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Definitions

• the invention relates to a virus particle comprising a lymphocytic choriomeningitis virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein derived from LCMV strain WE, and wherein the L segment comprises an open reading frame encoding an L protein derived from LCMV strain Clonel3.
• LCMV lymphocytic choriomeningitis virus
• L segment comprises an open reading frame encoding an L protein derived from LCMV strain Clonel3.
• the invention also relates to related host cells, methods of producing such virus particles, pharmaceutical compositions comprising such virus particles, and medical uses of such virus particles.
• LCMV belongs to the genus of Mammarenaviruses and can ubiquitously be isolated from mice and other rodents (Radoshitzky et al. (2020) in D. M. Knipe and P. M. Howley (eds.), Fields Virology). Intrauterine infection results in persistent infection of the offspring without clinical symptoms although the viral load may be very high (Radoshitzky et al. (2020) in D. M. Knipe and P. M. Howley (eds.), Fields Virology). Live virus is shed from infected animals lifelong via urine, saliva, nasal secretions and droppings resulting in symptomatic infection of exposed naive rodents.
• LCMV strains have been divided into four different lineages grouping the most commonly used laboratory strains Armstrong and WE into lineage I (Radoshitzky et al. (2020) in D. M. Knipe and P. M. Howley (eds.), Fields Virology). These laboratory strains have a low pathogenic potential for healthy humans but can cause substantial disease and death in susceptible animals. In line with the clinical manifestations caused in susceptible animals by the two strains and derivatives derived thereof the Armstrong strain is categorized as neurotropic in contrast to the WE strain that is categorized as viscerotropic.
• the LCMV genome consists of two negative stranded RNA segments named S(mall) and L(arge).
• S segment encodes the surface Glycoprotein (GP) and the Nucleocapsid protein (NP) while the L segment encodes the viral polymerase (LP) and the Z protein (Radoshitzky et al. (2020) in D. M. Knipe and P. M. Howley (eds.), Fields Virology).
• LCMV stain WE has a by default biosafety level (BSL) 3 classification, which would preclude the use of such products, as production and application of such strains under BSL 3 conditions is virtually impossible.
• BSL biosafety level
• the invention relates to a virus particle comprising a lymphocytic choriomeningitis virus (LCMV) S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40, wherein the glycoprotein comprises at least one mutated amino acid residue in comparison with the glycoprotein sequence set forth in SEQ ID NO: 4.
• LCMV lymphocytic choriomeningitis virus
• the invention also relates to a virus particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40, wherein the L protein comprises at position 1079 corresponding to the linear polypeptide sequence of SEQ ID NO: 40 an amino acid residue other than Lys.
• the invention also relates to a virus particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4 and an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO: 6, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40 and an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO: 38.
• the invention also relates to a host cell comprising an LCMV S segment and an LCMV L segment as comprised in a virus particle of the invention.
• the invention also relates to a host cell comprising cDNA of (a) an ORF encoding a glycoprotein, an ORF encoding an L protein, an ORF encoding a nucleoprotein, and an ORF encoding a Z protein as comprised in a virus particle of the invention; and/or (b) an LCMV S segment and an LCMV L segment as comprised in a virus particle of the invention.
• the invention also relates to a method of producing a virus particle of the invention comprising cultivating the host cell of the invention under conditions suitable for virus particle formation.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a virus particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the invention also relates to a virus particle comprising an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40 for use in therapy, in particular in the treatment of cancer.
• IHC tumor immunohistochemistry
• NP protein flow cytometry
• Figure 11 Bone marrow-derived dendritic cells.
• Figure 12 Treatment of Bl/6 mice with 2xlOE4 PFU/animal i.v., serum cytokines at days 1 and 3 p.i. Dotted line indicates lower detection limit.
• Figure 13 Treatment of Bl/6 mice with 2xlOE4PFU/animal i.v., serum cytokines at days 1 and 3 p.i. Dotted line indicates lower detection limit.
• Figure 18 ALT/AST serum levels upon i.v. treatment of B16 melanoma-bearing Bl/6 animals with 2xlOE4 PFU/animal 7 days p.i..
• Figure 37 FreeStyle 293-F suspension cells were infected with MOUO.OOl of LCMV strains WE-C113(L), P52-C113(L) or P52-WE(L) at a density of 1.5xl0 6 cells per mL. Supernatant was harvested on 12, 24, 30, 34, 40, 48, 72 and 96 hours post infection. LCMV titers in supernatant were analyzed via plaque-forming assay.
• Figure 38 Nucleic acid and amino acid sequences of the L segment, S segment, GPC, NP, ZP, and LP of LCMV strains A. WE, B. P52, C. Armstrong, and D. Clonel3. DETAILED DESCRIPTION
• pathogenicity of the WE strain may be consistently determined by elements located on the L segment, while the S segment of the WE and other LCMV strains is not contributing to pathogenicity.
