGAAAGAAGAACGUGCUGCGGCUGUGCUGCAAGAAG CUGAAGAUAUUCGCCAUGCCCAUGCAGGACAUCAAGAUGAUCC UGAAGAUGGUGCAGCUGGACAGCAUCGAGGACUUGGAGGUGA CCUGUACCUGGAAGCUGCCCACCCUGGCCAAGUUCAGCCCCU ACCUGGGGCAGAUGAUCAACCUGCGGCGGCUGCUGCUGAGCC ACAUACACGCCAGCAGCUACAUCAGCCCCGAGAAGGAGGAGCA GUACAUCGCCCAGUUCACCAGCCAGUUCCUGAGCCUGCAGUG CCUGCAAGCCCUGUACGUGGACAGCCUGUUCUUUCUGCGGG GCCGGCUGGACCAAUUACUGCGGCACGUGAUGAACCCACUGG AGACGCUGAGCAUCACCAACUGCCGCCUGAGCGAAGGCGACG UGAUGCACCUGAGCCAGAGCCCUAGCGUGAGCCAACUGAGCG UGCUGUCCCUGAGCGGCGUGAUGCUGACUGACGUGAGCCCC GAACCUCUGCAGGCCCUCCUGGAGCGGGCCUCUGCCACCCU GCAGGAUCUGGUGUUCGACGAGUGCGGCAUCACCGACGACCA GCUGCUAGCCCUGCUGCCUAGCCUGAGUCACUGCAGCCAGCU GACCACCCUGAGCUUCUACGGCAACAGCAUCUCCAUCAGCGC CCUGCAAAGCCUACUUCAGCACCUCAUCGGCCUGAGCAACCU CACCCACGUGCUGUACCCUGUGCCUCUGGAGAGCUACGAGGA CAUCCACGGCACCUUGCACUUGGAGCGGCUGGCCUAUCUGCA CGCCCGGCUGCGAGAGCUGCUGUGCGAGCUGGGCAGACCCA GCAUGGUGUGGCUGAGCGCCAACCCCUGCCCACACUGCGGG GACCGGACCUUCUACGACCCCGAGCCCAUCCUGUGCCCCUGC UUCAUGCCCAACUAAAGCUCCCCGGGGGCCUCGGUGGCCUAG CUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUU CCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 110 10 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGUCUCU CCUACAACACUUGAUUGGGCUGUCACUGCUCCAACAUCUGAU AGGCCUUAGCCUACUGCAACAUUUGAUCGGUCUUAGCCUCUU GCAACACCUCAUCGGCCUUUCCCUCUUACAACACCUGAUUGG CCUGAGCCUUCUGCAGCAUCUGAUCGGCCUCAGCCUGUUACA GCAUCUCAUUGGCUUGUCACUCCUGCAACACUUAAUCGGGCU GAGCCUCCUGCAGCACCUCAUAGGACUGUCCCUGCUACAGCA CCUGAUCGGACUGAGCUUGCUUCAGCACCUAAUCGGCCUGUC UCUGCUUCAACAUCUUAUUGGACUGUAAAGCUCCCCGGGGGC CUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGC CCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGA AUAAAGUCUGAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 111 11 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCCU GCUACAGCACUUGAUCGGCUUAGCUGCAAGCCUGUUGCAGCA CCUUAUUGGCCUGGCUGCUAGCUUAUUGCAACACCUCAUCGG UCUGGCAGCCUCACUGCUCCAGCACCUGAUCGGAUUAGCCGC UUCUCUCCUGCAACACUUAAUUGGCCUAGCUGCCAGCCUACU GCAACAUUUAAUCGGCUUGGCCGCCUCUCUGCUUCAGCAUCU GAUUGGACUCGCCGCCAGCCUCCUGCAGCAUUUGAUUGGCCU CGCCGCUAGCCUUCUGCAGCACUUAAUCGGACUGUAAAGCUC CCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGC P296PC00 / 3000181 -001977 PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 112 12 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCUU ACUGCAACAUCUGAUCGGGUUGGCAGCUGCAAGCUUAUUGCA ACAUUUGAUUGGCCUAGCCGCCGCUAGCCUACUGCAGCAUCU GAUUGGAUUAGCAGCCGCCUCCCUGCUACAACACCUGAUUGG GCUGGCAGCCGCAAGCCUGUUACAGCACCUGAUCGGCUUGGC CGCAGCCUCCUUGCUCCAACACUUGAUCGGACUGGCCGCCGC CAGCUUGCUUCAACACCUCAUCGGCCUGGCCGCUGCCAGUCU GUUGCAGCACUUAAUUGGCCUGGCUGCCGCCUCGCUCCUUC AGCACCUCAUUGGACUGUAAAGCUCCCCGGGGGCCUCGGUGG CCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 113 13 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAUCAG CGCCCUGCAAUCGCUGUUACAACACCUGAUAGGGCUUAGCAA UC UGACAC AU AUCAGCGCAU U AC AGAGC UU ACUCC AGC AC U UA AUCGGGCUGAGCAACCUCACCCACAUCUCAGCCCUGCAGAGC UUGCUCCAACAUCUGAUUGGCCUGAGUAACUUGACCCAUAUU AGCGCCCUUCAGAGCCUUCUCCAGCAUUUAAUAGGCCUGUCU AACCUGACUCACAUUAGCGCACUGCAAAGCCUGUUGCAGCAC CUGAUCGGCCUUAGCAACCUGACCCACUAAAGCUCCCCGGGG GCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCA GCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUU GAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 114 14 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGACUCU GUCGUUCUACGGGAACUCGAUCUCUAUAUCUGCCCUGCAAUC CCUUCUCCAGCAUCUAAUCGGACUGAGCAAUCUGACUCACGU GCUCUACCCCGUGCCACUGGAGAGUUAUACCUUAUCAUUCUA CGGUAACUCCAUCUCUAUCAGCGCACUUCAAAGCCUUCUGCA GCAUCUGAUCGGCCUGAGCAACCUGACACACGUCCUGUACCC GGUGCCCCUCGAGUCUUACACCCUCAGUUUCUACGGAAAUUC AAUCUCCAUCAGCGCCCUGCAGAGCUUGCUUCAACAUCUGAU UGGCCUGUCAAACCUUACACACGUGCUUUACCCCGUUCCCCU GGAAAGCUAUACACUGAGCUUCUACGGCAAUAGCAUUAGUAUU AGCGCACUGCAAAGCCUGCUCCAACACUUGAUAGGCCUGAGU AACCUGACCCACGUGCUGUAUCCCGUGCCGCUGGAGAGCUAC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCC UUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGU ACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 115 15 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCCU GCUUCAACAUCUGAUCGGGCUGAGCAAUCUGACACACGUCCU GUACCCUGUGCCCCUGGAGUCUUACACCUUGAGUUUCUACGG GAACAGCAUCAGUAUCAGUGCCCUGCAGUCGCUGCUGCAACA CCUAAUCGGCCUGAGCAACCUGACCCACGUACUGUACCCCGU GCCUCUGGAGAGCUAUACACUGAGCUUCUACGGUAACUCAAU CAGUAUUAGCGCCCUGCAAAGCUUGCUCCAACAUCUCAUCGG CCUCAGCAACCUUACCCACGUGCUGUAUCCCGUGCCCUUAGA GAGCUACAGCCUGAGCCACUGCAGCCAGCUGACCACCCUGUC AUUCUACGGCAAUAGCAUCUCUAUCAGCGCCCUCCAGAGCCU CCUCCAGCAUCUGAUUGGCCUGUAAAGCUCCCCGGGGGCCUC GGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCC P296PC00 / 3000181 -001977 UCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC PRAME-mRNA- GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG 116 16 UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAUCAG CGCACUGCAGUCUCUGCUGCAACAUCUGAUCGGUCUGUCGAA CCUGACACAUAUCAGCGCCCUGCAGAGCCUACUCCAACAUCU AAUCGGCCUGAGCAACUUGACCCAUAUUAGCGCCCUCCAAAG CCUGCUCCAGCACCUGAUCGGCCUUAGCAACCUCACCCAUAU CAGUGCCCUGCAAUCCCUCCUGCAGCACCUCAUUGGCCUCAG CAAUCUGACCCACGGCAACAGCAUCUCCAUCAGCGCUCUGCA AAGCCUCCUCCAGCAUCUGAUUGGCCUGUAAAGCUCCCCGGG GGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC PRAME-mRNA- AGGAAAUAGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGU 117 17 UUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCAUGAGCUUA CUGCAACAUCUGAUCGGGUUGGCAGCUGCAAGCUUAUUGCAA CAUUUGAUUGGCCUAGCCGCCGCUAGCCUACUGCAGCAUCUG AUUGGAUUAGCAGCCGCCUCCCUGCUACAACACCUGAUUGGG CUGGCAGCCGCAAGCCUGUUACAGCACCUGAUCGGCUUGGCC GCAGCCUCCUUGCUCCAACACUUGAUCGGACUGGCCGCCGCC AGCUUGCUUCAACACCUCAUCGGCCUGGCCGCUGCCAGUCUG UUGCAGCACUUAAUUGGCCUGGCUGCCGCCUCGCUCCUUCA GCACCUCAUUGGACUGUAAAGCUCCCCGGGGGCCUCGGUGG CCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC CCCUUCCUGCAGUUUGGGUUUGGGUGGUCUUUGAAUAAAGUC UGAGUGGGCGGC

