Improved viral particle entry into tumor cells

Abstract

This invention relates to a viral particle comprising a mutated lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said viral particle has an enhanced ability to enter tumor cells. The invention also relates to said mutated GP, nucleic acid molecule encoding said GP, host cell containing cDNA of said nucleic acid molecule or the genome of said viral particle, method of producing said viral particle, and medical uses.

Description

Cross-references to related applications

[0001] This application claims the benefit of priority to European patent application EP23194741.7, filed on August 31, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This invention relates to a viral particle comprising a mutated lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said viral particle has an enhanced ability to enter tumor cells. The invention also relates to said mutated GP, nucleic acid molecule encoding said GP, host cell containing cDNA of said nucleic acid molecule or the genome of said viral particle, method of producing said viral particle, and medical uses. Background Technology

[0003] For cancer patients who do not achieve a durable response with immune checkpoint inhibitors, oncolytic viruses (OVs) are a potential treatment option. OVs preferentially replicate in cancer cells, thus possessing the ability to promote immune responses within the tumor. The reasons for OV's tumor-specific proliferation are currently under investigation. Significantly enhanced anabolism makes cancer cells ideal hosts for viral replication. Furthermore, cancer cells do not express cellular autoimmunity due to the active suppression of cellular antiviral mechanisms by oncogenes. Specifically, overexpression of RAS can block the expression of antiviral effector protein kinase R (PKR). These factors may explain the accelerated viral replication, viral protein translation, and viral particle assembly in cancer cells. Whether other factors, including tumor-specific viral entry, also contribute to tumor-specific proliferation remains insufficiently understood.

[0004] Viral tropism refers to the infectivity of a virus for a given cell type. It is determined in part by its entry receptor. Typically, viruses attach to receptors expressed on target cells via their surface proteins and then enter the cell. Therefore, the expression of entry receptors in a variety of cell types and tissues allows viruses to exhibit broad tropism. Consistent with this, the binding of viral surface proteins to similar receptors in another species allows viral replication. Although malignant transformation regulates the expression and modification of many proteins, it remains unclear whether viral entry receptors are specifically overexpressed in malignant cells. Whether OVs can be modified to enhance their tumor tropism and reduce their tropism for healthy cells remains to be investigated.

[0005] Arenaviruses are enveloped and pleomorphic, ranging in diameter from 60 to 300 nm, and possess two single-stranded, double-sense RNA genomic segments. Some Old World arenaviruses (including lymphocytic choriomeningovirus (LCMV)) and members of clade C New World arenaviruses bind to the ubiquitously expressed cell receptor α-dystrophic glycan (DAG1). In the presence of heparan sulfate, the proteoglycan can function as an entry receptor. Recently, CD164 has been identified as an additional co-receptor that accelerates LCMV entry. Like other OVs, LCMVs preferentially proliferate in tumor cells, thereby inducing local immune activation. This immune activation modulates the tumor microenvironment and leads to the infiltration of several immune cells, which can subsequently activate anti-tumor mechanisms. However, due to the ubiquitous expression of the cell entry receptor DAG1, LCMVs can replicate in healthy organs, thus limiting their application in cancer therapy.

[0006] However, there is a need in the art to provide a sand-like virus, particularly LCMV, which has improved properties for cancer treatment. Summary of the Invention

[0007] This invention relates to a viral particle comprising a lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, which, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0008] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0009] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0010] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0011] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0012] (e) Spiral α3 defined by positions 187-199;

[0013] (f) Ring 3 defined by positions 200-226;

[0014] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0015] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0016] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0017] (j) The TM cytoplasmic domain defined at positions 439-498;

[0018] Compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4, the viral particle has an enhanced ability to enter tumor cells.

[0019] This invention also relates to a viral particle comprising a lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said glycoprotein, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, and compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein is selected from 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152, 153, 154, 155, 156, 163, 18 One or more of the following positions contain at least one mutated amino acid residue: 8, 198, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492, wherein the viral particle has an enhanced ability to enter tumor cells compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4.

[0020] The present invention also relates to an LCMV glycoprotein, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein contains at least one mutated amino acid residue defined for the viral particles of the present invention.

