WO2024245907A1 - Cancer antigens - Google Patents
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- WO2024245907A1 WO2024245907A1 PCT/EP2024/064280 EP2024064280W WO2024245907A1 WO 2024245907 A1 WO2024245907 A1 WO 2024245907A1 EP 2024064280 W EP2024064280 W EP 2024064280W WO 2024245907 A1 WO2024245907 A1 WO 2024245907A1
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- nucleic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/86—Lung
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/876—Skin, melanoma
Definitions
- Cancer antigens Introduction Cancer is a major global health problem and is one of the leading causes of death worldwide. Traditional cancer fa-eatments, such as surgery, chemotherapy, and radiation therapy, have limited efficacy and can cause significant side effects. Immunotherapy, which involves harnessing the power of the immune system to target cancer cells, has emerged as a promising new approach for the treatment of cancer. Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapies exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumour antigens, which are often proteins or other macromolecules.
- nucleic acid such as RNA
- RNA has emerged as a promising therapeutic tool for the treatment of cancer and other diseases, as it can be used to direct the production of proteins that can inhibit or otherwise reduce the growth or survival of cancer cells.
- the advantages of using RNA include transient expression and a non-transforming character - RNA does not need to enter the nucleus to be expressed and moreover cannot integrate into the host genome, thereby eliminating the risk of oncogenesis.
- nucleic acid e.g. RNA
- the object of the present invention is to provide neoantigens or tumour-associated antigens for cancer immunotherapy, in particular neoantigens ortumour-associated antigens provided by nucleic acid constructs.
- the object mentioned above is solved by the underlying description and the accompanying claims.
- the object of the invention is solved by providing novel tumour antigens, nucleic acids encoding such tumour antigens, and the use of these tumour antigens/nucleic acids in treating cancer in a subject such as NSCLC, HNSCC, or melanoma.
- tumour antigens are provided that are unexpectedly onginate from small open reading frames of long non- coding RNA (that, as the term suggests, have long been considered as being non-coding).
- the present invention inter alia relates to novel tumour or cancer antigens, in particular amino acid sequences and nucleic acid sequences, that may be used in cancer immunotherapy.
- the invention is directed to an artificial nucleic acid, preferably RNA, comprising at least one coding sequence encoding at least one tumour or cancer antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein encoded by a small open reading frame (smORF) of IncRNA, or a fragment or variant thereof; and/or at least one antigenic peptide selected or derived from a peptide or protein of a tumour neoantigen, or a fragment or van'ant thereof.
- smORF small open reading frame
- compositions comprising the artificial nucleic acid, preferably formulated in lipid-based carriers.
- methods of h-eating or preventing disorders, diseases, or conditions, and medical uses in particular cancer such as NSCLC, HNSCC, or melanoma.
- the invention is inter alia based on the surprising finding that peptides encoded by small open reading frames (smORF) of long non-coding RNA (IncRNA) can serve as effective tumour associated antigens.
- smORF small open reading frames
- IncRNA long non-coding RNA
- neoantigens have been identified that can serve as effective tumour specific antigens.
- These tumour antigens can be provided by an artificial nucleic acid, e.g. mRNA, for treating cancer, e.g.
- the present invention provides an artificial nucleic acid compnsing at least one coding sequence encoding at least one tumour or cancer antigen that comprises (a) at least one antigenic peptide seleded or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA); and/or (b) at least one antigenic peptide selected or derived from a peptide or protein of a tumour neoantigen.
- IncRNA long non-coding RNA
- the at least one antigenic peptide is selected or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA) selected from lnc-WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, or an immunogenic fragment or variant thereof.
- IncRNA long non-coding RNA
- the at least one antigenic peptide is selected from a peptide or protein of a tumour neoantigen is selected from NRAS, TiW1B, ECPAS, MAP2K1, TOMM22, GLB1, MAGE-A3, ATAD2, BRAF, EGFR, orTP53, or an immunogenic fragment or variant thereof.
- the present invention provides a pharmaceutical composition comprising at least one artificial nucleic acid as defined in the first aspect.
- the artificial nucleic acd preferably an RNA, may be formulated in LNPs.
- the present invention provides a tumour antigen or a composition antigen.
- the present invention provides an antibody, a T-cell, or a TCR that has been raised against or stimulated with any of at least one nucleic acid encoding a tumour antigen of the invention.
- the present invention provides a combination comprising at least two or a plurality of therapeutic modalities of any of the foregoing aspects.
- the present invention provides a kit or kit of parts comprising at least one artificial nucleic acid of the first aspect; at least one pharmaceutical composition of the second aspect; at least one tumour antigen or composition of the third aspect; and/or at least one antibody, T-cetl, or TCR of the fourth aspect.
- the invention provides methods of treating or preventing disease, disorder or condition and medical uses of the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or composition, the antibody, T- cell, or TCR, or the kit or kit of parts.
- the disease, disorder or condition is cancer, or any disease, disorder, or condition related to cancer, such as NSCLC, HNSCC or melanoma. Definitions For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitbns may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments. Percentages in the context of numbers should be understood as relative to the total number of the respective items.
- percentages should be understood as percentages by weight (wt.-%).
- About The term “about” is used when determinants or values do not need to be identical, i.e.100% the same and that e.g. the respective values or determinants may diverge by 1 % to 10%. Preferably, "about' means, that a determinant or values may diverge by +/-1%, +/-2%, +1-3%, +/-4%, +/-5%, +/ ⁇ %, +/-7%, +/-8%, +/-9%, +/-10%.
- Antiaenic peptide refers to a peptide derived from a (antigenic or immunogenic) protein which stimulates the body's adaptive or cellular immune system to provide an adaptive or cellular immune response. Therefore an antigenic/immunogenic peptide comprises at least one epitope or antigen selected or derived from a tumour antigen as defined herein.
- Cationic, cationizable The term “cationic” means that the respective structure, compound, or group, or atom bears a positive charge, either permanently or not permanently, e.g. in response to certain conditions such as pH.
- Cationic compound, polvcationic compound Where a respective strudure, compound, or group, or atom cames a plurality of positive charges, it may be referred to as potycationic.
- potycationic The terms as used herein must be understood as defined in W02021/156267 [p.88, line 12 to p.89, line 22].
- Coding sequence coding region, cds:
- coding sequence or “the corresponding abbreviation "cds” as used herein refers to a sequence of several nucleotide triplets that can be translated into a peptide or protein.
- a cds in the context of the present invention may be a DNA or RNA sequence consisting of several nucleotides that may be divided by three, which typically starts with a start codon and preferably terminates with a stop codon.
- the cds encodes at least one tumour antigen as defined herein.
- Derived from The term "derived from” as used herein in the context of a nucleic acid, i.e.
- nucleic acid derived from (another) nucleic acid means that the nucleic acid, which is derived from (another) nucleic acid, shares at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or is identical with the nucleic acid from which it is derived.
- the term "derived from” means that the amino acid sequence, which is derived from (another) amino add sequence, shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or is identical with the amino acid sequence from which it is derived.
- )itope The term “epitope” as used herein refers to T cell epitopes and B cell epitopes.
- T cell epitopes or parts of the antigenic peptides or proteins may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g.8,9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 to about 20 or even more amino acids.
- MHC class I molecules preferably having a length of about 8 to about 10 amino acids, e.g.8,9, or 10, (or even 11, or 12 amino acids)
- fragments as processed and presented by MHC class II molecules preferably having a length of about 13 to about 20 or even more amino acids.
- B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
- Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
- epitopes can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
- fragment as used herein in the context of a nucleic acid sequence (e.g. RNA or DNA) or an amino acid sequence may typically be a shorter portion of a reference sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment typically consists of a sequence that is identical to the corresponding stretch within the reference sequence.
- a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total reference molecule from which the fragment is derived.
- Identity of a sequence: The term "identity" as used herein in the context of a nucleic acid sequence, or an amino acid sequence, refers to the percentage to which two sequences are identical. To determine the percentage to ⁇ which Iwo sequences are identical, the sequences can be aligned (by also introducing gaps, if necessary) to be subsequently compared to one another.
- an immunogen is a peptide, polypeptide, or protein.
- RNA in vitro transcription The terms "RNA in vitro transchption” or “in vitro transcription” relate to a process where RNA is synthesized in a cell-free system in vitro.
- IVT RNA in vitro transcription
- the RNA is obtained by DNA-dependent in vitro transcription of an appropriate DNA template in the presence of a DNA-dependent RNA polymerases (e.g. T7, T3, SP6, or Syn5), ribonucleotide triphosphates (NTPs, and optionally modified NTPs); optionally, a cap analogue, MgCb, and a buffer (compnsing TRIS or HEPES, optionally DTT and/or spennidine).
- a DNA-dependent RNA polymerases e.g. T7, T3, SP6, or Syn5
- NTPs ribonucleotide triphosphates
- MgCb optionally modified NTPs
- buffer compnsing TRIS or HEPES, optionally DTT and
- Variant of a sequence:
- the term "variant" as used herein in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence.
- a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
- a variant of a nucleic acid sequence may at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence the variant is derived from.
- a van'ant may be a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
- a "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% nucleotide identity overs stretch of at least 30, 50,75 or 100 nudeotides of such nucleic acid sequence.
- the term "vanant” as used herein in the context of proteins or peptides is e.g.
- a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s) /substitution(s), such as one or more substituted, inserted and/or deleted amino acid(s).
- these fragments and/or variants have the same, or a comparable specific property. Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra).
- a variant of a protein may be a functional variant of the protein, which means that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 90% of the function of the protein it is derived from.
- a "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 30,50, 75 or 100 amino acids of such protein or peptide.
- NSCLC/Non-SmalI Cell Luna Cancer/Iung cancer Lung cancer, also known as lung carcinoma, is a malignant tumour that origin is based in cells of the lung.
- lung cancer is classified based on the type of cells the tumour is derived from; tumours derived from different cells progress and respond to treatment differently.
- NSCLC can be divided into different subgroups including lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSC) / squamous non-small-cell lung cancer (sqNSCLC).
- LAD lung adenocarcinomas
- LUSC lung squamous cell carcinomas
- sqNSCLC squamous non-small-cell lung cancer
- Head and Neck cancer Is a cancer that typically develops from tissues in the lip and oral cavity, larynx (throat), salivary glands, nose, sinuses, or skin of the face.
- Squamous-cell carcinoma is a type of cancer that typically compnses several different types of cancer that begin in squamous cells. These cells form on the surface of the skin, on the lining of hollow organs in the body, and on the lining of the respiratory and digestive tracts. Squamous-cell carcinoma comprise e.g. squamous head and neck cancer (e.g.
- Melanoma Is a type of skin cancer that develops from the pigment-producing cells known as melanocytes and is the most dangerous type of skin cancer. The outcome of melanoma depends on the stage at presentation. Types of melanomas include e.g. skin cutaneous melanoma (SKCM) or uveal melanoma.
- SKCM skin cutaneous melanoma
- SKCM can be divided into different subgroups, e.g. a BRAF subgroup and a NRAS subgroup.
- SEQ ID NOs e.g. amino acid sequences or nucleic acid sequences
- feature key i.e. “source” (for nucleic acids or proteins) or”misc_feature” (for nucleic acids) or “REGION” (for proteins)
- Nucleic Acid encoding at least one tumour antigen In a first aspect, the invention provides a nucleic acid encoding at least one tumour antigen.
- the nucleic acid encoding the at least one tumour antigen is an artificial nucleic acid.
- artificial nucleic acid refers to a nucleic acid that does not occur naturally.
- an artificial nucleic acid may be understood as a non-natural nucleic acid molecule.
- Such nucleic acid molecules may be non-natural due to its individual sequence (e.g. codon modified coding sequence, UTRs) and/or due to other modifications, e.g. structural modifications ofnucleotides.
- an artificial nucleic acid may be designed and/or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides.
- an artificial nucleic acid is a sequence that may not occur naturally, i.e. a sequence that differs from the wild type sequence or the naturally occurring sequence by at least one nudeotide.
- artificial nucleic acid is not restricted to mean “one single molecule” but is understood to comprise an ensemble of essentially identical nucleic acid molecules.
- artificial nucleic acid as used herein may relate to artificial DMA or artificial RNA.
- the artificial nucleic acid is selected from an artificial RNA.
- the artificial nucleic acid comprises at least one coding sequence encoding at least one tumour antigen.
- tumour antigen as used herein is intended to refer to tumour-associated antigens fTAAs), expressed by both normal and neoplastic tissue, and tumour-specific antigens CTSAs) including shared antigens, neoantigens, and unique antigens.
- tumour antigens can be loosely categorized as oncofetal (typically only expressed in fetal tissues and in cancerous somatic cells), oncoviral (encoded by tumorigenic transforming viruses), overexpressed/ accumulated (expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer-testis (expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted (expressed largely by a single cancer histotype), mutated (only expressed by cancer as a result of genetic mutation or alteration in transcription), posttranslationally altered (tumour-associated alterations in glycosylation, etc.), or idiotypic (highly polymorphic genes where a tumour cell expresses a specific "clonotype", i.e., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies).
- oncofetal typically only expressed in fetal tissues and in cancerous somatic
- tumour antigens may fall into more than one category.
- the tumour antigen (that is provided by the artificial nucleic acid) is produced in the cytosol upon administration of the artificial nucleic acid to a cell, tissue, or subject.
- the produced tumour antigen may be secreted or further processed.
- the administration of the artificial nucleic acid (e.g. RNA) to a cell, tissue, or subject leads to a translation of the at least one coding sequence into at least one tumour antigen.
- the term relates to the protein product that is generated from the artificial nucleic acid of the invention by translating the coding sequence of the nucleic acid into a protein.
- tumour antigen amino acid sequences that are produced/translated in the cytosol upon administration of the artificial nucleic acid to a cell, tissue, or subject.
- the artificial nucleic acid compnses at least one coding sequence encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant of such a peptide or protein.
- IncRNA long non-coding RNA
- the artificial nucleic acid comprises at least one coding sequence encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein of a tumour neoantigen, or an immunogenic fragment or variant of such a peptide or protein.
- tumour antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein of a tumour neoantigen, or an immunogenic fragment or variant of such a peptide or protein.
- immunogenic fragment or “immunogenic variant” has to be understood as any fragment/vanant of the corresponding peptide or protein capable of raising an immune response in a subject, preferably a human subject.
- the artificial nucleic acid compnses at least one coding sequence encoding at least one tumour antigen that comprises or consists of a) at least one amino acid sequence encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant thereof; and/or b) at least one amino acid sequence of a tumour neoantigen, or an immunogenic fragment or variant thereof.
- a tumour antigen that comprises or consists of a) at least one amino acid sequence encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant thereof; and/or b) at least one amino acid sequence of a tumour neoantigen, or an immunogenic fragment or variant thereof.
- the artificial nucleic acid comprises at least one coding sequence encoding at least one tumour antigen that comprises or consists of a) at least one antigenic peptide selected or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant thereof; and/or b) at least one antigenic peptide selected or derived from a peptide or protein of a tumour neoantigen, or an immunogenic fragment or variant thereof.
- IncRNA long non-coding RNA
- the artificial nucleic acid compnses at least one coding sequence encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA) or an immunogenic fragment or van'ant thereof.
- the artificial nucleic acid comprises at least one coding sequence encoding at least one tumour antigen that comprises or consists of at feast one antigenic peptide selected or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant thereof.
- long non-coding RNA and its abbreviation "IncRNA” relates to an RNA having a length of typically greater than 200 bases (up to e.g.10.0000 bases).
- IncRNA has been considered to have no protein encoding function.
- Research on long non-coding RNA in recent years has revealed that these molecules participate in various regulatory aspects of cells.
- long non-coding RNA Compared with mRNA, long non-coding RNA has the characteristics of low expression abundance but has stronger tissue and cell expression specificity.
- several IncRNAs have been found to encode for peptides with biologically significant function, e.g. micropeptides or putative small open reading frames (also herein referred to as "mini ORF” or "small ORF” (smORF)).
- IncRNA long non-coding RNA
- RNA species in the context of the invention encode peptides or proteins that can serve as suitable tumour antigens.
- peptides have been identified by the inventors that are produced by certain IncRNA species. More surprisingly, these peptides, encoded by IncRNA, show specificity for certain tumours (e.g. melanoma, NSCLC, HNSCC) and can raise an antigen specific immune response, e.g. these peptides can be recognized by the immune cells as being foreign, can be presented, and can raise an antigen specific immune response (e.g.
- the at least one antigenic peptide is selected from an amino acid sequence such as a peptide or protein encoded by a long non-coding RNA (IncRNA), or a (immunogenic) fragment or variant thereof, wherein the IncRNA comprises at least one smORF (encoding said peptide or protein) and wherein the IncRNA is characterized by an increased expression in cancer cells (e.g. melanoma, NSCLC, HNSCC) compared to non-cancer cells. Moreover, the IncRNA comprises at least one smORF.
- IncRNA long non-coding RNA
- the IncRNA comprises at least one smORF (encoding said peptide or protein) and wherein the IncRNA is characterized by an increased expression in cancer cells (e.g. melanoma, NSCLC, HNSCC) compared to non-cancer cells.
- the IncRNA comprises at least one smORF.
- the at least one antigenic peptide is selected from a peptide or protein encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or van'ant thereof, said IncRNA being selected from Inc- WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, or a fragment or variant of any of these, preferably an immunogenic fragment or variant of any of these.
- IncRNA long non-coding RNA
- the at least one antigenic peptide comprises an amino acid sequence encoded by a long non-coding RNA (IncRNA), or a fragment or variant thereof, said IncRNA being selected from lnc-WDR72-2:4, Inc- TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, or a fragment or variant of any of these, preferably an immunogenic fragment or van'ant of any of these.
- the terms "amino acid sequence encoded by” or “peptide or protein encoded by” or “antigen encoded by” in the context of a certain IncRNA species e.g.
- the respective amino acid sequence, peptide, protein, or antigen originates from the respective IncRNA species.
- the respective amino acid sequence, peptide, protein, or antigen that is encoded by a certain IncRNA species e.g. ZC3H8-6:1, WDR72-2:4, KCNMB2-AS1:4, or NTF3-5:5, or, in other words, that originates from a certain IncRNA species (e.g.
- nucleic acid of the invention e.g. an mRNA
- Preferred in the context of the invention are amino acid sequences such as peptide or proteins encoded by lnc-NTF3- 5:5, lnc-WDR72-2:4, KCNMB2-AS1:4, lnc-ZC3H8-6:1, orlnc-CLEC2D-9:1, oran immunogenic fragment or variant thereof, or nucleic acid sequences encoding amino acid sequences such as peptides or proteins encoded by Inc-NTFS- 5:5, lnc-WDR72-2:4, KCNMB2-AS1:4, lnc-ZC3H8-6:1, or lnc-CLEC2D-9:1, or fragments or variants thereof.
- amino acid sequences such as peptides or proteins encoded by lnc-NTF3-5:5, or an immunogenic fragment or van'ant thereof, or nucleic acid sequences encoding amino acid sequences such as peptides or proteins encoded by lnc-NTF3-5:5, or fragments or variants thereof.
- lnc-WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, and LINC00893:25 are predominantly expressed in cancer cells as shown by RNA sequencing (see Example sedion) and peptides encoded by these IncRNAs have been kJentified by mass spectrometry (see Example section).
- lnc-WDR72-2:4 is a long non-coding RNA that is transcribed from the following genomic location in humans: chr15,53513740,53540852,-.
- the naturally occurring (human) IncRNA sequence of lnc-WDR72-2:4 is according to SEQ ID NO: 512.
- the RNA sequence of ln&-WDR72-2:4 comprises at least two open reading frames (smORFs lncWDR722416421696 and lncWDR722415641696) that encode at least one amino acid sequence, e.g. at least one peptide that can serve as tumour antigen in the context of the invention.
- any peptide or protein, or immunogenic fragment or variant thereof, produced by lnc-WDR72-2:4 can serve as a suitable tumour antigen in the context of the invention.
- lnc-TRPC5-3:1 is a long non-coding RNA that is transcribed from the following genomic location in humans: chrX,111619483,111621517,-.
- the naturally occumng (human) IncRNA sequence of lnc-TRPC5-3:1 is according to SEQ ID NO: 516.
- the RNA sequence of lnc-TRPC5-3:1 comphses at least one open reading frame (smORF lncTRPC53116031801) that encodes at least one amino acid sequence, e.g. at least one peptide that can serve as tumour antigen in the context of the invention.
