Patent Grant
10933106
The present invention relates to a recombinant virus of the family Paramyxoviridae, comprising at least one expressible polynucleotide encoding a secreted activator of the immune response, to a polynucleotide encoding the same, and to a kit comprising the same. Moreover, the present invention relates to a method for treating cancer in a subject afflicted with cancer, comprising contacting said subject with a recombinant virus of the family Paramyxoviridae of the invention, and thereby, treating cancer in a subject afflicted with cancer.
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 wildtype measles virus, several non-pathogenic strains, including a vaccination strain, have been derived, which have been shown to be still 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.).
In general, immune response via T cell activation involves the integration of numerous signals at so-called immune checkpoints. Immune checkpoint inhibition is a novel paradigm in cancer immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD152 (Cluster of differentiation 152) and PD-L1 (Programmed cell death 1 ligand 1, also known as CD274 (cluster of differentiation 274) or B7 homolog 1 (B7-H1)) are key molecules in this process (Chen and Flies, Nat Rev Immunol. 2013, 13: 227-242). CTLA-4 is a co-inhibitory surface molecule on T cells which belongs to the CD28 receptor subfamily. It is induced upon initial recognition of a T cell's cognate antigen and constitutively expressed on regulatory T cells (Tregs) (Rudd et al., Immunol Rev. 2009, 229: 12-26; Walker and Sansom, Nat Rev Immunol. 2011, 11: 852-863). The physiological role of CTLA-4 is regulation of self-tolerance, which is illustrated by the lethal systemic immune hyperactivation phenotype of CTLA-4 knockout mice (Tivol et al., Immunity. 1995, 3: 541-547). As a central mediator of T cell inhibition, CTLA-4 has been implicated in immune tolerance of tumors. CTLA-4 blockade has been shown to enhance antitumor immunity in multiple preclinical and clinical studies (Egen et al., Nature Immunol. 2002, 3: 611-618; Ott et al., Clin Cancer Res. 2013, 19: 5300-5309). Similarly, PD-L1 is a surface glycoprotein which acts as a ligand for the T cell inhibitory factor PD-1. PD-L1 is broadly expressed on immune cells and healthy tissues and is induced by interferon-γ (Okazaki and Honjo, Trends Immunol. 2006, 27: 195-201). It mediates fetomaternal tolerance (Guleria et al., J Exp Med. 2005, 202: 231-237) and allograft tolerance after organ transplantation (Tanaka et al., J Immunol. 2007, 179: 5204-5210). In models of autoimmune diseases such as diabetes and encephalomyelitis, PD-L1 knockout leads to an aggravated phenotype (Keir et al., J Exp Med. 2006, 203: 883-895; Latchman et al., Proc Natl Acad Sci USA. 2004, 101: 10691-10696). PD-1/PD-L1 signaling is initiated after chronic antigen exposure, leading to T cell exhaustion (Barber et al., Nature. 2006, 439: 682-687). PD-L1 is overexpressed in various tumor entities and inhibits T cell-mediated anti-tumor immunity (Iwai et al., Proc Natl Acad Sci USA. 2002, 99: 12293-12297).
By antagonizing CTLA-4, PD-1 and PD-L1, anti-tumor immune effectors can be reinvigorated with unprecedented success in metastatic melanoma and other advanced-stage tumors (Hodi et al., N Engl J Med. 2010, 363: 711-723; Topalian et al. N Engl J Med. 2012, 366: 2443-2454; Brahmer et al. N Engl J Med. 2012, 366: 2455-2465). However, immune-related adverse events are frequent and tend to be severe in systemic immunotherapy (Quezada and Peggs, Br J Cancer. 2013, 108: 1560-1565.).
There is, thus, 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 a secreted activator of the immune response.
The terms “virus” and “virus of the family Paramyxoviridae” are known to the skilled person. Preferably, the virus of the family Paramyxoviridae is a member of the genus Morbillivirus. More preferably, the virus of the family Paramyxoviridae is a measles virus (MV), still more preferably a MV strain Edmonston A or B, or, most preferably, vaccine strain Schwarz (Edmonston A).
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 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.
As used herein, the term “activator of the immune response” relates to a compound which, when contacted with immune cells, causes at least one type of immune cell to be more active as compared to an immune cell of the same type not contacted with said compound. Preferably, said immune cell 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. 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. 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 helper T-cell or a Treg cell expressing CTLA-4 or a cytotoxic T-cell expressing PD-1.