[0555] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

[0556] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 99% to 10)%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% sequence identity to the sequence SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117. P296PC00 / 3000181 -001977 In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

[0557] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence is 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% identical to the sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.

[0558] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% sequence identity to the sequence of any of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0559] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0560] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence has 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% P296PC00 / 3000181 -001977 to 97%, 95% to 98%, or 95% to 99%, sequence identity to the sequence of any of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0561] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide, functional fragment, or variant thereof), wherein the nucleotide sequence is between 90% and 100% identical; between 91 % and 99% identical; between 92% and 98% identical; between 93% and 97% identical, or between 94% and 96% identical to the sequence of any of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0562] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence encoding a polypeptide (e.g., a full-length PRAME polypeptide), wherein the nucleotide sequence is 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 90% to 95%, 90% to 97%, 90% to 98%, 95% to 97%, 95% to 98%, or 95% to 99% identical to the sequence of SEQ ID NO: 101 or SEQ ID NO: 109.

[0563] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of this disclosure comprises from about 1,000 to about 100,000 nucleotides (e.g., from 1,000 to 2,500, from 1,000 to 2,600, from 1,000 to 2,700, from 1,000 to 2,800, from 1,000 to 2,900, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, or from 1,000 to 100,000).

[0564] In some embodiments, the polynucleotide of this disclosure (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF)encodinga PRAME epitope polypeptide, wherein the length of the nucleotide sequence (e.g., an ORF) is at least 500 nucleotides in length (e.g., at least or greater than about 500, 600, 700, 800, 900, 1,000, 1,050, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,635, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).

[0565] In some embodiments, the polynucleotide of this disclosure (e.g., a RNA, e.g., an mRNA) comprising a nucleotide sequence encoding a PRAME epitope polypeptide further comprises a 5'-UTR and / or a 3'-UTR. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of this disclosure comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, P296PC00 / 3000181 -001977 SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In a further embodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5' terminal cap (e.g., m7Gp-ppGm-A, CapO, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about 100 nucleotides in length). In some embodiments, the mRNA comprises a polyAtaiL In some instances, the polyAtail is 50-150 (SEQ ID NO: 94), 75-150 (SEQ ID NO: 95), 85-150 (SEQ ID NO: 96), 90-120 (SEQ ID NO: 97), 90-130 (SEQ ID NO: 98), or 90-150 (SEQ ID NO: 99) nucleotides in length. In some instances, the polyAtail is 100 nucleotides in length (SEQ ID NO: 92). In some instances, the polyA tail is protected (e.g., with an inverted deoxy-thymidine). In some instances, the poly A tail comprises A100-UCUAG-A20-inverted deoxy-thymidine. In some instances, the polyAtail is A100-UCUAG-A20-inverted deoxy-thymidine.

[0566] In some embodiments, the polynucleotide of this disclosure (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PRAME epitope polypeptide further comprises at least one nucleic acid sequence that is noncoding, e.g., a microRNA binding site. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of this disclosure further comprises a 5'-UTR and / or a 3' UTR. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of this disclosure comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In a further embodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5' terminal cap (e.g., m7Gp-ppGm-A, CapO, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about 100 nucleotides in length, e.g., A100-UCUAG-A20-inverted deoxy-thymidine). In some embodiments, the mRNA comprises a polyA tail. In some instances, the polyAtail is 50-150 (SEQ ID NO: 94), 75-150 (SEQ ID NO: 95), 85-150 (SEQ ID NO: 96), 90-120 (SEQ ID NO: 97), 90-130 (SEQ ID NO: 98), or 90-150 (SEQ ID NO: 99) nucleotides in length. In some instances, the polyAtail is 100 nucleotides in length (SEQ ID NO: 92). In some instances, the poly A tail is protected (e.g., with an inverted deoxy-thymidine). In some instances, the poly A tail comprises A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO: 100). In some instances, the polyAtail is A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO: 100).

[0567] In some embodiments, the polynucleotide of this disclosure (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PRAME epitope polypeptide is single stranded or double stranded.

[0568] In some embodiments, the polynucleotide of this disclosure comprising a nucleotide sequence (e.g., an ORF) encoding a polypeptide (e.g., a concatemeric PRAME epitope polypeptide) is DNA or RNA. In some embodiments, the polynucleotide of this disclosure is RNA. In some embodiments, the polynucleotide of this disclosure is, or functions as, a mRNA. In some P296PC00 / 3000181 -001977 embodiments, the mRNA comprises a nucleotide sequence (e.g., an ORF) that encodes the PRAME epitope polypeptide, and is capable of being translated to produce the encoded antigen binding polypeptide in vitro, in vivo, in situ or ex vivo.

[0569] In some embodiments, the polynucleotide of this disclosure (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PRAME epitope polypeptide, wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1 -methylpseudouracil or 5-methoxyuraciL In certain embodiments, all uracils in the polynucleotide are N1 -methylpseudouracils. In other embodiments, all uracils in the polynucleotide are 5-methoxyuracils. In some embodiments, the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 and / or a miRNA binding site that binds to miR-126.

[0570] Features of polynucleotides encoding concatemeric PRAME epitope polypeptides

[0571] In some embodiments of this disclosure, the polynucleotides comprise one or more of the following features:

[0572] Optimization of Nucleic Acid Sequence Intrinsic Properties

[0573] In some embodiments of this disclosure, the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence. For example, the nucleotide sequence (e.g., a RNA, e.g., an mRNA) can be sequence optimized for in vivo or in vitro stability. In some embodiments, the nucleotide sequence can be sequence optimized for expression in a given target tissue or cell. In some embodiments, the nucleic acid sequence is sequence optimized to increase its plasma halflife by preventing its degradation by endo and exonucleases.