[0021] The present invention also relates to a nucleic acid molecule encoding the LCMV GP of the present invention.

[0022] The present invention also relates to a viral particle comprising a glycoprotein (GP) of a sand virus and / or a nucleic acid encoding said GP, wherein said glycoprotein comprises at least one of the following amino acid residues:

[0023] (a) A mutated amino acid residue, or preferably an Asn residue, at the position corresponding to Ser 102 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position.

[0024] (b) A mutated amino acid residue, or preferably a Leu residue, at the position corresponding to Phe 122 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0025] (c) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Phe 129 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0026] (d) A mutated amino acid residue, or preferably a Gln residue, at the position corresponding to His 136 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a His residue at said position.

[0027] (e) A mutated amino acid residue, or preferably a Tyr residue, at the position corresponding to Asn 152 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0028] (f) A mutated amino acid residue at the position corresponding to Ser 153 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Pro, Phe or Tyr residue, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position.

[0029] (g) A mutated amino acid residue, or preferably an Asp residue, at the position corresponding to Asn 154 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0030] (h) A mutated amino acid residue at the His 155 position of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Leu or Tyr residue, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0031] (i) A mutated amino acid residue, or preferably a Glu residue, at the position corresponding to Lys 156 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0032] (j) A mutated amino acid residue, or preferably a Thr residue, at the position corresponding to Ala 211 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at said position.

[0033] (k) A mutated amino acid residue, or preferably an Ile residue, at the position corresponding to Thr 217 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0034] (l) A mutated amino acid residue, or preferably an Ile residue, at the position corresponding to Thr 218 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0035] (m) A mutated amino acid residue, or preferably an Arg residue, at the position corresponding to Lys 256 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0036] (n) A mutated amino acid residue, or preferably a Phe or Ile residue, at the position corresponding to Leu 260 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Leu residue at said position.

[0037] (o) A mutated amino acid residue, or preferably a His residue, at the position corresponding to Tyr 284 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Tyr residue at said position.

[0038] (p) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Ala 328 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at said position.

[0039] (q) A mutated amino acid residue, or preferably a Leu residue, at the position corresponding to Phe 332 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0040] (r) A mutated amino acid residue, or preferably an Ala residue, at the position corresponding to Val 342 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Val residue at said position.

[0041] (s) A mutated amino acid residue, or preferably a Lys residue, at the position corresponding to Arg 358 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Arg residue at said position; and

[0042] (t) The Ile residue at the position corresponding to Lys 492 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0043] The present invention also relates to a host cell containing the cDNA of the genome of the nucleic acid molecule of the present invention or the viral particle of the present invention.

[0044] The present invention also relates to a method for generating the viral particles of the present invention, comprising culturing the host cells of the present invention under conditions suitable for viral particle formation.

[0045] The present invention also relates to a pharmaceutical composition comprising the virus particles of the present invention, the GP of the present invention, the nucleic acid molecule of the present invention, or the sand-like virus particles of the present invention.

[0046] The present invention also relates to the virus particles of the present invention, the GP of the present invention, the nucleic acid molecules of the present invention, or the sand-like virus particles of the present invention, for use in treatment, preferably for the treatment of cancer.

[0047] This invention also relates to the use of any of the viral particles of this invention, the GP of this invention, the nucleic acid molecule of this invention, or the sand-like viral particles of this invention in the preparation of a medicament, wherein the medicament is preferably used for cancer treatment.

[0048] The present invention also relates to a method of treating a disease, comprising administering to a subject in need an effective amount of the virus particles of the present invention, the GP of the present invention, the nucleic acid molecules of the present invention, or the sand-like virus particles of the present invention, wherein the disease is preferably cancer.

[0049] The present invention also relates to a method for generating viral particles with enhanced ability to enter tumor cells, comprising:

[0050] (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0051] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0052] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0053] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0054] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0055] (e) Spiral α3 defined by positions 187-199;

[0056] (f) Ring 3 defined by positions 200-226;

[0057] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0058] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0059] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0060] (j) The TM cytoplasmic domain defined at positions 439-498; and

[0061] (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and

[0062] (iii) Optionally assess the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO: 4.