- lnc-CLEC2D-9:1 is a long non-coding RNA that is transcribed from the following genomic location in humans: chr12,9570760,9576267,+.
- the naturally occurring (human) IncRNA sequence of lnc-CLEC2D-9:1 is according to SEQ ID NO: 517.
- the RNA sequence oflnc-CLEC2D-9:1 comprises at least one open reading frame (smORF lncCLEC2D91412616) that encode at least one amino acid sequence, e.g.
- KCNMB2-AS1:4 is a long non-coding RNA that is transcribed from the following genomic location in humans: chr3,178525486,178860397,-.
- the naturally occurring (human) IncRNA sequence of KCNMB2-AS1:4 is according to SEQ ID NO: 513.
- RNA sequence of KCNMB2-AS1:4 comprises at least one open reading frame (smORF KCNMB2AS14633753) that encodes at least one amino acid sequence, e.g. at least one peptide that can serve as tumour antigen in the context of the invention.
- any peptide or protein, or immunogenic fragment or variant thereof, produced by KCNMB2-AS1:4 can serve as a suitable tumour antigen in the context of the invention.
- lnc-ZC3hl8-6:1 is a long non-coding RNA that is transcribed from the following genomic location in humans: chr2,111769354, 111776910,-.
- the naturally occurring (human) IncRNA sequence of lnc-ZC3H8-6:1 is according to SEQ ID NO: 514.
- the RNA sequence of lnc-ZC3hl8-6:1 comprises at least one open reading frame (smORF lncZC3hl86163144) that encodes at least one amino acid sequence, e.g. at least one peptide that can serve as tumour antigen in the context of the invention.
- any peptide or protein, or immunogenic fragment or variant thereof, produced by lno-ZC3hl8-6:1 can serve as a suitable tumour antigen in the context of the invention.
- lnc-NTF3-5:5 is a long non-coding RNA that is transcribed from the following genomic location in humans: chr12,5367810,5379568,+.
- the naturally occurring (human) IncRNA sequence of lnc-NTF3-5:5 is according to SEQ ID NO: 515.
- the RNA sequence of lnc-NTF3-5:5 comprises at least one open reading frame (smORF lncNTF35511951417) that encodes at least one amino acid sequence, e.g. at least one peptide that can sen/e as tumour antigen in the context of the invention.
- LINC00893:25 (encoding smORF LINC00893) is a long non-coding RNA that is transcribed from the following genomic location in humans: chrX,149483112,149532914,-.
- the naturally occurring (human) IncRNA sequence of LINC00893:25 is according to SEQ ID NO: 518.
- the RNA sequence of LINC00893:25 compnses at least one open reading frame (smORF LINC00893) that encodes at least one amino acid sequence, e.g.
- the at least one antigenic peptide encoded by the long non-coding RNA (IncRNA), or immunogenic fragment or van'ant of any of these comprises at least one T-cell epitope, preferably at least one CD8+ T cell epitope.
- the at least one antigenic peptide encoded by the long non-coding RNA (IncRNA), or immunogenic fragment or variant of any of these induce epitope-specific T cells in a subject, preferably epitope specific CD8+ T cells in a subject (in particular, a human subject).
- the at least one antigenic peptide encoded by the long non-coding RNA Inc- WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2 ⁇ S1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, or immunogenic fragment or variant of any of these induce epitope-specific T cells in a subject, preferably epitope specific CD8+ T cells in a subject (in particular, a human subject).
- T ⁇ ;ell epitope refers to a part or fragment of a protein or antigen that is recognized by a T cell when presented in the context of major histocompatibility complex (MHC) molecules, includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates.
- MHC proteins or molecules are important for signalling between tymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells.
- the proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-setf-antigens (e.g., fragments of invading microorganisms) to a T cell.
- self-antigens peptide fragments from the cell itself
- non-setf-antigens e.g., fragments of invading microorganisms
- the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective.
- binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas also longer and shorter peptides may be effective.
- the at least one antigenic peptide encoded by the IncRNA, or immunogenic fragment or variant thereof is an antigen or epitope displaying hlLA binding affinity or MhlC binding affinity.
- HLA human leukocyte antigen
- HLA human leukocyte antigen
- Human leukocyte antigen hlLA
- MHC major histocompatibility complex
- HLA genes are highly polymorphic, i.e. having may different alleles, which allows them to fine-tune the adaptive immune system of a subject.
- hlLA binding affinity or “MHC binding affinity” is to be understood as affinity of binding between a specific antigen and a specific MhlC allele.
- HLA type is to be understood as the complement of HLA gene alleles.
- the at least one antigenic peptide encoded by the IncRNA as specified herein is capable of binding to an antibody or a T-cell receptor.
- the antigenic peptide comprises at least one MhlCI or MHCII ligand.
- the at least one antigenic peptide encoded by a IncRNA as defined herein is a shared tumour associated antigen, in particular, a shared tumour associated antigen in NSCLC and/or melanoma.
- the at least one antigenic peptide (provided by the artificial nucleic acid) is seleded from a peptide or protein encoded by a long non-coding RNA (IncRNA) as defined herein and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-254,546-650,756-763, or an immunogenic fragment or variant of any of these.
- IncRNA long non-coding RNA
- the at least one antigenic peptide preferably comprises at least one amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-254,546-650,756-763, or an immunogenic fragment or variant of any of these.
- an immunogenic fragment of SEQ ID NOs: 124-254,546-650,756-763 has a shorter amino acid sequence as the respective reference sequence.
- an immunogenic fragment of SEQ ID NOs: 124-254, 546-650,756-763 is N-terminally truncated by e.g.1,5,10, or more amino acids and/or C-terminally truncated e.g.1,5, 10, or more amino acids.
- an immunogenic fragment still comprises at least one epitope, preferably at least one T-cell epitope, capable of raising an immune response.
- the at least one epitope has the size/length of an MhlC class I or class II epitope.
- the at least one epitope has the length of an MHC class I epitope.
- the at least one epitope has the length of an MHC class II epitope but comprises at least one MhlC class I epitope and could be processed during antigen processing during MHC:peptide complex loading.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide compnses an amino acid sequence such as a peptide or protein encoded by lnc-WDR72-2:4 (smORF lncWDR722416421696 orsmORF lncWDR722415641696), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA lnc-WDR72-2:4 comprises several predicted epitopes according to SEQ ID NOs 125-143,145-146, 546-571. Accordingly, the full-length polypeptides (smORF lncWDR722416421696 or lncWDR722415641696 according to SEQ ID NO: 124 or 144)or any fragment or variant thereof comprising epitopes as defined herein are particularly useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- WDR72-2:4 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 124-146,546-571, 756,757, or an immunogenic fragment or immunogenic van'ant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid 10 sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-146,546-571 ,756,757, or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 124-146,546-571,756,757, or a C-terminal truncation of SEQ ID NOs: 124-146,546-571,756,757.
- the immunogenic fragment of SEQ ID NOs: 15124-146,546-571,756,757 is N-terminally truncated by 1,2, 3, or4 amino acids and/or C-terminalty truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 124-146,546-571 , 756,757 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino adds, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by 20 lnc-WDR72-2:4 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 756, or an immunogenic fragment or immunogenic variant thereof.
- the at least one antigenic peptide comprises an amino acid sequence such as a peptide or protein 25 encoded by lnc-TRPC5-3;1 (smORF lncTRPC53116031801), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA TRPC5-3:1 comprises several predicted epitopes according to SEQ ID NOs 228-237,630-642.
- the full-length polypeptide (smORF lncTRPC53116031801 according to SEQ ID NO: 227) or any fragment or variant thereof comprising epitopes as defined herein are particularly useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- TRPC5-3:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 227-237,630-642,761, or an immunogenic fragment or immunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid 35 sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 227-237,630-642,761 , or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-terminal truncation SEQ ID NOs: 227-237,630-642, 761, or a C-terminal truncation of SEQ ID NOs: 227-237,630-642,761.
- the immunogenic fragment of SEQ ID NOs: 227- 237,630-642,761 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-terminally truncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 227-237, 630-642,761 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- TRPC5-3:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 761, or an immunogenic fragment or immunogenic vanant thereof.
- the at least one antigenic peptide comprises an amino acid sequence such as a peptide or protein encoded by lnc-CLEC2D-9:1 (smORF lncCLEC2D91412616), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA lnc-CLEC2D-9:1 comprises several predicted epitopes according to SEQ ID NOs 239-243,643-650.
- the full-length polypeptide (smORF lncCLEC2D91412616 according to SEQ ID NO: 238) or any fragment or variant thereof comprising epitopes as defined herein are particularly useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- CLEC2D-9:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 238-243,643-650, 762, or an immunogenic fragment or immunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 238-243,643-650,762, or an immunogenic fragment or immunogenic variant of any of these.
- An immunogenic fragment in that context may be an N-terminal truncation of SEQ ID NOs: 238-243,643-650,762, or a C-terminal truncation of SEQ ID NOs: 238-243,643-650,762.
- the immunogenic fragment of SEQ ID NOs: 238-243,643-650,762 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 238-243,643-650,762 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- CLEC2D-9:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 762, or an immunogenic fragment or immunogenic variant thereof.
- the at least one antigenic peptide comprises an amino acid sequence such as a peptide or 5 protein encoded by KCNMB2-AS1 :4 (smORF KCNMB2AS14633753), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA KCNMB2-AS1 :4 comprises several predicted epitopes according to SEQ ID NOs 148-161,572-589. Accordingly, the full-length polypeptide (smORF KCNMB2AS14633753 according to SEQ ID NO: 147) or any fragment or variant thereof comprising epitopes as defined herein are particulariy useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by KCNMB2- AS1:4 and compnses or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 147-161,572-589, 758, or an immunogenic fragment or immunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid 15 sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 147-161,572-589, 758, or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 147-161,572-589,758, or a C-tenninal truncation of SEQ ID NOs: 147-161,572-589,758.
- the immunogenic fragment of SEQ ID NOs: 147- 20161,572-589,758 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-terminally truncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 147-161 , 572-589,758 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by 25 KCNMB2-AS1:4 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 758, or an immunogenic fragment or immunogenk; van'ant of any of these.
- the at least one antigenic peptide comprises an amino acid sequence such as a peptkle or 30 protein encoded by lnc-ZC3H8-6:1 (smORF lncZC3H86163144), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA lnc-ZC3H8-6:1 comprises several predicted epitopes according to SEQ ID NOs 163-168,590-603.
- the full-length polypeptide (smORF lncZC3H86163144 according to SEQ ID NO: 162) or any fragment or variant thereof comprising epitopes as defined herein are particularly useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by Inc- ZC3H8-6:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 162-168,590-603,759, or an immunogenic fragment or immunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 162-168, 590-603,759, or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 162-168, 590-603,759, or a C-terminal truncation of SEQ ID NOs: 162-168.
- the immunogenic fragment of SEQ ID NOs: 162-168,590-603, 759 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-teminally truncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 162-168,590-603, 759 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by lnc-ZC3hl8-6:1 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 759, or an immunogenic fragment or immunogenic variant thereof.
- the at least one antigenic peptide comprises an amino acid sequence such as a peptide or protein encoded by lnc-NTF3-5:5 (smORF lncNTF35511951417), or an immunogenic fragment or variant thereof.
- the full-length polypeptides that are encoded by the IncRNA lnc-NTF3-5:5 comprises several predicted epitopes according to SEQ ID NOs 170-226,604-629.
- the full-length polypeptide (smORF lncNTF35511951417 according to SEQ ID NO: 169) or any fragment or variant thereof comprising epitopes as defined herein are particularly useful in the context of the invention.
- the at least one antigenic peptide is selected from a peptide or protein encoded by lnc-NTF3- 5:5 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 169-226,604-629,760, or an immunogenicfragmentorimmunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 169-226,604-629,760, or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 169-226, 604-629,760, or a C-terminal truncation of SEQ ID NOs: 169-226.
- the immunogenic fragment of SEQ ID NOs: 169-226,604-629, 760 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-terminally tmncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 169-226,604-629,760 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein encoded by lnc-NTF3-5:5 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 760, or an immunogenic fragment or immunogenic variant thereof.
- the at least one antigenic peptide compnses an amino acid sequence such as a peptide or protein encoded by LINC00893:25 (LINC00893), or an immunogenic fragment or variant thereof.
- the full-length polypeptide (smORF LINC00893 according to SEQ ID NO: 244) or any fragment or variant thereof comprising epitopes as defined herein are particulariy useful in the context of the invention. Accordingly, the full-length polypeptides that are encoded by the IncRNA LINC00893 (SEQ ID NO: 244) comprises several predicted epitopes according to SEQ ID NOs 245-254.
- the at least one antigenic peptide is selected from a peptide or protein encoded by LINC00893:25 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 244-254,763, or an immunogenic fragment or immunogenic variant of any of these.
- the at least one antigenic peptide preferably comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 244-254,763, or an immunogenic fragment or immunogenic variant of any of these.
- a fragment in that context may be an N-teminal truncation of SEQ ID NOs: 244-254, 763, or a C-terminal truncation of SEQ ID NOs: 244-254, 763.
- the immunogenic fragment of SEQ ID NOs: 244-254,763 is N-tenninally truncated by 1,2,3, or 4 amino acids and/or C-tenninally tmncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment ofSEQ ID NOs: 244-254, 763 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15aminoacids.
- the at least one antigenic peptide is selected from a peptide or protein enasded by LINC00893:25 and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 763, or an immunogenic fragment or immunogenic variant thereof.
- nucleic acid coding sequences are described that encode the herein specified antigenic peptides (encoded by the respective IncRNAs) that may be comprised in the at least one cds of the artificial nucleic acid.
- the at least one coding sequence comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a IncRNA, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 318-448,651 -755,764-771, or a fragment or a variant of any of these.
- a fragment has a length of at least 24 nucleotides.
- the nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a IncRNA is a codon modified cds, preferably wherein codon modified cds is selected from a C maximized cds (as further defined herein), a CAI maximized cds (as further defined herein), human codon usage adapted cds (as further defined herein), a G/C content modified cds (as further defined herein), and a G/C optimized cds (as further defined herein), or any combination thereof.
- the at least one codon modified coding sequence is a G/C optimized coding sequence.
- the at least one coding sequence comprises a GC optimized nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a IncRNA, wherein the nucleic acid sequence is identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 318-448, 651 -755,764-771 , or a fragment of any of these.
- a fragment has a length of at least 24 nucleotides.
- the at least one artificial nucleic acid comprises at least one cds comprising a nucleic acid sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-NTF3-5:5, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 363 ⁇ 20, 709-734,768, or a fragment or a variant of any of these, preferably SEQ ID NO 768, or a fragment or a variant thereof; or at least one cds comprising a nucleic acid sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, wherein the nucleic
- each row corresponds to suitable antigenic peptides or protein encoded by a long non-coding RNA (IncRNA).
- IncRNA RNA
- Column A provides a short description of the respective IncRNA that encodes the tumour antigens.
- Column B provides the SEQ ID NOs of respective full-length amino acid sequences encoded by the IncRNAs.
- Column C provides the amino acid SEQ ID NOs of respective fragments or epitopes.
- Column D provides SEQ ID NO of G/C optimized nucleic acid sequence encoding the full-length amino acid sequence of Column B.
- Column E provides SEQ ID NO of G/C optimized nucleic acid sequences encoding the respective fragments or epitopes of Column C.
- Table 1A Preferred antigens encoded by IncRNA (amino acid sequences and codina sequences) A Particularly preferred amino acid sequences and nucleic acid sequences in that context are provided in Table 1 B. Therein, each row corresponds to a particularly suitable antigenic peptides or protein encoded by a long non-coding RNA (IncRNA). Column A provides a short descn'ption of the respective IncRNA that encodes the tumour antigens.
- Column B provides the SEQ ID NOs of respective full-length amino acid sequence encoded by the IncRNA
- Column C provides the amino acid SEQ ID NOs of preferred fragments or epitopes.
- Column D provides SEQ ID NO of G/C optimized nucleic acid sequence encoding the full-length amino acid sequence of Column B.
- Column E provides SEQ ID NO of G/C optimized nucleic acid sequences encoding the preferred fragments or epitopes of Column C.
- Table 1B Particularly preferred antigens encoded bv IncRNA (amino acid sequences and coding sequences) B
- the at least one antigenic peptide comprises or consists of at least one amino acid sequence selected from Table 1A or 1B, columns B or C, or immunogenic fragments of any of these.
- the at least one coding sequence of the artificial nucleic acid of the invention comprises or consists of at least one nucleic acid sequence as provided in Table 1A or 1 B, columns D or E, or a fragment or variant of any of these.
- Tumour antigens selected from tumour neoantigens In preferred embodiments, the artificial nucleic acid comprises at least one coding sequence encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected or derived from a peptide or protein or a tumour neoantigen, or an immunogenic fragment or variant thereof.
- tumour neoantigen refers to a specific polypeptide generated by gene mutation of a tumour cell, which is presented to the cell surface by MHC molecules, in mammals HLA molecules, and thus recognized by a T lymphocyte to elicit an immune response.
- Neoantigens are therefore suitable to serve as markers for immunotherapy and further used to develop tumour neoantigen vaccines, because these neoantigens are generated by tumour cell mutation but not expressed in normal or healthy cells. Therefore, neoantigens may sen/e as promising targets for immunotherapy.
- the neoantigen is a shared neoantigen, preferably a shared neoantigen for NSCLC and/or melanoma cancer patients.
- the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen is selected from an NRAS neoantigen, TYW1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, DIP2B neoantigen, TRRAP neoantigen, RAC1 neoantigen, AP4B1 neoantigen, VWA5A neoantigen
- NRAS neoantigen For clarification, the terms “NRAS neoantigen”, “TYW1 B neoantigen”, “ECPAS neoantigen”, “MAP2K1 neoantigen”, “TOMM22 neoantigen”, “GLB1 neoantigen”, “MAGE-A3 neoantigen”, “ATAD2 neoantigen”, “BRAF neoantigen”, “EGFR neoantigen”, “TP53 neoantigen”, “DIP2B neoantigen”, “TRRAP neoantigen”, “RAC1 neoantigen”, “AP4B1 neoantigen”, “VWA5A neoantigen”, “CLTC neoantigen”, “RPL9 neoantigen”, “RPAP1 neoantigen”, “RPE65 neoantigen”, “PATZ1 neoantigen”, “PIK3CA ne
- the at least one antigenic peptide selected from a peptide or protein of an NRAS neoantigen, TYW1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, or an immunogenic fragment or variant of any of these.
- the at least one antigenic peptide selected from a peptide or protein of an NRAS neoantigen, TYW1B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, DIP2B neoantigen, TRRAP neoantigen, RAC1 neoantigen, AP4B1 neoantigen, VWA5A neoantigen, CLTC neoantigen, RPL9 neoantigen, RPAP1 neoantigen, RPE65 neoantigen, PATZ1 neoantigen comprises at least one T-cell eptope
- the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, or immunogenic fragment or variant thereof, in particular of an NRAS neoantigen, T/W1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, DIP2B neoantigen, TRRAP neoantigen, RAC1 neoantigen, AP4B1 neoantigen, VWA5A neoantigen, CLTC neoantigen, RPL9 neoantigen, RPAP1 neoantigen, RPE65 neoantigen
- the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, or immunogenic fragment or variant thereof is an antigen or epitope displaying HLA binding or MhlC binding affinity.
- the at least one antigenic peptide of a tumour neoantigen as specified herein bind to an antibody or a T-cell receptor.
- the antigenic peptide comprise an MhlCI or MHCII ligand/epitope.
- the at least one antigenic peptide (provided by the artificial nucleic acid) is selected from a peptide or protein of a tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 255-317, or an immunogenic fragment or variant of any of these.
- the at least one antigenic peptide (provided by the artificial nucleic acid) is selected from a peptide or protein of a tumour neoantigen and compnses or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 255-286, or an immunogenic fragment or variant of any of these.
- an immunogenic fragment of SEQ ID NOs: 255-317 has a shorter amino acid sequence as the respective reference sequence.
- An immunogenic fragment of SEQ ID NOs: 255-317 is N-terminally truncated by e.g.1, 55,10, or more amino acids and/or C-terminally truncated e.g.1,5,10, or more amino acids.
- an immunogenic fragment still comprises at least one epitope, preferably at least one T-cell epitope, capable of raising an immune response.