[0574] In other embodiments, the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.

[0575] In other embodiments, the sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation.

[0576] Nucleic Acid Sequences Optimized for Protein Expression

[0577] In some embodiments of this disclosure, the desired property of the polynucleotide is the level of expression of one or more polypeptides encoded by a codon optimized sequence. Protein expression levels can be measured using one or more expression systems. In some embodiments, expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells. In some P296PC00 / 3000181 -001977

[0578] embodiments, expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components. In other embodiments, the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.

[0579] In some embodiments, protein expression in solution form can be desirable. Accordingly, in some embodiments, a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form. Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products (i.e., fragments due to proteolysis, hydrolysis, or defective translation) can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).

[0580] Optimization of Target Tissue or Target Cell Viability

[0581] In some embodiments, the expression of heterologous proteins (e.g., therapeutic proteins) encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.

[0582] Accordingly, in some embodiments of this disclosure, the sequence optimization of a nucleic acid sequence disclosed herein, e.g., a nucleic acid sequence encoding one or more polypeps, can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.

[0583] Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art.

[0584] Reduction of an Undesired Immune and / or Inflammatory Response

[0585] In some cases, the administration of a sequence optimized nucleic acid encoding a one or more polypeptides can trigger an undesired immune response, which could be caused by the therapeutic agent itself (e.g., an mRNA). Accordingly, in some embodiments of the present disclosure the sequence optimization of nucleic acid sequence (e.g., RNA, e.g., an mRNA) disclosed P296PC00 / 3000181 -001977 herein can be used to decrease an immune or inflammatory response triggered by the administration of such a nucleic acid (e.g., the immune or inflammatory response caused by the nucleic acid itself).

[0586] In some cases, an undesired inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA. The term "inflammatory cytokine" refers to cytokines that are elevated in an inflammatory response. Examples of inflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine (C-X-C motif) ligand 1; also known as GROa, interferon-g (IFNg), tumor necrosis factor a (TNFa), interferon g-induced protein 10 (IP-10), or granulocyte-colony stimulating factor (G-CSF). The term “inflammatory cytokines” includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-13 (IL-13), interferon a (IFN-a).

[0587] Untranslated Regions

[0588] Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5' UTR) and after a stop codon (3' UTR) that are not translated. In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of this disclosure comprising an open reading frame (ORF) encoding one or more polypeptides further comprises a UTR (e.g., a 51UTR or functional fragment thereof, a 3' UTR or functional fragment thereof, or a combination thereof).

[0589] A UTR (e.g., 5' UTR or 3' UTR) can be homologous or heterologous to the coding region in a polynucleotide. In some embodiments, the UTR is homologous to the ORF encoding the one or more polypeptides. In some embodiments, the UTR is heterologous to the ORF encoding the one or more polypeptides.

[0590] In some embodiments, the polynucleotide comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.

[0591] In some embodiments, the 5' UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[0592] In some embodiments, the 5'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., N1 -methylpseudouracil or 5-methoxyuraciL

[0593] UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and / or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine P296PC00 / 3000181 -001977 methods. In some embodiments, a functional fragment of a 5' UTR or 3' UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.

[0594] Natural 5'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG (SEQ ID NO: 18), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5' UTRs also have been known to form secondary structures that are involved in elongation factor binding.

[0595] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide. For example, introduction of 51UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver. Likewise, use of 5'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CD11 b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A / B / C / D).

[0596] In some embodiments, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.

[0597] In some embodiments, the 5' UTR and the 3' UTR can be heterologous. In some embodiments, the 5' UTR can be derived from a different species than the 3' UTR.

[0598] Co-owned International Patent Application No. PCT / US2014 / 021522 (PubL No. WO / 2014 / 164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide as flanking regions to an ORF in certain embodiments.

[0599] Additional exemplary UTRs of the application include, but are not limited to, one or more 5'UTR and / or 3'UTR derived from the nucleic acid sequence of: a globin, such as an a- or p-globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translationalinitiation signal; a CYBA(e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-(3) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1 )), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter P296PC00 / 3000181 -001977 (e.g., hGLUTI (human glucose transporter 1)); an actin (e.g., human a or p actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5'UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the p subunit of mitochondrial H+-ATP synthase); a growth hormone e (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 a1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a p-F1-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen (e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1 (Coll A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low density lipoprotein receptor-related protein (e.g., LRP1 ); a cardiotrophin-like cytokine factor (e.g., Nnt1 ); calreticulin (Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plodl); and a nucleobindin (e.g., Nucbl).

[0600] In some embodiments, the 51UTR is selected from the group consisting of a pglobin 5' UTR; a 5'UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5' UTR; a hydroxysteroid (17-P) dehydrogenase (HSD17B4) 5' UTR; a Tobacco etch virus (TEV) 5' UTR; a Venezuelen equine encephalitis virus (TEEV) 5' UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5' UTR; a heat shock protein 70 (Hsp70) 5' UTR; a elF4G 5' UTR; a GLUT1 5' UTR; functional fragments thereof and any combination thereof.

[0601] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of this disclosure. In some embodiments, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some embodiments, variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.

[0602] Additionally, one or more synthetic UTRs can be used in combination with one or more nonsynthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.

[0603] UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and / or 3' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3' UTRs.

[0604] In some embodiments, the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5' UTR or 3' UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double beta-globin 3'UTR can be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in its entirety). P296PC00 / 3000181 -001977 The polynucleotides of this disclosure can comprise combinations of features. For example, the ORF can be flanked by a 5'UTR that comprises a strong Kozak translational initiation signal and / or a 3'UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail. A 5'UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different UTRs (see, e.g., US2010 / 0293625, herein incorporated by reference in its entirety).

[0605] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of this disclosure. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of this disclosure. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some embodiments, the polynucleotide of this disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et aL, Biochem. Biophys. Res. Commun. 2010 394(1 ):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide comprises an IRES instead of a 5' UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5' UTR in combination with a non-synthetic 3' UTR.

[0606] In some embodiments, the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, “TEE,” which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some embodiments, the 5' UTR comprises a TEE.

[0607] In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.

[0608] 5’-UTR Sequences

[0609] 5' UTR sequences are important for ribosome recruitment to the mRNAand have been reported to play a role in translation (Hinnebusch A, et aL, (2016) Science, 352:6292: 1413-6).

[0610] In some embodiments a polynucleotide, e.g., mRNA, comprises an open reading frame encoding one or more polypeptides, comprising, inter alia, a 5’ UTR. In an embodiment, the polynucleotide comprises: (a) a 5'-UTR (e.g., as provided in Table 4 or a variant or fragment thereof); (b) a coding region; and (c) a stop element and a 3'-UTR (e.g., as described herein), and LNP compositions comprising the same. In an embodiment, the polynucleotide comprises a 5'-UTR comprising a sequence provided in Table 4 or a variant or fragment thereof (e.g., a functional variant or fragment thereof). It will be understood that such 5’UTRs are incorporated into constructs not found in nature, e.g., such 5’ UTRs are synthetic, are altered in sequence from naturally occurring 5’UTRs, are truncated or lengthened versions of those found in nature, comprise chemically P296PC00 / 3000181 -001977 modified bases, are 5 of ORF sequences different from those which they may be found in nature, or the like.

[0611] In an embodiment, the 5' UTR comprises a sequence provided in Table 4 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 51UTR sequence provided in Table 4, or a variant or a fragment thereof.

