Patent Grant
11369669
The present invention relates to a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii). The present invention further relates to a polynucleotide encoding said recombinant virus of the family Paramyxoviridae and to a host cell comprising said recombinant virus of the family Paramyxoviridae and/or said polynucleotide encoding said recombinant virus of the family Paramyxoviridae. Moreover, the present invention relates to a method for activating immune cells with antitumor activity in a sample comprising cancer cells and to further means, methods, and uses related to the present invention.
Oncolytic viruses (OV) which replicate selectively in tumor cells are an emerging modality of cancer treatment. Aside from direct cytopathic effects and lysis of tumor cells, interactions of OV with the immune system can trigger systemic anti-tumor immunity. OV have been modified to express immunomodulatory transgenes to further enhance these effects (Melcher et al., Mol Ther. 2011, 19: 1008-1016). The vaccinia virus JX-594 and herpesvirus talimogene laherpavec (TVEC), both harboring GM-CSF, have shown promising results in clinical phase II and III trials (Heo et al., Nat Med. 2013, 19: 329-336 and Andtbacka et al. J Clin Oncol. 2013, 31, suppl; abstr LBA9008).
RNA viruses, in particular members of the family Paramyxoviridae like, e.g. measles virus, have also shown potential use in oncolysis. Viruses of the family Paramyxoviridae are negative-sense single-stranded RNA viruses and include human pathogens like, e.g. human parainfluenza viruses, mumps virus, human respiratory syncytial virus, and measles virus. From wild type measles virus, several non-pathogenic strains, including a vaccine strain, have been derived, which have been shown to remain oncolytic. The measles virus vaccine strain has been developed as a vector platform to target multiple tumor entities and several clinical trials are ongoing (Russell et al., Nat Biotechnol. 2012, 30: 658-670). Recently, the capacity of oncolytic MV encoding GM-CSF to support the induction of a specific anti-tumor immune response in terms of a tumor vaccination effect was demonstrated (Grossardt et al. Hum Gene Ther. 2013, 24: 644-654).
Tumor antigens, i.e. antigenic compounds associated with cancer cells, have been identified early in tumor research. Initially, the term “tumor antigen” was used to relate to antigens expressed by tumor cells relatively specifically, while the term “tumor specific antigen” was used to relate to structures exclusively found on tumors. However, this distinction was later given up in view of the vast diversity of expression profiles in tumors. Tumor antigens have been known as tumor markers, but also as targets useful for targeting cancer cells with high specificity. MV oncolytic specificity can be achieved by entry-targeting based on single-chain antibodies displayed on the viral attachment protein H blinded for its natural receptors CD46 and SLAM (Vongpunsawad et al. (2004), J Virol 78: 302; Nakamura et al. (2004), Nat Biotechnol 22: 331). To date, a wide variety of tumor antigen-specific MV have been generated including vectors with specificity for CD20, CD38, CEA, PSCA, PSMA, EGFR, EGFRvIII, Her2neu, HMWAA (cf. Hammond et al. (2001), J Virol 75(5): 2087. (PMID: 11160713); Peng et al. (2003), Blood 101(7): 2557 (PMID: 12433686); Allen et al. (2006), Cancer Res 66(24): 11840 (PMID: 17178881); Hasegawa et al. (2007), J Virol. 81(23): 13149 (PMID: 17804513); Ungerechts (2007), Cancer Res. 67(22): 10939 (PMID: 18006839); Liu et al. (2009), Prostate 69(10): 1128 (PMID: 19367568); Bossow et al. (2011), Cancer Gene Ther. 18(8): 598 (PMID: 21701532); Zaoui et al. (2012), Cancer Gene Ther. 19(3): 181-91 (PMID: 22076043); Kaufmann et al. (2013), J Invest Dermatol. 133(4): 1034 (PMID: 23223133)).
There is, however, still a need in the art for improved cancer therapies, in particular for improved oncolytic viruses.
Accordingly, the present invention relates to a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii).
As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.
Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, “particularly”, “more particularly”, “specifically”, “more specifically” or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by “in an embodiment of the invention” or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention. Moreover, if not otherwise indicated, the term “about” relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value±20%, more preferably ±10%, most preferably ±5%.
The term “recombinant virus”, as used herein, relates to a virus comprising a genome modified by biotechnological means as compared to known, naturally occurring, virus genomes. Preferably, the recombinant virus is a virus comprising a genome modified as compared to naturally occurring virus genomes. Preferred biotechnological means for modifying a viral genome are known to the skilled person and include any of the methods of molecular cloning, in particular recombinant DNA techniques including, without limitation, cleavage of DNA by restriction enzymes, ligation of DNA, polymerase chain reaction (PCR), cloning of viral genomes, and the like. It is understood by the skilled person that viruses of the family Paramyxoviridae have a single-stranded (−)-RNA as a genome. Accordingly, the genome of the recombinant virus of the present invention, preferably, is obtained by cloning an expression vector as described herein below comprising an expressible nucleotide sequence encoding said recombinant virus genome, followed by expressing said expressible nucleotide sequence encoding said recombinant virus in a permissive host cell. Alternatively, the recombinant virus genome may also be expressed in non-permissive host cells, e.g., preferably, from rodents or other higher eukaryotes. Preferably, the recombinant virus of the present invention is a recombinant virus of the family Paramyxoviridae, more preferably a recombinant Morbillivirus, most preferably, a recombinant measles virus (MV). As will be understood by the skilled person, the recombinant virus of the present invention may comprise further modifications as compared to a naturally occurring virus. Preferably, the recombinant virus comprises a polypeptide mediating a modified tropism and/or a polynucleotide encoding the same. More preferably, said polypeptide mediating a modified tropism is a fusion polypeptide of a viral membrane integral polypeptide or of a viral membrane associated polypeptide with a polypeptide mediating binding to a target, e.g. a cell, preferably a specific kind of cell, more preferably a cancer cell. Preferably, said fusion polypeptide comprises a viral hemagglutinin or a fragment thereof, preferably a membrane integral fragment thereof. Preferably, said fusion polypeptide comprises a single-chain antibody specifically binding to a target molecule, e.g. to Carcinoembryonic antigen (CEA) or CD20. Most preferably, said fusion polypeptide is a fusion polypeptide of a truncated viral hemagglutinin with an anti-CD20 single-chain antibody or with an anti-CEA single-chain antibody. Preferably, the recombinant virus comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 3 to 5.