• replacement of the L segment of the WE strain by the corresponding segment of another LCMV strain, such as the Armstrong strain, Clonel3 strain, and derivatives thereof are believed to result in a reassorted laboratory strain that does no longer display the original pathogenic profile including liver pathogenicity in NHPs and other susceptible animals.
• a virus strain comprising elements of the S segment of the LCMV WE strain or derivatives thereof, which are believed not to contribute to pathogenicity, and elements of the L segment of the strain Clone 13 or Armstrong or derivatives thereof, which are believed not to contribute to pathogenicity, have improved properties as compared to LCMV WE strain, in particular in tumor treatment.
• a virus strain is believed to no longer being able to induce LCMV specific disease including liver disease in NHPs, rodents or guinea pigs (Riviere et al. (1985) Journal of virology, 55: 704-09; Riviere et al.
• a reassorted virus according to the invention may have decreased pathogenic properties as compared to LCMV WE strain including liver pathogenicity (Examples 16, 17, 18, 19, and 35). These results do confirm that a virus particle of the invention may result in virus-based products that can safely be handled during manufacturing and clinical investigation as pathogenic properties described for wild-type LCMV strains are reduced, including liver pathogenesis and neurotropism (Examples 10, 21, 24).
• virus particles of the invention may demonstrate a strongly reduced potential to induce in vivo pathogenic cytokines in naive animals, and at the same time any signs of induced organ pathogenicity may be absent (Examples 12-17).
• virus particles comprising WE strain derived elements of the S segment and Clonel3 strain derived elements of the L segment but not virus particles comprising Clonel3 strain derived elements of the S segment and WE strain derived elements of the L segment showed attenuated replication in Example 8, supporting the hypothesis that pathogenicity of the WE and Clonel3 strain is determined by its L or S segment, respectively.
• the FVB mouse strain which exhibits a predisposition to viral induced pathologies (Schnell et al. (2012) PLoS Pathog, 8: el003073), succumbs to a hemorrhagic fever-like illness including thrombocytopenia and hepatocellular necrosis when infected with LCMV.
• the disease in FVB mice mimics LCMV disease in macaques and clinical signs of Argentine hemorrhagic fever (Schnell et al. (2012) PLoS Pathog, 8: el003073).
• infection with a virus particle of the invention did not result in neither one reduced animal weight nor liver pathology or any other signs of hemorrhagic fever-like illness in Examples 14-17.
• virus particles of the invention for clinical development is their retainment or even enhancement of strong anti-tumoral effects as described for the WE strain (Kalkavan et al. (2017) Nat Commun, 8: 14447) - despite their attenuation.
• a virus particle of the invention may be equipped with additional modifications to increase its tumor tropism (Examples 21-23). Such modifications may comprise mutations at positions 181 and/or 185 of the GP, in particular Arg 185 Trp and/or lie 181 Met.
• virus particles may exhibit strong anti-tumoral effects in murine tumor models (Example 25) but may bear the attenuated phenotype of the WE-Clonel3 strain from which it is derived (Examples 34 and 38).
• Pathogenicity of LCMV was reported as being mainly directed by the anti- viral functionality of the immune system and correlates with the strength of virus replication in infected organs (Lang et al. (2010) Cell Physiol Biochem, 26: 263-72).
• modified virus particles may result in a, compared to the WE strain, stronger early LCMV directed T cell response, the main anti-viral immune effector mechanism, and its organ distribution is controlled already early upon infection (Example 35).
• LCMV was described as being able to induce organ damage (e.g., liver) due to an overshooting and deregulated innate (Thl and pro-inflammatory cytokines) and adaptive (CD8 T cells) immune response (Lang et al. (2010) Cell Physiol Biochem, 26: 263- 72; Schnell et al. (2012) PLoS Pathog, 8: el 003073; Oldstone et al. (2016) Proceedings of the National Academy of Sciences, 115: E7814).
• the inventors of the present application have surprisingly found that - although a similar pattern of the cytokine response to the wild type LCMV strains Armstrong, Clonel3 and WE was observed - the strength of the immune response for a virus particle of the invention may be substantially lower. This observation is especially important for IFN-alpha, for which high cytokine levels early upon infection was linked to organ damage in respective animal models (Schnell et al. (2012) PLoS Pathog, 8: el003073).
• the virus particles of the invention in contrast, may induce especially early upon infection substantially lower IFN-alpha and IFN-gamma levels (Examples 12 and 13), which strongly points to a significantly lower potential for IFN-mediated detrimental effects (Baccala et al.
• pro-inflammatory cytokines IL-6 and TNF- alpha, as well as IL-10 may also be substantially decreased at different time points (days 1 and 3) upon infection, indicating enhanced virus control (Lang et al. (2010) Cell Physiol Biochem, 26: 263-72; Schnell et al. (2012) PLoS Pathog, 8: el003073).
• virus particles of the invention may exhibit a strongly attenuated phenotype and may show an even stronger in vivo attenuated immune phenotype, although retaining or even enhancing their anti-tumoral efficacy.
• the present invention relates to a virus particle comprising an LCMV S segment and an LCMV L segment.