[0612] In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 19. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 20. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 21. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 22. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 23. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 24. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 25. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 26. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 27. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 28. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 29. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 30. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 31. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 32. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 33. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 34. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 35. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 36. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 37. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 38. In an embodiment, the 51UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, P296PC00 / 3000181 -001977 97%, 98%, 99% or 100% identity to SEQ ID NO: 39. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 40. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 41. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 42. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 43. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 44. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 45. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 46. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 47. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 48. In an embodiment, the 5' UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 49.

[0613] In an embodiment, the 5' UTR comprises the sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the 5' UTR consists of the sequence of any one of SEQ ID NOs: 19-49 and 118-120.

[0614] In an embodiment, a 5' UTR sequence provided in Table 4 has a first nucleotide which is an A. In an embodiment, a 5' UTR sequence provided in Table 4 has a first nucleotide which is a G. In an embodiment, a 5' UTR sequence provided in Table 4 has two first nucleotides which are an AG. In an embodiment, a 5' UTR sequence provided in Table 4 has two first nucleotides which are a GA.

[0615] Table 4: 5' UTR sequences

[0616] SEQ ID Sequence Sequence

[0617] NO: name

[0618] 19 A1 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUC GCAACUAGCAAGCUUUUUGUUCUCGCC

[0619] 20 A5 GGAAAUCCCCACAACCGCCUCAUAUCCAGGCUCAAGAAUAGAGCUCAGU GUUUUGUUGUUUAAUCAUUCCGACGUGUUUUGCGAUAUUCGCGCAAAG CAGCCAGUCGCGCGCUUGCUUUUAAGUAGAGUUGUUUUUCCACCCGU UUGCCAGGCAUCUUUAAUUUAACAUAUUUUUAUUUUUCAGGCUAACCU ACGCCGCCACC

[0620] 21 A6 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAUCUCCCUGAG CUUCAGGGAGCCCCGGCGCCGCCACC

[0621] 22 A7 GGAAACCCCCCACCCCCGUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA AGAUCUCCCUGAGCUUCAGGGAGCCCCGGCGCCGCCACC

[0622] 23 A8 GGAGAACUUCCGCUUCCGUUGGCGCAAGCGCUUUCAUUUUUUCUGCU

[0623] ACCGUGACUAAG

[0624]

[0625] 24 A9 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC P296PC00 / 3000181 -001977 A11 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCC (Reference GCCACC

[0626] A2 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUC GCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC

[0627] A3 GGAAAUCGCAAAAUUUUCUUUUCGCGUUAGAUUUCUUUUAGUUUUCUU UCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC

[0628] A4 G GAAA U C G C A A A A (N2)X (N3)X C U (N4)X (N5)X C G C G U UAGA U U U C U U U U AG U U U U C U N6 N7 CAAC U AG CAAG C U U U U U G U U C U C G C C (N8 C C)x

[0629] (N2)x is a uracil and x is an integer from 0 to 5, e.g., wherein x =3 or 4; (N3)x is a guanine and x is an integer from 0 to 1;

[0630] (N4)x is a cytosine and x is an integer from 0 to 1;

[0631] (N5)x is a uracil and x is an integer from 0 to 5, e.g., wherein x =2 or 3; N6 is a uracil or cytosine;

[0632] N7 is a uracil or guanine;

[0633] N8 is adenine or guanine and x is an integer from 0 to 1.

[0634] A27 GGAAAAUUUUAGCCUGGAACGUUAGAUAACUGUCCUGUUGUCUUUAUA UACUUGGUCCCCAAGUAGUUUGUCUUCCAAA

[0635] A12 GGAAACUUUAUUUAGUGUUACUUUAUUUUCUGUUUAUUUGUGUUUCUU CAGUGGGUUUGUUCUAAUUUCCUUGGCCGCC

[0636] A13 GGAAAAUCUGUAUUAGGUUGGCGUGUUCUUUGGUCGGUUGUUAGUAU UGUUGUUGAUUCGUUUGUGGUCGGUUGCCGCC

[0637] A14 GGAAAAUUAUUAACAUCUUGGUAUUCUCGAUAACCAUUCGUUGGAUUUU AUUGUAUUCGUAGUUUGGGUUCCUGCCGCC

[0638] A15 GGAAAUUAUUAUUAUUUCUAGCUACAAUUUAUCAUUGUAUUAUUUUAGC UAUUCAUCAUUAUUUACUUGGUGAUCAACA

[0639] A16 GGAAAUAGGUUGUUAACCAAGUUCAAGCCUAAUAAGCUUGGAUUCUGG UGACUUGCUUCACCGUUGGCGGGCACCGAUC

[0640] A17 GGAAAUCGUAGAGAGUCGUACUUAGUACAUAUCGACUAUCGGUGGACA CCAUCAAGAUUAUAAACCAGGCCAGA

[0641] A18 GGAAACCCGCCCAAGCGACCCCAACAUAUCAGCAGUUGCCCAAUCCCAA CUCCCAACACAAUCCCCAAGCAACGCCGCC

[0642] A19 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGGUUGGGUA UCGUCGUGGGACUUGGAAAUUUGUUGUUUCC

[0643] A20 GGAAACUAAUCGAAAUAAAAGAGCCCCGUACUCUUUUAUUUCUAUUAGG UUAGGAGCCUUAGCAUUUGUAUCUUAGGUA

[0644] A21 GGAAAUGUGAUUUCCAGCAACUUCUUUUGAAUAUAUUGAAUUCCUAAUU CAAAGCGAACAAAUC UAC AAGCC AU AU ACC

[0645] A22 GGAAAUCGUAGAGAGUCGUACUUACGUGGUCGCCAUUGCAUAGCGCGC GAAAGCAACAGGAACAAGAACGCGCC

[0646] A23 GGAAAUCGUAGAGAGUCGUACUUAGAAUAAACAGAGUCGGGUCGACUUG UCUCUGAUACUACGACGUCACAAUC

[0647] A24 GGAAAAUUUGCCUUCGGAGUUGCGUAUCCUGAACUGCCCAGCCUCCUG AUAUACAACUGUUCCGCUUAUUCGGGCCGCC

[0648] A25 GGAAAUCUGAGCAGGAAUCCUUUGUGCAUUGAAGACUUUAGAUUCCUC UCUGCGGUAGACGUGCACUUAUAAGUAUUUG

[0649] A26 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGGUUGGGUA UCGUCGUGGGACUUGGAAAUUUGUUGCCACC

[0650] A28 GGAAAUUUUUUUUUGAUAUUAUAAGAGUUUUUUUUUGAUAUUAAGAAAA

[0651] UUUUUUUUUGAUAUUAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCC

[0652]

[0653] GCCACC P296PC00 / 3000181 -001977 46 A29 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAAAAAAAA AAAACC

[0654] 47 A30 GGAAAUCUCCCUGAGCUUCAGGGAGUAAGAGAGAAAAGAAGAGUAAGAA GAAAUAUAAGACCCCGGCGCCGCCACC

[0655] 48 A31 GCCRCC, wherein R= A or G

[0656] 49 A32 GGACUCACUAUUUGUUUUCGCGCCCAGUUGCAAAAA

[0657] 118 AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCU CGCAACUAGCAAGCUUUUUGUUCUCGCC

[0658] 119 GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCU CGCAACUAGCAAGCUUUUUGUUCUCGCC

[0659] 120 AGGAAAUAGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCU

[0660]

[0661] CGCAACUAGCAAGCUUUUUGUUCUCGCC

[0662] In an embodiment, the 51UTR comprises a variant of SEQ ID NO: 19. In an embodiment, the variant of SEQ ID NO: 19 comprises a nucleic acid sequence of:

[0663] G GAAAU C G CAAAA (N2)X (N3)X C U (N4)X (N5)X C G C G U UAGAU U U C U U U UAG U U U U C U N6 N7 C AAC U AG C AAG C U U U U U G U U C U C G C C (N8 C C)x (SEQ ID NO: 28), wherein:

[0664] (N2)x is a uracil and x is an integer from 0 to 5, e.g., wherein x =3 or 4;

[0665] (N3)x is a guanine and x is an integer from 0 to 1;

[0666] (N4)x is a cytosine and x is an integer from 0 to 1;

[0667] (N5)x is a uracil and x is an integer from 0 to 5, e.g., wherein x =2 or 3;

[0668] N6 is a uracil or cytosine;

[0669] N7 is a uracil or guanine;

[0670] N8 is adenine or guanine and x is an integer from 0 to 1.