[0063] The present invention also relates to a method for generating viral particles with enhanced tropism for tumor cells, comprising:

[0064] (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0065] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0066] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0067] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0068] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0069] (e) Spiral α3 defined by positions 187-199;

[0070] (f) Ring 3 defined by positions 200-226;

[0071] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0072] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0073] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0074] (j) The TM cytoplasmic domain defined at positions 439-498; and

[0075] (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and

[0076] (iii) Optionally assess the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells and / or non-tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a reference value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO: 4. Attached Figure Description

[0077] Figure 1 The amino acid positions in LCMV-GP identified as tumor-tropy. LCMV-WE wild-type was passaged in different human and mouse tumor cell lines with and without 5-fluorouracil (5FU). The virus was sequenced after 10–12 passages. For each cell type, the newly acquired mutation (MUT) and the original amino acid (WT) at that position are shown. Each setting was performed as 4–5 independent experiments. The number of experiments in which a specific mutation occurred is shown. Positions in loop #2 were not included in the analysis. A: Missense mutation in the stable signal peptide (SSP), B: Missense mutation in GP1, C: Missense mutation in GP2.

[0078] Figure 2 The LCMV-WE subtype P52 was stable and acquired additional mutations. LCMV-WE subtype P52 (I181M, R185W) was passaged (23-55x) with or without 5-fluorouracil (5FU). The resulting viruses were sequenced, and mutations in each cell line were shown.

[0079] Figure 3 : Mutations observed after proliferation of the LCMV-WE subtype YF13.

[0080] Figure 4A. Tumor-tropy mutations accumulate in specific structural regions (structural hotspots). The figure shows the known domains of the LCMV glycoprotein (Hastie et al., Nat Struct Mol Biol 23, 513-521 (2016) and the RCSB protein database: https: / / www.rcsb.org / structure / 5INE (release 2016-04-20)). Mutations in the corresponding domains and inter-domain linker sequences are shown. Loop #2 is omitted as it is not part of the analysis. Mutation frequencies for each domain and its linker sequences are given. B. The significance between low mutation frequencies (n=2–13) and high mutation frequencies (n=13–2) of the domains was calculated using the Mann-Whitney test. The arithmetic mean (12.85%) of the two lowest significance values ​​(p = 0.0003108) was used as the cutoff value. C. The mutation frequencies of domains with low mutation frequency (n=7) and domains with high mutation frequency (n=8) are shown, and the Mann-Whitney test is performed (0.0003108).

[0081] Figure 5 Mutations within different domains of LCMV-GP. The total number of AS sites categorized by structural region and the number of AS sites we identified as tumor-loving in the rapid evolution platform are shown. Ring #2 was not included in the analysis. * indicates a significant difference in domain mutation frequencies as determined by the chi-square test.

[0082] Figure 6 Tumor-loving regions accumulate in specific functional regions (functional hotspots). Known functional regions of LCMV-GP are shown. Ring #2 is omitted because it was not involved in the analysis. Mutations in the corresponding functional regions are shown. Mutation frequencies for each region are given.

[0083] Figure 7 Rapid Evolution Platform Mutations Accelerate Entry into A549 Cells: A: Raw FACS blot of LCMV-WE wild-type A549 cell entry assay. Time indicates the addition of monensin (which blocks viral entry). B: Entry assay of recombinant virus carrying randomly selected mutations from the rapid evolution platform (n=6 / mutation). Structural regions where mutations are located are shown. Mutant virus compared to recombinant WT control-WE-CL13(L)h2.r3 (black solid line) (colored dashed line). C: Entry assay of recombinant virus carrying mutations not identified in the rapid evolution platform but located within structural regions (n=6 / mutation). D: Entry assay of recombinant virus carrying mutations not identified in the rapid evolution platform but located within assumed hotspot regions (n=2 / mutation).