• the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the S segment may also comprise an open reading frame encoding a nucleoprotein.
• the nucleoprotein may have at least 97% sequence identity to SEQ ID NO: 6.
• the L segment may also comprise an L segment.
• the L segment may comprise an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO: 38.
• the virus particle of the invention is preferably an arenavirus particle, more preferably a lymphocytic choriomeningitis virus (LCMV) particle.
• LCMV lymphocytic choriomeningitis virus
• the wild-type arenavirus genomic segments and ORFs are known in the art.
• the arenavirus genome consists of an S segment and an L segment.
• the S segment carries the ORFs encoding the GP and the NP.
• the L segment encodes the L protein and the Z protein. Both segments are flanked by the respective 5’ and 3’ UTRs.
• the vims particles of the invention preferably comprise genomic segments that correspond to the genomic segments of a wild-type arenavirus. This means that the S segment carries the ORFs encoding the GP and the NP and that the L segment encodes the L protein and the Z protein.
• the genes of the vims particles of the disclosure are preferably located at their natural positions.
• the vims particle of the invention thus preferably has a bi-segmented genome.
• the genome of the vims particle of the invention preferably consists of one L segment and one S segment as described herein.
• Illustrative examples of LCMV S segments are shown in SEQ ID NOs: 1, 11, 21, and 31.
• Illustrative examples of LCMV L segments are shown in SEQ ID NOs: 2, 12, 22, and 32.
• LCMV “WE”, “strain WE”, “WE strain”, or the like refers to an LCMV having the genomic segments as shown in SEQ ID NOs: 1 and 2.
• LCMV “P52”, “P52-WE”, “strain P52”, “P52 strain”, or the like refers to a variant/derivative of strain WE, which has the genomic segments as shown in SEQ ID NOs: 11 and 12.
• LCMV “Armstrong”, “strain Armstrong”, “Armstrong strain”, or the like refers to LCMV strain Armstrong 53b, which has the genomic segments as shown in SEQ ID NOs: 21 and 22.
• LCMV “Clonel3”, “Cl 13” “strain Clonel3”, “Clonel3 strain”, or the like refers to an LCMV which has the genomic segments as shown in SEQ ID NOs: 31 and 32.
• glycoprotein refers to an LCMV-derived glycoprotein, which is considered to mediate receptor binding and membrane fusion.
• Illustrative examples of glycoproteins are shown in SEQ ID NOs: 4, 14, 24, and 34.
• Illustrative examples for genes that encode a glycoprotein are shown in SEQ ID NOs: 3, 13, 23, and 33.
• L protein refers to an LCMV- derived RNA polymerase L.
• L proteins are shown in SEQ ID NOs: 10, 20, 30, and 40.
• genes that encode an L protein are shown in SEQ ID NOs: 9, 19, 29, and 39.
• nucleoprotein refers to an LCMV-derived nucleoprotein.
• nucleoproteins are shown in SEQ ID NOs: 6, 16, 26, and 36.
• genes that encode a nucleoprotein are shown in SEQ ID NOs: 5, 15, 25, and 35.
• Z protein or “ZP”, which are used interchangeably, refer to an LCMV- derived small RING finger protein Z.
• Z proteins are shown in SEQ ID NOs: 8, 18, 28, and 38.
• genes that encode a Z protein are shown in SEQ ID NOs: 7, 17, 27, and 37.
• a virus particle of the invention comprises a glycoprotein, wherein the glycoprotein may comprise at least one mutated amino acid residue in comparison with the glycoprotein sequence set forth in SEQ ID NO: 4.
• the glycoprotein may comprise at least one mutated amino acid residue at position 181 and/or 185 in comparison with the glycoprotein sequence set forth in SEQ ID NO: 4. It is however preferred that the glycoprotein comprises a mutation at both positions in comparison with SEQ ID NO: 4. Preferred mutations at these positions are selected from Arg 185 Trp and lie 181 Met.
• a virus particle thus can have any one or preferably both mutations.
• the presence of the one or two above-mentioned mutations in the glycoprotein can improve the function of the LCMV (e.g., anti-tumoral activities), independent from the presence of other mutations, in particular of other mutations in other genes or proteins of LCMV.
• a virus particle of the invention comprises an L protein, wherein the L protein may comprise at position 1079 corresponding to the linear polypeptide sequence of SEQ ID NO: 40 an amino acid residue other than Lys.
• the amino acid residue at this position is preferably a non- basic amino acid residue, more preferably a neutral hydrophilic amino acid residue, even more preferably an Asn or Gin, most preferably a Gin.
• amino acid typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (lie or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired
• Naturally occurring residues are divided into groups based on common side- chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine.
• substitutions may entail exchanging a member of one of these classes for another class.
• a virus particle of the invention comprises an S segment and an L segment, wherein the S segment is preferably derived from LCMV strain WE or a variant thereof and wherein the L segment is preferably derived from LCMV strain Clonel3 or a variant thereof.
• a variant of the L segment may have at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity to the sequence of the original L segment.