[0671] In an embodiment (N2)x is a uracil and x is 0. In an embodiment (N2)x is a uracil and x is 1. In an embodiment (N2)x is a uracil and x is 2. In an embodiment (N2)x is a uracil and x is 3. In an embodiment, (N2)x is a uracil and x is 4. In an embodiment (N2)x is a uracil and x is 5.

[0672] In an embodiment, (N3)x is a guanine and x is 0. In an embodiment, (N3)x is a guanine and x is 1.

[0673] In an embodiment, (N4)x is a cytosine and x is 0. In an embodiment, (N4)x is a cytosine and x is 1.

[0674] In an embodiment (N5)x is a uracil and x is 0. In an embodiment (N5)x is a uracil and x is 1. In an embodiment (N5)x is a uracil and x is 2. In an embodiment (N5)x is a uracil and x is 3. In an embodiment, (N5)x is a uracil and x is 4. In an embodiment (N5)x is a uracil and x is 5.

[0675] In an embodiment, N6 is a uracil. In an embodiment, N6 is a cytosine.

[0676] In an embodiment, N7 is a uracil. In an embodiment, N7 is a guanine.

[0677] In an embodiment, N8 is an adenine and x is 0. In an embodiment, N8 is an adenine and x is 1. In an embodiment, N8 is a guanine and x is 0. In an embodiment, N8 is a guanine and x is 1. P296PC00 / 3000181 -001977 In an embodiment, the 51UTR comprises a variant of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 50% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 60% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 70% identity to the corresponding non-variant sequence of anyone of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 80% identityto the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 90% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 95% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 96% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 97% identity to the corresponding non-variant sequence of anyone of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 98% identityto the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a sequence with at least 99% identity to the corresponding non-variant sequence of any one of SEQ ID NOs: 19-49 and 118-120.

[0678] In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 5%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 10%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 20%. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 30%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 40%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 50%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 P296PC00 / 3000181 -001977 comprises a uridine content of at least 60%. In an embodiment, the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 70%. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises a uridine content of at least 80%.

[0679] In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises at least 2, 3, 4, 5, 6 or 7 consecutive uridines (e.g., a polyuridine tract). In an embodiment, the polyuridine tract in the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises at least 1 -7, 2-7, 3-7, 4-7, 5-7, 6-7, 1 -6, 1 -5, 1 -4, 1 -3, 1 -2, 2-6, or 3-5 consecutive uridines. In an embodiment, the polyuridine tract in the variant of anyone of SEQ ID NOs: 19-49 and 118-120 comprises 4 consecutive uridines. In an embodiment, the polyuridine tract in the variant any one of SEQ ID NOs: 19-49 and 118-120 comprises 5 consecutive uridines.

[0680] In an embodiment, the variant of any one of SEQ ID NOs: 19-49 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 polyuridine tracts. In an embodiment, thevariantof anyoneof SEQ ID NOs: 19-49 and 118-120 comprises 3 polyuridine tracts. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises 4 polyuridine tracts. In an embodiment, the variant of any one of SEQ ID NOs: 19-49 and 118-120 comprises 5 polyuridine tracts.

[0681] In an embodiment, one or more of the polyuridine tracts are adjacent to a different polyuridine tract. In an embodiment, each of, e.g., all, the polyuridine tracts are adjacent to each other, e.g., all of the polyuridine tracts are contiguous.

[0682] In an embodiment, one or more of the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides. In an embodiment, each of, e.g., all of, the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18.

[0683] 19, 20, 30, 40, 50 or 60 nucleotides.

[0684] In an embodiment, a first polyuridine tract and a second polyuridine tract are adjacent to each other.

[0685] In an embodiment, a subsequent, e.g., third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth, polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides from the first polyuridine tract, the second polyuridine tract, or any one of the subsequent polyuridine tracts.

[0686] In an embodiment, a first polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides from a subsequent polyuridine tract, e.g., a second, third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth polyuridine tract. In an embodiment, one or more of the subsequent polyuridine tracts are adjacent to a different polyuridine tract.

[0687] In an embodiment, the 51UTR comprises a Kozak sequence, e.g., a GCCRCC nucleotide sequence (SEQ ID NO: 48) wherein R is an adenine or guanine. In an embodiment, the Kozak sequence is disposed at the 31end of the 5'UTR sequence. P296PC00 / 3000181 -001977

[0688] In an embodiment, the polynucleotide comprising a 5’ UTR sequence disclosed herein comprises a coding region which encodes for a payload, e.g., a therapeutic or prophylactic payload (e.g., one or more polypeptides).

[0689] In some embodiments, the polynucleotide (e.g., mRNA) comprising a 5’ UTR sequence disclosed herein is formulated as an LNP. In an embodiment, the LNP composition comprises: (i) an ionizable lipid, e.g., an amino lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0690] In other embodiments, the LNP compositions of the disclosure are used in a method of treating a disease or disorder.

[0691] In some embodiments, an LNP composition comprising a polynucleotide disclosed herein encoding a therapeutic payload or prophylactic payload, e.g., one or more target polypeptides, can be administered with one or more other therapeutic agents, e.g., as described herein.

[0692] Stop elements and 3’-UTRs

[0693] Translational stop codons, UAA, UAG, and UGA, are an important component of the genetic code and signal the termination of translation of an mRNA. During protein synthesis, stop codons interact with protein release factors and this interaction can modulate ribosomal activity thus having an impact translation (Tate WP, et aL, (2018) Biochem Soc Trans, 46(6):1615-162).

[0694] 31UTR sequences have been shown to influence translation, half-life, and subcellular localization of mRNAs (Mayr C., Cold Spring Harb Persp Biol 2019 Oct 1;11 (10):a034728).

[0695] Disclosed herein, inter alia, is a polynucleotide, e.g., mRNA, comprising an open reading frame encoding one or more polypeptides, which polynucleotide has a stop element in combination with a 3' UTR that confers an increased half-life, increased expression and / or increased activity of the one or more polypeptides encoded by said polynucleotide, or of the polynucleotide itself. In an embodiment, a polynucleotide disclosed herein comprises: (a) a 5'-UTR; (b) a coding region; and (c) a stop element and 3'-UTR (e.g., as described herein), and LNP compositions comprising the same.