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 BTLA receptor signaling, TIM3 receptor signaling, or, more preferably of CTLA-4 receptor signaling or 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 CTLA-4 antagonist, a PD-1 antagonist, a CD80 antagonist, a CD86 antagonist, or 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 herein in the accompanying Examples.
Preferably, the activator of the immune response is an antagonist as described above selected from the list of molecule types consisting of a peptide aptamer, an anticalin, a Designed Ankyrin Repeat Protein (DARPin), an inhibitory peptide, and, preferably, an antibody.
In the context of this invention, a “peptide aptamer” is a peptide specifically binding its interaction partner and having the activity of activating the immune response as specified herein above, preferably, the activity of being an antagonist of CTLA-4, PD-1, CD80, CD86, and/or PD-L1 as specified herein above. Peptide aptamers, preferably, are peptides comprising 8-80 amino acids, more preferably 10-50 amino acids, and most preferably 15-30 amino acids. They can e.g. be isolated from randomized peptide expression libraries in a suitable host system like baker's yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998). A peptide aptamer, preferably, is a free peptide; it is, however, also contemplated by the present invention that a peptide aptamer is fused to a polypeptide serving as “scaffold”, meaning that the covalent linking to said polypeptide serves to fix the three-dimensional structure of said peptide aptamer to one specific conformation. More preferably, the peptide aptamer is fused to a transport signal, in particular a peptide export signal.
As used herein, the term “anticalin” relates to an artificial polypeptide derived from a lipocalin specifically binding its interaction partner. Similarly, a “Designed Ankyrin Repeat Protein” or “DARPin”, as used herein, is an artificial polypeptide comprising several ankyrin repeat motifs and specifically binding its interaction partner. The anticalins and the DARPins of the present invention have the activity of activating the immune response as specified herein above, preferably, the activity of being an antagonist of CTLA-4, PD-1, CD80, CD86, and/or PD-L1 as specified herein above.
As used herein, the term “inhibitory peptide” relates to any chemical molecule comprising at least one peptide having the activity of activating the immune response as specified herein above, preferably, the activity of being an antagonist of CTLA-4, PD-1, CD80, CD86, and/or PD-L1 as specified herein above. Preferably, the inhibitory peptide comprises a peptide having an amino acid sequence corresponding to an amino acid sequence of at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least 13, at least 14, or at least 15 consecutive amino acids comprised in a CTLA-4, a PD-1, a CD80, a CD86, and/or a PD-L1 polypeptide. Preferably, the inhibitory peptide 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 CTLA-4, a PD-1, a CD80, a CD86, and/or a PD-L1 polypeptide. Moreover, also encompassed are variants of the aforementioned inhibitory peptides. Such variants have at least the same essential biological activity as the specific inhibitory peptides. Moreover, it is to be understood that a 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 sequence of the specific inhibitory peptides. 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 reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the peptide, 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. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Moreover, the variants referred to herein include fragments of the specific inhibitory peptides or the aforementioned types of variants as long as these fragments and/or variants have the essential biological activity as referred to above. Such fragments may be or be derived from, e.g., degradation products or splice variants of the inhibitory peptides. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation or myristylation.
Preferably, the inhibitory peptide comprises further amino acids which may serve e.g. as immunogens, as a tag for purification or detection or as a linker. In a preferred embodiment of the inhibitory peptide of the present invention, said inhibitory peptide further comprises an immunogenic peptide. The term “immunogenic peptide” refers to a stretch of amino acids which is added to or introduced into the inhibitory peptide of the invention. Preferably, the immunogenic peptide shall be added C- or N-terminally to the inhibitory peptide of the present invention. In another preferred embodiment of the inhibitory peptide of the present invention, said inhibitory peptide further comprises a detectable tag. The term “detectable tag” refers to a stretch of amino acids which are added to or introduced into the inhibitory peptide of the invention. Preferably, the tag shall be added C- or N-terminally to the inhibitory peptide of the present invention. The said stretch of amino acids shall allow for detection of the inhibitory peptide 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. More preferably, the inhibitory peptide comprises further amino acids which may serve as mediators of cell entry, i.e., preferably, the inhibitory peptide further comprises at least one cell-penetrating peptide (CPP). CPPs are well known in the art and include, e.g., Penetratins, HIV-tat-related peptides, Transportans, and the like, see, e.g. Nasrollahi et al., Chem Biol Drug Des. 2012, 80: 639-646.