• a virus particle disclosed herein may have an L segment that comprise or preferably consists of a sequence that has at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or
• a variant of the S segment may have at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity to the sequence of the original S segment.
• a virus particle disclosed herein may have an S segment that comprise or preferably consists of a sequence that has at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NO: 1 or 11.
• a virus particle of the invention comprising an LCMV preferably comprises S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4 and an open reading frame encoding a nucleoprotein having at least 97% sequence identity to SEQ ID NO: 6, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40 and an open reading frame encoding a Z protein having at least 90% sequence identity to SEQ ID NO: 38.
• An open reading frame encoding a glycoprotein disclosed herein may encode a sequences that has at least about 98%, preferably at least about 98.5%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.5%, preferably at least about 99.7% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NO: 4 or 14.
• An open reading frame encoding a glycoprotein disclosed herein may comprise a sequence that has at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NO: 3 or 13.
• An open reading frame encoding an L protein disclosed herein may encode a sequences that has at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NO: 30 or 40.
• An open reading frame encoding an L protein disclosed herein may have at least about 83%, preferably at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or is preferably identical, to a sequence set forth in SEQ ID NO: 29 or 39
• An open reading frame encoding a nucleoprotein disclosed herein may encode a sequence that has at least about 98%, preferably at least about 98.5%, preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a sequence set forth in any one of in SEQ ID NO: 6 or 16.
• An open reading frame encoding a nucleoprotein disclosed herein may have at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a sequence set forth in SEQ ID NO: 5 or 15.
• An open reading frame encoding a Z protein disclosed herein may encode a sequence that has at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is preferably identical, to a sequence set forth in SEQ ID NO: 28 or 38.
• An open reading frame encoding a Z protein disclosed herein may have at least about 84%, preferably at least about 85%, preferably at least about 86%, preferably at least about 87%, preferably at least about 88%, preferably at least about 89%, preferably at least about 90%, preferably at least about 91%, preferably at least about 92%, preferably at least about 93%, preferably at least about 94%, preferably at least about 95%, preferably at least about 96%, preferably at least about 97%, preferably at least about 98% preferably at least about 99%, preferably at least about 99.1%, preferably at least about 99.2%, preferably at least about 99.3%, preferably at least about 99.4%, preferably at least about 99.5%, preferably at least about 99.6%, preferably at least about 99.7%, sequence identity, or that is identical, to a sequence set forth in SEQ ID NO: 27 or 37.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 98.5% sequence identity to SEQ ID NO: 4 or 14, with SEQ ID NO: 14 being preferred, and an open reading frame encoding a nucleoprotein having at least 98.5% sequence identity to SEQ ID NO: 6 or 16, with SEQ ID NO: 16 being preferred, and wherein the L segment comprises an open reading frame encoding an L protein having at least 95% sequence identity to SEQ ID NO: 30 or 40, with SEQ ID NO: 40 being preferred, and an open reading frame encoding a Z protein having at least 95% sequence identity to SEQ ID NO: 28 or 38, with SEQ ID NO: 38 being preferred.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 98% sequence identity to SEQ ID NO: 4 or 14, with SEQ ID NO: 14 being preferred, and an open reading frame encoding a nucleoprotein having at least 98% sequence identity to SEQ ID NO: 6 or 16, with SEQ ID NO: 16 being preferred, and wherein the L segment comprises an open reading frame encoding an L protein having at least 98% sequence identity to SEQ ID NO: 30 or 40, with SEQ ID NO: 40 being preferred, and an open reading frame encoding a Z protein having at least 98% sequence identity to SEQ ID NO: 28 or 38, with SEQ ID NO: 38 being preferred.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 99% sequence identity to SEQ ID NO: 4 or 14, with SEQ ID NO: 14 being preferred, and an open reading frame encoding a nucleoprotein having at least 99% sequence identity to SEQ ID NO: 6 or 16, with SEQ ID NO: 16 being preferred, and wherein the L segment comprises an open reading frame encoding an L protein having at least 99% sequence identity to SEQ ID NO: 30 or 40, with SEQ ID NO: 40 being preferred, and an open reading frame encoding a Z protein having at least 99% sequence identity to SEQ ID NO: 28 or 38, with SEQ ID NO: 38 being preferred.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having a sequence set forth in SEQ ID NO: 4 or 14, with SEQ ID NO: 14 being preferred, and an open reading frame encoding a nucleoprotein having a sequence set forth in SEQ ID NO: 6 or 16, with SEQ ID NO: 16 being preferred, and wherein the L segment comprises an open reading frame encoding an L protein having a sequence set forth in SEQ ID NO: 30 or 40, with SEQ ID NO: 40 being preferred, and an open reading frame encoding a Z protein having a sequence set forth in SEQ ID NO: 28 or 38, with SEQ ID NO: 38 being preferred.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having a sequence set forth in SEQ ID NO: 4, and an open reading frame encoding a nucleoprotein having a sequence set forth in SEQ ID NO: 6, and wherein the L segment comprises an open reading frame encoding an L protein having a sequence set forth in SEQ ID NO: 40, and an open reading frame encoding a Z protein having a sequence set forth in SEQ ID NO: 38.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having a sequence set forth in SEQ ID NO: 14, and an open reading frame encoding a nucleoprotein having a sequence set forth in SEQ ID NO: 16, and wherein the L segment comprises an open reading frame encoding an L protein having a sequence set forth in SEQ ID NO: 40, and an open reading frame encoding a Z protein having a sequence set forth in SEQ ID NO: 38.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having a sequence set forth in SEQ ID NO: 4, and an open reading frame encoding a nucleoprotein having a sequence set forth in SEQ ID NO: 6, and wherein the L segment comprises an open reading frame encoding an L protein having a sequence set forth in SEQ ID NO: 30, and an open reading frame encoding a Z protein having a sequence set forth in SEQ ID NO: 28.