[0084] Figure 8 The mutations resulted in widespread infectivity in cancer cells rather than healthy cells. A: Infection assays were performed on different human cancer (n=6), primary neurons (n=4), and myotube (n=2) cells using 10 different chimeric (WE-CL13-chim) viruses carrying one point mutation at the corresponding site (MOI = 0.1, 16 hours). Note that no infection was detected in neurons and myotubes. B: The infection rates (infectivity assays) of different tumor cell lines were plotted against the infection rates of other tumor cell lines. A comparison between melanoma and lung adenocarcinoma cell lines is shown. The infection rate of neurons was also plotted against the infection rate of MaMel86a. The average ratio for each mutant virus is given (n=6). C: Spider plots show the fold increase in proliferation of the tested mutations in various tumor cells and neurons. The average ratio for each mutant virus is given (n=6). D: Infectivity assays of mutant viruses carrying the point mutation F129S, tested in cell lines H1975, MaMel51, MaMel86A, and A549. Infection rates are shown in bar charts. The infection rate of the WT control virus is shown as a dashed line (n=6). E: Infectivity assays of mutant viruses carrying the aforementioned point mutation, tested in H1975 and A549 cells (n=6 / mutation). F: Infectivity assays of mutant viruses carrying the aforementioned point mutation, tested in cell lines H1975, A549, Gist-T1, MaMel51, MaMel86A, and neurons. Infectivity rates are shown in bar charts. The infection rate of the WT control virus is shown as a dashed line (n=6 / tumor cell line; n=4 for neurons). G: Infectivity assays of wild-type LCMV-WE and mutant virus strains with the 211T mutation in LCMV-GP, tested in B16F10 melanoma cells. Analysis of infected cells 48 hours later (n=6). H: Infectivity assays of wild-type LCMV-WE and mutant virus strains with the 492I mutation in LCMV-GP, tested in Mamel51 melanoma cells. Analysis of infected cells 48 hours later (n=6). I: Immunofluorescence of LCMV-NP cells of the stated cell types infected 24 hours after infection with LCMV strains 8B10-D2 and A10D, and control virus. One of three representative stainings is shown.

[0085] Figure 9Infection assays were performed on different human cancer and healthy cells carrying different recombinant viruses with optimized combinations (MOI = 0.1, 16 hours). Mutations within LCMV-GP for each viral strain are presented. The human cancer cell lines selected for testing infectivity were A549, H1975, MaMel86A, and MaMel51. The mouse cell lines tested were B16F10, TrampC2, MC-38, and MOPC (n = 6–7).

[0086] Figure 10 The novel mutations altered the viral entry mechanism. Randomly selected recombinant viruses carrying mutations from a rapid evolution platform were tested in entry assays in A549 cells and A549 cells lacking the known entry receptor α-dystrophin of LCMV. Mutations within LCMV-GP for each viral strain are presented (n=6).

[0087] Figure 11 A: Infectivity assays of wild-type LCMV-WE virus and chimeric WE-Cl13 virus carrying the LCMV-WE wild-type glycoprotein, LCMV-WE nucleoprotein, and the L-segment of LCMV clone 13, tested in primary human neurons and the tumor cell lines (n = 3-6 / virus strain). B: Infectivity assays of WE-CL13 chimeric virus carrying the LCMV-WE wild-type glycoprotein and WE-CL13 chimeric virus carrying the point mutation in LCMV-GP, tested in the tumor cell lines (n = 6 / virus strain). C: Tumor growth in mice carrying B16F10-OVA tumors treated on day 0 with wild-type LCMV-WE virus, LCMV-WE strain carrying the point mutation 211T in LCMV-GP, and WE-CL13 chimeric virus carrying the point mutation (n = 3-5 / group). D: Tumor growth in mice carrying MC38-OVA tumors, treated on day 0 with a WE-CL13 chimeric virus carrying the point mutation (n = 3-5 / group). The right figure shows the tumor size after the mice were sacrificed on day 9 (n = 5 / group).

[0088] Figure 12 Mutations at positions 155, 156, and 256 of the LCMV-GP gene were introduced into the glycoprotein of the arena virus Lunk. These correspond to positions 152, 153, and 253. The ability to replicate in different cell lines was analyzed in infectivity assays. Data showed a relative increase in infectivity compared to WT virus (n=6–10).