As used herein, the term “activator of the immune response” relates to a compound which, when contacted with a mixture of immune cells and immune-response inducing cells, e.g. cancer cells, causes at least one type of immune cell to be more active as compared to an immune cell of the same type comprised in the same mixture but lacking said compound. Preferably, the immune cell activated is a cell mediating a response increasing a subject's resistance to an antigen, i.e. preferably, said immune cell is not a tolerance-mediating immune cell. Preferably, the immune cell activated by the activator of the immune response is a T-cell, more preferably a helper T-cell or a cytotoxic T-cell. Most preferably, the immune cell activated by the activator of the immune response is a cytotoxic T-cell expressing PD-1. Measures of immune cell activity are known to the skilled person and include, preferably, expression of activation markers, production of antibodies, excretion of cytokines, and release of cytotoxins, e.g. perforin, granzymes, and/or granolysin. As used herein, a tumor antigen does not have the activity of being an activator of the immune response; thus, preferably, a tumor antigen is not an activator of the immune response.
Preferably, the activator of the immune response is an activatory chemokine, preferably an activatory interleukin, more preferably IL-12. Also preferably, the activator of the immune response is an antagonist of a signaling pathway causing at least one type of immune cell to become inhibited. Accordingly, preferably, the activator of the immune response is a ligand for an immune checkpoint blockade protein. More preferably, the activator of the immune response is a ligand for an immune checkpoint blockade protein. Still more preferably, the activator of the immune response is an inhibitor of PD-1 receptor signaling. It is understood by the skilled person that signaling through a receptor signaling pathway can be inhibited by either preventing the receptor from being activated, or by preventing the signal generated by the activated receptor from being further transmitted. Accordingly, preferably, the activator of the immune response is a PD-L1 antagonist, the term “antagonist” relating to a compound binding to the molecule the effect of which is antagonized and through said binding preventing said molecule from interacting with its native binding partner in a productive, i.e. signaling-inducing, way. Preferred assays for said activity are described e.g. in WO 2015/128313 A1.
The term “immune response”, as used herein, relates to any protective response of the body of a subject to an antigen involving activity of at least one type of leukocyte, preferably lymphocyte, and/or of at least one antigen-recognizing macromolecule. Preferably, the immune response comprises inactivation of said antigen by production of antigen-specific antibodies (humoral immune response). More preferably, the immune response comprises lysis of foreign cells or of body cells, preferably of cancer cells, presenting said antigen (cell-mediated immune response).
The term “modulating the immune response”, as used herein, relates to inducing a change in, preferably an activation of, the response of a subject's adaptive immune system. Accordingly, the term “modulating the immune response”, preferably, relates to (i) newly inducing an immune response of a subject to an antigen, (ii) to inducing an increase in the quality or intensity of the immune response of a subject to an antigen, and/or to (iii) redirecting an existing immune response to a target cell of interest, preferably a cancer cell, more preferably a cancer cell comprising a tumor antigen. Preferably, modulating an immune response includes or is presenting an antigen or epitope on a cell in the context of at least one of major histocompatibility complex (MHC) class I (MHC-I) and MHC class II (MHC-II) molecules. Preferably, the modulation is an activation, i.e. leads to an enhanced response to the antigen. Also preferably, modulating an immune response includes or is increasing the frequency of antigen-specific T-cells in the blood of a subject. More preferably, modulating an immune response is increasing the frequency of antigen-specific CD-4+ and/or CD-8+ T-cells in the blood of said subject. Most preferably, modulating an immune response is increasing the frequency of activated antigen-specific CD-4+ and/or CD-8+ T-cells in the blood of said subject. Preferably, said modulating an immune response against an antigen induces killing of cells expressing said antigen by the immune system of the subject. More preferably, the term relates to inducing regression of at least one cancer comprising cells expressing said antigen. It is to be understood that inducing an immune response may not be effective in all subjects; e.g. in case a single specific epitope of a polypeptide is used as an antigen, a subject may lack MHC molecules suitable for effectively displaying said epitope. The skilled person knows how to improve immune reaction in such case, e.g. by including further epitopes into the recombinant virus of the family Paramyxoviridae.
The term “antigen” is known to the skilled person and relates to a chemical compound modulating an immune response in a host organism, wherein said host organism, preferably, is a vertebrate organism, preferably is a mammal, more preferably a human or an experimental animal, in particular a rat, a mouse, a rabbit, a guinea pig, a hamster, a sheep, a goat, a horse, a cow, a donkey, most preferably is a human.
Preferably, the antigen is an antigenic polypeptide. The term “antigenic polypeptide”, as used herein, relates to a polypeptide comprising at least one antigenic epitope. Preferably, said antigenic epitope is a glycan portion of a polypeptide. More preferably, said antigenic epitope is a peptide and comprises a sequence of at least four, preferably at least five, more preferably at least six, most preferably at least seven amino acids. Also preferably, said antigenic epitope is a peptide and comprises a sequence of from four to 15, preferably of from five to twelve, more preferably of from six to ten, most preferably of from seven to nine amino acids. Preferably, the antigenic epitope is a T-cell epitope. A T-cell epitope, as is known to the one skilled in the art, is a contiguous sequence of amino acids comprised in a peptide, which can be bound to a MHC class I or class II molecule to be presented on the surface of a cell (MHC-I) or of a professional antigen presenting cell (MHC-II). The skilled artisan knows how to predict immunogenic peptides presented on MHC-I or MHC-II (Nielsen et al., (2004), Bioinformatics, 20 (9), 1388-1397), Bordner (2010), PLoS ONE 5(12): e14383) and how to evaluate binding of specific peptides (e.g. Bernardeau et al., (2011), J Immunol Methods, 371(1-2): 97-105). Preferably, the antigenic epitope is an MHC-II epitope.
The term “cancer”, as used herein, relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body. Preferably, also included by the term cancer is a relapse. Thus, preferably, the cancer is a solid cancer, a metastasis, or a relapse thereof.
Preferably, the cancer is selected from the list consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, waldenström macroglobulinemia, and wilms tumor. More preferably, the cancer is a solid cancer, a metastasis, or a relapse thereof. Most preferably, the cancer is a tumor derived from malignant melanoma, head and neck cancer, hepatocellular carcinoma, pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastric carcinoma, colorectal carcinoma, a lymphoma or a leukemia.