• a preferred virus particle comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having a sequence set forth in SEQ ID NO: 14, and an open reading frame encoding a nucleoprotein having a sequence set forth in SEQ ID NO: 16, and wherein the L segment comprises an open reading frame encoding an L protein having a sequence set forth in SEQ ID NO: 30, and an open reading frame encoding a Z protein having a sequence set forth in SEQ ID NO: 28.
• a virus particle of the disclosure preferably does not comprise a heterologous ORF.
• a “heterologous ORF” in this context refers to an ORF from an organism other than an LCMV and/or a ORF encoding an artificial or synthetic protein.
• nucleic acid may generally refer to DNA or RNA.
• DNA and RNA differ - among others - in their nucleobases.
• the complementary base to adenine in DNA is thymine, whereas in RNA, it is uracil.
• t the corresponding base to adenine is denoted as “t” throughout the application, which - depending on its context - may refer to thymine (in DNA) or uracil (in RNA).
• nucleic acid sequence may encompass the sequence of a coding strand and may also encompass the sequence of a strand that is complementary to the coding strand.
• Percent (%) sequence identity with respect to sequences disclosed herein is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are pair-wise identical with the amino acid residues or nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The same is true for nucleotide sequences disclosed herein. For determining sequence identity, uracil (e.g. in RNA) may be considered to be identical to thymine (e.g. in DNA).
• the term “attenuation” as used herein, relates to a reduced capacity of a virus to replicate within a given host (cell), a reduced capacity of replicating in a healthy organ, and/or a reduced capacity of inducing cytokines.
• a virus particle of the invention is preferably attenuated.
• infectious as used herein, relates to a virus’ capacity to infect, i.e., enter, a given host cell.
• a virus particle of the invention is preferably infectious.
• replication competent means that a virus has the ability to amplify and express its genetic material in infected cells and being able to produce further progeny in normal cells that are not genetically engineered.
• replication competent viruses do not require host cells that were engineered to express a viral gene for being capable of replication.
• a virus particle of the invention is preferably replication competent.
• pathogenic as used herein, relates to a virus’ capacity to cause disease, i.e., harm to an organism.
• a virus particle of the invention may be capable of promoting a reduction of growth of a tumor that is at least as strong or even stronger as compared to LCMV strain WE and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2.
• the virus particle may be capable of promoting a stronger reduction of growth of a cold tumor as compared to LCMV strain WE and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 1.
• a virus particle of the invention may be capable of inducing an adaptive immune activation that is at least as strong or even stronger as compared to LCMV strain WE and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 3.
• a virus particle of the invention may be capable of promoting a reduction of growth of a warm tumor that is at least as strong as compared to LCMV strain WE and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 4.
• a “hot” or “warm” tumor which is used interchangeably, relates to a tumor having a T-cell-inflamed phenotype. Such tumors show signs of inflammation, meaning the tumor has already been infiltrated by T cells to fight the cancerous cells. Examples of cancers that are typically hot tumors include melanoma, bladder cancer, kidney cancer, head and neck cancer, and non-small cell lung cancer.
• a “cold tumor” relates to a tumor having a non-T-cell-inflamed phenotype, which has low T cell infiltration or which have not been infiltrated with T cells.
• cancers that are typically cold tumors include breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma.
• hot tumors and cold tumors are described in detail in Gajewski et al. (2017) Adv Exp Med Biol. 1036:19-31 and Maleki Vareki (2016) Journal for ImmunoTherapy of Cancer 6:157.
• a virus particle of the invention may be capable of inducing decreased levels of an interferon upon (preferably systemic) administration compared to LCMV strain WE, Armstrong, Clone 13 and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 12.
• the interferon is preferably selected from the group consisting of IFN-a nd IFN-
• a virus particle of the invention may be capable of inducing a lower concentration of one or more Thl- and pro-inflammatory cytokines compared to LCMV strain WE, Armstrong, Clone 13 and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 13.
• the cytokine is preferably selected from the group consisting of IL-6, IL-10, TNFa, and IFNy
• a virus particle of the invention may be capable of inducing a lower concentration of one or more Thl- and pro-inflammatory cytokines compared to LCMV strain WE, Armstrong, Clone 13 and/or a virus particle comprising the same S segment as the virus particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 13.