[0089] Figure 13LCMV-GP expressing the mutant H155Y was expressed on vesicular stomatitis virus (VSV). The infection rates of wild-type chimeric virus (LCMV-GP / VSV) and mutant chimeric virus (LCMV-GP-mut / VSV) were analyzed in MC57 fibrosarcoma cells (n=3). Detailed Implementation

[0090] The inventors of this application have surprisingly discovered a method for identifying mutations that regulate the tropism of LCMV on tumor cells. By applying this method to primary and subcultured cell cultures, at least 95 missense mutations have been identified. The generation of recombinant LCMV carrying these mutations reveals that LCMV entry into tumor cells is primarily influenced by these mutations. By combining these mutations, viruses with accelerated tumor tropism, limited replication in healthy cells, and beneficial antitumor activity have been generated.

[0091] Viral therapy is an emerging pillar of cancer treatment, clinically applied to various cancer types. A major challenge in viral therapy is designing a virus that exhibits effective antitumor activity without side effects. Enhancing tumor tropism without affecting viral replication in healthy tissues would address this issue. By passaged lymphocyte choriomeningitis virus (LCMV) in human and mouse cancer cells, the inventors of this application identified regions within the LCMV proglycoprotein polyprotein (GPC) that accelerate tumor cell-specific entry. Combinations of mutations in different GPC regions have synergistic and / or additive effects on tumor cell proliferation. Therefore, modifications to these regions in the LCMV-GPC increase entry into tumor cells, restrict replication in healthy cells, and thus demonstrate proprietary efficacy in a mouse cancer model. Mechanistically, it is believed that many selected recombinant viruses do not depend on α-dystrophic glycan (DAG-1) as a primary entry receptor. Consistent with this, the introduction of mutated LCMV-GP into other viruses (such as the vesicular stomatitis virus vector system) also accelerated their entry into tumor cells, indicating that this novel tumor-specific entry mechanism is applicable to other therapeutic viruses, not just LCMV. In summary, by utilizing the biological principles of mutation and selection, the inventors of this application can identify functional LCMV-GPC regions that alter the viral tropism for cancer cells. Recombinant viruses carrying these mutations exhibit accelerated and more specific replication in tumor cells and induce strong anti-tumor immunity.

[0092] The application of mutated viruses in cancer treatment is proprietary because they have accelerated site-specific replication and immune activation, and limited proliferation in healthy tissues.

[0093] Therefore, the present invention relates to a viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO:4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO:13 (preferably SEQ ID NO:4), wherein said mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions: (a) a chain β1 N-terminus defined by positions 59-89, wherein said at least one mutant amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein said at least one mutant amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) a helical α1 and / or α-terminus defined by positions 114-147. 2, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) loop 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (e) helix α3 defined by positions 187-199; (f) loop 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) H1 and H2 domains defined by positions 1-58; and / or (j) TM cytoplasmic domain defined by positions 439-498. Preferably, the viral particles have an enhanced ability to enter tumor cells compared to reference viral particles containing the wild-type glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4). Preferably, the reference viral particles differ only in the sequence of the glycoprotein.

[0094] The present invention also relates to a viral particle comprising a lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said glycoprotein, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or compared with the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4). 4) In comparison, the WE glycoprotein of the mutated LCMV strain is selected from 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152, 153, 154, 155, 156, 163, 188, 1 The virus particle contains at least one mutated amino acid residue at one or more of the following positions: 98, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492. Preferably, the virus particle has an enhanced ability to enter tumor cells compared to a reference virus particle containing the wild-type glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4). Preferably, the reference virus particle differs only in the sequence of the glycoprotein.

[0095] The virus particles disclosed herein are preferably sand virus particles, and more preferably lymphocytic choroid plexus meningitis virus (LCMV) particles.

[0096] Wild-type arenavirus genome segments and ORFs are known in the art. In particular, the wild-type arenavirus genome typically consists of an S segment and an L segment. The S segment carries ORFs encoding GP and NP. The L segment encodes the L and Z proteins. The flanking sides of each segment are their respective 5' and 3' UTRs.

[0097] The viral particles of the present invention preferably comprise genomic segments corresponding to genomic segments of wild-type arenavirus. This means that the S segment carries ORFs encoding GP and NP, and the L segment encodes L and Z proteins. In the S segment, the ORF encoding the glycoprotein is controlled by the 5' untranslated region (UTR), and the nucleoprotein is controlled by the 3' UTR. In the L segment, the ORF encoding the L protein is controlled by the 3' UTR, and the ORF encoding the Z protein is controlled by the 5' UTR. Therefore, the genome of the viral particles of the present disclosure is preferably located in its native position. Therefore, the viral particles of the present invention preferably have a dual-segment genome. The genome of the viral particles of the present invention preferably consists of one L segment and one S segment as described herein. Illustrative examples of the LCMV S segment are shown in SEQ ID NO: 1, 11, and 14. Illustrative examples of the LCMV L segment are shown in SEQ ID NO: 2 and 15.