The term “tumor antigen”, as used herein, relates to any antigen comprised by a cancer cell. Thus, the term tumor antigen includes antigens comprised by cancer cells and by one or more non-cancer cell types, e.g. CD19, CD20, CD22, CD30, and CD33. Preferably, the tumor antigen is a tumor-associated antigen, i.e. an antigen expressed by cancer cells, but not by normal cells of the same type as the cancer cells in said subject. Also preferably, the tumor antigen is a tumor-specific antigen, i.e. an antigen expressed by cancer cells, but not by normal, i.e. non-cancer, cells of said subject at the given development stage. Also preferably, the tumor antigen is a neoantigen, i.e. an antigen not encoded in the germ line of said subject; preferably, said neoantigen is an antigen encoded by the tumor genome or an infectious agent, in particular a virus, preferably a tumorigenic virus, more preferably a papillomavirus, an Epstein-Barr virus, Hepatitis B virus, or Hepatitis C virus; or said neoantigen is a mutein comprised by a cancer cell, i.e. a polypeptide variant produced after a mutation of the encoding gene in the genome of said subject has occurred. As specified herein above, an antigen, and also a tumor antigen, has the biological activity of modulating an immune response in a host organism. Preferably, the tumor antigen has the biological activity of modulating an immune response in a human. Also preferably, the tumor antigen has the biological activity of modulating an immune response in the subject from which the cells comprising said tumor antigen are derived from. Preferably, the tumor antigen is a biological macromolecule, more preferably a polypeptide. Preferably, the tumor antigen is a human tumor antigen.
Preferably, the tumor antigen is selected from the group consisting of L-dopachrome-tautomerase (TRP2), melanocyte protein PMEL (gp100), HPV E6/7, MAGE 1, MAGE 3, NY-ESO, androgen receptor (AR), BCL-1, calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial cell adhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogen receptors (mER), FAP HER2/neu, human high molecular weight melanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52, melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesterone receptor (PGR), prostate specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33. More preferably, the tumor antigen is HPV E6/7, MAGE 1, MAGE 3, NY-ESO, TRP2 or gp100, more preferably is TRP2. The open reading frame encoding human TRP2 is disclosed herein as SEQ ID NO: 1, the amino acid sequence of human TRP2 is disclosed as SEQ ID NO:2.
Preferably, the recombinant virus of the family Paramyxoviridae comprises an expressible polynucleotide encoding a fragment of a tumor antigen. As used herein, the term “fragment of a tumor antigen” relates to a substructure of a tumor antigen having the biological activity of modulating an immune response as specified herein above. Thus, the fragment of a tumor antigen, preferably, is an antigenic polypeptide as specified herein, comprising at least a subsequence of said tumor antigen polypeptide; preferably, the fragment of a tumor antigen comprises at least one antigenic epitope in such case, more preferably at least one epitope which is antigenic in the subject the cells comprising the tumor antigen are derived from. Preferably, a fragment of a tumor antigen is or is derived from, e.g., a degradation product or a splice variant of the tumor antigen. Preferably, the fragment of a tumor antigen comprises, preferably consists of: (i) a fragment of a human papillomavirus (HPV) E6 polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. of a HPV16 E6 (Genbank Acc No: NP_041325.1 GI: 9627104), (ii) a fragment of a HPV E7 polypeptide, preferably of an E7 polypeptide of a high-risk HPV, e.g. of a HPV16 E7 (Genbank Acc No: NP_041326.1 GI: 9627105); (iii) TRP2, preferably human TRP2 (preferably. encoded by Genbank Acc No: NM_001922.4 GI: 1015809739), (iv) cancer/testis antigen 1B (CTAG1B, also referred to as NY-ESO, preferably encoded by Genbank Acc No: NM_001327.2 GI: 215272337, or (v) an arbitrary combination of any of (i) to (iv).
Preferably, the recombinant virus of the family Paramyxoviridae comprises an expressible polynucleotide encoding a variant of a tumor antigen and/or of a fragment of a tumor antigen. As used herein, the term “variant” of a tumor antigen relates to an antigen being non-identical to said tumor antigen having the activity of modulating the immune response. Thus, as used herein, the term polypeptide “variant” relates to any chemical molecule comprising at least one polypeptide or fusion polypeptide as specified elsewhere herein, having the indicated activity, but differing in primary structure from said polypeptide or fusion polypeptide. Thus, the polypeptide variant, preferably, is a mutein having the indicated activity. Preferably, the polypeptide variant comprises a peptide having an amino acid sequence corresponding to an amino acid sequence of 5 to 200, more preferably 6 to 100, even more preferably 7 to 50, or, most preferably, 8 to 30 consecutive amino acids comprised in a polypeptide as specified above. Moreover, also encompassed are further polypeptide variants of the aforementioned polypeptides. Such polypeptide variants have at least essentially the same biological activity as the specific polypeptides. Moreover, it is to be understood that a polypeptide variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the amino acid sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the polypeptide, the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Polypeptide variants referred to herein may be allelic variants or any other species specific homologs, paralogs, or orthologs; moreover, the polypeptide variants referred to herein include fragments of the specific polypeptides or the aforementioned types of polypeptide variants as long as these fragments and/or variants have the biological activity as referred to above. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristylation, by including non-natural amino acids, and/or by being peptidomimetics. Preferably, the variant of the tumor antigen or fragment of a tumor antigen comprises, preferably consists of, at least one of a variant of (i) a HPV E6 polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. of a HPV16 E6 (Genbank Acc No: NP_041325.1 GI: 9627104), (ii) a HPV E7 polypeptide, preferably of an E7 polypeptide of a high-risk HPV, e.g. of a HPV16 E7 (Genbank Acc No: NP_041326.1 GI: 9627105); (iii) TRP2, preferably human TRP2 (preferably. encoded by Genbank Acc No: NM_001922.4 GI: 1015809739), (iv) cancer/testis antigen 1B (CTAG1B, also referred to as NY-ESO, preferably encoded by Genbank Acc No: NM_001327.2 GI: 215272337), or (v) an arbitrary combination of any of (i) to (iv).
Preferably, the tumor antigen, fragment of a tumor antigen, or variant of tumor antigen is a fusion polypeptide comprising further amino acids or polypeptides. More preferably, the fusion polypeptide further comprises a detectable tag. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into the fusion polypeptide. Preferably, the tag is added C- or N-terminally to the fusion polypeptide. The said stretch of amino acids shall allow for detection of the fusion polypeptide by an antibody which specifically recognizes the tag or it shall allow for forming a functional conformation, such as a chelator or it shall allow for visualization by fluorescent tags. Preferred tags are the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag or GFP-tag. These tags are all well known in the art. In a further preferred embodiment, said fusion protein comprises a peptide or polypeptide comprising the amino acid sequence of a further tumor antigen or of an activator of the immune response.