- the at least one epitope has the size/Iength of an MhlC class I or class II epitope.
- the at least one epitope has the length of an MHC class I epitope.
- the at least one epitope has the length of an MHC class II epitope but comprises at least one MHC class I epitope and could 10 be processed during antigen processing during Mh)C:peptide complex loading.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of an NRAS tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 1585%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 267-272, or immunogenic fragments or variants of any of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a NRAS tumour neoantigen, wherein the antigenic peptide compnses or consists of at least one of the amino acid sequences being identical or at least 80% identical to any 20 one of SEQ ID NOs: 267-272, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 267-272 or a C-terminal truncation of SEQ ID NOs: 267-272.
- the immunogenic fragment ofSEQ ID NOs: 267-272 is N-terminally truncated by 1, 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 267-272 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a 25 typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a TYW1 B tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID 30 NOs:273,274, or an immunogenic fragment or immunogenic variant of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that compnses or consists of at least one antigenic peptide selected from a peptide or protein of a TYW1 B tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 273,274, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 273, 274 or a C-terminal truncation of SEQ ID NOs: 273,274.
- the immunogenic fragment of SEQ ID NOs: 273,274 is N-terminally truncated by 1 , 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino adds.
- An immunogenic fragment of SEQ ID NOs: 273,274 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is seleded from a peptide or protein of a ECPAS tumour 5 neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 275, or an immunogenic fragment or immunogenic variant thereof.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a ECPAS tumour neoantigen, wherein the antigenic 10 peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 275, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 275 or a C-terminal truncation of SEQ ID NOs: 275.
- the immunogenic fragment of SEQ ID NOs: 275 is N-terminally truncated by 1,2, 3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino adds.
- An immunogenic fragment of SEQ ID NOs: 275 15 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein ofaMAP2K1 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 2085%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:276,277,or an immunogenic fragment or immunogenic variant of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that compnses or consists at least one antigenic peptide selected from a peptide or protein of a MAP2K1 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any 25 one of SEQ ID NOs:276,277, or an immunogenic fragment or variant of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 276, 277 or a C-terminal truncation of SEQ ID NOs: 276,277.
- the immunogenic fragment of SEQ ID NOs: 276,277 is N-terminally truncated by 1 , 2,3, or 4 amino acids and/or C-temninally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 276,277 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a TOMM22 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID 35 NOs: 278,279, or an immunogenic fragment or immunogenic vanant of these.
- the artificial nucleic ackJ comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a TOMM22 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 278,279, or an immunogenic fragment or variant of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 278, 279 or a C-terminal truncation of SEQ ID NOs: 278,279.
- the immunogenic fragment of SEQ ID NOs: 278,279 is N-terminally truncated by 1 , 52,3, or 4 amino acids and/or C-terminally truncated by 1,2,3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 278,279 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a GLB1 tumour 10 neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:280,281,or an immunogenic fragment or immunogenic variant of any of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a GLB1 tumour neoantigen, wherein 15 the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 280,281, or an immunogenic fragment or variant of these.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 280, 281 or a C-terminal truncation of SEQ ID NOs: 280,281.
- the immunogenic fragment of SEQ ID NOs: 280, 281 is N-terminally tmncated by 1, 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID 20 NOs: 280,281 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a MAGE-A3 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 2585%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:282,283,or an immunogenic fragment or immunogenic variant of these.
- the artificial nucleic ackl comprises at least one cds encoding at least one tumour antigen that compnses or consists of at least one antigenic peptide selected from a peptide or protein of a MAGE-A3 tumour neoantigen, wherein the antigenic peptide compnses or consists of at least one of the amino acid sequences being identical or at least 80% identical to any 30 one of SEQ ID NOs:282,283, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 282, 283 or a C-terminal truncation of SEQ ID NOs: 282,283.
- the immunogenic fragment of SEQ ID NOs: 282,283 is N-terminally truncated by 1 , 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 282,283 still compnses at least one epitope, preferably a T-cell epitope, capable of raising an immune response. 35
- a typical immunogenic fragment in that context has the see of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a ATAD2 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 284-286, or an immunogenic fragment or immunogenic vanant of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a ATAD2 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 284-286, or an immunogenic fragment or van'ant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 284-286 or a C-terminal truncation of SEQ ID NOs: 284-286.
- the immunogenic fragment ofSEQ ID NOs: 284-286 is N-terminally truncated by 1, 2,3, or 4 amino acids and/or C-terminally tmncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 284-286 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- a typical immunogenic fragment in that context has the size of 5 to 20 amino acids, or 8 to 15 amino acids.
- the at least one antigenic peptide is selected from a peptide or protein of a BRAF tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 262-266, or an immunogenic fragment or immunogenic van'ant of any of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a BRAF tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 262-266, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 262-266 or a C-temninal truncation of SEQ ID NOs: 262-266.
- the immunogenic fragment of SEQ ID NOs: 262-266 is N-terminally truncated by 1, 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 262-266 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- the at least one antigenic peptide is selected from a peptide or protein of a EGFR tumour neoantigen and compnses or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 255-257, or an immunogenic fragment or immunogenic variant of any of these.
- the artificial nucleic ackl comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a EGFR tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 255-257, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 255-257 or a C-terminal truncation of SEQ ID NOs: 255-257.
- the immunogenic fragment ofSEQ ID NOs: 255-257 is N-terminally truncated by 1, 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 255-257 still comprises at least one epitope, preferably a T ⁇ ;ell epitope, capable of raising an immune response.
- the at least one antigenic peptide is selected from a peptide or protein of a TP53 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 258-261, or an immunogenic fragment or immunogenic vanant of any of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a TP53 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 258-261, or an immunogenic fragment or van'ant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 258-261 or a C-terminal truncation of SEQ ID NOs: 258-261.
- the immunogenic fragment of SEQ ID NOs: 258-261 is N-terminally truncated by 1 , 2, 3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 258-261 still comprises at least one epitope, preferably a Toll epitope, capable of raising an immune response.
- the at least one antigenic peptide is selected from a peptide or protein of a DIP2B, TRRAP, RAC1, AP4B1,VWA5A, CLTC, RPL9, RPAP1, RPE65, PATZ1, PIK3CA, NFE2L2, or CNOT9 tumour neoantigen and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 287- 317, or an immunogenic fragment or immunogenic variant of any of these.
- the artificial nucleic acid comprises at least one cds encoding at least one tumour antigen that comprises or consists of at least one antigenic peptide selected from a peptide or protein of a DIP2B, TRRAP, RAC1, AP4B1, VWA5A, CLTC,RPL9, RPAP1, RPE65, PATZ1 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 287-317, or an immunogenic fragment or variant thereof.
- a fragment in that context may be an N-terminal truncation of SEQ ID NOs: 287-317 or a C-terminal truncation of SEQ ID NOs: 287-317.
- the immunogenic fragment ofSEQ ID NOs: 287-317 is N-terminally truncated by 1, 2,3, or 4 amino acids and/or C-terminally truncated by 1,2, 3, or 4 amino acids.
- An immunogenic fragment of SEQ ID NOs: 287-317 still comprises at least one epitope, preferably a T-cell epitope, capable of raising an immune response.
- nucleic acid coding sequences are described that encode the herein specified antigenic peptides of the tumour neoantigen as defined herein that may be comprised in the at least one coding sequence of the artificial nucleic acid of the invention.
- the at least one coding sequence compnses a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-511, or a fragment or a van'ant of any of these.
- a fragment has a length of at least 24 nucleotides.
- the at least one coding sequence comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-480, or a fragment or a variant of any of these.
- a fragment has a length of at least 24 nucleotides.
- the nucleic acid sequence encoding at least one antigenic peptide from a tumour neoantigen is a codon modified cds, preferably wherein codon modified cds is selected from a C maximized cds (as defined herein), a CAI maximized cds (as defined herein), human codon usage adapted cds (as defined herein), a G/C content modified cds (as defined herein), and a G/C optimized cds (as defined herein), or any combination thereof.
- the at least one codon modified cds is a G/C optimized cds.
- the at least one coding sequence compnses a GC optimized nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-511, or a fragment of any of these.
- a fragment has a length of at least 24 nucleotides.
- Preferred amino acki sequences and nucleic acid sequences in that context are provided in Table 2.
- each row corresponds to a suitable antigenic peptides of a tumour neoantigen.
- Column A provides a short descnption of the respective tumour neoantigen.
- Column B provides protein (amino acid sequence) SEQ ID NOs of preferred antigenic peptides of the tumour neoantigens.
- Column C provides SEQ ID NO of the corresponding G/C optimized nucleic acid sequences encoding the preferred antigenic peptides of the tumour neoantigens.
- the at least one antigenic peptide comprises or consists of at least one amino acid sequence selected from Table 2, column B, or immunogenic fragments of any of these.
- the at least one antigenic peptide comprises or consists of at least one amino acid sequence selected from Table 2, column B, rows 1 to 11, or immunogenic fragments of any of these.
- the at least one coding sequence of the artificial nucleic acid of the invention comprises or consists of at least one nucleic acid sequence as provided in Table 2, column C.
- the at least one coding sequence of the artificial nucleic acid of the invention comprises or consists of at least one nucleic acid sequence as provided in Table 2, column C, rows 1 to 11, or immunogenic fragments thereof. Further peptide or protein features
- the at least one coding sequence encodes 2 to 10 different tumour antigens as specified herein, preferably 2 to 5 different tumour antigens as specified herein.
- the coding sequence may encode multiple tumour antigens that are provided in form of a fusion construct.
- the respective different tumour antigens are selected to obtain an increased coverage in a certain cancer disease, e.g. NSCLC, HNSCC, or melanoma.
- the coverage of all the different tumour antigens is at a value of above 0.5, preferably above 0.6, more preferably above 0.7.
- a “different tumour antigen” in the context of the invention relates to a difference on amino acid sequence level and has to be understood as a difference in at least one amino acid position or having a different amino acid sequence length. Accordingly, a “different tumour antigen” may be derived from the same tumour neoantigen but the "different tumour antigen” should differ in at least one amino acid position or length.
- the at least one coding sequence may encode 2 to 10 different tumour antigens, preferably 2 to 5 different tumour antigens, wherein the different tumour antigens are selected from a peptide or protein encoded by a long non- coding RNA (IncRNA) as defined herein and/or from a peptide or protein of a tumour neoantigen as defined herein, or an immunogenic fragment or variant of any of these.
- Suitable amino acid and nucleic acid sequences in that context are defined above and may inter alia be taken from Table 1A and B and Table 2.
- the different tumour antigens are selected to cover different HLA types.
- the different tumour antigen are selected from at least one tumour antigen as defined herein that comprises at least one epitope that binds to HLA-A type, at least one tumour antigen as defined herein that comprises at least one epitope binds to HLA-B type, at least one tumour antigen as defined herein that comprises at least one epitope binds to HLA-Ctype.
- the different tumour antigens are separated by a linker element.
- the coding sequence may encode multiple different tumour antigens that are provided in form of a fusion construct, wherein the different tumour antigens in the fusion construct are separated by linker elements. Suitably, such linkers are non-immunogenic.
- Suitable linker elements may be selected from SEQ ID NOs: 2937, 76400-76418,77018-77058 of published PCT patent application W02019008001.
- the at least one cds encodes at least one additional amino acid sequence selected from at least one immune response activating signal transduction protein, preferably at least one transmembrane domain and/or at least one cytoplasmic domain derived from said at least one immune response adivating signal transduction protein.
- said immune response activating signal transduction protein is located in the external side of plasma membranes and readily and recurrently internalized to the endosomal pathways, preferably to target the encoded and translated antigens to desired intracellular pathways, which preferably intersect MHC class I and in particular also MhtC class II pathways, for effective delivery ofantigenic sequences to MHC class I and MhlC class II processing compartments (as described in W02019008001 and fully cooperated herein by reference).
- the immune response activating signal transduction protein is selected from CTLA4.
- the at least one immune response activating signal transduction protein comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NO: 76636 of published PCT patent application W02019008001 or a fragment or variant of any of these.
- the at least one coding sequence encodes at least one additional amino acid sequence selected from at least one T-helper epitope. Suitable T-helper epitopes may be selected from SEQ ID NOs: 3083-3294 of published PCT patent application W02019008001.
- the at least one coding sequence encodes at least one additional amino acid sequence selected from at least one signal peptide.
- Suitable signal peptides may be selected from ofSEQ ID NOs: 1-156,76948-76951 of published PCT patent application W02019008001.
- the at least one signal peptkle is selected from CTLA4. and embodiments
- suitable features and embodiments referring to artificial nucleic acid sequences of the invention are provided and described in detail (e.g. type of nucleic acid, structure of nucleic acid, elements of nucleic acid, modification of nucleic acid etc.).
- said features defining nucleic acid sequences may also apply to any nucleic acid sequence in any other aspect of the invention (e.g.
- Suitable coding sequences the artificial nucleic acid of the invention compnses at least one coding sequence encoding at least one tumour antigen as defined herein.
- any coding sequence encoding at least one tumour antigen as defined herein, or fragments and variants thereof may be understood as suitable coding sequence and may therefore be comprised in the nucleic acid of the invention.
- the artificial nucleic acid is a modified and/or stabilized nucleic acid.
- the artificial nucleic acid may thus be provided as a "stabilized nucleic acid” that is to say a nucleic acid showing improved resistance to in vivo degradation and/or a nucleic acid showing improved stability in vivo, and/or a nucleic acid showing improved translatability in vivo.
- the nucleic acid is an RNA.
- the artificial nucleic of the present invention may be provided as a "stabilized nucleic acid", preferably a “stabilized RNA”.
- suitable modifications/adaptations are described that are capable of "stabilizing" the nucleic acid, preferably the RNA.
- the artificial nucleic acid comprises at least one codon modified cds.
- the amino acid sequence encoded by the at least one codon modified coding sequence is not being modified compared to the amino acid sequence encoded by the corresponding wild type or reference coding sequence.
- the term "codon modified coding sequence” relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type or reference coding sequence.
- a codon modified cds in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo.
- the at least one cds is a codon modified cds, wherein the codon modified cds is selected from a C maximized cds (as further defined in W02021239880 [p.122, lines 33 to 39] which is hereby incorporated by reference); a CAI maximized cds (as further defined in W02021239880 [p.123, lines 33 to 44] which is hereby incorporated by reference); a human codon usage adapted cds (as further defined in W02021239880 [p.123, lines 7 to 17] which is hereby incorporated by reference); a G/C content modified cds (as further defined in W02021239880 [p.123, lines 19 to 31] which is hereby hereby a
- the nucleic acid comprising the codon modified coding sequence When transfected into mammalian host cells, the nucleic acid comprising the codon modified coding sequence has a stability of between 12-18 hours, or greater than 18 hours, e.g., 24,36, 48,60,72, or greater than 72 hours and is capable of being expressed by the mammalian host cell.
- the artificial nucleic acid comprising the codon modified coding sequence 5 is translated into protein, wherein the amount of protein is at least comparable to, or preferably at least 10% more than, or at least 20% more than, or at feast 30% more than, or at least 40% more than, or at least 50% more than, or at least 100% more than, or at least 200% or more than the amount of protein obtained by a naturally occurring or wild type or reference coding sequence transfected into mammalian host cells.
- the artificial nucleic acid may be modified, wherein the G/C content of the at least 10 one coding sequence may be optimized compared to the G/C content of the corresponding wild type or reference coding sequence ("G/C optimized coding sequence").
- Optimized in that context refers to a coding sequence wherein the G/C content is preferably increased to the essentially highest possible G/C content.
- the generation of a G/C content optimized nucleic acid sequence may be carried out using a method according to W02002098443. In this context, the disclosure ofW02002098443 is included in its full scope in the present invention.
- the at least one codon modified coding sequence is a G/C optimized coding sequence.
- Such G/C optimized nucleic acid sequences are provided in Table 1Aor 1 B or Table 2.
- the at least one cds of the artificial nucleic acid, preferably the RNA has a G/C content of at least about 50%, 55%, or 60%.
- the at least one cds of the nucleic acid has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 2068%, 69%, or 70%.ln particulariy preferred embodiments, the artificial nucleic acid may be modified, wherein the G/C content of the at least one cds may be optimized compared to the G/C content of the corresponding wild type or reference cds ("G/C optimized coding sequence").
- the artificial nucleic acid of the invention preferably the RNA, comprises at least one untranslated region (UTR) or UTR element.
- UTR untranslated region
- the terms "untranslated region” or “UTR” or “UTR element' refer to a part of a nucleic acid molecule typically located 5' 30 or 3' of a coding sequence.
- An UTR is not translated into protein.
- An UTR may be part of the nucleic acid, e.g. an RNA.
- An UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites, promotor elements etc.
- the artificial nucleic acid compnses a coding sequence and a 5'-UTR and/or 3-UTR.
- UTRs may ha ⁇ iour regulatory sequence elements that determine RNA turnover, stability, and bcalization.
- 35 UTRs may harbour sequence elements that enhance translation. In medical applications, translation of the nucleic acid into at least one peptide or protein is of paramount importance to therapeutic efficacy.
- the artificial nucleic acid comprises at least one untranslated region (UTR) preferably selected from at least one 55'-UTR and/or at least one 3'-UTR.
- UTR untranslated region
- Said 5'-UTRs or 3'-UTRs may be derived from naturally occurring genes or may be synthetically engineered.
- the artificial nucleic acid compnses at least one coding sequence as defined herein operably linked to at least one 3'-UTR and/or at least one 5'-UTR.
- the at least one untranslated region (UTR) is selected from at least one heterologous 5-UTR and/or at least one heterologous 3'-UTR.
- the term "heterologous" sequence or UTR as used herein is intended to refer to a nucleic acid sequence or UTR that is not from the same gene or the same genomic fusion or the same naturally occurring transcript (e.g. IncRNA). Accordingly, heterologous sequences or UTRs may be derivable from the same organism (e.g. human) or from a different organism.
- the artificial nucleic acid of the invention comprises at least one 3-UTR.
- the terms "S'-untranslated region" or "3'-UTR” refer to a part of a nucleic acid located 3' (i.e. downstream) of a cds and which is not translated into protein.
- a S'-UTR may be part of a nucleic acid tocated between a cds and an (optional) terminal poly(A) sequence.
- a 3'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites.
- the artificial nucleic acid comprises at least one 3'-UTR, which may be derivable from a gene that relates to 20 an RNA with enhanced hatf-life (i.e. that provides a stable RNA).
- the S'-UTR comprises one or more of a polyadenylation signal, a binding site for proteins that affect a nucleic acid stability of location in a cell, or one or more miRNA or binding sites for miRNAs.
- the artificial nucleic acid comprises at least one 3'-UTR, wherein the at least one 3'-UTR comprises or consists of a nucleic acid sequence derived or selected from a 3-UTR of a gene selected from PSMB3, 25 ALB7, alpha-globin, beta-globin, ANXA4, CASP1, COX6B1, FIG4, GNAS, NDUFA1, RPS9, SLC7A3 or TUBB4B, or from a homolog, a fragment, or variant of any one of these genes.
- the at least one S'-UTRthat is derived or selected from PSMB3, ALB7, alpha-globin, beta- globin, ANXA4, CASP1, COX6B1 , FIG4, GNAS, NDUFA1, RPS9, SLC7A3 or TUBB4B comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 3095%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 66-95,112-123, or a fragment or a variant of these.
- the at least one 3-UTR comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 96-111, or a fragment or a variant of any of these.
- the artificial nucleic acid comprises a 3'-UTR derived or selected from a PSMB3 gene, wherein the at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 66,67, 112-123, preferably SEQ ID NO: 67, or a fragment or a van'ant of any of these.
- the artificial nucleic acid of the invention comprises at least one 5'-UTR.
- S'-untranslated region refers to a part of a nucleic acid located 5' (i.e. "upstream") of a cds and which is not translated into protein.
- a 5-UTR may be part of a nucleic acid bcated 5' of the cds.
- a 5'-UTR starts with the transcriptional start site and ends before the start codon of the cds.
- a 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding 10 sites, miRNA binding sites etc.
- the 5'-UTR may be modified, e.g.
- the artificial nucleic acid compnses at least one 5'-UTR, which may be derivable from a gene that relates to an RNA with enhanced hatf-life (i.e. that provides a stable RNA).
- the S'-UTR comprises one or more of a binding site for proteins that affect a nucleic acid stability 15 or nucleic acid location in a cell, or one or more miRNA or binding sites for miRNAs (as defined above).