[0696] Disclosed herein, inter alia, is a polynucleotide encoding a polypeptide comprising, inter alia, a 3’ UTR. In an embodiment, a polynucleotide disclosed herein comprises: (a) a 5’-UTR (e.g., as provided in Table 4 or a variant or fragment thereof); (b) a coding region; and (c) a stop element and 3’-UTR (e.g., as provided in Table 5 or a variant or fragment thereof), and LNP compositions comprising the same. In an embodiment, the polynucleotide comprises a 3’-UTR comprising a sequence provided in Table 5 or a variant or fragment thereof (e.g., a functional variant or fragment thereof). It will be understood that such 3’UTRs are incorporated into constructs not found in nature, e.g., such 3’ UTRs are synthetic, are altered in sequence from naturally occurring 3’UTRs, are truncated or lengthened versions of those found in nature, comprise chemically modified bases, are 3’ of ORF sequences different from those which they may be found in nature, or the like. P296PC00 / 3000181 -001977 In an embodiment, the 31UTR comprises a sequence provided in Table 5 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in Table 5, or a variant or a fragment thereof. In an embodiment, the 31UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyone of SEQ ID NO: 50-58 and 121.

[0697] Table 5: 3' UTR sequences (stop cassette is italicized; miR binding sites are boldened)

[0698] SEQ Sequence Sequence

[0699] ID NO information

[0700] 50 D1 UAA4GCL / CCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCC CCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0701] 51 D2 UAAGL / CL / AAGCUGGAGCCUCCUGAGAGACCUGUGUGAACUAUUG AGAAGAUCGGAACAGCUCCUUACUCUGAGGAAGUUGGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0702] 52 D3 L / AAAGCL / CCCCGGGGCAAACACCAUUGUCACACUCCAGCCUCGG UGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACAC UCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCC CCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC

[0703] (miR122 binding sites in bold)

[0704] 53 D4 UAAAGCUCCCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGAG CCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAUU GUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0705] (miR-142-3p and miR122 binding sites in bold))

[0706] 54 D5 L / AAAGCL / CCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCC CCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGC

[0707] (miR122 binding sites in bold)

[0708] 55 D6 L / AAGCCCCL / CCGGGGCAAACACCAUUGUCACACUCCAGCCUCGG UGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACAC UCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCC CCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC

[0709] (miR122 binding sites in bold)

[0710] 56 D7 UAAGCCCCL / CCGGGGUCCAUAAAGUAGGAAACACUACAGCCUCG GUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAAC ACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCC CCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC

[0711] (miR-142-3p and miR126-3p binding sites in bold)

[0712] 57 D8 UAAGCCCCL / CCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGA GCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAU UGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0713] (miR-142-3p and miR122 binding sites in bold)

[0714] 58 D9 UAAGCCCCL / CCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCC CCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGC P296PC00 / 3000181 -001977

[0715] (miR122 binding sites in bold)

[0716] 121 UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCAGUUUGGGUU UGGGUGGUCUUUGAA UAAA GUCUGA GUGGGCGGC

[0717] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0718] UAA4GCL / CCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGC CCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0719] (SEQ ID NO: 50) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 50).

[0720] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 50 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0721] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 50 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0722] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 50 or a variant or fragment thereof.

[0723] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 50 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 50.

[0724] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized): UAAGL / CL / AAGCUGGAGCCUCCUGAGAGACCUGUGUGAACUAUUGAGAAGAUCGGAACAGCUCCU UACUCUGAGGAAGUUGGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 51 ) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 51 ). P296PC00 / 3000181 -001977 In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 51 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0725] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 51 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0726] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 51 or a variant or fragment thereof.

[0727] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 51 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 51.

[0728] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized): UA4AGCL / CCCCGGGGCAAACACCAUUGUCACACUCCAGCCUCGGUGGCCUAGCUUCUUGCCCC UUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGU ACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 52) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 52).

[0729] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 52 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the P296PC00 / 3000181 -001977 increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0730] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 52 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0731] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 52 or a variant or fragment thereof.

[0732] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 52 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 52.

[0733] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0734] UAA4GCL / CCCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGC UUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUC CUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 53) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 53.

[0735] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 53 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0736] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 53 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0737] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 53 or a variant or fragment thereof. P296PC00 / 3000181 -001977 In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 53 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 53.

[0738] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized): UAA4GCL / CCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC (SEQ ID NO: 54) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 54.

[0739] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 54 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0740] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 54 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0741] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 54 or a variant or fragment thereof.

[0742] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 54 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 54.

[0743] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0744] L / AAGCCCCL / CCGGGGCAAACACCAUUGUCACACUCCAGCCUCGGUGGCCUAGCUUCUUG CCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUCCUGCAC CCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

[0745] (SEQ ID NO: 55) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 55. P296PC00 / 3000181 -001977 In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 55 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0746] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 55 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0747] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 55 or a variant or fragment thereof.

[0748] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 55 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 55.

[0749] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0750] UAAGCCCCUCCGGGGUCCAUAAAGUAGGAAACACUACAGCCUCGGUGGCCUAGCUUCUUG CCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG

[0751] C (SEQ ID NO: 56) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 56).

[0752] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 56 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the P296PC00 / 3000181 -001977 increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0753] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 56 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0754] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 56 or a variant or fragment thereof.

[0755] In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 56 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 56.

[0756] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0757] UAAGCCCCUCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGC UUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAGCCCCUCCUCCCCUUC CUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 57) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 57.

[0758] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 57 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0759] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 57 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0760] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 57 or a variant or fragment thereof. P296PC00 / 3000181 -001977 In an embodiment, the polynucleotide comprises a 3' UTR sequence provided in SEQ ID NO: 57 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3' UTR sequence provided in SEQ ID NO: 57.

[0761] In an embodiment, the polynucleotide comprises a stop element and 3’-UTR, wherein the sequence is (stop element is italicized):

[0762] UAAGCCCCUCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGC CCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGA AUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 58) or a variant or fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, six, or more nucleotides of nucleotides of SEQ ID NO: 58.

[0763] In an embodiment, the polynucleotide having a 3’ UTR sequence provided in SEQ ID NO: 58 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0764] In an embodiment, the polynucleotide having a 3' UTR sequence provided in SEQ ID NO: 58 or a variant or fragment thereof, results in an increased level and / or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0765] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3' UTR, has a different 31UTR, or does not have a 3' UTR of SEQ ID NO: 58 or a variant or fragment thereof.

[0766] In an embodiment, the pol...

Claims

P296PC00 / 3000181 -001977CLAIMS1. A method for inducing or enhancing an immune response in a subject in need thereof, comprising administering to said subject:a. a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ); andb. an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail.

2. The method of claim 1, wherein the polypeptide encoded by the mRNA comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 repeats, about 5-15 repeats, about 7-14 repeats, about 8-13 repeats, or about 9-12 repeats of the PRAME epitope.

3. The method of claim 2, wherein the polypeptide comprises no alanine linker residues.

4. The method of claim 2, wherein the polypeptide comprises an alanine linker linking adjacent repeats of the PRAME epitope.

5. The method of claim 4, wherein the alanine linker comprises a single alanine reside.

6. The method of claim 4, wherein the alanine linker comprises at least two alanine residues.

7. The method of claim 4, wherein the alanine linker comprises at least three alanine residues.

8. The method of claim 1, wherein the polypeptide comprises 12 repeats of the PRAME epitope.

9. The method of claim 8, wherein the polypeptide consists of 12 repeats of the PRAME epitope.

10. The method of claim 8, wherein the polypeptide comprises the amino acid sequence of MSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHL IGLSLLQHLIGLSLLQHLIGLSLLQHLIGLSLLQHLIGL (SEQ ID NO: 2).P296PC00 / 3000181 -001977 11. The method of claim 1, wherein the polypeptide comprises 9 repeats of the PRAME epitope, wherein adjacent repeats are linked by two alanine residues.

12. The method of claim 11, wherein the polypeptide consists of the 9 repeats of the PRAME epitope and the alanine residues linking the adjacent repeats.