As used herein, the term “antibody” relates to a soluble immunoglobulin from any of the classes IgA, IgD, IgE, IgG, or IgM, having the activity of activating the immune response as specified herein above, preferably, the activity of being an antagonist of CTLA-4, PD-1, CD80, CD86, and/or PD-L1 as specified herein above. Antibodies against said polypeptides can be prepared by well known methods using a purified polypeptide 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 polypeptides of the invention by proteolytic digestion, may be a synthetic peptide, or may be recombinantly expressed. Preferably, the peptide used as an antigen is located at or close to the interaction site in one of the C80/CTLA-4, CD86/CTLA-4, and PD-L1/PD-1 receptor complexes. Suitability of an antibody thus generated as an activator of the immune response can be tested by the assay as described herein in the Examples. Preferably, the antibody of the present invention is a monoclonal antibody, a human or humanized antibody or primatized, chimerized or fragment thereof. More preferably, the antibody is a single chain antibody or an antibody fragment, such as Fab, scFab. 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 not cross react with other polypeptides or peptides) to a polypeptide 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 Köhler 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.
Preferably, the activator of the immune response as described herein above is a polypeptide expressible from a single transcription unit. Accordingly, preferably, the activator of the immune response is a polypeptide or a fusion polypeptide. More preferably, the activator of the immune response is a single chain antibody or a single chain Fab polypeptide. Still more preferably, the activator of the immune response is an antagonistic anti-CTLA-4 single chain antibody, most preferably comprising the amino acid sequence of SEQ ID NO:1, preferably encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2; or an antagonistic anti-PD-L1 antibody, most preferably comprising the amino acid sequence of SEQ ID NO:3, preferably encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:4.
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, in case the activator of the immune response is a peptide or polypeptide, said secretion 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 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. Most preferably, the signal peptide is or comprises an amino acid sequence of SEQ ID NO: 5.
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, the 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 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, the expressible polynucleotide comprises the nucleic acid sequence of SEQ ID NO:2, encoding a polypeptide comprising SEQ ID NO:1, or comprises the nucleic acid sequence of SEQ ID NO:4, encoding a polypeptide comprising SEQ ID NO:3.
The term “polynucleotide”, as used in accordance with the present invention, encompasses variants of the aforementioned specific polynucleotides. Moreover, it is to be understood that the polypeptides having amino acid sequences of the polypeptides of the present invention may also be encoded due to the degenerated genetic code by more than one species of polynucleotide. The polynucleotide variants, preferably, comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a peptide or polypeptide having the activity as specified herein. Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, 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. 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 the polypeptides or peptides of the present invention. Conserved domains of the polypeptides or peptides of the present invention may be identified by a sequence comparison of the nucleic acid sequence of the polynucleotide or of the amino acid sequence of the polypeptides as specified above. Suitable PCR conditions are well known in the art. As a template, DNA or cDNA from appropriate cells 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 nucleic acid sequences detailed above. 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 as described herein above. A polynucleotide comprising a fragment of any of the aforementioned nucleic acid sequences and encoding a polypeptide or peptide comprising or consisting of the domains conferring the biological activities of a polypeptide of the present invention is also encompassed as a polynucleotide of the present invention. 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 polynucleotide, preferably, is DNA including cDNA, or RNA. The term encompasses single as well as double stranded polynucleotides. Also included by the term polynucleotide, preferably, are chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified ones such as biotinylated polynucleotides. The polynucleotides of the present invention either essentially consist of the aforementioned nucleic acid sequences or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid sequences as well.
The term “polynucleotide encoding a recombinant virus”, as used herein, relates to a polynucleotide comprising a nucleic acid sequence or nucleic acid sequences required 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, 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, as described herein above and below in the Examples. Preferably, the polynucleotide encoding a recombinant virus comprises or consists of the nucleic acid sequence of SEQ ID NO: 6, 7, 8, or 9. 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.
The term “cytokine” is known to the skilled person and relates to any one of a group of peptides released by cells and affecting the state or behaviour of other or the same cells. Preferably, the cytokine is a chemokine, an interferon, an interleukin, a lymphokine, or a tumor necrosis factor. More preferably, the cytokine is GM-CSF (Genbank Acc NO: AAA52121.1 GI:181146, preferably encoded by Genbank Acc NO: M10663.1 GI:181145) or Interleukin-12 (p35 subunit, Genbank Acc NO: AAD16432.1 GI:4323579; p40 subunit, Genbank Acc NO: AAG32620.1 GI:11192035.)