- the artificial nucleic acid comprises at least one 5'-UTR, wherein the at least one S'-UTR comprises a nucleic acid sequence derived or selected from a 5'-UTR of gene selected from HSD17B4, RPL32, AIG1, 20 alpha-globin, ASAH1, ATP5A1 , COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31, RPL35A, SLC7A3, TUBB4B, or UBQLN2, or from a homolog, a fragment or variant of any one of these genes,
- the at least one 5'-UTR comprises or consist of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 46-63, or a fragment or a variant of any of these.
- the nucleic acid comprises a 5'-UTR derived or selected from a HSD17B4 gene wherein the at least one heterologous 5-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12,13,64, 65, preferably SEQ ID NO: 13, or a fragment or a variant of any of these.
- the at least one nucleic acid compnses at least one cds operably linked to a 3'- UTR element and/or a S'-UTR element selected from the S'-UTR/S'-UTR combinations (S'UTR/3'UTR) provided in W02021239880 [p.127, line 35 to p.128, line 2], which is hereby incorporated by reference.
- the at least one heterologous 5-UTR is selected from HSD17B4 and the at least one heterologous 3' UTR is selected from PSMB3.
- the artificial nucleic acid preferably the RNA compnses at least one cds as defined herein encoding at least one tumour antigen as defined herein, wherein said cds is operably linked to a HSD17B45'-UTR and a PSMB33'-UTR (HSD17B4/PSMB3 (a-1)). It has been shown by the inventors that this embodiment is particularly beneficial for expressing the tumour antigen in human cells.
- the nucleic acid e.g. the RNA, is monocistronic, bicistronic, or mutticistronic.
- the nucleic acid, preferably the RNA is monocistronic.
- the artificial nucleic acid comprises a ribosome binding site, also referred to as "Kozak sequence", identical to or at least 80%, 85%, 90%, 95% identical to any one of SEQ ID NOs: 1 or 2, or sequences GCCGCCACC (DMA), GCCGCCACC (RNA), GCCACC (DNA), GCCACC (RNA), ACC (DNA) or ACC (RNA), or fragments or variants of any of these.
- the "Kozak sequence” comprises or consists of RNA sequence ACC.
- DNAand RNA constructs In particularly preferred embodiments, the artificial nucleic acid of the invention is an isolated nucleic acid.
- isolated nucleic acid does not comprise a cell or a subject that comprises said nucleic acid but relates to the artificial nucleic acid as an isolated molecule or ensemble of isolated molecules.
- the "isolated nucleic acid” can be an artificial nucleic acid isolated or purified from a cell (e.g. cell curture, bacterial culture), or can be an artificial nucleic acid (e.g. RNA) isolated from an RNA in vitro transcription.
- the artificial nucleic acid of the invention is a therapeutic nucleic acid. Accordingly, the artificial nucleic acid is suitably used in a therapeutic context, in particular to provide a therapeutic modality for providing at least one tumour antigen according to the invention.
- the artificial nucleic acid of the invention is selected from a DNA or an RNA. In embodiments, the artificial nucleic acid of the invention is selected from a DMA.
- the DNA may be any type of DNA that comprises a cds as defined herein including any type of single stranded DNA, any type of double stranded DNA, any type of linear DMA, and any type of circular DNA.
- a suitable DNA in the context of the invention may be selected from bacterial plasmid, an adenovims, a poxvims, a parapoxivirus (orf virus), a vaccinia virus, a fowlpox virus, a herpes virus, an adeno-associated virus (AAV), an alphavirus, a lentivirus, a lambda phage, a tymphocytic choriomeningitis virus and a Listeria sp, Salmonella sp.
- the DNA a viral DMA, preferably an MV DNA.
- the artificial nucleic acid of the invention is an RNA.
- the RNA may be any type of RNA that comprises a cds as defined herein including any type of single stranded RNA, double stranded RNA, linear RNA, and circular RNA.
- the RNA may be any type of RNA that comprises a cds as defined herein including any type of single stranded RNA, any type of double stranded RNA, any type of linear RNA, and any type of circular RNA, wherein the RNA is not a IncRNA.
- the RNA is selected from mRNA, circular RNA, replicon RNA or setf-replicating RNA, or viral RNA, preferably mRNA or a circular RNA.
- the RNA is not a IncRNA.
- the artificial nucleic acd of the invention is not a IncRNA selected from lnc-WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, lnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, which means that the RNA of the invention may of course comprise a coding sequence encoding a peptide encoded by lnc-WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1 :4, tnc-ZC3H8-6:1, lnc-NTF3-5:5, or LINC00893:25, but the RNA of the invention is not identical in sequence and length to lnc-V ⁇ DR72-2:4, lnc-TRPC5- 3:1,
- the RNA is a circular RNA.
- a "circular RNA” (circRNAs) is an RNA connected to form a circle and therefore does not comprise a 3' or 5' terminus. Said circRNA comprises at least one cds as defined herein.
- circRNA construct designs can be taken from W02023073228, claims 1 to 51, hereby incorporated by reference.
- the RNA is a replicon RNA or setf-replicating RNA.
- Such constructs may encode replicase elements derived from e.g. alphaviruses (e.g. SR/, SIN, VEE, or RRV) and a cds as defined herein.
- the RNA is selected from an mRNA.
- the artificial nucleic acid of the invention is an mRNA, suitably an isolated mRNA.
- An mRNA is preferred in the context of the invention to provide tumour antigens according to the invention because mRNA allows for regulated dosage, transient expression, complete degradation of the mRNA after protein synthesis, and do not pose the risk of insertional mutations.
- the artificial nucleic acid, preferably the RNA comprises about 50 to about 20000 nudeotides, or about 500 to about 10000 nucleotides, or about 1000 to about 10000 nucleotides, or preferably about 1000 to about 5000 nucleotides or about 2000 to about 5000 nucleotides.
- the artificial nucleic acid comprises at least one poly(N) sequence, e.g. at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or combinations thereof.
- the artificial nucleic acid e.g. the RNA, comprises at least one poly(A) sequence.
- the artificial nucleic acid comprises least two, three, or more poly(A) sequences.
- the terms "poly(A) sequence” or "poly(A) tail” refer to a sequence up to 1000 adenosines typically located at the 3'-end of a linear RNA.
- a poly(A) sequence is homopolymeric.
- a poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine.
- the at least one poly(A) sequence may comprise about 20 to about 500 adenosine nucleotides, about 40 to about 250 adenosine nucleotides, about 60 to about 250 adenosine nucleotides, preferably about 60 to about 150 adenosine nucleotides.
- the length of the poly(A) sequence may be at least about or even more than about 50, 64,75,100,200, 300,400, or 500 adenosines, preferably consecutive adenosine nucleotides.
- the at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), preferably about 100 consecutive adenosine nucleotides.
- the at least one nucleic acid comprises at least one interrupted poty(A) sequence, wherein the poly(A) sequence is intermpted by non-adenosine nucleotides, preferably by about 10 non-adenosine (N10) nucleotides.
- a poly(A) sequence A30-N10-A70 is preferred.
- the poly(A) sequence as defined herein may be located directly at the 3' terminus of the artificial nucleic acid, preferably the RNA.
- the 3'-terminal nucleotide (that is the last 3-terminal nucleotide in the potynucleotide chain) is the S'-terminal A nucleotide of the at least one poly(A) sequence.
- the term "directly located at the 3' terminus" has to be understood as being located exactly at the 3' terminus - in other words, the 3' terminus of the nucleic acid consists of a poly(A) sequence terminating with an A.
- having a 3' terminus ending on an adenosine nucleotide decreases the induction of interferons, e.g. IFNalpha, by the RNA of the invention if for example administered as a medicament to a human.
- interferons e.g. IFNalpha
- the artificial nucleic acid of the invention e.g.
- the RNA comprises a poly(A) sequence of about 100 consecutive adenosine nucleotides, wherein said poty(A) sequence is located directly at the 3' terminus of the RNA, optionally wherein the 3' terminal nucleotide is an adenosine
- the artificial nucleic acid e.g. the RNA
- hSL in the context of the invention may be located in the 3' region.
- histone stem-loop hSL refers to a nucleic acid sequences that forms a stem-loop secondary structure predominantly found in histone mRNAs.
- Histone stem-loop sequences/structures may suitably be selected from hSL sequences as disclosed in W02012019780.
- a hSL sequence that may be used herein may be derived from formulae (I) or (II) of W02012019780.
- the artificial nucleic acid may comprise at least one hSL sequence derived from the specific formulae (la) or (lla)ofW02012019780.
- the at least one histone stenn-loop sequence comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3 or 4, or a fragment or variant of any of these.
- the histone stem-loop sequence comprises or consists of a nucleic acid sequence according to SEQ ID NO: 4, or a fragment or thereof.
- the artificial nucleic acid comprises a 3'-terminal sequence element. The 3-terminal sequence element represents the 3' terminus of the RNA.
- a S'-terminal sequence element may comprise at least one poly(N) sequence as defined herein and, optionally, at least one hSL as defined herein.
- the artificial nucleic acid comprises at least one S'-terminal sequence element compnsing or consisting of an RNA sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-11, or a fragment or variant of these sequences.
- the artificial nucleic acid comprises a 3'-terminal sequence element comprising a hSL as defined herein followed by a poly(A) sequence comprising about 100 consecutive adenosines.
- the artificial nucleic acid comprises a S'-terminal sequence element comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or 6, or a fragment or van'ant thereof.
- the artificial nucleic acid comprises a S'-terminal sequence element comprising or consisting of a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequences AGGAGA, GGGAGA, GGGAAA, AGAAUA, AGAUUA, GAUGGG or GGGCG, or a fragment or variant of these sequences, preferably AGGAGA.
- a S'-terminal sequence element may comprise e.g. a binding site for T7 RNA polymerase.
- the first nucleotide of said S'-terminal start sequence may preferably comprise a 2'0 methylation, e.g.2'0 methylated guanosine or a 2'0 methylated adenosine.
- Modified nudeotides In various embodiments, the artificial nucleic acid, preferably the RNA, is modified, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.
- a modified nucleic acid or RNA may compnse nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications.
- a backbone modification in the context of the invention is a modification in which phosphates of the backbone of the nucleotides of the RNA are chemically modified.
- a sugar modification in the context of the invention is a chemical modification of the sugar of the nudeotides of the RNA.
- a base modification in the context of the invention is a chemical modification of the base moiety of the nucleotides of the RNA.
- nudeotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation. Accordingly, in preferred embodiments, the nucleic acid, preferably the RNA of the invention comprises at least one modified nudeotide.
- the at least one modified nucleotide is selected from pseudouridine, Nl-methylpseudouridine, Nl-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thicKJihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 ⁇ nethyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.
- the nucleic acid is an RNA that comprises at least one modified nucleotide, preferably a modified nucleotide selected from pseudouridine ( ⁇ i) or N1 -methylpseudouridine (m1 i
- essentially all, e.g. essentially 100% of the uracil in the coding sequence (or the full nucleic ack) sequence have a chemical modification, preferably a chemical modification in the 5-position of the uracil.
- 100% of the uracil in the full nucleic acid sequence preferably the RNA sequence are substituted with a modified nucleotide, preferably Nl-methylpseudouridine (m1 ⁇ y).
- 100% of the uracil in the full nucleic acid sequence, preferably the RNA sequence are substituted with pseudouridine (i(J).
- the artificial nucleic acid, preferably the RNA does not comprise chemically modified nucleotides.
- a 5'-cap structure as defined below is typically not considered to be a chemically modified nucleotide.
- the artificial nucleic acid preferably the RNA, comprises a sequence that consists only of G, C, A and U nucleotides and therefore does not comprise modified nucleotides, and optionally comprises a S'-cap structure.
- RNA constructs that do not comprise chemically modified nucleotides may be beneficial in the context of cancer therapies as stronger T-cell responses can be induced by providing the cancer antigens via non ⁇ nodified RNA (compared to m1 qj or i)J modified RNA).
- the artificial nucleic acid, preferably the RNA of the invention does not compnse N1 - methylpseudouridine (m141) substituted positions or pseudouridine ( ⁇ i) substituted positions.
- the artificial nucleic acid is an RNA that comprises a 5'-cap structure.
- a 5'-cap structure suitably stabilizes the nucleic acid and/or enhances expression of the encoded tumour antigen and/or reduces the stimulation of the innate immune system after administration.
- the artificial nucleic acid preferably the RNA, comprises a 5'-cap structure, preferably m7G, capO, cap1 , cap2, a modified capO or a modified cap1 structure.
- the term "5'-cap structure" as used herein will be recognized and understood by the person of ordinary skill in the art and is e.g.
- RNA RNA RNA cleavage region
- 5-cap structures which may be suitable in the context of the present invention are capO (methylation of the first nucleobase, e.g.
- cap1 additional methylation of the ribose of the adjacent nucleotide of m7GpppN
- cap2 additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN
- cap3 additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN
- cap4 additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN
- ARCA anti-reverse cap analogue
- modified ARCA e.g.
- a 5'-cap (capO or cap1) structure may be formed in chemical RNA synthesis or in RNA in vitro transcription (co- transcriptional capping) using cap analogues.
- cap analogue as used herein will be recognized and understood by the person of ordinary skill in the art and is e.g.
- Non-polymerizable means that the cap analogue will be incorporated only at the S'-terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' ⁇ lirection by a template- dependent polymerase, particularly, by template-dependent RNA polymerase.
- a cap1 structure is generated using a cap analogue as disclosed in W02017053297, W02017066793, W02017066781, W02017066791, W02017066789, W02017066782, W02018075827, W02017066797, and W02023007019.
- cap structures derivable from the structure disclosed in claim 1 -5 of W02017053297 may be suitably used to co-transcriptionally generate a cap1 structure.
- any cap structures as defined in claim 1 to claim 37 of W02023007019 may be suitably used to generate a cap1.
- the S'-cap structure may suitably be added co-transcriptionally using tri-nucleotide cap analogue as defined herein, preferably in an RNA in vitro transcription reaction as defined herein.
- the artificial nucleic acid, preferably the RNA of the invention comprises a cap1 structure or a modified cap1 structure.
- the cap1 structure is formed via co-transcriptional capping using tri-nudeotide cap analogues m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG.
- a particulariy preferred cap1 analogue in that context is m7G(5')ppp(5')(2'OMeA)pG.
- the cap1 structure is a modified cap1 structure and is formed using co-transcriptional capping using tri-nucleotide cap analogue 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.
- the 5'-cap structure may be formed via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes and/or 2'-0 methyltransferases) to generate capO, cap1 or cap2 shnctures.
- the artificial nucleic acid is an RNA that provides at least one coding sequence encoding at least one tumour antigen as defined herein that is produced after administration to a cell or subject.
- RNA of the invention preferably comprises are for example a 5' Cap structure as defined herein, a 5' UTR as defined herein, a 3' UTR as defined herein, hSL as defined herein, poly(A) sequence as defined herein, and optional chemical modifications as defined herein.
- the RNA is preferably an in vitro transcribed RNA (e.g. an in vitro transcribed mRNA).
- the nucleotide mixture i.e. the fraction of each nucleotide in the mixture
- the nucleotide mixture used for RNA in vitro transchption reactions is optimized for the given RNA sequence, preferably as described W02015188933.
- the nucleic acid of the invention is an in vitro t'anscribed RNA, preferably wherein RNA in vitro transcnption has been performed in the presence of a sequence optimized nucleotide mixture.
- the artificial nucleic acid of the invention is a purified RNA, preferably a purified mRNA.
- the RNA of the invention has been purified by at least one step of purification
- purified RNA or refers to RNA which has a higher purity after certain purification steps than the starting material (e.g. the crude IVT RNA).
- Typical impurities comprise peptides, proteins, spermidine, BSA, abortive RNA fragments, dsRNA, free nucleotides, DNA, buffer components etc. It is desirable in this regard for the "degree of RNA purity” to be as close as possible to 100%.
- “purified RNA” as used herein has a degree of purity of more than 75%, 80%, 85%, 90%, or 95%. The degree of purity may be determined by an analytical HPLC.
- the nucleic acid is a purified RNA that has been purified by at least one step of (RP)-hlPLC, AEX, size exclusion chromatography (SEC), hydroxyapatite chromatography, tangential flow filtration (TFF), filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
- the RNA has been purified using RP-HPLC (preferably as described in W02008077592) and/orTFF (preferably as described in W02016193206) and/or oligo d(T) purification (preferably as described in W02016180430) to e.g.
- the RNA has an integrity of at least 60%, 70%, 80%, 90%.
- the term "RNA integrity" generally describes whether the complete RNA sequence is present. The RNA integrity can be determined by RP-HPLC and may be based on determining the area under the peak of the expected full-length RNA in a chromatogram. It is desirable in this regard for the "degree of RNA integrity" to be as close as possible to 100%.
- the nucleic acid, preferably the RNA is suitable for use in treatment or prevention of a disease, disorder or condition, preferably a tumour or cancer disease, disorder, or condition or any disease, disorder, or condition related to tumour or cancer.
- the term "cancer” also comprises cancer metastases.
- Preferred nucleic acid constructs In various embodiments, the artificial nucleic acid comprises at least the following elements: A) a 5'-cap structure, preferably as specified herein; B) at least one cds encoding at least one tumour antigen as defined herein; C) a 5'-UTR and/or a 3'-UTR, preferably as specified herein; D) at least one poly(A) sequence, preferably as specified herein.
- the artificial nucleic acid preferably the RNA
- the artificial nucleic acid preferably the RNA
- the nucleic acid is an mRNA that comprises the following sequence elements in 5'-to3'-direction: A) a 5'-cap structure, preferably a cap1 structure; B) a 5'-UTR that compnses or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13, or a fragment or a variant of any of these; C) a cds that comprises a nucleic acid sequence encoding at least one antigenic peptide seleded or derived from a peptide or protein encoded by a long non-coding RNA (IncRNA), wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9
- IncRNA
- the nucleic acid is an mRNA that comprises the following sequence elements in 5-to3'-direction: A) a 5'-cap structure, preferably a cap1 structure; B) a 5'-UTR that compn'ses or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13, or a fragment or a variant of any of these; C) a coding sequence that comprises a nucleic acid sequence encoding at least one antigenic peptide selected or derived from a peptide or protein of a neoantigen as defined herein, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
- composition comprising at least one nucleic acid encoding a tumour antigen:
- the invention provides a pharmaceutical composition comprising at least one artificial nucleic acid encoding at least one tumour antigen as defined in the first aspect.
- features and embodiments described in the context of the first aspect have to be read on and have to be understood as suitable embodiments of the pharmaceutical composition of the second aspect and vice versa.
- a "composition” refers to any type of composition in which the specified ingredients (e.g. nucleic acid encoding at least one tumour antigen) may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient.
- the composition may be a dry composition such as a powder, a granule, or a solid lyophilized form. Alternatively, the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed fom.
- the at least one artificial nucleic acid of the pharmaceutical composition is selected from an RNAas further defined in the first aspect.
- the at least one nucleic acid of the pharmaceutical composition is selected from an mRNA as further defined in first aspect.
- the pharmaceutical composition comprises a plurality or at least more than one nucleic acid species (e.g. RNA species), preferably wherein each nucleic acid species encodes a different tumour antigen.
- the pharmaceutical composition as defined herein may comprise 2 to 10 nudeic acid species each as defined in the first aspect, preferably 2 to 5 nucleic acid species each as defined in the first aspect, wherein each of the 2 to 10 nucleic acid species, preferably each of the 2 to 5 nucleic acid species, encode at least one different tumour antigen.
- the respective nucleic acid species encoding different tumour antigens are selected to obtain an increased coverage in a certain cancer disease, e.g. NSCLC or melanoma.
- the coverage of all the different tumour antigens is at a value of above 0.5, preferably above 0.6, more preferably above 0.7.
- the different tumour antigens are seleded to cover different HLA types. Accordingly, the different tumour antigen are selected from at least one tumour antigen as defined herein that comprises at least one epitope that binds to hlLA-A type, at least one tumour antigen as defined herein that comprises at least one epitope binds to HLA-B type, at least one tumour antigen as defined herein that comprises at least one epitope binds to hlLA-C type.
- the at least one artificial nucleic acid preferably the at least one RNA of the pharmaceutical composition, is formulated with a pharmaceutically acceptable carrier or excipient.
- the at least one nucleic acid preferably the at least one RNA is complexed or associated with at least one further compound to obtain a formulated composition.