13. The method of claim 11, wherein the polypeptide comprises the amino acid sequence of MSLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASLLQHLIGLAASL LQHLIGLAASLLQHLIGLAASLLQHLIGL (SEQ ID NO: 3).

14. The method of claim 1, wherein the polypeptide comprises 9 repeats of the PRAME epitope, wherein adjacent repeats are linked by three alanine residues.

15. The method of claim 14, wherein the polypeptide consists of the 9 repeats of the PRAME epitope and the alanine residues linking the adjacent repeats.

16. The method of claim 14, wherein the polypeptide comprises the amino acid sequence of MSLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIG LAAASLLQHLIGLAAASLLQHLIGLAAASLLQHLIGL (SEQ ID NO: 4).

17. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLI GLSNLTHISALQSLLQHLIGLSNLTH (SEQ ID NO: 5).

18. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of MTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHV LYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIG LSNLTHVLYPVPLESY (SEQ ID NO: 6).

19. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of MSLLQHLIGLSNLTHVLYPVPLESYTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYTLSFYG NSISISALQSLLQHLIGLSNLTHVLYPVPLESYSLSHCSQLTTLSFYGNSISISALQSLLQHLIGL (SEQ ID NO: 7).

20. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of MISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLIGLSNLTHISALQSLLQHLI GLSNLTHGNSISISALQSLLQHLIGL (SEQ ID NO: 8).P296PC00 / 3000181 -00197721. The method of any one of the preceding claims, wherein the polypeptide is about 50-150 amino acids in length, or about 80-120 amino acids in length, or about 90-110 amino acids in length.

22. The method of any of the preceding claims, wherein the mRNA comprises one or more of the following: a 5'-UTR comprising the sequence of any one of SEQ ID NOs: 19-49, and a 3' UTR comprising the sequence of any one of SEQ ID NOs: 50-58.

23. The method of claim 1, wherein the mRNA comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

24. The method of claim 1, wherein the mRNA comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

25. The method of any one of the preceding claims, wherein said antigen binding protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a CDRal comprising or consisting of an amino acid sequence according to SEQ ID NO: 122, and a CDRa3 comprising or consisting of the amino acid sequence according to SEQ ID NO: 124, and wherein the second polypeptide comprises a CDRbl comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 125, and a CDRb3 comprising or consisting of the amino acid sequence accordingto SEQ ID NO: 127, optionally wherein the CDRal, CDRa3, CDRbl and / or CDRb3 sequence(s) may comprise one, two or three amino acid mutations.

26. The method of claim 25, wherein the first polypeptide further comprises a CDRa2 comprising or consisting of the amino acid sequence according to SEQ ID NO: 123 or 134.

27. The method of any one of the preceding claims, wherein said first polypeptide comprises or consists of a variable domain VA.

28. The method of claim 27, wherein said VAdomain comprises or consists of a TCR a-chain variable domain (Va).

29. The method of claim 28, wherein said TCR a-chain variable domain (Va) comprises or consists of an amino acid sequence accordingto SEQ ID NO: 128, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence accordingto SEQ ID NO: 128.

30. The method of claim 25, wherein the second polypeptide further comprises a CDRb2 comprising or consisting of the amino acid sequence according to SEQ ID NO: 126.P296PC00 / 3000181 -00197731. The method of claim 30, wherein said second polypeptide comprises or consists of a variable domain VB.

32. The method of claim 31, wherein said VBdomain is a TCR p-chain variable domain (Vp).

33. The method of claim 32, wherein said TCR p-chain variable domain (Vp) comprises or consists of an amino acid sequence according to SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO: 130.

34. The method of any one of the preceding claims, wherein said antigen binding protein further comprises a TCR constant domain comprising or consisting of an amino acid sequence according to SEQ ID NO: 129 or 131, or an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO: 129or 131.

35. The method of any one of the preceding claims, wherein said antigen binding protein is antigen binding protein is a TCR or a fragment or derivative thereof.

36. The method of anyone of the preceding claim, wherein the antigen binding protein is capable of activating said host cell.

37. The method of any one of the preceding claims, wherein said host cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

38. The method of any one of the preceding claims, wherein the host cell is provided in the form of a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.

39. The method of claim 38, wherein said excipient is selected from DMSO, Dextran 40, and dextrose.

40. The method of any one of the preceding claims, wherein the mRNA is provided in the form of a pharmaceutical composition optionally comprising at least one pharmaceutically acceptable excipient.

41. The method of claim 40, further comprising a lipid nanoparticle (LNP).

42. The method of claim 41, wherein the LNP comprises an ionizable lipid.P296PC00 / 3000181 -001977 43. The method of claim 42, wherein the ionizable lipid has a chemical structure selected from anyone of (a)-(p):R2HTTTM'XR3(I)or a salt thereof, wherein:R1is -OH, -NRN-C4. IO cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”);RNis H or C1-6 alkyl;RNis H or C1-6 alkyl;RN” is H or C-i-6 alkyl;o is 1, 2, 3, or 4;n is 4, 5, 6, 7, or 8;m is 4, 5, 6, 7, or 8;M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;R2aR2bor-(Ci-6alkylene)-(C3-8 cycloalkyl)-Ci.6alkyl;R2ais -H or C1-10 alkyl;R2bis -H or C1-10 alkyl;R2cis C1-8 alkyl or C2.8alkenyl;R3ais H or C1-10 alkyl;R3bis H or C1-8 alkyl; andR3cis C1-10 alkyl or C2-8alkenyl,P296PC00 / 3000181 -001977R3a(H)or a salt thereof, wherein:R1is -OH;o is 2, 3, or4;n is 4, 5, 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2cis C4-8alkyl;R3ais C7-10 alkyl; andR3cis C3-5 alkyl,or(c)R2CR3a(HI)or a salt thereof, wherein:R1is NRN-C4.10cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”); RNis H;RN’ is C-i-2 alkyl;RN”is H;o is 2, 3, or4;n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2ais C7-10 alkyl;P296PC00 / 3000181 -001977R2cis C4-6 alkyl;R3ais C1-3 alkyl; andR3cis C4-6 alkyl,or(d)(IV)or a salt thereof, wherein:R1is OH;o is 2, 3, or4;n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2bis C3-5 alkyl;R2cis C2-4 alkyl;R3ais C7.10alkyl; andR3cis C4-6 alkyl,(V)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula I; and R3ais C1-8alkyl.(f)(VI)P296PC00 / 3000181 -001977or a salt thereof, wherein:o, M, M’, R2cand R3care as defined for Formula (I); andR3ais Ci-8alkyl.(VII)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula I; and R3ais Ci-8alkyl.(h)(VIII)or a salt thereof, wherein:o, M, M’, R2cand R3care as defined for Formula (I); andR3ais Ci-8alkyl.(i)R2CR3a(IX)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula (I); R2ais a Cva alkyl; andR3ais Ci-8alkyl,(j)P296PC00 / 3000181 -001977(X)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined forformula I; R2bis a Cva alkyl; andR3ais Ci-8alkyl,or(k)(XI)or a salt thereof, wherein:R1, o, M, M’, R2c, and R3care as defined forformula I;R2ais a Ci-8alkyl; andR3ais Ci-8alkyl,or(I)or a salt thereof, wherein:R1, o, M, M’, R2c, and R3care as defined forformula I;R2ais a Ci-8alkyl; andR3ais Ci-8alkyl,or(m)P296PC00 / 3000181 -001977R2aI-I H V R3aR3c(XIII)or a salt thereof, wherein:R1, o, M, M’, R2c, and R3care as defined forformula I;R2ais a Ci-8alkyl; andR3ais C1-8alkyl,or(XIV)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for formula I;R2bis a Ci-8alkyl; andR3ais Ci-8alkyl,orR2b(XV)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for formula I;R2bis a Ci-8alkyl; andR3ais Ci-8alkyl,orP296PC00 / 3000181 -001977R2b(XV)or a salt thereof, wherein:o, M, M’, R2c, and R3care as defined for formula I;R2ais a Ci-8alkyl; andR3ais C1-8alkyl,or N-oxides, salts, or isomers of each thereof.