• the cytokine is preferably selected from the group consisting of IL-6, IL-10, TNFa, and IFNy.
• a virus particle of the invention may be less pathogenic in a mouse compared to LCMV strain WE, preferably as measured in an FVB/N mouse in an assay as essentially described in Example 14.
• a virus particle of the invention may be capable of inducing a reduced degree of liver pathology in a mouse compared to LCMV strain WE and/or a virus particle comprising the same S segment as the vims particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 18.
• a vims particle of the invention may be capable of inducing a reduced degree of thrombocytopenia in a mouse compared to LCMV strain WE and/or a vims particle comprising the same S segment as the vims particle and an L segment as shown in SEQ ID NO: 2 or no detectable thrombocytopenia in a mouse, such as an FVB/N mouse, preferably as measured in an assay as essentially described in Example 15.
• a vims particle of the invention may show reduced replication in healthy cells such as neuron cells compared to LCMV strain WE and/or a vims particle comprising the same S segment as the vims particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 24.
• a vims particle of the invention may show increased replication in tumor cells compared to LCMV strain WE, preferably as measured in an assay as essentially described in Example 24.
• a vims particle of the invention may show reduced replication in a healthy organ compared to LCMV strain WE and/or a vims particle comprising the same S segment as the vims particle and an L segment as shown in SEQ ID NO: 2, preferably as measured in an assay as essentially described in Example 35.
• the present invention also relates to a host cell comprising an LCMV S segment and an LCMV L segment as described for the vims particle of the invention.
• the present invention also relates to a host cell comprising cDNA of an ORF encoding a glycoprotein as described for the vims particle of the invention, an ORF encoding an L protein as described for the vims particle of the invention, an ORF encoding a nucleoprotein as described for the vims particle of the invention, and an ORF encoding a Z protein as described for the vims particle of the invention.
• the present invention also relates to a host cell comprising cDNA of an LCMV S segment and an LCMV L segment as described for the vims particle of the invention.
• a cDNA described herein can be incorporated into a plasmid.
• the cDNA described herein can be part of or can be incorporated into a DNA expression vector and optionally introduced into a host cell.
• a cDNA described herein or plasmid or vector comprising the cDNA preferably comprises a promoter.
• promoters include an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T7 promoter, an SP6 promoter or a T3 promoter.
• a host cell can be any host cell suitable for cloning, expression, propagation, or production of the virus particle.
• a host cell can be prokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HEK cells, BHK cells, HeLa cells or CHO cells) or primary mammalian cells.
• Preferred host cells include HEK cells, in particular HEK293 cells, such as HEK293T (CVCL 0063) or FreeStyle 293-F (CVCL D603).
• FreeStyle 293-F cells are commercially available, e.g., from Thermo Fisher Scientific Inc. (Catalogue number 12338026).
• Preferred host cells also include BHK cells, such as BHK-21 cells (CVCL 1914).
• Preferred host cells also include WHO Vero RCB 10-87 cells (ATCC CCL 81).
• the present invention also relates to a method of producing a virus particle of the invention.
• the method comprises cultivating a host cell of the invention under conditions suitable for virus particle formation.
• the method of producing the virus particle can further comprise introducing into a host cell the cDNA described herein.
• the method of producing the virus particle can also comprise recovery and/or purification of the virus particle. Such recovery and/or purification methods are well-known to those skilled in the art.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a virus particle disclosed herein.
• the virus particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient or carrier.
• a carrier may be e.g., be selected from the group consisting of water, aqueous saline solution, aqueous buffer solution, cell culture medium and combinations of at least two of the foregoing carriers.
• the present invention also relates to a virus particle disclosed herein for use in therapy.
• the virus particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the virus particle disclosed herein may be for use in the treatment of a cancer or tumor.
• the present invention also relates to a use of a virus particle disclosed for the manufacture of a medicament.
• the virus particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the medicament is preferably for the treatment of a cancer or tumor.
• the present invention also relates to a method of treating a disease comprising administering to a subject a virus particle disclosed herein.
• the subject is preferably in need thereof.
• the virus particle is preferably administered in an effective amount.
• the virus particle preferably comprises an LCMV S segment and an LCMV L segment, wherein the S segment comprises an open reading frame encoding a glycoprotein having at least 97% sequence identity to SEQ ID NO: 4, and wherein the L segment comprises an open reading frame encoding an L protein having at least 90% sequence identity to SEQ ID NO: 40.
• the disease is preferably a cancer or tumor.
• a "subject” is a vertebrate, preferably a mammal, more preferably a human.
• the term "mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as mice, sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys and etc., to name only a few illustrative examples.
• the mammal herein is human.
• the cancer or tumor may be a human or murine cancer or tumor, with a human cancer or tumor being preferred.
• an "effective amount” is an amount sufficient to effect beneficial or desired results.