As used herein, the term “host cell” relates to a vertebrate cell. Preferably, the cell is a mammalian cell, more preferably, a mouse, rat, cat, dog, hamster, guinea pig, sheep, goat, pig, cattle, or horse cell. Still more preferably, the host cell is a primate cell. Most preferably, the host cell is a human cell. Preferably, the host cell is a tumor cell, more preferably a cancer cell.
Advantageously, it was found in the work underlying the present invention that measles virus can be engineered to express polypeptides destined for secretion and that these polypeptides are efficiently secreted during viral replication in the cell. Moreover, it was found that by administering measles virus expressing a secreted molecule preventing shutdown signaling to T-cells, the immune response to cancer cells can be improved and, in particular, tolerance induction by the tumor microenvironment can be alleviated. In contrast to methods of the prior art, no systemic treatment with the activator of the immune system is required.
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 a polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to the present invention.
The present invention also relates to a medicament comprising the recombinant virus of the family Paramyxoviridae of the present invention and 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, methylester, HCl, sulfate, chloride and the like. The pharmaceutical compositions are, preferably, administered 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, nonimmunogenic 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 treating cancer in a subject afflicted with cancer, comprising
a) contacting said subject with a recombinant virus of the family Paramyxoviridae according to the present invention, and
b) thereby, treating cancer in a subject afflicted with cancer.
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). 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.
The term “treatment” refers to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a 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 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 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 used herein, the term “subject” relates to a vertebrate. Preferably, the subject is a mammal, more preferably, a mouse, rat, cat, dog, hamster, guinea pig, sheep, goat, pig, cattle, or horse. Still more preferably, the subject is a primate. Most preferably, the subject is a human. Preferably, the subject is afflicted with a disease caused or aggravated by an insufficient response of the immune response of said subject, more preferably, the subject is afflicted with cancer.
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.
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, 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, waldenstrom macroglobulinemia, and wilms tumor. More preferably, the cancer is a solid cancer, a metastasis, or a relapse thereof. Most preferably, the cancer is a solid superficial tumor derived from head and neck cancer, malignant melanoma or cutaneous T cell lymphoma.
The present invention further relates to an in vitro method for activating immune cells in a sample comprising cancer cells and immune cells, comprising
a) contacting said sample comprising cancer cells and immune cells with a recombinant virus of the family Paramyxoviridae according to the present invention, and
b) thereby, activating immune cells comprised in said sample.
The method for activating immune cells may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing the recombinant virus of the family Paramyxoviridae for step a), administering further activating compounds, e.g. cytokines, to the immune cells in step b), or separating immune cells from cancer cells after step b). Moreover, one or more of said steps may be performed by automated equipment.
The present invention also relates to a recombinant virus of the family Paramyxoviridae according to the present invention for use in medical treatment.
Moreover, the present invention relates to a recombinant virus of the family Paramyxoviridae for use in treatment of inappropriate cell proliferation.
The term “inappropriate cell proliferation” relates to any proliferation of cells of a subject which is not appropriate to the physiological state of said subject and/or to the tissue context of said cells. Preferably, inappropriate cell proliferation is caused or aggravated by an inhibition of the immune system, more preferably of T-cells. More preferably, inappropriate cell proliferation is cancer.
The present invention further relates to a kit comprising at least the recombinant virus of the family Paramyxoviridae housed in a container.
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 such the 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.
Summarizing the findings of the present invention, the following embodiments are preferred:
A recombinant virus of the family Paramyxoviridae, comprising at least one expressible polynucleotide encoding a secreted activator of the immune response.
The recombinant virus of the family Paramyxoviridae of embodiment 1, wherein said recombinant virus is a recombinant Morbillivirus, preferably, a recombinant measles virus (MV).
The recombinant MV of embodiment 2, wherein the recombinant MV is derived from MV strain Edmonston A or B, preferably vaccine strain Schwarz (Edmonston A).
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 3, wherein the secreted activator of the immune response is a ligand for an immune checkpoint blockade protein.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 4, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against CTLA-4.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 5, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against CTLA-4 comprising the amino acid sequence of SEQ ID NO:1.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 6, wherein the at least one expressible polynucleotide encoding a secreted activator of the immune response comprises the nucleic acid sequence of SEQ ID NO:2.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 4, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against PD-L1.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 4, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against PD-L1 comprising the amino acid sequence of SEQ ID NO:3.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 4, wherein the at least one expressible polynucleotide encoding a secreted activator of the immune response comprises the nucleic acid sequence of SEQ ID NO:4.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 10, wherein the at least one expressible polynucleotide encoding a secreted activator of the immune response is comprised in the polynucleotide encoding the recombinant virus of the family Paramyxoviridae.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 11, further comprising a second expressible polynucleotide encoding a second secreted activator of the immune response.
The recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 12, wherein said second expressible polynucleotide encoding a secreted activator of the immune response is a cytokine or a second antagonist of an inhibitory factor of a T-cell or an antagonist of a negative immune regulator of the tumor-immune microenvironment.
A polynucleotide encoding the recombinant virus of the family Paramyxoviridae according of any one of embodiments 1 to 13.
The polynucleotide of embodiment 14, wherein said polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 6, 7, 8, or/and 9.
A medicament comprising the recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 13 and/or the polynucleotide of embodiment 14 or 15, and at least one pharmacologically acceptable excipient.
A method for treating cancer in a subject afflicted with cancer, comprising
The method of embodiment 17, wherein said cancer is a solid cancer, a metastasis, or a relapse thereof.
The method of embodiment 17 or 18, wherein treating cancer is reducing tumor burden.
The method of any one of embodiments 17 to 19, wherein said cancer is malignant melanoma, head and neck cancer, hepatocellular carcinoma, pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastric carcinoma, colorectal carcinoma, lymphomas or leukemias.
An in vitro method for treating activating immune cells in a sample comprising cancer cells and immune cells, comprising
a) contacting said sample comprising cancer cells and immune cells with a recombinant virus of the family Paramyxoviridae of any one of embodiments 1 to 13 and/or with a polynucleotide according to embodiment 14 or 15, and
b) thereby, activating immune cells comprised in said sample.
A recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 13 for use in medical treatment.
A recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 13 and/or a polynucleotide according to embodiment 14 or 15 for use in treatment of inappropriate cell proliferation.
The recombinant virus of the family Paramyxoviridae for use of embodiment 24, wherein treatment of inappropriate cell proliferation is cancer treatment.
Kit comprising at least the recombinant virus of the family Paramyxoviridae according to any one of embodiments 1 to 13 and/or with a polynucleotide according to embodiment 14 or 15 housed in a container.
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.
Construction of Recombinant MV Genomes in DNA Plasmids
The genome of the measles vaccine strain Schwarz (Genbank Acc NO: AF266291.1 GI:9181912) was cloned into a pUC19-based plasmid. For later generation of viral particles from a DNA plasmid in a transfected mammalian host cell line, the 5′-end of the MV leader was fused to the CMV minimal promoter, and the 3′-end of the MV trailer is followed by the Hepatitis Delta virus ribozyme sequence and a eukaryotic polyA signal (note: with respect to the natural 3′→5′-orientation of negative-strand (−)RNA viruses, the sequence of the DNA copy is annotated in the usual 5 ‘→3’-orientation; this corresponds to the viral sequence in antigenomic (+)RNA orientation; the same condition applies for the cloned viral genome with respect to the direction of the CMV promoter-driven transcription through RNA polymerase II). An additional MV-specific transcription unit (ATU) was inserted into the 3′-untranslated region (UTR) of the H gene. The H-ATU consists of viral transcription control elements—a copy of gene end signal from the N gene and gene start signal of the P gene—and the unique cloning site MauBI for insertion of transgenic open reading frames (ORF).
The coding sequences for the claimed immunomodulatory transgenes were cloned into a mammalian expression vector, providing a secretion signal and a HA-tag at the N-terminus as well as a myc-tag at the C-terminus. The respective ORFs were excised as 5 ‘-MluI 3’-AscI fragments and inserted into the MV H-ATU plasmid via the compatible MauBI site, leading to the novel vectors (
Generation and Propagation of Recombinant MV
Recombinant MV particles were generated from cDNA constructs according to Martin et al. (J Virol. 2006; 80: 5708-5715) with slight modifications. Vero cells (5×105 per 6-well) were transfected with 5 μg of the recombinant MV plasmid, together with 500 ng N, 100 ng P and 500 ng L expression plasmids using FugeneHD at a ratio of 3:1. Four to six days after transfection, cell culture supernatants were transferred onto fresh cells. To prepare virus stocks, Vero cells (African green monkey, normal kidney) were infected at a MOI of 0.03 and incubated at 37° C. for 36 to 48 hours. Viral particles were harvested by one freeze/thaw cycle and centrifugation from their cellular substrate resuspended in Opti-MEM (Invitrogen). Virus preparations can be further purified by GMP-complying protocols for ultracentrifugation or tangential flow filtration. All following infection experiments were performed with viral stocks from the third passage. Titers were determined by 50% tissue culture infectious dose (TCID50) titration on Vero cells. For generation and propagation of fully re-targeted viruses, all procedures were done analogously using Vero-αHis cells (Nakamura et al., Nat Biotechnol 2005; 23: 209-214).