[0098] Some strains of LCMV are part of this invention. As used herein, LCMV “WE”, “strain WE”, “WE strain”, etc., refer to LCMV having the genomic regions shown in SEQ ID NO: 1 and 2. As used herein, LCMV “P52”, “P52-WE”, “strain P52”, “P52 strain”, etc., refer to variants / derivatives of strain WE having the genomic regions shown in SEQ ID NO: 11 and 2. As used herein, LCMV “clone 13”, “Cl13”, “strain clone 13”, “clone 13 strain”, etc., refer to LCMV having the genomic regions shown in SEQ ID NO: 14 and 15.

[0099] The terms “glycoprotein,” “GP,” “G protein,” “glycoprotein complex,” and “GPC” are used interchangeably to refer to LCMV-derived glycoproteins, which are considered to mediate receptor binding and membrane fusion. Illustrative examples of glycoproteins are shown in SEQ ID NO: 4, 13, and 17. Illustrative examples of genes encoding glycoproteins are shown in SEQ ID NO: 3, 12, and 16.

[0100] The terms “L protein” and “LP” are used interchangeably to refer to LCMV-derived RNA polymerase L. Illustrative examples of L proteins are shown in SEQ ID NO: 10 and 23. Illustrative examples of genes encoding L proteins are shown in SEQ ID NO: 9 and 22.

[0101] The terms “nucleoprotein,” “N protein,” and “NP,” used interchangeably, refer to LCMV-derived nucleoproteins. Illustrative examples of nucleoproteins are shown in SEQ ID NO: 6 and 19. Illustrative examples of genes encoding nucleoproteins are shown in SEQ ID NO: 5 and 18.

[0102] The terms “Z protein” or “ZP” are used interchangeably to refer to the LCMV-derived small ring finger protein Z. Illustrative examples of Z proteins are shown in SEQ ID NO: 8 and 21. Illustrative examples of genes encoding Z proteins are shown in SEQ ID NO: 7 and 20.

[0103] The term "amino acid" or "amino acid residue" generally refers to an amino acid having its recognized definition in the art, such as those selected from the following: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile 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), but modified, synthetic, or rare amino acids may be used as needed. Based on general side-chain properties, naturally occurring residues are classified into the following groups: (1) hydrophobic: methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues affecting chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, substitution may require replacing members of one of these categories with members of another.

[0104] As used herein, in the context of proteins, polypeptides, peptides, or amino acid sequences, the term "mutation" or "mutated amino acid (residue)" includes amino acid substitutions, deletions, insertions, and / or one or more chemical modifications to an amino acid or nucleotide sequence. Such substitutions can be conserved, meaning that an amino acid residue is replaced by a chemically similar amino acid residue. Examples of conserved substitutions are substitutions between the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. Mutations can also include substitutions by their respective D-steroisomers or by amino acids other than the 20 naturally occurring amino acids (e.g., ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, and valine). However, substitutions with naturally occurring amino acids are preferred.

[0105] The term "nucleic acid" as used herein can generally refer to either DNA or RNA. The difference between DNA and RNA lies in their nucleic acid bases. The complementary base of adenine in DNA is thymine, while in RNA it is uracil. For simplicity, the corresponding base of adenine is represented as "t" in this application, which, depending on the context, can refer to either thymine (in DNA) or uracil (in RNA).

[0106] As used in the context of nucleic acids, the term "encoding" (or "encode(s)") refers to a nucleic acid having a sequence that can be translated into a specific amino acid sequence encoded by that nucleic acid. A nucleic acid sequence can encompass the sequence of the coding strand or the sequence of a strand complementary to the coding strand.