• An effective amount can be administered in one or more administrations.
• the cancer or tumor may be any cancer or tumor disclosed herein. Generally, the cancer or tumor can be selected from the group consisting of carcinoma, melanoma, blastoma, lymphoma and sarcoma.
• carcinoma in the context of the present disclosure should be understood to mean a malignant neoplasia of epithelial origin.
• a carcinoma is preferably selected from the group consisting of anal carcinoma, bronchial carcinoma, lung carcinoma, endometrial carcinoma, gallbladder carcinoma, bladder carcinoma, hepatocellular carcinoma, testicular carcinoma, colon carcinoma, colorectal carcinoma, rectal carcinoma, laryngeal carcinoma, esophageal carcinoma, gastric carcinoma, breast carcinoma, renal carcinoma, ovarian carcinoma, pancreatic carcinoma, pharyngeal carcinoma, oropharyngeal carcinoma, prostate carcinoma, thyroid carcinoma and cervical carcinoma.
• sarcoma in the context of the present disclosure should be understood to mean a malignant neoplasia of mesodermal origin.
• a sarcoma can be selected from the group consisting of angiosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, Kaposi sarcoma, liposarcoma, leiomyosarcoma, malignant fibrous histiocytoma, neurogenic sarcoma, osteosarcoma and rhabdomyosarcoma.
• melanoma in the context of the present disclosure should be understood to mean a malignant neoplasia of melanocytic origin.
• lymphocytic origin a malignant neoplasia of lymphocytic origin.
• blastoma in the context of the present disclosure should be understood to mean a malignant neoplasia of embryonic origin.
• the cancer or tumor to be treated may be a cold tumor.
• Preferred cold tumors include breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma.
• the cancer or tumor to be treated may be a hot tumor.
• Preferred hot tumors include melanoma, bladder cancer, kidney cancer, head and neck cancer, and non-small cell lung cancer.
• Preferred cancers or tumors to be treated include melanoma, colon carcinoma, fibrosarcoma, pancreas cancer, thyroid carcinoma, lung cancer, adenocarcinoma, and gastrointestinal cancer.
• the virus particles disclosed herein can be administered via any suitable route that is known to the skilled person. Administration generally includes enteral and parenteral administration. Parenteral administration can include local administration, such as intramuscular, intraperitoneal, subcutaneous, or intratumoral administration. Alternatively, parenteral administration can include systemic administration, in particular intravenous administration, such as via injection or infusion.
• any of the terms “comprising”, “consisting essentially of' and “consisting of' may be replaced with either of the other two terms.
• the term “comprising” is meant to provide explicit support also for “consisting essentially of' and “consisting of, the term “consisting essentially of' is meant to provide explicit support also for “comprising” and “consisting of, the term “consisting of' is meant to provide explicit support also for "consisting essentially of' and “comprising”.
• MC57G (CVCL 4985) is a murine fibrosarcoma cell line, which show robust LCMV replication.
• Tramp-C2 is a murine prostate gland cell line (CVCL 3615).
• B16 (CVCL F936) and B16-F10 (B16F10, CVCL 0159) are murine melanoma cell lines.
• B16-Ova represents the respective B16 cell line expressing ovalbumin as antigen.
• MC-38 (MC38, CVCL B288) is a murine colon adenocarcinoma cell line.
• JAWSII is a murine, spontaneously immortalized cell line, which can be differentiated into dendritic like cells by treatment with GM-CSF (CVCL_3727).
• BMDCs bone marrow derived dendritic cells
• C643 (CVCL 5969) is a human anaplastic thyroid carcinoma cell line.
• NCI-H1975 (H1975, CVCL 1511) is a human lung adenocarcinoma cell line.
• A549 is human adenocarcinoma cell line (CVCL 0023).
• FTC133 is a human thyroid carcinoma cell line (CVCL 1219).
• GIST-T1 is a human gastrointestinal tumor cell line derived from pleural effusion (CVCL 4976).
• Hek293T (CVCL 0063) is a human embryonic kidney cell line, which shows robust LCMV replication.
• FreeStyle 293-F (CVCL D603) is a derivative of Hek293 cells, propagated in Freestyle Medium. FreeStyle 293-F cells are the desired producer cell line for LCMV production.
• UKE-Ma-Mel-51 (Mamel51, CVCL_A186) and UKE-Ma-Mel-86a (Ma-Mel-86a, CVCL A221) are human primary melanoma cell lines derived from lymph node metastasis provided by Prof. Dr. Paschen, Stamm fur Dermatologie, UK Essen. Primary human neurons were differentiated from human neuronal progenitor cells (NPC) kindly provided by Prof. Dr. Gopalakrishnan, HHU Dusseldorf. SkMM (Human Skeletal muscle myoblasts, Lonza CC-2580) are human primary skeletal myoblasts.
• Myotubes were differentiated from SkMM in the presence of SkGM-2, Horse Serum, Glutamine, Gentamicin and Dexamethasone. Differentiation was verified by staining with Anti-Myosin 4 AK (eBioscience, Clone MF-20).