The term “expressible polynucleotide”, as used herein, relates to a polynucleotide operatively linked to at least one expression control sequence causing transcription of the nucleic acid sequence comprised in said polynucleotide to occur, preferably in eukaryotic cells or isolated fractions thereof, preferably into a translatable mRNA or into a viral genome. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They, preferably, comprise regulatory sequences ensuring initiation of transcription and, optionally, poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the aforesaid at least one expression control sequence is an expression control sequence of a (−)strand RNA virus, more preferably of a Paramyxovirus as described herein above, most preferably of an MV. Thus, preferably, at least one expression control sequence comprises a (−)strand RNA viral regulatory sequence ensuring initiation of transcription (consensus “gene start signal”, preferably consensus MV “gene start signal”) and termination signals (consensus “gene stop signal”, preferably, consensus MV “gene stop signal”) ensuring termination of transcription and stabilization of the transcript. It is known in the art that production of viral particles in permissive host cells can be initiated by transfecting into said permissive host cells one or more expressible DNA constructs encoding (i) a recombinant viral anti-genome, (ii) the viral L gene, (iii) the viral P gene, and (iv) the viral N gene. It is also understood by the skilled person that, once a viral genome and the aforesaid viral genes were expressed in said host cell, replication and assembly of viral particles occurs in the cytoplasm of the host cell and is, therefore, solely dependent on viral regulatory signals. Preferably, production of viral particles in permissive host cells may also be initiated by transfecting into said permissive host cells one or more expressible DNA (i) a construct encoding a recombinant viral anti-genome and introducing into said permissive host cells the polypeptide products of (ii) the viral L gene, (iii) the viral P gene, and (iv) the viral N gene. The term polynucleotide, as used herein, preferably encompasses polynucleotide variants as specified elsewhere herein. Preferably, the expressible polynucleotide encoding the polynucleotides of the invention, preferably (i) a tumor antigen, (ii) a fragment of a tumor antigen, or (iii) a variant of (i) or (ii), is comprised in the genome of the recombinant virus of the family Paramyxoviridae in a region corresponding to the region intervening the H and the L gene of measles virus. Preferably, the expressible polynucleotide comprises further coding sequences. More preferably, the expressible polynucleotide encodes an activator of the immune response. In an embodiment, the expressible polynucleotide encodes at least one further of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii). As will be understood, the further polypeptides encoded may be encoded as separate polypeptides or as fusion polypeptides comprising at least one, preferably at least two, more preferably at least three of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii). Thus, preferably, the expressible polynucleotide may encode a multitude of antigenic epitopes of tumor antigens as one or more fusion polypeptide(s). In a further embodiment, said activator of the immune response comprises, preferably is, a secreted soluble activator of the immune response, preferably an immunoglobulin or fragment thereof, more preferably a secreted immunoglobulin or fragment thereof.
As used herein, the term “immunoglobulin” relates to a polypeptide being a soluble immunoglobulin, preferably an antibody from any of the classes IgA, IgD, IgE, IgG, or IgM, preferably having the activity of binding, more preferably specifically binding, a molecule of interest. Immunoglobulins against antigens of interest can be prepared by well-known methods using, e.g., a purified molecule of interest or a suitable fragment derived therefrom as an antigen. A fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either from one of the molecules of interest by proteolytic digestion, may be a synthetic peptide, or may be obtained by recombinant expression. Preferably, a peptide of a molecule of interest used as an antigen is located at the exterior of a cell expressing the molecule of interest; i.e. preferably, the epitope the binding domain interacts with, preferably, is an extracellular domain. Preferably, the immunoglobulin of the present invention is a monoclonal antibody, a human or humanized antibody or primatized, chimerized antibody or a fragment thereof, so long as they exhibit the desired binding activity as specified elsewhere herein. Also comprised as antibodies of the present invention are a bispecific antibody, a synthetic antibody, or a chemically modified derivative of any of these. Preferably, the antibody of the present invention shall specifically bind (i.e. does only to a negligible extent or, preferably, not cross react with other polypeptides or peptides) to a molecule of interest as specified above. Specific binding can be tested by various well known techniques. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques originally described in Kohler and Milstein, Nature. 1975. 256: 495; and Galfré, Meth. Enzymol. 1981, 73: 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. As will be understood by the skilled person, a molecule of interest, bound by an immunoglobulin of the present invention, may also be an Fc receptor or a complement protein binding an Fc part of an antibody; accordingly, the immunoglobulin preferably is an Fc domain of an antibody, more preferably a soluble Fc domain of an antibody, most preferably a secreted soluble Fc domain of an antibody. Preferably, said antibody the Fc domain is derived from is an IgG, more preferably an IgG1, most preferably a human IgG1.
“Immunoglobulin fragments” comprise a portion of an intact immunoglobulin, preferably of an antibody, in an embodiment, comprise the antigen-binding region thereof. Examples of antibody fragments and fusion proteins of variable regions include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; single-domain-antibodies (VHH), also known as nanobodies, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. “Fv” is the minimum antibody fragment which contains a complete antigen-binding site. Preferably, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs, also referred to as complementarity determining regions (CDRs)) of each variable domain interact to define an antigen-binding site. Collectively, the six HVRs of one scFv confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 0 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9 (2003) 129-134; and Hollinger et al., PNAS USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9 (2003) 129-134.
The term “secreted”, as used herein, relates to a compound being transferred from the interior of a host cell to the exterior of said host cell by a mechanism intrinsic to said host cell. Preferably, secretion of a polypeptide or fusion polypeptide is mediated by a, preferably eukaryotic, signal peptide mediating import of said peptide or polypeptide into the lumen of the endoplasmic reticulum and, more preferably, by the absence of retention signals. Signal peptides causing secretion of peptides or polypeptides are known in the art. Preferably, the signal peptide is an IL-12 signal peptide. Also preferably, the signal peptide is or comprises an Ig leader sequence. More preferably, the signal peptide is or comprises a human Ig leader sequence. Still more preferably, the signal peptide is or comprises a matching leader sequence, i.e. a leader sequence selected from the same Ig kappa subgroup as the variable light chain of the antibody, preferably, of the single-chain antibody.