44. The method of claim 42 or 43, wherein the ionizable lipid is a compound selected from the group consisting of:or N-oxides, salts, or isomers thereof.

45. The method of any of the preceding claims, wherein the mRNA comprises a 5' terminal cap, optionally wherein the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-P296PC00 / 3000181 -001977guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof.

46. The method of any of the preceding claims, wherein the mRNA comprises a poly-A region, optionally wherein the poly-A region 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 nucleotides in length, or at least about 100 nucleotides in length, or optionally wherein the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length.

47. The method of any of the preceding claims, wherein the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.

48. The method of claim 47, wherein the mRNA comprises at least one chemically modified nucleobase, wherein the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ip), N1 -methylpseudouracil (ml ip), 1 -ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof, optionally wherein at least about 25%, 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%, at least about 95%, at least about 99%, or 100% of the uracils are N1 methylpseudouracils.

49. The method of claim 47, wherein the mRNA comprises at least one chemically modified nucleobase, wherein the at least one chemically modified nucleobase is N1- methylpseudouracil (ml ip), optionally wherein at least about 25%, 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%, at least about 95%, at least about 99%, or 100% of the uracils are N1 methylpseudouracils.

50. The method of any of the preceding claims, wherein the mRNA comprises a 5’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length (SEQ ID NO: 92), and wherein all uracils of the polynucleotide are N1 -methylpseudouracils51. The method or use of any one of the preceding claims, wherein said immune response is a T cell response.

52. The method of any one of the preceding claims, wherein the method is for treating a proliferative disease.P296PC00 / 3000181 -00197753. The method of any one of the preceding claims, wherein said proliferative disease is cancer.

54. The method of claim 53, wherein said cancer is a PRAME-positive cancer.

55. The method of claim 54 claims, wherein the PRAME-positive cancer expresses PRAME peptide SLLQHLIGL (SEQ ID NO: 1) in a complex with a major histocompatibility complex (MHO) protein on cell surface.

56. The method or use of any one of claims 53 to 55, wherein said cancer is selected from cutaneous melanoma (CM) and synovial sarcoma (SS).

57. The method any one of claims 53 to 56, wherein the cancer is an unresectable and / or metastatic cancer.

58. The method of any one of the preceding claims, wherein the mRNA and the host cell are administered separately from one another.

59. The method of any one of the preceding claims, wherein the mRNA and the host cell are administered simultaneously or sequentially.

60. The method of any one of the preceding claims, wherein the mRNA or the pharmaceutical composition comprising the same is administered to the subject via intravenous (IV), subcutaneous, intramuscular (IM), or intradermal administration.

61. The method of any one of the preceding claims, wherein the host cell or the pharmaceutical composition comprising the same is administered to the subject via intravenous (IV) administration.

62. The method of any one of the preceding claims, comprising lymphodepletion prior to administering the host cell.

63. The method of any one of the preceding claims, wherein lymphodepletion comprises a lymphodepletion treatment at least 1, 2, 3 or 4 consecutive days before administering said host cell.P296PC00 / 3000181 -001977 64. The method of any one of the preceding claims, further comprising administering IL-2 to said subject.

65. The method of claim 64, wherein IL-2 is administered after administering said host cell.

66. The method of claim 64 or 65, wherein IL-2 is administered at low doses, preferably at 1 million IU or less.

67. The method of any of claims 64 to 66, wherein IL-2 is administered daily or twice daily.

68. The method of any of claims 64 to 67, wherein IL-2 is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

69. The method of claim 58, wherein IL-2 is administered daily for days 1 -5 after administering said host cell, and / or twice daily at days 6-10, preferably at a dose of 1 million IU.

70. The method of any one of the preceding claims, wherein said host cell is administered to a total dose of 1 -10x109host cells, preferably of 1 -5x109host cells, such as up to 5 x 109host cells to the subject.

71. The method of any one of the preceding claims, comprising administering said mRNA after administering said host cell and optionally IL-2.

72. The method of any one of the preceding claims, comprising administering said mRNA 2-4 weeks after administering said host cell or optionally IL-2.

73. The method of any one of the preceding claims, comprising administering said mRNA every 2-4 weeks for up to 52 weeks or less.

74. The method of any one of the preceding claims, comprising the following steps:a. administering said host cell(s) on Day 1,b. administering IL-2 on Days 2-11; andc. administering said mRNA on Day 15 or later.

75. The method of claim 74, whereinhost cells are administered intravenously at a dose of 1 -10 x 109cells per patient;P296PC00 / 3000181 -001977 IL-2 is administered subcutaneously at a dose of 1 million IU or less once daily on Day 2-6 and twice daily on Day 7-11;mRNA is administered intramuscularly in the form of LNPs; and / ormRNA is administered for up to 12 cycles each having a length of about 28 days, further wherein said mRNA is administered on Day 1 and Day 15 of Cycle 1, and on Day 1 of Cycle 2-12.

76. A method for treating a proliferative disease in a subject in need thereof, comprising administering to said subject:a. a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ); andb. an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail.

77. A combination comprising:a. a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1 ); andb. an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail.

78. A kit comprisinga. a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); andb. an mRNA encoding a PRAME antigenic peptide concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail;c. optionally packaging material; andP296PC00 / 3000181 -001977 d. optionally a label or packaging insert contained within said packaging material indicating that said combination is effective for a method of treating cancer or for use in the treatment of cancer.

79. A combination comprising:a. at least one nucleic acid(s) and / or vector(s) encoding an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b. an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail.

80. A kit comprisinga. at least one nucleic acid(s) and / or vector(s) encoding an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1); and b. an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail;c. optionally packaging material; andd. optionally a label or packaging insert contained within said packaging material indicating that said combination is effective for a method of treating cancer or for use in the treatment of cancer.

81. The combination or kit of any one of claims 77 to 80 for use as a medicament.

82. The combination or kit of any one of claims 77 to 81 for use in a method of inducing or enhancing an immune response in a subject in need thereof, comprising administering said host cell and said mRNA to said subject.

83. The combination or kit of any one of claims 77 to 82 for use in a method of treating a proliferative disease, comprising administering said mRNA and said host cell to a subject in need thereof.P296PC00 / 3000181 -00197784. A host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1) for use in a method of treating a proliferative disease, wherein treatment comprises administering to a subject in need thereof a) said host cell, and b) an mRNA encoding a PRAME epitope concatemeric polypeptide, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail85. An mRNA encoding a PRAME epitope concatemeric polypeptide for use in a method of treating a proliferative disease, wherein the polypeptide comprises at least one PRAME epitope having the sequence of SLLQHLIGL (SEQ ID NO: 1), wherein the mRNA comprises one or more of the following: a 5’ UTR, a 3’ UTR, a nucleotide cap, and a poly A tail, for use in a method of treating a proliferative disease, wherein said treatment comprises administering to a subject in need thereof a) said mRNA, and b)a host cell comprising an antigen binding protein capable of specifically binding to a Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide having the sequence of SLLQHLIGL (SEQ ID NO: 1) to a subject in need thereof.

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