• BHK-21 cells represent a spontaneous immortalized fibroblast hamster cell line (CVCL_1914).
• the LCMV strain WE was obtained from the laboratory of Prof. Zinkernagel (Experimental Immunology, Zurich, Switzerland) and has been propagated in L929 cells or BHK-21 cells since 2008.
• the LCMV strains Clonel3 and Armstrong 53b were obtained from S. Basta, Queens University and R. M. Zinkernagel, University of Zurich, respectively.
• the LCMV reassortants WE-C113(L), P52-C113(L), P52-WE(L) and C113-WE(L) were rescued by transient transfection entirely from plasmids as described (Flatz et al. (2006) Proc Natl Acad Sci USA 103(12): 4663- 4668).
• the viruses consist of the S-Segment of the LCMV strain WE or WE-derived P52 and the L-Segment of the LCMV strains Clonel3 or WE. Viruses were propagated on BHK-21 or Freestyle 293F cells.
• LCMV recombinant and reassortant viruses were generated entirely from plasmids.
• BHK-21 cells were transiently transfected with plasmids coding for the LCMV S segment, L segment as well as helper plasmids coding for the L polymerase and the nucleoprotein.
• the LCMV reassortant WE-C113(L) was generated using a WE S segment and the Clone 13 L segment plasmid.
• the LCMV P52-C113(L) was rescued using a LCMV P52 S segment plasmid, which harbors two coding mutations in the glycoproteins, I181M and R185W, and a Clonel3 L segment plasmid.
• the LCMV P52-WE(L) was generated using a LCMV P52 S segment plasmid and a LCMV WE L segment plasmid.
• the C113-WE(L) was generated using a LCMV Clone 13 S segment plasmid and a WE L segment plasmid.
• Immunohistofluorescence was used to detect LCMV and immune cell distribution in tumor tissues of LCMV treated or control treated tumor bearing mice. Tumor biopsies were cut into 7 pm thick sections and stained with a fluorochrome-labelled anti-LCMV-NP antibody (clone VL4), and antibody against CD8 (eBioscience) and visualized with a fluorescence microscope (Keyence) and photographed with an integrated CCD camera.
• LCMV infected plaques were stained with anti LCMV-NP antibody (clone VL4). Plaques were counted and infectious particles were determined as virus titer per mL of supernatant or per organ.
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• LCMV therapy immune cells were analyzed via flow cytometry (LSR Fortessa, BD). Thrombocytes were detected by their specific size and granularity. T cells were stained with specific fluorescent labelled antibodies for CD8, CD4, CD3 (eBioscience). LCMV specific T cells were stained using tetramer staining (NIH, Tetramer Facility).
• LCMV strain WE-C113(L) shows a strong tumor growth inhibiting effect in the immunogenic warm MC38 tumor model. This is a model for a warm tumor.
• LCMV strain WE-C113(L) shows lack of neurotropism but simultaneously enhanced replication in susceptible tumor cells such as the murine fibrosarcoma cell line MC57G and robust replication on human cancer cell lines A549 and FTC133 compared to LCMV strain WE.
• Bone marrow derived dendritic cells were infected with LCMV strains WE and WE- 013. Supernatants were harvested and analyzed for Interferon alpha protein via commercial Interferon alpha ELIS A-Kit ( Figure 11).
• Tumor draining lymph node resident cytotoxic, antiviral CD8 + GP33-tetramer positive T cells were stained via fluorescent labeled antibodies (eBioscience, NIH) and analyzed via flow cytometry on day seven after infection (Figure 29).
• LCMV strain P52-C113(L) shows a clear tendency towards an enhanced T cell accumulation in tumor tissue compared to LCMV strains WE, WE-C113(L) and control treated tumors.
• LCMV strains WE-C113(L) and P52-C113(L) induce a stronger T helper cell infiltration into tumor tissue compared to tumors treated with wildtype LCMV strain WE or control treated.
• LCMV reassortant P52-C113(L) displays a decreased replication in healthy organs by simultaneously showing measurable replication in tumor tissue.
• MC38 tumor cells were subcutaneously transplanted into C57BL/6 mice. After visible tumor formation mice were intravenously treated with 2xl0 4 PFU of LCMV strains WE, WE- C113(L) or P52-C113(L). On day eleven after infection tumors were dissected an LCMV replication was analyzed via focus forming assay ( Figure 36).
• FreeStyle 293-F suspension cells were infected with MOUO.OOl of LCMV strains WE- C113(L), P52-C113(L) or P52-WE(L) at a density of 1.5xl0 6 cells per mL. Supernatant was harvested on 12, 24, 30, 34, 40, 48, 72 and 96 hours post infection. LCMV titers in supernatant were analyzed via focus forming assay ( Figure 37). [0241] Thereby it was proven that FreeStyle 293-F cells show enhanced replication of the LCMV reassortants WE-CL13(L) and P52-C113(L) and display an ideal producer cell line for the production of LCMV reassortants.

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