- a formulation in that context may have the function of a transfection agent.
- a formulation in that context may also have the function of protecting the nucleic acid from degradation, e.g. to allow storage, shipment, etc.
- the at least one nucleic acid, preferably the at least one RNA is formulated with at least one compound, e.g. peptides, proteins, lipids, polysaccharides, and/or polymers.
- the at least one artificial nucleic acid preferably the at least one RNA is formulated with at least one cationic (cationic or preferably ionizable) or polycationic compound (cationic or preferably ioneable).
- the at least one artificial nucleic acid, preferably the at least one RNA is complexed or associated with or at least partially complexed or partially associated with one or more cationic (cationic or preferably ionizable) or polycationic compound.
- cationic or polycationic compound refers to a charged molecule, which is positively charged at a pH value ranging from about 1 to 9, at a pH value ranging from about 3 to 8, at a pH value ranging from about 4 to 8, at a pH value ranging from about 5 to 8, more preferably at a phi value ranging from about 6 to 8, even more preferably at a pH value ranging from about 7 to 8, most preferably at a physiological pH, e.g. ranging from about 7.2 to about 7.5.
- a cationic component e.g.
- a cationic peptide, cationic protein, cationic polymer, cationic polysaccharide, cationic lipid may be any positively charged compound or polymer which is positively charged under physiological conditions.
- a "cationic or polycationic peptide or protein” may contain at least one positively charged amino acid, or more than one positively charged amino acid, e.g. selected from Arg, hlis, Lys or Om. Accordingly, "polycationic" components are also within the scope exhibiting more than one positive charge under the given conditions.
- the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
- the at least one artificial nucleic acid, preferably the at least one RNA of the pharmaceutical composition is formulated in lipid-based carriers.
- the term "iipid-based carriers" encompass lipid-based delivery systems for nucleic acid (e.g. RNA) that comprise a lipid component.
- a lipid-based carrier may additionally comprise other components suitable for encapsulating/incorporating/complexing a nucleic acid (e.g. RNA) including a cationic or polycatonic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations thereof.
- a nucleic acid e.g. RNA
- the at least one nucleic acid, preferably the RNA may completely or partially be incorporated or encapsulated in a lipid- based carrier, wherein the at least one nucleic acid may be located in the interior space of the lipid-based carrier, within the lipkJ layer/membrane of the lipid-based carrier, or associated with the exterior of the lipid-based carrier.
- nucleic acid into lipid-based carriers
- encapsulation refers to the essentially stable combination of nucleic ackl, preferably RNA with one or more lipids into lipid- based carriers (e.g. larger complexes or assemblies) preferably without covalent binding of the nucleic acid.
- the lipid- based carriers - encapsulated nucleic acid e.g. RNA
- nucleic acid e.g. RNA
- incorporation as the nucleic acid (e.g. RNA) is preferably contained within the interior of the lipid-based earners.
- the purpose of incorporating or encapsulating nucleic acid into lipid-based carriers may be to protect the nucleic acid from an environment which may contain enzymes, chemicals, or conditions that degrade the nucleic acid (e.g. RNA).
- lipid-based carriers may promote the uptake of the nucleic acid and their release from the endosomal compartment, and hence, may enhance the therapeutic effect of the nucleic acid (e.g. RNA) when administered to a cell or a subject.
- the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polyplexes, and/or nanoliposomes.
- the lipid-based carriers are lipid nanoparticles (LNPs).
- the lipid nanoparticles encapsulate the at least one nucleic acid, preferably the at least one RNA of the invention.
- the lipid-based carriers comprise at least one or more lipids selected from at least one aggregation-reducing lipid, at least one cationic lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analogue, or any combinations thereof.
- the lipid-based carriers comprise (i) an aggregation-reducing lipid, (ii) a cationic lipid or ionizable lipid, and (iii) a neutral lipid/phospholipid or a steroid/steroid analogue.
- the lipid-based earners comprise an (i) aggregation-reducing lipid, (ii) a cationic lipid or ionizable lipid, (iii) a neutral lipid or phospholipid, (iv) and a steroid or steroid analogue.
- Aggregation reducinci lipids / polymer conjugated lipids In preferred embodiments, the lipid-based carriers comprise at least one aggregation reducing lipid or moiety.
- aggregation reducing moiety refers to a molecule comprising a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
- aggregation reducing lipid refers to a molecule comprising both a lipid portion and a moiety suitable of reducing or preventing aggregation of the lipid-based carriers.
- the lipid-based carriers may undergo charge-induced aggregation, a condition which can be undesirable for the stability of the lipid-based carriers. Therefore, it can be desirable to include a compound or moiety which can reduce aggregation, for example by stencally stabilizing the lipid-based carriers.
- Such a steric stabilization may occur when a compound having a sterically bulky but uncharged moiety that shields or screens the charged portions of a lipid-based carriers from close approach to other lipid-based carriers in the composition.
- lipids which may comprise a lipid bearing a sterically bulky group which, after formation of the lipid-based carrier, is preferably located on the exterior of the lipid- based carrier.
- Suitable aggregation reducing groups include hydrophilic groups, e.g. monosialoganglioside GM1, potyamide oligomers (PAO), or certain polymers, such as poly(oxyalkylenes), e.g., poly(ethylene glycol) or poly(propylene glycol).
- PEO potyamide oligomers
- Lipids comprising a polymer as aggregation reducing group are herein referred to as "polymer conjugated lipid”.
- polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion, wherein the polymer is suitable of reducing or preventing aggregation of lipid-based carriers comprising the RNA.
- a polymer has to be understood as a substance or material consisting of very large molecules, or macromolecules, composed of many repeating subunits.
- a suitable polymer in the context of the invention may be a hydrophilic polymer.
- An example of a polymer conjugated lipid is a PEGylated or PEG ⁇ ;onjugated lipid.
- the lipid-based carriers compnse an aggregation reducing lipid selected from a polymer conjugated lipid.
- the polymer conjugated lipid is a PEG ⁇ onjugated lipid (or PEGylated lipid, PEG lipid).
- the average molecular weight of the PEG moiety in the PEG- conjugated lipid preferably ranges from about 500 to about 8,000 Dattons (e.g., from about 1,000 to about 4,000 Dattons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Dattons.
- a PEG ⁇ »njugated lipid may be selected or derived from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG 2000), C10-PEG2K, or Cer8-PEG2K.
- a PEG-conjugated lipid may also be selected or derived from formula (IVa) of W02018078053.
- a suitable PEG-conjugated lipid of said formula (IVa) has the chemical term 2[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide, also referred to as ALC-0159.
- the tipid-based carriers comprise an aggregation reducing lipid, wherein the aggregation reducing lipid is not a PEG-conjugated lipid.
- the aggregation reducing lipid may suitably be selected from a PEG-less lipid, e.g. a PEG-less polymer conjugated lipid.
- the aggregation reducing lipid is a PEG-free lipid that comprises a polymer different from PEG.
- a PEG-free lipid in the context of the invention may be selected or derived from a POZ-lipid.
- the POZ lipids or respectively preferred polymer conjugated lipids are described in W02023031394, the full disclosure herewith incorporated by reference.
- the disclosure relating to polymer conjugated lipids as shown in any one of claims 1 to 8 ofW02023031394 are incorporated by reference.
- the polymer conjugated lipid is a PEG-free lipid selected from a POZ-lipid.
- the polymer conjugated lipid is a "POZ-lipid", which preferably is defined as a compound according to formula (POZ): [hi] - [linker] - [M], wherein [H] is a homopolymer moiety compnsing at least one potyoxazoline (POZ) monomer unit A wherein R is C1-9 alkyl or C2-9 alkenyl, preferably C1 ,and n has a mean value ranging from 2 to 200, preferably from 20 to 100, more preferably from 24 to 26 or 45 to 50; [linker] is an optional linker group; [M] is a lipid moiety.
- POZ potyoxazoline
- the aggregation-redudng lipid is selected or derived from PMOZ 1, PMOZ 2, PMOZ 3, PMOZ 4, or PMOZ 5 ofW02023031394.
- the aggregation-reducing lipid is selected or derived from PMOZ4 according to or derived from the follcwing formula:
- the at least one aggregation-reducing lipid is selected from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K, or a POZ-lipid such as PMOZ4.
- the at least one aggregation-reducing lipid is selected from ALC-0159.
- the lipid-based carriers comprise at least one cationic or ionizable lipid.
- the cationic or ionizable lipid of the lipid-based earners may be cationisable or ionizable, i.e. it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher phi values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
- the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
- the lipid-based carriers comprise a cationic or ionizable lipid that preferably carries a net positive charge at physiological pH, more preferably the cationic or ionizable lipid comprises a tertiary nitrogen group or quaternary nitrogen group. Accordingly, in preferred embodiments, the lipid-based carriers comprise a cationic or ionizable lipid selected from an amino lipid, preferably wherein the amino lipid comprises a tertiary amine group.
- cationic or ionizable lipids may be selected from the lipids disclosed in W02018078053 (i.e. lipids derived from formula I, II, and III of W02018078053, or lipids as specified in claims 1 to 12 of W02018078053), the disclosure of W02018078053 hereby incorporated by reference in its entirety.
- lipids disclosed in Table 7 of W02018078053 e.g. lipids derived from formula 1-1 to 1-41
- lipids disclosed in Table 8 of W02018078053 e.g. lipids derived from formula 11-1 to II-36
- the lipid-based carriers of the pharmaceutical composition comprise a cationic lipid selected or derived from structures 111-1 to III-36 ofTable 9 of W02018078053. Accordingly, formula 111-1 to III-36 of W02018078053, and the specific disclosure relating thereto, are herewith incorporated by reference. In embodiments, the lipid-based carriers comprise a cationic lipid selected or derived from formula 111-3 of W02018078053.
- a preferred lipid of said formula III-3 has the chemical term ((4-hydroxybutyl)azanediyl)bis (hexane- 6,1-diyl)bis(2-hexyldecanoate), also referred to as ALC-0315 i.e. CAS Number 2036272-5M.
- Further suitable cationic lipids may be selected or derived from cationic lipids according to PCT claims 1 to 14 of W02021123332, or table 1 of W02021123332, the disclosure relating to claims 1 to 14 or table 1 ofW02021123332 herewith incorporated by reference.
- suitable cationic lipids may be selected or derived from cationic lipids according Compound 1 to Compound 27 (C1 - C27) of Table 1 of W02021123332.
- the lipid-based earners comprise a cationic lipid selected or derived from (COATSOME®SS-EC) SS- 33/4PE-15 (see C23 in Table 1 of W02021123332).
- the lipid-based carriers comprise a cationic lipid selected or derived from HEXA-CSDE-PipSS (see C2 in Table 1 ofW02021123332).
- the lipid-based carriers comprise a cationic lipid selected or derived from compound C26 as disclosed in Table 1 of W02021123332.
- the lipid-based carriers comprise a cationic lipid selected or derived from 9-Heptadecanyl 8- ⁇ (2- hydroxyethyl)[6oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate, also referred to as SM-102.
- the lipid-based carrier preferably the LNP, comprises a cationic lipid selected or derived from above mentioned compound C26, SM-102, SS-33/4PE-15, or HEXA-CSDE-PipSS.
- the lipid-based carrier preferably the LNP, comprise a cationic lipid selected or derived from ALC-0315.
- Neutral lipids In preferred embodiments, the lipid-based carriers (e.g. LNPs) comprise at least one neutral lipid or phospholipid.
- neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionicform at physiological pH.
- Neutral lipids may be selected from DHPC, DHPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1 -trans PE, SOPE, transDOPE, 1,2 ⁇ fiphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), DPhyPS (1,2 ⁇ liphytanoyl-sn- glycero-3-phospho-L-serine), or mixtures thereof.
- DPhyPE 1,2 ⁇ fiphytanoyl-sn-glycero-3-phosphoethanolamine
- DPhyPS 1,2 ⁇ liphytanoyl-sn- glycero-3-phospho-L-serine
- the lipid-based carriers comprise a neutral lipid selected or derived from DSPC, DHPC, DPhyPE, or DPhyPS. Preferred in that context is DSPC.
- Steroids, steroid analogues or sterols In preferred embodiments, the lipid43ased earners comprise a steroid, sterokl analogue or sterol. In embodiments, the steroid, steroid analogue or sterol is derived or selected from cholesterol, cholesteryl hemisuccinate (ChlEMS), or any derivate of these. In preferred embodiments, the lipid-based carriers comphse cholesterol.
- the lipid-based earners preferably the LNPs, compnsing the at least one nucleic acid, preferably the at least one RNA, comprise (i) at least one cationic lipid or ionizable lipid, preferably as defined herein; (ii) at least one or two (e.g. two different) neutral lipids or phospholipids, preferably as defined herein; (iii) at least one steroid or steroid analogue, preferably as defined herein; and (iv) at least one aggregation reducing lipid, preferably as defined herein.
- the lipid-based carriers comprising the at least one nucleic acid, preferably the at least one RNA
- the lipid-based carriers comprise (i) at least one cationic lipid selected or derived from C26, SS-33/4PE-15, HEXA-CSDE-PipSS, SM-102; (ii) at least one or two (e.g.
- lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
- the lipid-based carriers preferably the LNPs, compnsing the at least one nucleic acid, preferably the at least one RNA, comprise (i) at least one cationic lipid selected or derived from ALC-0315; (ii) at least one neutral lipid selected or derived from DSPC; (iii) at least one steroid or steroid analogue seleded or derived from cholesterol; and (iv) at least one aggregation reducing lipid selected or derived from ALC-0159; and wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
- the nucleic acid e.g. the RNA
- the lipid-based carriers comprising the at least one nucleic acid, preferably the at least one RNA compnse (i) a cationic lipid selected from C26; (ii) a neutral lipid selected from DPhyPE and a neutral lipid selected from DPhyPS; (iii) a steroid or steroid analogue seteded from cholesterol; and (iv) an aggregation reducing lipid selected from a POZ lipid as defined herein, preferably PMOZ 4; and wherein the lipid- based carriers encapsulate the nucleic acid (e.g. the RNA).
- RNA compnse a cationic lipid selected from C26; (ii) a neutral lipid selected from DPhyPE and a neutral lipid selected from DPhyPS; (iii) a steroid or steroid analogue seteded from cholesterol; and (iv) an aggregation reducing lipid selected from a POZ lipid as defined herein,
- the cationic lipids (as defined herein), neutral lipid (as defined herein), steroid or steroid analogue (as defined herein), and/or aggregation reducing lipid (as defined herein) may be combined at various relative ratios.
- the lipid-based carriers preferably the LNPs, comprise (i) to (iv) in a molar ratio of about 20-60% cationic lipid or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analogue, and about 0.5-15% aggregation reducing lipid e.g.
- the lipid-based earners comprise (i) to (iv) in a molar ratio of about 45- 55% cationic lipid or ionizable lipid, about 5-15% neutral lipid, about 35-45% steroid or steroid analogue, and about 0.5- 2.5% aggregation reducing lipid e.g. polymer conjugated lipid, preferably wherein the lipid-based carriers encapsulate the at least one nucleic acid, preferably the at least one RNA.
- the lipid-based carriers preferably the LNPs, comprise (i) to (iv) in a molar ratio of about 47-51% cationic lipid or ionizable lipid, about 8-12% neutral lipid, about 38-42% steroid or steroid analogue, and about 0.75-1.75% aggregation reducing lipid e.g. polymer conjugated lipid, preferably wherein the lipid-based carriers encapsulate the at least one nucleic acid, preferably the at least one RNA.
- the lipid-based carriers preferably the LNPs comprising the at least one nucleic acid, preferably the at least one RNA, 03mprise (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analogue selected from cholesterol; and (iv) at least one aggregation reducing lipid selected from ALC-0159 and wherein the lipid-based carriers encapsulate the nucleic acid (e.g.
- RNA preferably wherein i) to (iv) are in a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analogue, and about 1.7% aggregation reducing lipid, preferably wherein the lipid-based carriers encapsulate the nucleic acid (e.g. the RNA).
- the lipid-based earner preferably the LNP comprising the at least one nucleic acid molecule, preferably RNA
- the amount of lipid comprised in the lipid-based earners may be selected taking the amount of the nucleic acid cargo into account. In one embodiment, these amounts are selected such as to result in an N/P ratio of the lipid-based carriers encapsulating the nucleic acid in the range of about 0.1 to about 50.
- the N/P ratio is defined as the mole ratio of the nitrogen atoms ("N") of the basic nitrogen-containing groups of the lipid to the phosphate groups ("P") of the nucleic ac'id which is used as cargo.
- the N/P ratio may be calculated on the basis that, for example, 1 pg nucleic acid typically contains about Snmol phosphate residues, provided that the nucleic acid exhibits a statistical distribution of bases.
- the "N"-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and - if present - cationisable groups.
- the N/P ratio can be in the range of about 1 to about 50. In preferred embodiments, the range is from about 5 to about 20. In some embodiments, the N/P ratio is at about 17. In some embodiments, the N/P ratio is at about 14. In some embodiments, the N/P ratio is at about 6.
- the pharmaceutical composition comprises lipid-based carriers (encapsulating nucleic acid, preferably RNA as defined herein) that have a defined size (particle size, homogeneous size distribution).
- the size of the lipid-based carrier is typically described herein as Z-average size.
- Z-average size refers to the mean diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PDI), which is dimensionless.
- DLS dynamic light scattering
- DLS dynamic light scattering
- the lipid-based carriers preferably the LNPs
- the lipid-based carriers have a Z-average size of less than 400nm, preferably less than 300nm, more preferably less than 200nm.
- the lipid-based carriers, preferably the LNPs have a Z-average size ranging from about 50nm to about 2CX)nm, preferably about 50nm to about 150nm, more preferably about 50nm to about120nm.
- the composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% lipid-based carriers that have a particle size exceeding about 500nm.
- the composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % LNPs that have a particle size smaller than about 20nm.
- the lipid-based carriers exhibit a zeta potential in the range of -50 mV to +50 mV, preferably -25 mV to +25 mV, more preferably -10 mV to +10 mV, most preferably -5 mV to +5 mV.
- the polydispersity index (PDI) of the lipid-based carriers, preferably the LNPs is in the range of 0.1 to 0.5.
- the polydispersity index (PDI) value is less than about 0.3, preferably of less than about 0.2.
- the PDI is determined by dynamic light scattering.
- at least 70%, 80%, 90%,95% of the nucleic acid (e.g. RNA) is encapsulated in lipid-based carriers such as LNPs. The percentage of encapsulation may be determined by a RiboGreen assay as known in the art.
- the plurality of lipid-based carriers have a lamellar morphology and/or a bilayer morphology.
- at least 80%, 85%, 90%, 95% of the lipid-based carriers have a spherical morphology.
- the surface of the lipid-based carriers preferably the LNPs, is uncharged at pH 7.
- Administration of the pharmaceutical composition or the nucleic acid Suitably, upon administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, the at least one tumour antigen is produced in an amount sufficient for inducing an antigen specific immune response in said cell, tissue, or subject.
- the administration is an intramuscular, intratumoral, or intravenous administration, preferably an intramuscular, intratumoral, or intravenous injection.
- the encoded tumour antigens are produced, preferably in an amount sufficient for inducing an antigen specific immune response in said cell, tissue, or subject.
- the encoded tumour antigens are produced and induce an increased immunogenicity in the subject.
- the encoded tumour antigens upon intramuscular, intratumoral, or intravenous administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, are produced and induce an epitope-specific or antigen-specific CD8+ T cells in the subject.
- the encoded tumour antigens upon intramuscular, intratumoral, or intravenous administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, are produced and induce an epitope-specific or antigen-specific C04+ T cells in the subject
- the encoded tumour antigens upon intramuscular, intratumoral, or intravenous administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, are produced and induce humoral immunity, e.g.
- the encoded tumour antigens upon intramuscular, intratumoral, or intravenous administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, are produced and increase IFN- gamma production by CD8+ T cells upon exposure to the encoded tumour antigens in the subject.
- the encoded tumour antigens upon intramuscular, intratumoral, or intravenous administration of the pharmaceutical composition or nucleic acid to a cell, tissue, or subject, the encoded tumour antigens are produced and increase the presentation of the encoded tumour antigens on MHC molecules in the subject.
- presentation of the encoded polypeptide or peptide relates to higher amount and/or van'ety of immunogenic and/or stable peptides presented via MHC class I and II molecules after protein degradation by the proteasomal machinery, and thus cellular immunity, e.g. T cell activation based thereon.