[0107] The “sequence identity percentage (%)” for sequences disclosed herein is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to those in a reference sequence after sequence alignment and the introduction of gaps (if necessary to obtain the maximum sequence identity percentage), and without considering any conserved substitutions as part of sequence identity. Alignments used to determine the amino acid sequence identity percentage can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. The same applies to the nucleotide sequences disclosed herein. For the purpose of determining sequence identity, uracil (e.g., in RNA) can be considered identical to thymine (e.g., in DNA).

[0108] As used herein, the term "attenuated" refers to a reduced ability of a virus to replicate within a given host (cell), a reduced ability to replicate in a healthy organ, and / or a reduced ability to induce cytokines. Preferably, the viral particles of the present invention are attenuated.

[0109] As used herein, the term "infectious" refers to viral infection, i.e., the ability to enter a given host cell. Preferably, the viral particles of the present invention are infectious.

[0110] The term "replicating capability" refers to the ability of a virus to amplify and express its genetic material in infected cells and to produce further progeny in unengineered normal cells. Specifically, a replicating virus can replicate without requiring engineered host cells to express its viral genes. Preferably, the viral particles of the present invention are replicating.

[0111] As used in this article, the term "pathogenicity" refers to the ability of a virus to cause disease, that is, the ability of a virus to harm an organism.

[0112] The term "entry" refers to the ability of a virus to enter a given target cell. Viral entry can be measured substantially as described in Example 10B. This measurement preferably includes the following steps: pre-incubating the target cells with the virus (multiple of infection of 0.1) at 4°C. After incubation for 1 hour, heating the cells to 37°C. After heating the cells to 37°C, adding monensin to the culture at different time points (i.e., 0 min, 20 min, 60 min, or 180 min). The cells are then incubated for an additional 16 hours. After this incubation period, the viral protein in each cell is determined by staining the cells with anti-LCMV-NP antibody and analyzing them in a flow cytometer. The ability to enter the cell is directly related to the percentage of cells infected in this assay.

[0113] The term "infectivity" refers to the ability of a virus to enter cells, replicate within cells, and produce viral proteins. Viral infectivity can be determined substantially as described in Example 10C. This assay preferably includes the following steps: incubating target cells with the virus (multiple of infection of 0.01-0.1) at 37°C for 16 hours. After this incubation time, the viral proteins in each cell are determined by staining the cells with anti-LCMV-NP antibody and analyzing them in a flow cytometer. The ability of the virus to proliferate in a specific target cell culture is directly related to the percentage of cells infected in this assay.

[0114] The term "RNA replication" refers to the ability of a virus to replicate its viral RNA within a cell.

[0115] The term "proliferation" refers to the increase in viral particles.

[0116] The term "tropism" refers to the infectivity of viral particles for a given cell type. Tropicism is at least partially related to the ability of a virus to enter a given cell type. The term "tumor tropism" refers to tropism in the context of tumor cells.

[0117] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions included in the following regions: (a) the N-terminus of chain β1 defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) (e) a ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (f) a helix α3 defined by positions 187-199; (g) a ring 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; and / or (h) an N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365.

[0118] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions of helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0119] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions in the N-helix defined by positions 313-373 in (b), wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0120] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions in the chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0121] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions in (d) of the ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0122] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions of chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0123] The virus particles disclosed herein are preferably virus particles in which the glycoprotein of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4) contains at least one mutated amino acid residue at one or more positions in the ring 3 defined by positions 200-226 in (f).

[0124] The virus particles disclosed herein are preferably virus particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the virus particle contains at least one mutated amino acid residue at one or more positions in the H1, H2 domains defined by positions 1-58.

[0125] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more positions in the TM cytoplasmic domain defined by positions 439-498 in (h); and / or

[0126] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue at one or more of the α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more of the positions selected from 245-251 and 253-258.

[0127] The viral particles disclosed herein are preferably viral particles in which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of the viral particle contains at least one mutated amino acid residue in any one or any combination of regions (a), (b), (c), (d), (e), (f), (g), (h) and / or (i) as defined above.

[0128] The viral particles of this disclosure preferably comprise a glycoprotein and / or a nucleic acid encoding the glycoprotein, wherein the glycoprotein has at least about 80%, preferably at least about 85%, 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 99.5%, preferably at least about 99.7% sequence identity with the glycoprotein sequence shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4).