MV-Mediated Expression of Secretable Antibodies
Human melanoma cells Mel888 were seeded into a six-well plate (1.5×105 per well) and infected with variant viruses at MOI of 1. Twenty-four hours after infection, supernatants were collected and passed through a 0.2 μm filter. Antibodies were precipitated using Protein A Sepharose and detected by immunoblot with an anti-HA antibody (
Binding of Secretable Antibodies to their Respective Cognate Antigens
Vero cells were seeded in six-well plates (2×105 cells per well) and infected at MOI of 3 with the indicated viruses. 36 hours after infection, cell culture supernatants were collected and passed through a 0.2 μm filter. Nunc Maxisorp 96-well plates were coated with 100 ng recombinant protein each of CTLA-4 and PD-L1, respectively. Wells were blocked with FBS and a dilution series of equal volumes of supernatants of cells equally treated and infected with MV H-sαY variants were added to the ELISA plates (
To determine viral growth kinetics in one-step growth curves, human melanoma cells Mel888 were seeded into a six-well plate (1×105 per well) and infected with the indicated MV vectors at an MOI of 3. At designated time points, cells were harvested and progeny viral particles were determined by titration assays (
To address the cytolytic effect of MV vectors encoding secretable anti-CTLA-4 and anti-PD-L1 antibodies against human melanoma cells, in vitro infection experiments were performed with Sk-Mel28 and Mel888 cells for qualitative evaluation via microscopic inspection. Syncytia formation on human cell lines was delayed compared to the simian producer cell line Vero. Nevertheless, by 48 hours after infection MV H-sαCTLA-4 and MV H-sαPD-L1 had spread across the entire cell layer. Cytopathic effects were as pronounced as those caused by the control virus MV-EGFP.
Cytopathic effects of oncolytic MV on human and murine melanoma cell lines were quantified by cell viability assays. Human melanoma cells Mel888 were seeded into a six-well plate (1×105 per well) and infected with the indicated MV vectors at an MOI of 1. At designated time points after infection, cell viability was determined using the colorimetric XTT assay (
Oncolytic efficacy of MV expressing secretable antibodies was assessed in a xenograft model of human melanoma (
MV treatment led to a significant delay in tumor progression (
MV treatment led to a significant survival benefit (
In this immunodeficient model, MV encoding sαCTLA-4 or sαPD-L1 were both as efficient as a parental control virus for oncolysis of human melanoma.
Therapeutic efficacy of immunovirotherapy in vivo was assessed in a syngeneic immunocompetent murine melanoma model (
Treatment with MV expressing the secretable antibody variants led to a delay in tumor progression in both cases (
While reduced tumor volumes at early time points did not prolong overall survival (
To investigate possible mechanisms of immunomodulatory effects by MV-mediated checkpoint blockade, tumor-infiltrating lymphocytes were characterized by flow cytometry (
Treatment with MV H-sαCTLA-4 and MV H-sαPD-L1 led to a favorable immune profile with lower abundance of negative regulatory T cells and, particularly in the case of MV H-sαPD-L1, to a higher abundance of cytotoxic T cells in treated tumors. The data suggest that MV-mediated blockade of CTLA-4 as well as of PD-L1 leads to beneficial reduction of regulatory T cells, whereas only PD-L1 inhibition led to a statistically significant increase in cytotoxic T cells.
The present application is a U.S. national stage of, and claims the priority benefit of, International Patent Application Serial No. PCT/EP2015/053801, filed Feb. 24, 2015 and U.S. Patent Application Ser. No. 61/994,353, filed Feb. 25, 2014, the text and drawings of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/053801 | 2/24/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/128313 | 9/3/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20150250837 | Nolin | Sep 2015 | A1 |
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| Grote et al. (2003) Cancer Research, vol. 63, 6463-6468. |
| Combredet et al. (2003) J. Virol., vol. 77(21), 11546-11554. |
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| European Patent Office, Written Opinion of the International Searching Authority, dated Jun. 25, 2015. |
| Number | Date | Country | |
|---|---|---|---|
| 20170065650 A1 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61944353 | Feb 2014 | US |