- the presentation of the encoded polypeptide or peptide on MHC class I and II molecules is increased on cells, comprising immune cells (e.g., T cells), antigen- presenting cells (e.g., dendritic cells, macrophages, engineered antigen-presenting cells), MhlC class 1-expressing cells, MhlC class 11-expressing cells, or any combination thereof.
- the administration of the pharmaceutical composition or nucleic acid results in increased immunogenicity presentation on MHC molecules of the encoded tumour antigen that is increased by between about 0.1 % and about 100% (e.g., about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100%) when compared to a subject without the administered nucleic acid/composition.
- about 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 100% when compared to a subject without the administered nucleic acid/composition.
- the administration of the pharmaceutical composition or nucleic acid results in immunogenicity or presentation on MHC molecules of the encoded tumour antigen that is increased by about 2-fold to about 100-fold (e.g., about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold,) when compared to a subject without the administered nucleic acid/composition.
- about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold, when compared to a subject without the administered nucleic acid/composition.
- tumour antigen / a composition of tumour antigen provides a peptide tumour antigen or a composition compnsing at least one peptide tumour antigen.
- first aspect the nucleic acid of the invention
- second aspect pharmaceutical composition of the invention
- the tumour antigen compnses at least one antigenic peptide selected from a peptide or protein encoded by a long non-coding RNA (IncRNA) and/or at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen.
- IncRNA long non-coding RNA
- the at least one antigenic peptide encoded by the long non-coding RNA in particular lnc-WDR72-2:4, lnc-TRPC5-3:1, lnc-CLEC2D-9:1, KCNMB2-AS1:4, tnc-ZC3H8-6:1, Inc-NTFS- 5:5, or LINC00893:25, or an immunogenic fragment or van'ant of any of these.
- RNA long non-coding RNA
- Preferred peptide tumour antigens are provided in Table 1A and 1 B (column B or C).
- the at least one antigenic peptide is selected from a peptide or protein encoded by a long non-coding RNA (IncRNA) and comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-146,546-571, 756,757, or an immunogenic fragment or variant of any of these.
- IncRNA long non-coding RNA
- the at least one antigenic peptide is selected from a peptide or protein of a tumour neoantigen, in particular selected from NRAS neoantigen, TYW1B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, DIP2B neoantigen, TRRAP neoantigen, RAC1 neoantigen, AP4B1 neoantigen, VWA5A neoantigen, CLTC neoantigen, RPL9 neoantigen, RPAP1 neoantigen, RPE65 neoantigen, PATZ1 neo
- the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs:255- 286, or an immunogenic fragment or immunogenic variant of any of these.
- composition comprising at least one tumour antigen refers to any type of composition in which the specified peptide tumour antigens may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient.
- the composition may be a dry composition such as a powder, a granule, or a solid lyophilized form. Alternatively, the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed (e.g. suspended or emulsified) form.
- the at least one tumour antigen of the composition is formulated with a pharmaceutically acceptable carrier or excipient.
- the composition comprises a plurality or at least more than one different peptide tumour antigen as defined herein.
- the composition as defined herein may comprise 2 to 10 different peptide tumour antigens as defined herein, preferably 2 to 5 different peptide tumour antigens as defined herein.
- An antibody, a T-cell, or a TCR In a fourth aspect, the invention provides an antibody, a T ⁇ ;ell, or a T-cell receptor fTCR) that has been raised against or stimulated with any tumour antigen or with any nucleic acid of any of the foregoing aspects.
- the antibody is a therapeutic antibody that has been raised against any of the tumour antigens of the invention.
- the antibody has been raised against any peptide tumour antigen provided in Table 1 A and B (columns B or C) or any peptide tumour antigens provided in Table 2 (column C).
- the T-cell is a therapeutic T-cell that has been stimulated with any tumour antigen or with any nucleic acids of any of the foregoing aspects.
- the T-cell has been stimulated with any tumour antigen or with any nucleic acid provided in Table 1A or 1 B or Table 2.
- the TCR has been raised against any of the tumour antigens of the invention (e.g. antigen peptide- loaded on MHC).
- the TCR has been raised against any peptide tumour antigen provided in Table 1A or 1 B (column B or C) or any peptide tumour antigens provided in Table 2 (column C).
- a combination in a fifth aspect, provides a combination comprising at least two or a plurality of therapeutic modalities of the foregoing aspects.
- features and embodiments described in the context of the first aspect (the nucleic acid of the invention) or the second aspect (pharmaceutical composition of the invention) or the third aspect (tumour antigen) or the fourth aspect (antibody, T-cell, TCR) have to be read on and have to be understood as suitable embodiments of the combination of the fifth aspect.
- the combination compnses at least two therapeutic modalities selected from a) at least one or more artificial nucleic acids as defined in the first aspect; and/or b) at least one or more pharmaceutical composition as defined in the second aspect; and/or c) at least one or more tumour antigen or composition as defined in the third aspect; and/or d) at least one or more antibody, T-cell, or TCR as defined in the fourth aspect;
- the combination of the invention comprises at least one or more than one therapeutic modality as defined herein, preferably 2,3, 4,5,6,7,8, or even more therapeutic modalities as defined herein.
- the therapeutic modalities may be administered in a spatially separated and/or timely staggered manner.
- kits or kit of parts In a sixth aspect, the invention provides a kit or kit of parts.
- features and embodiments described in the context of the first aspect (the nucleic acid of the invention) or the second aspect (pharmaceutical composition of the invention) or the third aspect (tumour antigen) or the fourth aspect (antibody, T-cell, TCR) or the fifth aspect (combination) have to be read on and have to be understood as suitable embodiments of the kit or kit of parts of the sixth aspect.
- the kit or kit of parts comprises i) at least one or more artificial nucleic acids as defined in the first aspect; and/or ii) at least one or more pharmaceutical composition as defined in the second aspect; and/or iii) at least one or more tumour antigen or composition as defined in the third aspect; and/or iv) at least one or more antibody, Toll, or TCR as defined in the fourth aspect;
- the kit or kit of parts comprises a liquid vehicle for solubilising.
- the kit or kit of parts comprises technical instructions providing information on administration and dosage of the components. The technical instructions of said kit may contain information about administration and dosage and patient groups. Such kits, preferably kits of parts, may be applied e.g.
- the present invention relates to the medical use of the therapeutic agents of the foregoing aspects.
- features and embodiments described in the context of the first aspect (the nucleic acid of the invention) or the second aspect (pharmaceutical composition of the invention) or the third aspect (tumour antigen) or the fourth aspect (antibody, T-cell, TCR) or the fifth aspect (combination) or the kit or kit of parts of the sixth aspect have to be read on and understood as suitable embodiments of any of the medical uses presented in the follcwing aspects.
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth asped for use as a medicament e.g. for treating or preventing a disease, disorder, or condition in a subject.
- the use may be for human medical purposes and for veterinary medical purposes, preferably for human medical purposes.
- the use may be for human medical purposes, in particular, for young infants, newboms, immunocompromised recipients, pregnant and breast-feeding women, and elderiy people.
- the use may be for human medical purposes for subjeds that have cancer. Medical use in treating or [jreventins cancer
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing cancer in a subject, or any disease, disorder, or condition related to cancer.
- features and embodiments described in the context of any of the foregoing aspects may be read on and have to be understood as suitable embodiments of the present aspect.
- cancer refers to a neoplasm characterized by the uncontrolled and usually rapid proliferation of cells that tend to invade surrounding tissue and to metastasize to distant body sites.
- the term encompasses benign and malignant neoplasms. Malignancy in cancers is typically characterized by anaplasia, invasiveness, and metastasis; whereas benign malignancies typically have none of those properties.
- the terms includes neoplasms characterized by tumour growth as well as cancers of blood and lymphatic system.
- cancer metastases e.g. cancer metastases associated with HNSCC, NSCLC, or melanoma.
- the treating or preventing cancer in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the cancer of the patient is analyzed by e.g. sequencing.
- the nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the antibody, T-cell, or TCR, the combination, the kit or kit of parts is administered by intramuscular, intratumoral, or intravenous administration, in particular injection.
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing squamous cell carcinoma.
- an artificial nucleic acid of the first aspect a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing squamous cell carcinoma.
- the squamous cell carcinoma is selected from squamous cell carcinomas (LUSC) / squamous NSCLC (sqNSCLC) or head and neck squamous cell carcinoma (hlNSCC).
- the treating or preventing squamous cell carcinoma in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the squamous cell carcinoma of the patient is analyzed by e.g. sequencing.
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing Non-small cell lung cancer (NSCLC) in a subject, or any disease, disorder, or condition related to NSCLC.
- NSCLC Non-small cell lung cancer
- the use relates to treatment of NSCLC subgroups including lung adenocarcinomas (LUAD) and/or lung squamous cell carcinomas (LUSC).
- the use relates to treatment of squamous cell carcinomas (LUSC) / sqNSCLC.
- LUSC squamous cell carcinomas
- sqNSCLC squamous cell carcinomas
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts comprises a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, lnc-TRPCS-3:1, KCNMB2-AS1:4, lnc-NTF3-5:5, or lnc-ZC3H8-6: 1, or an immunogenic fragment or variant of any of these; and/or b) at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen selected from a BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, PIK3CA neoantigen, or NFE2L2 neoantigen, or an immunogenic fragment or variant of any of these, preferably selected from an EGFR ne
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, Inc- TRPC5-3:1, KCNMB2-AS1:4, lnc-NTF3-5:5, or lnc-ZC3H8-6:1, or an immunogenic fragment or variant of any of these.
- the nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the antibody, T-cell, or TCR, the combination, the kit or kit of parts is administered by intramuscular, intratumoral, or intravenous administration, in particular intramuscular injection.
- the treating or preventing NSCLC in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the NSCLC cancer of the patient is analyzed by e.g. sequencing.
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth asped for use as a medicament in treating or preventing head and neck squamous cell carcinoma (HNSCC) in a subject, or any disease, disorder, or condition related to HNSCC.
- HNSCC head and neck squamous cell carcinoma
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, KCNMB2-AS1:4, lnc-NTF3-5:5, or lnc-ZC3H8-6: 1, or an immunogenic fragment or variant of any of these
- the nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the antibody, T-cell, or TCR, the combination, the kit or kit of parts is administered by intramuscular, intratumoral, or intravenous administration, in particular intramuscular injection.
- the treating or preventing HNSCC in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the hlNSCC cancer of the patient is analyzed by e.g. sequencing. Medical use in treating or preventing melanoma
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing melanoma in a subject, or any disease, disorder, or condition related to melanoma.
- the use relates to treatment of skin cutaneous melanoma (SKCM) or uveal melanoma.
- the use relates to treatment of melanoma BRAF subgroup and/or NRAS subgroup.
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts compnses a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-TRPC5-3:1 or lnc-CLEC2D-9:1, or an immunogenic fragment or variant of any of these; and/or b) at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen selected from an NRAS neoantigen, T/W1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, DIP2B neoantigen,
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-TRPC5-3:1 or Inc- CLEC2D-9:1, or an immunogenic fragment or variant of any of these.
- the nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the antibody, T-cell, or TCR, the combination, the kit or kit of parts is administered by intramuscular, intratumoral, or intravenous administration, in particular intramuscular injection.
- the treating or preventing melanoma in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the melanoma of the patient is analyzed by e.g., sequencing. Medical use in treatiny or preventina further cancer indications In further aspects, the present invention relates to the medical use of the therapeutic agents of the foregoing aspects in treating or preventing further cancer indications.
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing Adrenocortical carcinoma, Bladder Urothelial Carcinoma, Breast invasive carcinoma, Cervical carcinoma (Cervical squamous cell carcinoma and endocervical adenocarcinoma), Colon adenocarcinoma, Kidney renal papillary cell carcinoma, Lung adenocarcinoma, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, Rectum adenocarcinoma, Sarcoma (in particular synoval Sarcoma), Skin Cutaneous Melanoma, Thymoma, Thyroid carcinoma, Hepatocellular carcinoma, Uterine
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing Bladder Urothelial Carcinoma, Cervical carcinoma (Cervical squamous cell carcinoma and endocervical adenocarcinoma), Thymoma, or Uterine Corpus Endometrial Carcinoma in a subject, or any related disease, disorder, or condition related, wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by KCNMB2- AS1:4, or an immunogenic
- the invention provides an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, orTCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth asped for use as a medicament in treating or preventing Ovanan serous cystadenocarcinoma, Testicular Germ Cell Tumors, Thymoma, or Uterine cardnosarcoma in a subject, or any related disease, disorder, or condition related, wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-NTF3-5:5, or an immunogenic fragment or variant thereof.
- the invention provides an artificial nucleic acid of the first asped, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T ⁇ ;ell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing Bladder Urothelial Carcinoma, Colon adenocarcinoma, Esophageal carcinoma, or Stomach adenocarcinoma in a subject, or any related disease, disorder, or condition, wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, or an immunogenic fragment or van'ant thereof.
- the invention provides an artificial nucleic acid of the first asped, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens ofthe third aspect, an antibody, T-cell, orTCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect for use as a medicament in treating or preventing Skin Cutaneous Melanoma, Testicular Germ Cell Tumors, or Uveal Melanoma in a subject, or any related disease, disorder, or condition, wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour anhgens, the combination, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-CLEC2D- 9:1, or an immunogenic fragment or vanant thereof.
- the present invention relates to a method of treating or preventing a disease, disorder or condition.
- embodiments relating to the previous aspects may likewise be read on and be understood as suitable embodiments of method of treatments of the invention.
- specific features and embodiments relating to method of treatments as provided herein may also apply for medical uses of the invention and vice versa.
- Preventing (inhibiting) or treating a disease relates to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as an infection or cancer.
- Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
- ameliorating refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease.
- the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a sbwer progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease.
- a "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
- the present invention relates to a method of treating or preventing a disease, disorder or condition, wherein the method comprises applying or administering to a subject in need thereof an effective amount of an artificial nucleic acid of the first aspect, a pharmaceutical composition of the second aspect, a tumour antigen or a composition of tumour antigens of the third aspect, an antibody, T-cell, or TCR of the fourth aspect, a combination of the fifth aspect, or a kit or kit of parts of the sixth aspect.
- the disease, disorder or condition is cancer, e.g. any disease, disorder or condition associated with cancer as defined herein.
- the cancer is NSCLC.
- the cancer is squamous cell carcinoma, preferably selected from squamous cell carcinomas (LUSC) / squamous non- small-cell lung cancer (sqNSCLC) or head and neck squamous cell carcinoma (hlNSCC).
- the cancer is melanoma.
- Preferred embodiments in that context are inter alia provided in the section "Medical use in treating or preventing squamous cell carcinoma” or “Medical use in treating or preventing A/SCLC” or “Medical use in treating or preventing HNSCC' or “Medical use in treating or preventing melanoma” or “Medical use in treating or preventing further cancer indications” outlined in the context of the aspect "medical uses”.
- the nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the antibody, T-cell, or TCR, the combination, the kit or kit of parts is administered by intramuscular, intratumoral, or intravenous administration, in particular injection.
- the treating or preventing cancer in a subject is a personalized treatment. Accordingly, prior to a treatment with the respective therapeutic agents, the cancer of the patient is analyzed by e.g. sequencing.
- Item List Preferred embodiments of the present invention are provided in the following numbered list of items (Items 1-116): 1.
- An artificial nucleic acid comprising at least one coding sequence encoding at least one tumour antigen that comprises or consists of a) at least one antigenic peptide selected from a peptide or protein encoded by a long non-coding RNA (IncRNA), or an immunogenic fragment or variant thereof; and/or b) at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, or an immunogenic fragment or van'ant thereof.
- IncRNA long non-coding RNA
- IncRNA long non-coding RNA
- the artificial nucleic acid of items 1 to 2, wherein the at least one antigenic peptide encoded by the long non- coding RNA (IncRNA) comprises at least one T-cell epitope, preferably at least one CD8+ T cells epitope. 4.
- the artificial nucleic acid of items 1 to 3, wherein the at least one antigenic peptide selected from a peptide or protein encoded by a long non-coding RNA (IncRNA) comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-254,546-650,756-763, or an immunogenic fragment or variant of any of these. 5.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by Inc- WDR72-2:4, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 124-146,546-571,756,757, or an immunogenic fragment or van'ant of any of these. 6.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by Inc- TRPC5-3:1, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 227-237, 630-642,761, or an immunogenic fragment or van'ant of any of these. 7.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by Inc- CLEC2D-9:1, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 238-243, 643-650,762, or an immunogenic fragment or variant of any of these.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by KCNMB2- AS1:4, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 147-161, 572-589,758, or an immunogenic fragment or variant of any of these.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by Inc- ZC3hl8-6:1, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one ofSEQ ID NOs: 162-168,590-603, 759,or an immunogenic fragment or variant of any of these. 10.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-NTF3- 5:5, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 169-226, 604-629,760, or an immunogenic fragment or variant of any of these. 11.
- the artificial nucleic acid of items 1 to 4 comprising at least one coding sequence encoding at least one tumour antigen that comprises at least one antigenic peptide selected from a peptide or protein encoded by LINC00893:25, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one ofSEQ ID NOs: 244-254, 763,or an immunogenic fragment or variant of any of these. 12.
- the at least one coding sequence comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a IncRNA, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 318448,651.755,764- 771, or a fragment or a van'ant of any of these. 13.
- the artificial nucleic acid of item 12 wherein the at least one coding sequence comprises a GC optimized nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a IncRNA, wherein the nucleic acid sequence is identical or at least 80% identical to any one ofSEQ ID NOs: 318-448,651 -755,764-771 , or a fragment of any of these. 14.
- tumour neoantigen is selected from an NRAS neoantigen, TYW1B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, DIP2B neoantigen, TRRAP neoantigen, RAC1 neoantigen, AP4B1 neoantigen, VWA5A neoantigen, CLTC neoantigen, RPL9 neoantigen, RPAP1 neoantigen, RPE65 neoantigen, PATZ1 neoantigen, PIK3CA neoantigen, N
- the artificial nucleic acid of item 14, wherein the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen comprises at least one T-cell epitope, preferably at least one CD8+ T cells epitope.
- the artificial nucleic acid of item 14 or 15, wherein the at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 255-317, or an immunogenic fragment or immunogenic variant of any of these. 17.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a NRAS tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 267-272, or an immunogenic fragment or variant of any of these. 18.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein ofaTYWIBtumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs:273,274,or an immunogenic fragment or variant of these. 19.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a ECPAS tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NO: 275, or an immunogenic fragment or variant thereof.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein ofaMAP2K1 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 276,277, or an immunogenic fragment or vanant of these. 21.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a TOMM22 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 278,279, or an immunogenic fragment or variant of these. 22.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a GLB1 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 280,281 , or an immunogenic fragment or van'ant of these.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a MAGE-A3 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs:282,283,or an immunogenic fragment or variant of these. 24.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a BRAF tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 262-266, or an immunogenic fragment or variant of any of these. 26.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a EGFR tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 255-257, or an immunogenic fragment or variant of any of these. 27.
- the artificial nucleic acid of items 14 to 16 comprising at least one coding sequence encoding at least one antigenic peptide selected from a peptide or protein of a TP53 tumour neoantigen, wherein the antigenic peptide comprises or consists of at least one of the amino acid sequences being identical or at least 80% identical to any one of SEQ ID NOs: 258-261, or an immunogenic fragment or variant of any of these.
- the at least one coding sequence comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-511, or a fragment or a variant of any of these.
- artificial nucleic acid of item 28 wherein the at least one coding sequence comprises a GC optimized nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 80% identical to any one of SEQ ID NOs: 449-511, or a fragment of any of these.
- artificial nucleic acid of item 30 wherein the different tumour antigens are separated by a linker element.
- artificial nucleic acid of any one of the preceding items wherein the at least one coding sequence encodes at least one additional amino acid sequence selected from at least one immune response activating signal transduction protein.
- artificial nucleic acid of any one of the preceding items wherein the at least one coding sequence encodes at least one additional amino acid sequence selected from at least one T-helper epitope.
- artificial nucleic acid of any one of the preceding items wherein the at least one coding sequence encodes at least one additional amino acid sequence selected from at least one signal peptide.
- the at least one coding sequence is a codon modified coding sequence, preferably wherein codon modified coding sequence is selected from a C maximized coding sequence, a CAI maximized coding sequence, human codon usage adapted coding sequence, a G/C content modified coding sequence, and a G/C optimized coding sequence, or any combination thereof, artificial nucleic acid of item 35, wherein the at least one codon modified coding sequence is a G/C optimized coding sequence.