Advantageously, it was found in the work underlying the present invention that recognition of tumor antigens not recognized by the immune system can be induced by presenting said tumor antigens in the context of a virus infection. Without wishing to be bound by theory, it is presumed that expression of tumor antigens in the context of a measles virus infection breaks tumor-induced tolerance and makes and immune response to the tumor antigen possible. Moreover, it was found that the expression of tumor antigens on tumor cells induced by measles virus infection increases infiltration and persistence of T-cells in tumors.
The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.
The present invention further relates to polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to the present invention
The term “polynucleotide” is understood by the skilled person to relate to a polymer composed of a series of contiguous nucleotides; the term encompasses single as well as double stranded polynucleotides. Preferably, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified ones such as biotinylated polynucleotides and polynucleotides comprising modified nucleotides. Preferably, the nucleotides comprised in the polynucleotide are naturally occurring nucleotides. Preferably, the polynucleotide is RNA, including mRNA, or DNA, including cDNA. More preferably, the polynucleotide is DNA. Preferably, the nucleotides comprised in the polynucleotide comprise, preferably comprise exclusively, the bases adenine, guanine, cytosine, and thymine in case the polynucleotide is DNA; also preferably, the nucleotides comprised in the polynucleotide comprise, preferably comprise exclusively, the bases adenine, guanine, cytosine, and uracil in case the polynucleotide is RNA. The polynucleotides of the present invention either consist of, essentially consist of, or comprise the indicated nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well. The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form.
The term “polynucleotide encoding a recombinant virus”, as used herein, relates to a polynucleotide comprising a nucleic acid sequence or nucleic acid sequences sufficient for generating a virus particle or a virus-like particle in a host cell. It is understood by the skilled person that a virus is constituted by a polynucleotide genome and at least one kind of capsid polypeptide. Accordingly, the polynucleotide encoding a recombinant virus of the present invention, preferably, comprises a recombinant virus genome. As will be understood by the skilled person, in case the polynucleotide encoding a recombinant virus is comprised in a virus according to the present invention, i.e. a virus of the family Paramyxoviridae, the polynucleotide is (−)strand RNA. It is also understood by the skilled person that in case the polynucleotide is DNA comprised in a host cell, at least an RNA-dependent RNA polymerase activity will additionally be required to produce viral particles from said DNA polynucleotide. Preferably, the polynucleotide encoding a recombinant virus comprises or consists of the nucleic acid sequence as specified elsewhere herein. As annotated herein, the sequence of the DNA copy of negative-strand (−)RNA viruses is annotated in the usual 5′→3′-orientation; this corresponds to the viral sequence in antigenomic (+)RNA orientation with respect to the natural 3′→5′-orientation of negative-strand (−)RNA viruses. Preferably, the polynucleotide encoding a recombinant virus is based on Measles virus strain Edmonston (Moraten vaccine), Genbank Acc No: AF266287.1 GI: 9181873. More preferably, the polynucleotide encoding a recombinant virus comprises, preferably consists of, the nucleic acid sequence of any one of SEQ ID NOs: 3 to 5.
The term polynucleotide, preferably, includes polynucleotide variants. The term “polynucleotide variant”, as used herein, relates to a variant of a polynucleotide related to herein comprising a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and/or deletion, wherein the polynucleotide variant shall have the activity as specified for the specific polynucleotide. Preferably, said polynucleotide variant is an ortholog, a paralog or another homolog of the specific polynucleotide. Also preferably, said polynucleotide variant is a naturally occurring allele of the specific polynucleotide. Polynucleotide variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific polynucleotides, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6× sodium chloride/sodium citrate (=SSC) at approximately 45° C., followed by one or more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depending on the type of nucleic acid between 42° C. and 58° C. in aqueous buffer with a concentration of 0.1× to 5×SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42° C. The hybridization conditions for DNA:DNA hybrids are preferably for example 0.1×SSC and 20° C. to 45° C., preferably between 30° C. and 45° C. The hybridization conditions for DNA:RNA hybrids are preferably, for example, 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55° C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (=base pairs) in length and a G+C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR-based techniques such as mixed oligonucleotide primer-based amplification of DNA, i.e. using degenerated primers against conserved domains of a polypeptide of the present invention. Conserved domains of a polypeptide may be identified by a sequence comparison of the nucleic acid sequence of the polynucleotide or the amino acid sequence of the polypeptide of the present invention with sequences of other organisms. As a template, DNA or cDNA from bacteria, fungi, or plants preferably, from animals may be used. Further, variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the specifically indicated nucleic acid sequences. Moreover, also encompassed are polynucleotides which comprise nucleic acid sequences encoding amino acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences specifically indicated. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))), which are part of the GCG software packet (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991)), are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments.
A polynucleotide comprising a fragment of any of the specifically indicated nucleic acid sequences is also encompassed as a variant polynucleotide of the present invention. The fragment shall still encode a polypeptide or fusion polypeptide which still has the activity as specified. Accordingly, the polypeptide encoded may comprise or consist of the domains of the polypeptide of the present invention conferring the said biological activity. A fragment as meant herein, preferably, comprises at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of any one of the specific nucleic acid sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the specific amino acid sequences.
The present invention further relates to a host cell comprising the recombinant virus of the family Paramyxoviridae according to the present invention and/or the polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to the present invention.
As used herein, the term “host cell” relates to a vertebrate cell. Preferably, the cell is a vertebrate cell, preferably is a mammalian cell, more preferably a human cell or a cell of an experimental animal, in particular a rat, a mouse, a rabbit, a guinea pig, a hamster, a sheep, a goat, a horse, a cow, a donkey cell, most preferably is a human cell. Preferably, the host cell is a cancer cell, more preferably a tumor cell.
Further, the present invention relates to a medicament comprising
(a) (i) a recombinant virus of the family Paramyxoviridae according to the present invention,
(b) at least one pharmacologically acceptable excipient.
The terms “medicament” and “pharmaceutical composition”, as used herein, relate to the compounds of the present invention and optionally one or more pharmaceutically acceptable carrier, i.e. excipient. The compounds of the present invention can be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, methyl ester, HCl, sulfate, chloride and the like. The pharmaceutical compositions are, preferably, administered locally, topically or systemically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, or parenteral administration as well as inhalation. A preferred route of administration is intra-tumoral administration. However, depending on the nature and mode of action of a compound, the pharmaceutical compositions may be administered by other routes as well. For example, polynucleotide compounds may be administered in a gene therapy approach by using viral vectors or viruses or liposomes.
Moreover, the compounds can be administered in combination with other drugs either in a common pharmaceutical composition or as separated pharmaceutical compositions wherein said separated pharmaceutical compositions may be provided in form of a kit of parts. The compounds are, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
The excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The excipient employed may be, for example, a solid, a gel or a liquid carrier. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. The diluent(s) is/are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenic stabilizers and the like.