- the nucleic acid comprises at least one untranslated region (UTR) preferably selected from at least one heterologous 5'-UTR and/or at least one heterologous 3'-UTR.
- artificial nucleic acid of item 37 wherein the at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence selected or from a 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, beta- globin, ANXA4, CASP1, COX6B1, FIG4, GNAS, NDUFA1, RPS9, SLC7A3 or TUBB4B, or from a homolog, a fragment or a variant of any one of these genes, preferably wherein the at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%
- the at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence selected or from a 3-UTR of a PSMB3 gene, wherein the at least one heterologous 3-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 66, 67, 112-123, preferably SEQ ID NO: 67, or a fragment or a variant of any of these.
- the at least one heterologous 5'-UTR compnses or consists of a nucleic acid sequence seleded or from a 5'-UTR of a gene selected from HSD17B4, RPL32, AIG1, alpha- globin, ASAH1, ATP5A1, COX6C, DPYSL2, MDR, MP68, NDUFA4, NOSIP, RPL31, RPL35A, SLC7A3, TUBB4B, orUBQLN2, orfroma homolog, afragment or variant of any one of these genes, preferably wherein the at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12-45,64,65, or a fragment or
- the at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence selected or from a 5'-UTR of a HSD17B4 gene, wherein the at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12,13,64,65, preferably SEQ ID NO: 13, or a fragment or a variant of any of these.
- rtificial nucleic acid of items 37 to 41 wherein the at least one heterologous 5'-UTR is selected from HSD17B4 and the at least one heterologous 3' UTR is selected from PSMB3.
- the artificial nucleic acid is selected from a DNA or an RNA, preferably an RNA.
- rtificial nucleic acid of any one of the preceding items wherein the artificial nucleic acid is an RNA selected from mRNA, circular RNA, replicon RNA, or viral RNA.
- the artificial nucleic acid preferably the RNA
- the artificial nucleic acid comprises at least one poly(A) sequence, preferably wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosine nucleotides.
- artificial nucleic acid of item 46 wherein the at least one poly(A) sequence comprises about 60 to about 150 adenosine nudeotides, preferably about 100 adenosine nucleotides.
- the artificial nucleic acid preferably the RNA, comprises at least one poly(C) sequence and/or at least one miRNA binding site and/or at least one histone stem-loop sequence.
- the artificial nucleic acid preferably the RNA
- the artificial nucleic acid comprises at least one histone stem-bop sequence
- said histone stem-loop sequence comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 3 or 4, or a fragment or variant of these.
- artificial nucleic acid of any one of the preceding items wherein the artificial nucleic add is an RNA that comprises at least one modified nucleotide, preferably a modified nucleotide selected from pseudouridine (ip) or Nl-methylpseudouridine (m1ip).
- artificial nucleic acid of any one of the preceding items wherein the artificial nucleic acid is a modified RNA wherein each uracil is substituted by a modified nucleotide.
- artificial nucleic acid of items 1 to 50 wherein the nucleic acid is an RNA that does not comprise a modified nucleotide.
- artificial nucleic acid of any one of the preceding items wherein the artificial nucleic acid is an RNA that compnses a 5' ⁇ ap structure.
- artificial nucleic acid of item 55 wherein the S'-cap structure is selected from a cap1 stmcture or a modified cap1 structure.
- artificial nucleic acid of any one of the preceding items, wherein the artificial nucleic acid is an vitro transcribed RNA.
- the artificial nucleic acid is a purified RNA, preferably wherein the RNA has been purified by at least one step of RP-hlPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow through chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.
- artificial nucleic acid of any one of the preceding items, wherein the artificial nucleic acid, preferably the RNA has an integrity of at least 50%, preferably of at least 60%, more preferably of at least 70%, most preferably of at least 80%.
- artificial nucleic acid of any one of the preceding items wherein the artificial nucleic acid, preferably the RNA, is suitable for use in treatment or prevention of a disease, disorder or condition, preferably a tumour disease, disorder or condition.
- artificial nucleic acid of any one of the preceding items, wherein the artificial nucleic acid, preferably the RNA comprises the following sequence elements preferably in 5'- to S'-direction: A) a 5'-cap structure; B) a 5'-UTR, preferably selected from a S'-UTR of a HSD17B4 gene; C) a coding sequence encoding at least one tumour antigen; D) a 3'-UTR, preferably selected from a 3'-UTR of a PSMB3 gene; E) optionally, a histone stem-loop; and F) a poly(A) sequence, preferably comprising about 100 A nucleotides.
- the artificial nucleic acid of items 1 to 61, wherein the artificial nucleic acid is an mRNA that comprises the following sequence elements in 5'-to S'-direction: A) a 5'-cap structure; B) a 5-UTR that comprises or consists of a nucleic acid sequence being identical or at least 80% identical to SEQ ID NO: 13, or a fragment or a variant thereof; C) a coding sequence that comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein encoded by a long non-coding RNA (IncRNA), wherein the nucleic acid sequence is identical or at least 80% identical to any one of SEQ ID NOs: 318-448, 651 -755,764-771, or a fragment or a variant of any of these; D) a 3'-UTR that comprises or consists of a nucleic acid sequence being identical or at least 80% identcal to SEQ ID NO: 67, or a fragment or a variant
- the artificial nucleic acid of items 1 to 61, wherein the artificial nucleic acid is an mRNA that comprises the following sequence elements in 5'- to 3' ⁇ lirection: A) a 5'-cap structure; B) a 5'-UTR that comprises or consists of a nucleic acid sequence being identical or at least 80% identical to SEQ ID NO: 13, or a fragment or a variant thereof; C) a coding sequence that comprises a nucleic acid sequence encoding at least one antigenic peptide selected from a peptide or protein of a tumour neoantigen, wherein the nucleic acid sequence is identical or at least 80% identical to any one of SEQ ID NOs: 449-511, or a fragment or a variant of any of these; D) a 3'-UTR that comprises or consists of a nucleic acid sequence being identical or at least 80% identfcal to SEQ ID NO: 67, or a fragment or a variant thereof; E) optionally, a histone stem
- a pharmaceutical composition comprising at least one artificial nucleic acid compnsing at least one coding sequence encoding at least one tumour antigen as defined by any one of the items 1 to 63.
- the pharmaceutical composition of item 64 comprising a plurality of artificial nucleic acids each encoding at least one different tumour antigen.
- the pharmaceutical composition of item 64 or 65 wherein the at least one artificial nucleic acid, preferably the RNA, is formulated in at least one cationic or polycationic compound. .
- the pharmaceutical composition of item 66 wherein the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combinations.
- the pharmaceutical composition of item 68 wherein the lipid-based carriers are selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipo-polyplexes, and/or nanoliposomes. .
- the pharmaceutical composition of item 71, wherein the aggregation reducing lipid is a polymer conjugated lipid selected from a PEG-conjugated lipid or a PEG-free lipid.
- composition of items 68 to 77 wherein the lipid-based carriers, preferably the LN Ps, comprise (i) at least one cationic lipid, preferably as defined in items 74 or 75; (ii) at least one neutral lipid, preferably as defined in item 76; (iii) at least one steroid or steroid analogue, preferably as defined in item 77; and (iv) at least one aggregation reducing lipid, preferably as defined in items 72 to 73 .
- the lipid-based carriers preferably the LN Ps, comprise (i) at least one cationic lipid, preferably as defined in items 74 or 75; (ii) at least one neutral lipid, preferably as defined in item 76; (iii) at least one steroid or steroid analogue, preferably as defined in item 77; and (iv) at least one aggregation reducing lipid, preferably as defined in items 72 to 73 .
- tumour antigen or a composition of item 86 wherein the tumour antigen comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 124-254, 546-650, 756-763, or an immunogenic fragment or immunogenic variant of any of these.
- tumour antigen or a composition of item 86, wherein the tumour antigen comprises or consists of at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 255-317, or an immunogenic fragment or immunogenic vanant of any of these.
- tumour antigen or a composition of items 86 to 88, wherein the composition comprises a plurality of tumour antigens and, optionally, wherein the antigens are formulated in a pharmaceutically acceptable carrier.ntibody, a T-cell, or a TCR that has been raised against or stimulated with any of a tumour antigens or nucleic acids as defined in any one of the preceding items.
- mbination comprising at least two or a plurality of therapeutic modalities selected from a) at least one artificial nucleic acid of items 1 to 62; and/or b) at least one pharmaceutical composition of items 63 to 85; and/or c) at least one tumour antigen or composition items 86 to 89; and/or d) at least one antibody, T-cell, or TCR of item 90; or kit of parts comprising at least one artificial nucleic acid of any one of items 1 to 62, and/or at least one pharmaceutical composition of any one of items 63 to 85, and/or at least one tumour antigen or a composition of tumour antigens of terns 86 to 89, and/or at least one antibody, a T-cell, or a TCR of item 90, optionally comprising a liquid vehicle for solubilising, and optionally comprising technical instructions providing information on administration and dosage of the components.
- rtificial nucleic acid of any one of items 1 to 62 a pharmaceutical composition of any one of items 63 to 85, a tumour antigen or a composition of tumour antigens of items 86 to 89, an antibody, T-cell, or TCR of item 90, a combination of item 91, or a kit or kit of parts of item 92 for use as a medicament.
- rtificial nucleic acid of any one of items 1 to 62 a pharmaceutical composition of any one of items 63 to 85, a tumour antigen or a composition of tumour antigens of items 86 to 89, an antibody, T-cell, or TCR of item 90, a combination of item 91, or a kit or kit of parts of item 92 for use as a medicament in treating or preventing cancer in a subject, or any disease, disorder, or condition related to cancer.
- rtificial nucleic acid of any one of items 1 to 62 a pharmaceutical composition of any one of items 63 to 85, a tumour antigen or a composition of tumour antigens of items 86 to 89, an antibody, T-cell, or TCR of item 90, a combination of item 91, or a kit or kit of parts of item 92, for use as a medicament in treating or preventing squamous cell carcinoma in a subject, or any disease, disorder, or condition related to squamous cell carcinoma, preferably wherein the squamous cell carcinoma is selected from LUSC / squamous non-small-cell lung cancer (sqNSCLC) or head and neck squamous cell carcinoma (HNSCC).
- sqNSCLC squamous non-small-cell lung cancer
- HNSCC head and neck squamous cell carcinoma
- rtificial nucleic acid of any one of items 1 to 62 a pharmaceutical composition of any one of items 63 to 85, a tumour antigen or a composition of tumour antigens of items 86 to 89, an antibody, T-cell, or TCR of item 90, a combination of item 91, or a kit or kit of parts of item 92, for use as a medicament in treating or preventing NSCLC in a subject, or any disease, disorder, or condition related to NSCLC.
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises (a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, lnc-TRPC5-3:1, KCNMB2-AS1:4, Inc- NTF3-5:5, or lnc-ZC3H8-6:1, or an immunogenic fragment or vanant of any of these; and/or (b) at least one antigenic peptide selected from a peptide or protein of a BRAF neoantigen, EGFR neoantigen, TP53 neoantigen, PIK3CA neoantigen, NFE2L2 neoantigen, preferably selected from EGFR neoantigen, orTP53 neoant
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, KCNMB2-AS1:4, lnc-NTF3-5:5, or Inc- ZC3hl8-6:1, or an immunogenic fragment or variant of any of these. .
- the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, the combination, or the kit or kit of parts comprises (a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-CLEC2D-9:1, lnc-TRPC5-3:1, or LINC00893:25, or an immunogenic fragment or variant of any of these; and/or (b) at least one antigenic peptide selected from a peptide or protein from an NRAS neoantigen, T/W1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neo
- a method of treating or preventing a disease, disorder or condition comprising applying or administering to a subject in need thereof an effective amount of the artificial nucleic acid of items 1 to 62, pharmaceutical composition of items 63 to 85, tumour antigen or a composition of tumour antigens of items 86 to 89, antibody, T-cell, or TCR of item 90, combination of item 91, or kit or kit of parts of item 92.
- the method of item 101, wherein the disease, disorder or condition is cancer. 103.
- cancer is squamous cell carcinoma, preferably selected from squamous non- small-cell lung cancer (sqNSCLC) or head and neck squamous cell carcinoma (HNSCC).
- the cancer is NSCLC and wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts is comprises (a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-WDR72-2:4, lnc-TRPC5-3:1, KCNMB2-AS1:4, lnc-NTF3-5:5, or Inc- ZC3H8-6:1, or an immunogenic fragment or variant of any of these; and/or (b) at least one antigenic peptide selected from a peptide or protein from a BRAF neoantigen, EGFR neo
- the cancer is melanoma and wherein the at least one tumour antigen that is provided by the artificial nucleic acid, the pharmaceutical composition, the tumour antigen or a composition of tumour antigens, or the kit or kit of parts comprises (a) at least one antigenic peptide selected from a peptide or protein encoded by lnc-CLEC2D-9:1 or lno-TRPC5-3:1, or an immunogenic fragment or variant of any of these; and/or (b) at least one antigenic peptide selected from a peptide or protein from an NRAS neoantigen, TiW1 B neoantigen, ECPAS neoantigen, MAP2K1 neoantigen, TOMM22 neoantigen, GLB1 neoantigen, MAGE-A3 neoantigen, ATAD2 neoantigen, BRAF neoantigen, DIP2B neoantigen
- Figure 1 shows an exemplary neoantigen identification workflow.
- Immunogenicity prediction neolM
- MHC presentation prediction MhlCnuggets & neoMS. ** Taking into account host gene expression, sample coverage, HLA coverage/distribution, MHC presentation ability and immunogenicity, etc.
- Figure 2 shows the high tumour specificity of the IncRNAtranscnpt lnc-CLEC2D-9:1 (corresponding smORF: lncCLEC2D91412616).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in melanoma and low to absent expression in healthy tissues (except in the immune-privileged tissue testis).
- Figure 3 shows the high tumour specificity of the IncRNA transcript lnc-TRPC5-3:1 (corresponding smORF: lncTRPC53116031801).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in melanoma and low to absent expression in healthy tissues.
- Figure 4 shows the high tumour specificity of the IncRNA transcript lnc-TRPC5-3:1 (corresponding smORF: lncTRPC53116031801).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in NSCLC (both in lung adenocarcinoma, LUAD, and lung squamous cell carcinoma, LUSC) and low to absent expression in healthy tissues.
- Figure 5 shows the high tumour specificity of the IncRNA transcript lnc-WDR72-2:4 (corresponding smORFs: lncWDR722415641696 and lncWDR722416421696).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in NSCLC (lung squamous cell carcinoma, LUSC) and low to absent expression in healthy tissues.
- Figure 6 shows the high tumour specificity of the IncRNA transcript KCNMB2-AS1:4 (corresponding smORF: KCNMB2AS14633753).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in NSCLC (lung squamous cell carcinoma, LUSC) and low to absent expression in healthy tissues.
- FIG 7 shows the high tumour specificity of the IncRNA transcript lnc-NTF3-5:5 (corresponding smORF: lncNTF35511951417).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in NSCLC (lung squamous cell carcinoma, LUSC) and low to absent expression in healthy tissues.
- Figure 8 shows the high tumour specificity of the IncRNA transcript lnc-ZC3hl8-6:1 (corresponding smORF: lncZC3H86163144).
- RNAseq data obtained from the TCGA (tumour tissue) and GTEX (healthy tissue) database shows high target expression in NSCLC (both in lung adenocarcinoma, LUAD, and lung squamous cell carcinoma, LUSC) and low to absent expression in healthy tissues.
- Figure 9 shows the near absence of the smORF lncTRPC53116031801 's translated peptide in healthy tissues.
- Mass spectrometry data shows very low to absent levels (asterisks) in various healthy tissues in comparison to different highly abundant controls (e.g. housekeeping genes; dots).
- Figure 10 shows the in vitro antigen validation of melanoma neoantigens (peptide pool 1) in a DC-T cell assay (further details see Example 6.1, Table E3-E5). IFN-gamma readouts of pan T-cells after two stimulation rounds (R1 and R2) were performed for 10 PBMC donors (D1 -D10).7 donors showed an antigen specific T cell response to at least one of the peptides of pool 1. Recall and memory responses were observed in 1 and 1 donor, respectively.
- Figure 11 shows the in vitro antigen validation of melanoma neoantigens (peptide pool 2) in a DC-T cell assay (further details see Example 6.1, Table E3-E5).
- IFN-gamma readouts of pan T-cells after two stimulation rounds were performed for 10 PBMC donors (D1 -D10).7 donors showed an antigen specific T cell response to at least one of the peptides of pool 2. A memory response was observed in 2 donors.
- Figure 12 shows the in vitro antigen validation of melanoma cancer antigens (peptide pool 3) in a DC-T cell assay (further details see Example 6.1, Table E3-E5).
- IFN-gamma readouts of pan T-cells after two stimulation rounds were performed for 10 PBMC donors (D1-D10). All donors showed an antigen specific T cell response to at least one of the peptides of pool 3.
- FIG. 13 shows the in vitro antigen validation of melanoma neoantigens (peptide pool 4) in a DC-T cell assay (further details see Example 6.1, Table E3-E5). IFN-gamma readouts of pan T-cells after two stimulation rounds (R1 and R2) were performed for 10 PBMC donors (D1 -D10).8 donors showed an antigen specific T cell response to at least one of the peptides of pool 4. Recall and memory responses were observed in 2 and 1 donors, respectively.
- Figure 14 shows the in vitro antigen validation of melanoma neoantigens (peptide pool 5) in a DC-T cell assay (further details see Example 6.1, Table E3-E5). IFN-gamma readouts of pan T-cells after two stimulation rounds (R1 and R2) were performed for 10 PBMC donors (D1-D10).4 donors showed an antigen specific T cell response to peptide pool 5. Recall and memory responses were observed in 1 and 2 donors, respectively.
- Figure 15 shows an overview of all recall and memory responses observed in Example 6.1/ Figures 10-14.
- Figure 16 shows the in vitro antigen validation of the indicated smORFs in a DC-T cell assay (further details see Example 6.2, Table E6-E8). IFN-gamma readouts of pan T-cells after two stimulation rounds (R1 and R2) were performed for various PBMC donors.
- Figure 17 shows the in vitro antigen validation of the indicated melanoma neoantigens in a DC-T cell assay (further details see Example 6.3, Table E9-E1 OA). IFN-gamma readouts of pan T-cells after two stimulation rounds (R1 and R2) were performed for various PBMC donors.
- Figure 18 shows the in vivo immunogenicity of an LNP-formulated mRNA vaccine R12300 encoding two smORFs (that are amongst others expressed in melanoma) in CB6F1 hybrid mice.
- Mice were vaccinated intramuscularly with 5
- mice were sacrificed and splenocytes were isolated. Splenocytes were restimulated with a 15-mer peptide library for lncCLEC2D91412616 or DMSO as control and analyzed by flow cytometry.
- FIG. 19 shows the in vivo immunogenicity of an LNP-formulated mRNA vaccine R12301 encoding multiple smORFs (that are amongst others expressed in NSLSC) in CB6F1 hybrid mice. Mice were vaccinated intramuscularly with 5
- mice were sacrificed and splenocytes were isolated. Splenocytes were individually restimulated with peptide libraries for each smORF (15-mer libraries covering the complete antigens) or DMSO as control and analyzed by flow cytometry.
- the magnitude of the CD8+ (top) and CD4+ (bottom) T cell responses against the 4 encoded smORF peptides KCNMB2AS14633753, lncNTF35511951417, lncWDR722415641696 and !ncZC3H86163144(NKZW-smORFs) is shown as percentage of IFN-Y+ TNF+ cells of CD8+ or CD4+ T cells, respectively. Median values are plotted.
- Example 1 Antigen discovery and selection methods The goal of the example was to identify cancer antigens that produce epitopes in patients with NSCLC or melanoma.
- tumor-specifc antigens derived from somatic mutations (neoepitopes) and tumor-associated antigens encoded by small open reading frames (smORFs) identified in long non ⁇ oding RNAs (IncRNAs).
- smORFs small open reading frames
- IncRNAs tumor-associated antigens encoded by small open reading frames (smORFs) identified in long non ⁇ oding RNAs
- IncRNAs tumor-associated antigens encoded by small open reading frames (smORFs) identified in long non ⁇ oding RNAs (IncRNAs).
- smORFs/lncRNAs showed a well defined tumour-associated expression and were only detected in low levels in normal tissue, using transcriptomics and proteomics.
- MhlC major histocompatibility complex
- Discovery/ of neoepttopes Sequence alignment map (BAM) files from the cancer genome atlas (TCGA) database were used to detect tumour- specific variants.
- the lncR ⁇ WsmORFs were kept that show low expression in normal tissue, using transcriptomics (GTEx W) and mass spectrometry (PXD010154).
- the immunogenicily score (neoim_score) is generated using a machine learning algorithm and reflects the probability that an ep'rtope is immunogenic.
- the RNAseq expression is given in TPMs; TPMs normalize RNAseq reads by the length of each gene and sequencing depth and are commonly used to compare expression levels in different samples.
- the expected coverage of the neoantigens was calculated by taking the product of the population frequency of the mutation and the HLA allele frequencies predicted to bind the epitope.
- Example 2 Antigen discovery and selection of melanoma and NSCLC neoantigens The goal of the experiment was to discover melanoma and NSCLC cancer antigens derived from tumour-specific neoantigens. The resulting top hits are summarized in Table 3. Table 3 shows identified melanoma and NSCLC neoantigens.
- M/melanoma in this context furthermore specifically means skin cutananeous melanoma (SKCM). 7ab/e_3; Identjfiedsuitable melanoma and NSCLC neoantiaens
- SKCM skin cutananeous melanoma
- 7ab/e_3 Identjfiedsuitable melanoma and NSCLC neoantiaens
- Example SLAntigen discovery and selection of melanoma and NSCLC antigens encoded by IncRNAs The goal of the experiment was to discover melanoma and NSCLC cancer antigens derived from IncRNA-smORFs. The resulting top hits are summarized in Table 4. Table 4 shows identified melanoma and NSCLC antigens encoded by IncRNA transcripts.
- M/melanoma in this context furthermore specifically means skin cutananeous melanoma (SKCM).
- Table 4 Identified suitable melanoma and NSCLC antigens encoded by IncRNA transcripts Additionally, data from mass-spectrometry studies including 50 melanoma patients (PXD009630 - melanoma) and 57 lung cancer patients (PXD002612 - lung adenocarcinoma) confirmed that the preferred smORFs are indeed translated. In this context, the frequency of patients where the smORFs' translated peptides could be detected in the tumor samples by mass spectrometry are given in Table 4A.
- Example 4 High tumour specificity of preferred IncRNAs The goal of the experiment was to comparatively analyze the expression of preferred IncRNA transcripts (Table E1) in tumour and healthy tissue. To this end, RNAseq expression data of the TCGA (tumour tissue) and GTEX (healthy tissue) database was analyzed. Furthermore, mass-spectrometry data was analyzed.
- Table El Expression of preferred IncRNA transcripts in melanoma/NSCLC and healthy tissues n.a., not available; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma Furthermore, low to absent expression in healthy tissues of the preferred smORFs' translated peptides was confirmed in mass spectrometry analyses. Exemplary data is shown for lncTRPC53116031801 in Figure 9. Overall, the data suggests high and frequent tumour expression and bw/absent healthy tissue expression for the preferred IncRNA and derived smORFs.
- Example 5 Expression of IncRNA transcripts in the thymys The goal of the experiment was to analyze the expression of the preferred IncRNA transcripts (Table E1) in the thymus.
- antigen expression in the thymus is associated with central tolerance and thus ideally should be low to absent for target antigens employed in vaccines.
- transcriptomics analyses were performed on publicly available datasets. Results Exemplary data from three datasets depicted in Table E2 show only low to absent expression of the IncRNA transcnpts in the thymus. This suggests a low likelihood for central tolerance of smORFs derived from these IncRNA transcripts, such as the preferred smORFs stated in Table E1.
- Example 6 Antigen validation - in vitro immunoflenicity The goal of the experiment was to analyse the in vitro immunogenicity of different melanoma and NSCLC cancer antigens derived from tumour-specific neoantigens and IncRNA-derived smORFs described by Examples 1-5. Peptides comprising the epitopes of interest were synthesized according to standard procedures. The amino acid sequences of the peptides are given in the respective Examples. Peptide pools (Examples 6.1 and 6.2) or single peptides (Example 6.3 and 6.4) were used. Immunogenicity of test peptides was assessed using an in vitro DC-T cell assay.
- cryopreserved PBMCs from multiple (healthy) donors of a series of HLA types were retrieved from cryo-storage and monocytes were isolated.
- Cultured monocytes were differentiated into immature dendritic cells (iDCs) using a cytokine cocktail (GM-CSF and IL- 4).
- the iDCs were loaded with the test peptides and further matured with CD40L to mature dendritic cells (mDC).
- mDCs were co-cultured with previously isolated autologous pan T cells for 8 days in the presence of a cytokine cocktail. During co-cutture, the medium and cytokines were regularly refreshed.
- round 1 and round 2 the pan T cells were harvested and re- stimulated with peptide loaded monocytes on IFN-y FluoroSpot plates.
- the FluoroSpot plates were developed according to the Manufacturer's protocol. The number of IFN-y secreting pan T cells were measured using the Mabtech IRISTM FluoroSpot Reader. Furthermore, moDC quality control was performed using flow cytometry.
- DMSO fetal sulfate
- MART Myelin- oligodendrocyte glycoprotein
- CEF/CEFBA positive controls
- Materials need to be sterile, endotoxin levels should be below 0.1 EU/mg and protein concentration s 1 mg/ml.
- IFNy+ spot forming units (SFU) were measured with a Fluorospot assay for each R1 and R2 and the delta SFU (dSFU) was calculated as the absolute difference in spots between the tested smORF-derived peptides and the respective DMSO control condition after normalizing the SFU/well to SFU/million.
- Example 6.1 and 6.2 a response was considered a "recall response” if the following criteria was met: i) dSFU for R1 was at least 30, and ii) dSFU R2 was at least 2-fold higher compared to dSFU R1.
- Example 6.3 single peptides
- a response was considered a "recall response” if the following criteria was met: i) dSFU for R1 was at least 5, and ii) dSFU R2 was at least 2-fold higher compared to dSFU R1.
- a response was considered a "memory response" if the following criteria was met: i) dSFU for R1 was at least 100, and ii) dSFU R2 was 0.1 - 2.0 fold compared to dSFU R1. It should be noted that failure to meet the above criteria in a given condition does not necessarily mean that there was no recall or memory response at all. Rather, it may be that a recall or memory response was of a lesser magnitude than the above thresholds or incompatible with the above criteria that were chosen for uniform batch analysis.
- Example 6.1 Antigen validation - in vitro immunofleniaty of melanoma cancer antigens - pooled peptides
- the goal of the experiment was to analyse the in vitro immunogenicity of different melanoma cancer antigens derived from tumour-specific neoantigens and encoded by IncRNA-derived smORFs.
- cryopreserved PBMCs from 10 multiple (healthy) donors with a series of different hlLA types (see Table E3) were tested with 5 drtferent peptide pools (see Table E4) derived from melanoma cancer antigens.
- Table E3 Overview of PBMC donors used in Example 6.1_and corresponding HLA alleles
- Table E4 Overview of melanoma peptide pools used in Example 6.1 Results: The in vitro immunogenicity of melanoma cancer antigens using the peptfcle pools stated in Table E4 is shown in Figures 10-14. The graphs show IFN-gamma readouts of the assay described in the present example. Summaries of the recall and memory responses defined according to the above-described criteria for uniform batch analysis are shown in Figure 15 and Table E5.
- PBMC donors showed an antigen specific T cell response to at least one of the peptides of pool 3.4 PBMC donors showed a recall response to at least one of the peptides of pool 3.4 PBMC donors showed a memory response to at least one of the peptides of pool 3 (see also Figure 15/Table E5).
- 8 PBMC donors showed an antigen specific T cell response to at least one of the peptides of pool 4.2 PBMC donors showed a recall response to at least one of the peptides of pool 4.1 PBMC donor showed a memory response to at least one of the peptides of pool 4 (see also Figure 15/Table E5).
- PBMC donors showed an antigen specific T cell response to at least one of the peptides of pool 5.1 PBMC donor showed a recall response to at least one of the peptides of pool 5.
- 2 PBMC donors showed a memory response to at least one of the peptides of pool 5 (see also Figure 15/Table E5).
- every peptide pool comprises at least one cancer antigen that could lead to an immune response in human T cells. Both recall and memory responses could be observed in this experimental setup.
- Example 6.2 _Antiflen validation - inwtroimmunogenicjty of NSCLC and melanoma cancer antigens - pooled peptides
- the goal of the experiment was to analyse the in vitro immunogenicity of different NSCLC and melanoma cancer antigens derived from tumour-specrfic neoantigens and encoded by lncRNA ⁇ )erived smORFs.
- cryopreserved PBMCs from 10 multiple (healthy) donors with a sen'es of different HLA types (see Table E6) and 8 different peptide pools (see Table E7) derived from NSCLC and melanoma cancer antigens were tested.
- Table E6 Overview ofPBMC donors used in Example 6.2 and corresponding HLA alleles
- Table E7 Overview of NSCLC/melanoma peptide pods used in Example 6.2
- Results Exemplary in vitro immunogenicity results for the NSCLC/melanoma cancer antigens and the peptide pools stated in Table E7 are shown in Figure 15.
- the graph shows IFN-gamma readouts of the assay described in the present example.
- Recall and memory responses shown in Figure 15 are furthermore summarized in Table E8.
- Table E8 Summary of recall and memory responses shown in Fisure 16
- the condition Pool 8/Donor D9 in Example 6.2 showed an immune response characterized by a dSFU R1 and a dSFU R2 of 21.25 and 236, respectively.
- IncRNA encoded peptides (lncCLEC2D91412616, KCNMB2AS14633753, lncZC3H86163144, lncNTF35511951417 and lncWDR722415641696) were immunogenic. Notably, the observed in vitro immunogenicity of IncRNA encoded peptides was comparable or even superior (lncNTF35511951417 and lncWDR722415641696) to the well-known immunogenic TAA MART-1 , further underscoring their promise as cancer targets.
- ExamBle 6.3 Antigen validation - in vitro immunoflenicity of melanoma cancer antigens - single peptides
- the goal of the experiment was to analyse the in vitro immunogenicity of different melanoma cancer antigens derived from tumour-specific neoantigens and encoded by IncRNAs. Cryopreserved PBMCs from 5 (healthy) donors with a series of different hlLA types (see Table E9) and 9 single peptides derived from melanoma cancer antigens (see Table E10) were tested.
- Table E9 Overview ofPBMC donors used in Example 6.3 and corresponding HLA alleles
- Table E10 OveM'ew of melanoma single peptides In future experiments further single peptides from pools 1 and 2 of Table E7 are tested. Results: Exemplary in vitro immunogenicity results for the melanoma cancer antigens using the single peptides stated in Table E10 are shown in Figure 17. The graph shows IFN-gamma readouts of the assay described in the present example. Recall responses shown in Figure 17 are furthermore summarized in Table E1 OA.
- Example 6.4 Antigen validation - in vitro immunoqenicity of NSCLC and melanoma cancer antigens • single rtides The goal of the experiment is to analyse the in vitro immunogenicity of different NSCLC cancer antigens derived from tumour-specific neoantigens and encoded by IncRNAs. In this assay, cryopreserved PBMCs from multiple (healthy) donors with a sen'es of different HLA types are tested with selected single NSCLC peptides from Table E7.
- Example 7 Antigen validation - immunopeptidomics To further study and confirm epitope presentation on tumour cell MhlC-1/11, immunopeptidomics analyses are performed.
- Example 8 Preparation of nucleic acid encoding cancer antigens
- the present example provides methods of obtaining RNAs of the invention as well as methods of generating compositions of the invention comprising nucleic acid(s), in particular RNA, formulated in lipid-based carriers. ExampjeSJ.
- DNA sequences encoding cancer antigens of the invention were prepared and used for subsequent RNA in vitro transcription reactions. Some DNA sequences were prepared by modifying the wild type or reference encoding DNA sequences by introducing a G/C optimized coding sequence for stabilization and expression optimization. Sequences were introduced into a pUC derived DMA vector to compnse a stabilizing UTR sequences and a stretch of adenosines and an optional histone stem-loop (hSL). The obtained plasmid DNA templates were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA templates were extracted, purified, and linearized using a type II restriction enzyme.
- RNA in vitro transcription from plasmid DNA templates Linearized DMA templates were used for DNA dependent RNA in vitro transcription (IVT) using T7 RNA polymerase in the presence of a sequence optimized nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (for Cap1: m7G(5')ppp(5')(2'OMeA)pG; TriLink) under suitable buffer conditions.
- IVTT DNA dependent RNA in vitro transcription
- RNA IVT reaction was subjected to purification steps comprising RP-HPLC.
- Example 8.3 Preparation of lipid-based carriers encapsulatina the mRNA LNPs used in the working examples were prepared using the NanoAssemblrTM microfluidic system (Precision NanoSystems Inc., Vancouver, BC) according to standard protocols which enables controlled, bottom-up, molecular self-assembly of nanoparticles via custom-engineered microfluidic mixing chips that enable millisecond mixing of nanoparticle components at a nanolitre scale.
- NanoAssemblrTM microfluidic system Precision NanoSystems Inc., Vancouver, BC
- lipid nanoparticle compositions For preparation of lipid nanoparticle compositions the following excipients / lipids the following excipients / lipids were used: (i) ionizable lipid: VitE-C4DE-Pip-thioether as described herein and in Table 1 of published patent application W02021123332 (compound C26); (ii) cholesterol (Avanti Polar Lipids; Alabaster, AL) as described herein; (iii) neutral tipid / phospholipid "DPhyPE” (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; Avanti Polar Lipids; Alabaster, AL) as described herein; (iv) phosphatidylserine DPhyPS (1,2-diphytanoyl-sn-glycero-3-phospho-L-serine; Avanti Polar Lipids; Alabaster, AL, 850408P) as described herein; and (v) a polymer
- LNP compositions were furthermore characterized as described in Table E13.
- Table E13 LNP compositions
- the lipids were solubilized in alcoholic solution (ethanol) according to standard procedures.
- LNPs were prepared by mixing appropriate volumes oflipid stock solutions for cholesterol, phospholipids, and polymer conjugated lipid in ethanol buffer (20 mg/ml in EtOH); the cationic lipid C26 was solubilized in 30 mg/ml t-butanol and added to the ethanol premix of lipids. Subsequently, this ethanol mix of lipids was combined with an aqueous phase (50 mM sodium acetate, pH 4.0) containing 1g/L of mRNA.
- aqueous phase 50 mM sodium acetate, pH 4.0
- mRNA as indicated in the working examples was diluted to 0.05 to 0.2 mg/ml in 50 mM acetate buffer, pH 4.
- Syringe pumps were installed into inlet parts of the NanoAssemblrTM (Precision NanoSystems Inc., Vancouver, BC) and used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (volA/ol) with total flow rates from about 14 ml/min to about 18 ml/min.
- lipid nanoparticles were filtered through a 0.2 pm pore sterile filter. Lipid nanoparticle particle diameter size was from about 90 nm to about 140 nm as determined by quasi-elastic light scattering using a Malvem Zetasizer Nano (Malvem Instmments Ltd.; UK).
- Example 9 Translation ofsmORF encoding mRNA and stability of the translated protein Translation ofsmORF encoding mRNA (mRNA constructs described in Table E11) and stability of the expressed protein was measured according to standard procedures known in the art using western blot and flow cytometry.
- HEK293T cells 400000 cells/well, 200000 cells/ml
- Lipofectamin 1 pg/ml mRNA using Lipofectamin
- the amount of the expressed protein was - after 4h and 16-18h incubation with and without Carfilzomib - measured by FACS using antibodies to the CTLA4 domains or HA-tag.
- HeLa cells were transfected with mRNA, supplemented or not with Carfilzomib 2h post-treatment, and harvested and lysed 18h post-treatment. Subsequently, the amount of the expressed protein was assessed by Western blot using an antibody to the hlA-tag. Using FACS and Western Blot, translation of mRNA constructs encoding single smORFs or several smORFs as well as the stability of their encoded fusion proteins could be confirmed via CTLA-4 or HA staining.
- Helper epitope(s)-Linker(GS4)-TMD/TMCD(CTLA4)-3'UTR-PolyA Variant R12301 (mRNA encoding for six smORFs expressed in NSCLC): 5'UTR-SP(CTLA4)-Linker(GS4)-smORF2- Linker(GS4)-smORF3-Linker(GS4)-smORF5-Linker(GS4)-smORF6-Linker(GS4)-smORF7-Linker(GS4)-smORF4- Linker(GS4)-Helperepitope(s)-Linker(GS4)-TMD/TMCD(CTLA4)-3'UTR-PolyA
- Example 11 Inyiyo immunogenicit ⁇ ofmRNA constructs encoding for smORFs
- R12300 and R12301 constructs were produced and formulated as described in Example 8 and tested as follows.
- the constructs were applied to female CB6F1 hybrid mice on days 0, 7 and day 14 intramuscularly (i.m.; musculus tibialis) with doses ofSpg LNP-formulated mRNA (R12300, R12301 or PpLuc control mRNA R8730).
- mice 21 days after the first mRNA administration, mice were sacrificed, and spleens were collected for further analysis. Cancer antigen-specific cellular responses in splenocyte samples obtained in this step were measured as antigen- specific T cell activation. This was analyzed by intracellular cytokine staining and subsequent analysis by flow cytometiy according to standard protocols as follows: isolated splenocytes were individually restimulated with peptide libraries for each smORF (15-mer libraries covering the complete antigens) at 1 pg/ml final peptide concentration in the presence of anti-CD28 (BD Biosciences, San Jose, USA) for 6 hours with the addition ofGolgiPlug (BD Biosciences) after 1h.
- peptide libraries for each smORF (15-mer libraries covering the complete antigens) at 1 pg/ml final peptide concentration in the presence of anti-CD28 (BD Biosciences, San Jose, USA) for 6 hours with the addition ofGolgiPlug (BD Bioscience
- Unstimulated splenocytes were treated the same way, but supplemented with DMSO instead of the peptide cocktail. Additional controls were splenocytes stimulated with PMA/ionomycin (no anti-CD28; PMA and ionomycin from Sigma- Aldrich; Merck KGaA, Darmstadt, Germany) (positive control) and splenocytes which were left unstained by fluorophore- conjugated antibodies (negative control). After stimulation, splenocytes were stained with surface and intracellular, fluorophore-conjugated antibodies and analysed by flow cytometry. CDS and CD4 responses are shown as IFN- gamma/TNF-alpha double-positive CDS and CD4 T cells.
- Exemplary data for the construct R12300 are shown in Figure 18.
- the data shows that the mRNA R12300 raised potent CDS (top) and CD4 (bottom) responses against the lncCLEC2D91412616 smORF.
- Exemplary data for the construct R12301 and the T-cell responses against the NKZW-smORFs are shown in Figure 19.
- the data shows that R12301 raised potent CDS (top) and CD4 (bottom) responses.
- Example 12 Broad IncRNA indication screening The goal of the experiment was to analyze whether the IncRNAs KCNMB2-AS1:4, lnc-NTF3-5:5, lnc-WDR72-2:4, Inc- ZC3hl8-6:1 and lnc-CLEC2D-9:1 are (over)expressed in indications beyond melanoma and NSCLC. To this end, all indications in the TCGA tumor database were analyzed.
- Table E14 Examples of tumor expression values and applied expression thresholds Table E15 summarizes indications in the TCGA database that (over)expressed at least one of the five studied IncRNAs as well as the proportion of patients in a given indication that (over)expressed the one or more IncRNAs. As can be seen in this table, the five IncRNAs are widely expressed across a multitude of indications and to high extents within those indications (ranging from 26-94% of patients in a given study/indication).
- the four IncRNAs Inc- KCNMB2-AS1:4, KCNMB2-AS1:4, WDR72-2:4, lnc-ZC3H8-6:1 are particularly (over)expressed in squamous cell carcinomas, namely LUSC, HSNCC, cervical carcinoma and esophageal carcinoma.
- squamous cell carcinomas namely LUSC, HSNCC, cervical carcinoma and esophageal carcinoma.
- lnc-ZC3hl8-6:1 is expressed in synovial sarcomas.
- Table El 5 Broad IncRNA indication screening Numbers show the proportion of patients in a given indication considered to express the indicated IncRNA.
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