A therapeutically effective dose refers to an amount of the compounds to be used in a pharmaceutical composition of the present invention which prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 to 1000 μg for a polypeptide or polynucleotide, or 104-108 viral particles for a virus or a virus-like particle; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Progress can be monitored by periodic assessment. The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from one to four times daily up to a non-limited number of days. Specific pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound referred to herein above in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent. For making those specific pharmaceutical compositions, the active compound(s) will usually be mixed with a carrier or the diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper or other suitable containers or vehicles. The resulting formulations are to be adapted to the mode of administration, i.e. in the forms of tablets, capsules, suppositories, solutions, suspensions or the like. Dosage recommendations shall be indicated in the prescribers or users instructions in order to anticipate dose adjustments depending on the considered recipient.
The present invention further relates to a method for activating immune cells with antitumor activity in a sample comprising cancer cells and immune cells, comprising
The method for activating immune cells of the present invention, preferably, may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing a sample for step a), or administration of additional compounds to the immune cells, e.g. immunostimulatory compounds before or during step b). Moreover, one or more of said steps may be performed by automated equipment. The method for activating immune cells of the present invention, preferably, is an in vitro method.
The term “contacting”, as used in the context of the methods of the present invention, is understood by the skilled person. Preferably, the term relates to bringing a compound, e.g. a virus, a sample, or a subject of the present invention in physical contact with a further compound and thereby allowing the compound and the further compound to interact.
The term “immune cells”, as used herein, relates cells mediating an immune response in a subject. Preferably, the immune cell is a leukocyte, preferably a lymphocyte. Preferably, the immune cell is a B-cell; more preferably, the immune cell is a T-cell, still more preferably a cytotoxic T cell or a T helper cell.
The present invention further relates to a method for treating cancer in a subject afflicted with cancer, comprising
1. a) contacting said subject with
The method of treatment of the present invention, preferably, may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to localizing a tumor and/or diagnosing cancer for step a), or administration of additional medication for step b). More preferably, the method of treatment further comprises the steps of the method for activating immune cells as specified elsewhere herein, and the further step of administering said activated immune cells with antitumor activity to said subject. Moreover, one or more of said steps may be performed by automated equipment. The method of the present invention, preferably, is an in vivo method of treatment. Preferably, in the method of treatment, the cancer is a solid cancer, a metastasis, or a relapse thereof.
The term “treating” refers to ameliorating the diseases or disorders referred to herein or the symptoms accompanied therewith to a, preferably, significant extent. Said treating as used herein also includes an entire restoration of the health with respect to the diseases or disorders referred to herein. It is to be understood that treating as used in accordance with the present invention may not be effective in all subjects to be treated. However, the term shall, preferably, require that a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 10%, at least 20% at least 50% at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. Preferably, treating cancer is reducing tumor burden in a subject. As will be understood by the skilled person, effectiveness of treatment of e.g. cancer is dependent on a variety of factors including, e.g. cancer stage and cancer type. Preferably, treating cancer is reducing tumor burden.
Moreover, the present invention relates to a preparation of activated immune cells with antitumor activity obtained or obtainable by the method for activating immune cells of the present invention.
Furthermore, the present invention relates to the use of
Also, the present invention relates to a recombinant virus of the family Paramyxoviridae according to the present invention and/or a polynucleotide according to the present invention and/or a host cell according to the present invention for use in medical treatment. The present invention further relates to a recombinant virus of the family Paramyxoviridae according to the present invention and/or a polynucleotide according to the present invention and/or a host cell according to the present invention for use in treatment of inappropriate cell proliferation. Preferably, treatment of inappropriate cell proliferation is cancer treatment.
The present invention also relates to a kit comprising
The term “kit”, as used herein, refers to a collection of the aforementioned components. Preferably, said components are combined with additional components, preferably within an outer container. The outer container, also preferably, comprises instructions for carrying out a method of the present invention. Examples for components of the kit as well as methods for their use have been given in this specification. The kit, preferably, contains the aforementioned components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, e.g., a user's manual for applying the recombinant virus of the family Paramyxoviridae with respect to the applications provided by the methods of the present invention. Details are to be found elsewhere in this specification. Additionally, such user's manual may provide instructions about correctly using the components of the kit. A user's manual may be provided in paper or electronic form, e.g., stored on CD or CD ROM. The present invention also relates to the use of said kit in any of the methods according to the present invention.
Also, the present invention relates to a method for selecting (i) a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding a tumor antigen or a fragment or variant thereof, (ii) a polynucleotide encoding the recombinant virus of (i), and/or (iii) a host cell comprising the recombinant virus according to (i) and/or a polynucleotide according to (ii) for treating a subject suffering from cancer, said method comprising
The method for selecting of the present invention, preferably, may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., a cancer sample for step a), or administration of a recombinant virus, a polynucleotide and/or a host cell comprising an expressible polynucleotide encoding a tumor antigen or a fragment or a variant thereof to a cancer sample after step b). Moreover, one or more of said steps may be performed by automated equipment. The method of the present invention, preferably, is an in vitro method, preferably an in vitro method aiding in taking a treatment decision.
As used herein, the term “detecting a tumor antigen” relates to detecting the presence of a tumor antigen in and/or on the surface of a cell, preferably cancer cell. Preferably, detection is qualitative, more preferably semi-quantitative, most preferably quantitative detection. Preferably, detecting is immunologically detecting, preferably by contacting a, preferably isolated, cancer sample or a fraction thereof with at least one antibody binding to the tumor antigen; preferably, said binding is specific binding. As used herein, the term “specific binding” relates to a binding in which other compounds which are not the tumor antigen are bound with an affinity at least 100 fold, more preferably at least 1000 fold, most preferably at least 104 fold lower as compared to the affinity for the tumor antigen. Preferred methods of immunologically detecting a tumor antigen are known in the art and include in particular in-situ immunostaining, ELISA and related methods, and immunoblots. As will be understood by the skilled person, expression of a tumor antigen may also be detected by detecting an RNA encoding said tumor antigen expressed by a tumor cell.
Selection of the agent for treating a subject suffering from cancer, i.e. the specific selection of recombinant virus, polynucleotide and/or host cell comprising an expressible polynucleotide encoding a tumor antigen or a fragment or a variant thereof, will depend on the specific circumstances. Preferably, if it is found that cancer cells in a cancer sample express a tumor antigen, an agent increasing expression of said tumor antigen will be selected. On the other hand, in case it is found that the cancer cells express a mutant form of a polypeptide (e.g. a neoantigen), preferably, an agent inducing expression of said mutant form will be elected. Thus, preferably the tumor antigen or fragment or variant thereof encoded by the expressible polynucleotide comprised in the recombinant virus, the polynucleotide and/or the host cell selected in step b) shares at least one epitope with the tumor antigen detected in step a). Also preferably, the tumor antigen or fragment or variant thereof encoded by the expressible polynucleotide comprised in the recombinant virus, the polynucleotide and/or the host cell selected in step b) is or is derived from the tumor antigen detected in step a).
In view of the above, the following embodiments are particularly proposed:
1. A recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii).
2. The recombinant virus of the family Paramyxoviridae of embodiment 1, wherein said tumor antigen is selected from the group consisting of L-dopachrome-tautomerase (TRP2), melanocyte protein PMEL (gp100), HPV E6/7, MAGE 1, MAGE 3, NY-ESO, androgen receptor (AR), BCL-1, calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial cell adhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogen receptors (mER), FAP HER2/neu, human high molecular weight melanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52, melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesterone receptor (PGR), prostate specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33. More preferably, the tumor antigen is HPV E6, HPVE7, MAGE 1, MAGE 3, NY-ESO, TRP2 or gp100, more preferably is TRP2.
3. The recombinant virus of the family Paramyxoviridae of embodiment 1 or 2, wherein said tumor antigen is a human tumor antigen.
4. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 3, wherein said fragment comprises at least five, preferably at least six, more preferably at least seven, even more preferably at least eight, most preferably at least nine amino acids, wherein preferably, said amino acids correspond to a contiguous sequence comprised in the amino acid sequence of said tumor antigen.
5. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 4, wherein said fragment comprises at least one antigenic epitope of said tumor antigen.
6. The recombinant virus of the family Paramyxoviridae of embodiment 5, wherein said epitope is an MHC class I epitope and/or an MHC class II epitope.
7. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 6, wherein said fragment is selected from the group consisting of (i) a fragment of a human papillomavirus (HPV) E6 polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. of a HPV16 E6 (Genbank Acc No: NP_041325.1 GI: 9627104), (ii) a fragment of a HPV E7 polypeptide, preferably of an E7 polypeptide of a high-risk HPV, e.g. of a HPV16 E7 (Genbank Acc No: NP 041326.1 GI: 9627105); (iii) TRP2, preferably human TRP2 (preferably. encoded by Genbank Acc No: NM_001922.4 GI: 1015809739), (iv) cancer/testis antigen 1B (CTAG1B, also referred to as NY-ESO, preferably encoded by Genbank Acc No: NM_001327.2 GI:215272337, an (v) an arbitrary combination of any of (i) to (iv).
8. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 7, wherein said tumor antigen, fragment or variant thereof comprises, preferably consists of, at least one of (i) a HPV E6 polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. of a HPV16 E6 (Genbank Acc No: NP 041325.1 GI: 9627104), (ii) a HPV E7 polypeptide, preferably of an E7 polypeptide of a high-risk HPV, e.g. of a HPV16 E7 (Genbank Acc No: NP 041326.1 GI: 9627105); (iii) TRP2, preferably human TRP2 (preferably. encoded by Genbank Acc No: NM 001922.4 GI: 1015809739), (iv) cancer/testis antigen 1B (CTAG1B, also referred to as NY-ESO, preferably encoded by Genbank Acc No: NM_001327.2 GI: 215272337), and (v) an arbitrary combination of any of (i) to (iv).
9. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 8, wherein said expressible polynucleotide encodes at least one further of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii).
10. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 9, wherein said expressible polynucleotide is comprised in the genome of said recombinant virus, preferably in a region corresponding to the region intervening the H and the L gene of measles virus.
11. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 10, further comprising at least one expressible polynucleotide encoding a further activator of the immune response.
12. The recombinant virus of the family Paramyxoviridae of embodiment 11, wherein said further activator of the immune response comprises, preferably is, an immunoglobulin or fragment thereof.
13. The recombinant virus of the family Paramyxoviridae of embodiments 11 or 12, wherein said further activator of the immune response comprises, preferably is, a secreted soluble activator of the immune response, preferably IL-12.
14. The recombinant virus of the family Paramyxoviridae of any one of embodiments 11 to 13, wherein said further activator of the immune response comprises, preferably is, a secreted immunoglobulin or fragment thereof.
15. The recombinant virus of the family Paramyxoviridae of any one of embodiments 11 to 14, wherein said further activator of the immune response comprises, preferably is, a single-chain antibody or a nanobody.
16. The recombinant virus of the family Paramyxoviridae of any one of embodiments 11 to 15, wherein said further activator of the immune response comprises, preferably is, a secreted soluble anti-PD-1/PD-L1 antibody.
17. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 16, wherein said recombinant virus is a recombinant Morbillivirus, preferably, a recombinant measles virus (MV).
18. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 17, wherein said recombinant MV is derived from MV strain Edmonston A or B, preferably B, more preferably from MV vaccine strain Schwarz/Moraten.
19. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 18, wherein said at least one expressible polynucleotide is comprised in a polynucleotide encoding the recombinant virus of the family Paramyxoviridae.
20. The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 19, wherein said polynucleotide encoding the recombinant virus of the family Paramyxoviridae comprises, preferably consists of, the nucleic acid sequence of any one of SEQ ID NOs: 3 to 5.
21. A polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20.
22. The polynucleotide according to embodiment 21, wherein said polynucleotide comprises, preferably consists of, the nucleic acid sequence any one of SEQ ID NOs: 3 to 5.
23. A host cell comprising the recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20 and/or the polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to embodiment 21 or 22.
24. The host cell of embodiment 23, wherein said host cell is a cancer cell.
25. A medicament comprising
(a) (i) a recombinant virus of the family Paramyxoviridae according of any one of embodiments 1 to 20,
(b) at least one pharmacologically acceptable excipient.
26. A method for activating immune cells with antitumor activity in a sample comprising cancer cells and immune cells, comprising
(i) a recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20,
(ii) a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (i), preferably a polynucleotide according to embodiment 21 or 22;
(iii) a host cell comprising the recombinant virus of the family Paramyxoviridae according to (i) and/or a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (ii); preferably a host cell according to embodiment 23 or 24; or
(iv) any combination of (i) to (iii); and thereby,
27. The method of embodiment 26, wherein said method is an in vitro method.
28. A method for treating cancer in a subject afflicted with cancer, comprising
(i) a recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20,
(ii) a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (i), preferably a polynucleotide according to embodiment 21 or 22;
(iii) a host cell comprising the recombinant virus of the family Paramyxoviridae according to (i) and/or a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (ii); preferably a host cell according to embodiment 23 or 24; or
(iv) any combination of (i) to (iii); and, thereby,
29. The method of embodiment 28, wherein said cancer is a solid cancer, a metastasis, or a relapse thereof.
30. The method of embodiment 28 or 29, wherein treating cancer is reducing tumor burden.
31. The method of any one of embodiments 28 to 30, wherein said cancer is malignant melanoma, head and neck cancer, hepatocellular carcinoma, pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastric carcinoma, colorectal carcinoma, a lymphoma or a leukemia.
32. The method of any one of embodiments 28 to 31, wherein said method further comprises the steps of the method for activating immune cells with antitumor activity in a sample according to embodiment 26, and the further step of administering said activated immune cells with antitumor activity to said subject.
33. A preparation of activated immune cells with antitumor activity obtained or obtainable by the method of embodiment 26 or 27.
34. Use of
(i) a recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20,
(ii) a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (i), preferably a polynucleotide according to embodiment 21 or 22;
(iii) a host cell comprising the recombinant virus of the family Paramyxoviridae according to (i) and/or a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (ii); preferably a host cell according to embodiment 23 or 24; or
(iv) any combination of (i) to (iii);
35. A recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20 and/or a polynucleotide according to embodiment 21 or 22 and/or a host cell according to embodiment 23 or 24 for use in medical treatment.
36. A recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 20 and/or a polynucleotide according to embodiment 21 or 22 and/or a host cell according to embodiment 23 or 24 for use in treatment of inappropriate cell proliferation.
37. The recombinant virus of the family Paramyxoviridae for use of embodiment 36, wherein treatment of inappropriate cell proliferation is cancer treatment.
38. A kit comprising
(i) a recombinant virus of the family Paramyxoviridae according of any one of embodiments 1 to 20,
(ii) a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (i), preferably a polynucleotide according to embodiment 21 or 22;
(iii) a host cell comprising the recombinant virus of the family Paramyxoviridae according to (i) and/or a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to (ii); preferably a host cell according to embodiment 23 or 24; or
(iv) any combination of (i) to (iii);
39. A method for selecting (i) a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding a tumor antigen or a fragment or variant thereof, (ii) a polynucleotide encoding the recombinant virus of (i), and/or (iii) a host cell comprising the recombinant virus according to (i) and/or a polynucleotide according to (ii) for treating a subject suffering from cancer, said method comprising
a) detecting at least one tumor antigen expressed by cancer cells in a cancer sample of said subject; and,
b) based on the determination of step a), selecting a recombinant virus, a polynucleotide and/or a host cell comprising an expressible polynucleotide encoding a tumor antigen or a fragment or a variant thereof.
40. The method of embodiment 39, wherein the tumor antigen or fragment or variant thereof encoded by the expressible polynucleotide comprised in the recombinant virus, the polynucleotide and/or the host cell selected in step b) shares at least one epitope with the tumor antigen detected in step a).
41. The method of embodiment 39 or 40, wherein the tumor antigen or fragment or variant thereof encoded by the expressible polynucleotide comprised in the recombinant virus, the polynucleotide and/or the host cell selected in step b) is or is derived from the tumor antigen detected in step a).
42. The method of any one of embodiments 39 to 41, wherein the recombinant virus of (i) is a recombinant virus according to any one of embodiments 1 to 20,
wherein the polynucleotide of (ii) is a polynucleotide according to embodiment 21 or 22, and/or wherein the host cell of (iii) is a host cell according to embodiment 23 or 24.
All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.
The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.
Recombinant measles viruses encoding the tumor-associated antigen (TAA) trp2 have been generated according to known methods (MV-trp2,
Vero cells were transduced with parental MV or MV encoding trp2 with a multiplicity of infection (MOI)=3. At designated time points, cells were harvested and progeny viral particles were determined in titration assays by serial dilution. Insertion of the tumor-associated antigen does not impair viral replication and cytotoxic effects (
RNA was extracted from Vero cells infected with parental MV or MV encoding trp2. cDNA synthesis was performed using oligo-dT primers. PCR was performed with trp2-specific primers. Cells infected with parental MV or MV encoding trp2 were lysed in RIPA buffer for protein extraction. After SDS PAGE of cell lysates, Western blot analysis was performed with trp2-specific antisera. Beta-actin was detected as a loading control. A melanosome preparation was used as a positive control. Tumor-antigen expression in infected cells was confirmed both on the mRNA- and protein level (
Trp2-specific T cells were co-cultured with MC38-hCD46 cells infected with MV-trp2 or parental MV or with B16 cells or stimulated with the immunodominant trp2 peptide. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. MV encoding the trp2 tumor antigen (TAA) activate TAA-specific T cells: Co-culture of trp2-specific T cells with murine cells infected with MV-trp2 leads to significantly higher IFN-gamma secretion than co-culture with parental MV (
1×106 B16ova_hCD46 cells were implanted into the flank of C57BL/6J mice (day 0). Starting on day 10, mice received intratumoral injections of 1×106 cell infectious units (ciu) of MV-trp2, 1×106 ciu of parental MV in a total volume of 100 μL or 100 μL carrier fluid (mock). Mice were sacrificed on day 23 and spleens were extracted for ELISPOT analysis.
Splenocytes of mice from different treatment groups were co-cultured with MC38-hCD46 cells infected with MV-trp2 or parental MV. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. Splenocytes of mice from different treatment groups were co-cultured with B16 cells. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. It was found that intratumoral injection of MV-trp2 can enhance TAA-specific and tumor-specific immunity (
Thus, in the present invention, an oncolytic MV vaccine was generated to elicit a tumour antigen-specific immune response. With this approach, the MV vector encoded tumour antigen is expressed in the context of viral danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) which serve as “natural adjuvants”. Furthermore, MV-mediated oncolysis functions as an in situ tumour vaccine: MV infection causes immunogenic cell death within the tumour accompanied by release of tumour-associated antigens, resulting in efficient antigen cross-presentation and epitope spread.
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| 6896881 | Russell | May 2005 | B1 |
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| Number | Date | Country | |
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| 20180110848 A1 | Apr 2018 | US |