[0129] In the viral particles of this disclosure, preferably, one or more positions containing at least one mutated amino acid residue are selected from positions 102, 122, 129, 132, 136, 152, 153, 154, 155, 156, 211, 217, 218, 256, 260, 284, 328, 332, 342, 358, and 492. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4).

[0130] In the viral particles of this disclosure, preferably, the at least one mutated amino acid residue is selected from Ser 102→Asn, Phe 122→Leu, Phe 129→Ser, Lys 132→Arg, His 136→Gln, Asn 152→Tyr, Ser 153→Pro, Phe or Tyr, Asn 154→Asp, His 155→Leu or Tyr, Lys 156→Glu, Ala 211→Thr, Thr 217→Ile, Thr 218→Ile, Lys 256→Arg, Leu 260→Phe or Ile, Tyr 284→His, Ala 328→Ser, Phe 332→Leu, Val 342→Ala, Arg 358→Lys and Lys 492→ Ile, including combinations thereof. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4).

[0131] In the viral particles of this disclosure, preferably, compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the GP contains the following mutated amino acid residue groups:

[0132] (a) Lys 492→Ile;

[0133] (b) Ala 211→Thr;

[0134] (c) Lys 260→Phe and Lys 492→Ile;

[0135] (d) Ser 153 → Pro and Lys 492 → Ile;

[0136] (e) Phe 122→Leu, His 136→Gln and Ser 153→Pro;

[0137] (f) Phe 122→Leu and Lys 492→Ile;

[0138] (g) Phe 122→Leu, His 136→Gln, Ser 153→Pro and Lys 492→Ile;

[0139] (h) Glu 255→Gly, Lys 492→Ile;

[0140] (i) Ser 153→Pro and Arg 358→Lys;

[0141] (j) Ser 153→Pro, Lys 256→Arg, Leu 260→Phe and Lys 492→Ile;

[0142] (k) Ser 153→Phe, Glu 255→Gly and Leu 260→Phe;

[0143] (l) Ser 153→Phe, Glu 255→Gly and Leu 260→Phe;

[0144] (m) Lys 156→Glu and Lys 492→Ile;

[0145] (n) Ser 153→Pro, Lys 256→Arg and Leu 260→Phe;

[0146] (o) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Lys 256→Arg, and Lys 492→Ile; or

[0147] (p) Glu 379→Asn and Lys 492→Ile.

[0148] In the viral particles of this disclosure, preferably, the at least one mutated amino acid residue is selected from Ser 102→Asn, Phe 122→Leu, His 136→Gln, Ser 153→Pro or Phe, Lys 156→Glu, Ala 211→Thr, Lys256→Arg, Leu 260→Phe, Tyr 284→His, Ala 328→Ser, Phe 332→Leu, Val 342→Ala, Arg358→Lys, Lys 492→Ile, including combinations thereof. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4).

[0149] The viral particles disclosed herein may be equipped with additional modifications. Such modifications may include mutations at positions 181 and / or 185 of the GP, particularly Arg 185→Trp and / or Ile 181→Met. Viral particles with such modifications may result in stronger antitumor activity, such as a stronger early LCMV-guided T-cell response, compared to strains containing wild-type amino acids (Ile 181 and Arg 185) at positions 181 and 185. Preferred viral particles may comprise a glycoprotein having at least one mutated amino acid residue at positions 181 and / or 185 compared to the glycoprotein sequence shown in SEQ ID NO: 4. However, it is preferred that the glycoprotein contains mutations at both positions compared to SEQ ID NO: 4. Preferred mutations at these positions are selected from Arg 185→Trp and Ile 181→Met or Val, more preferably from Arg 185→Trp and Ile 181→Met. Such mutations can be combined with other mutations disclosed herein, such as mutations that improve entry into tumor cells.

[0150] In the viral particles of this disclosure, preferably, compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, GP contains the following mutated amino acid residue group:

[0151] (a) Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0152] (b) Ile 181→Met, Arg 185→Trp, and Ala 211→Thr;

[0153] (c) Ile 181→Met, Arg 185→Trp, Lys 260→Phe, and Lys 492→Ile;

[0154] (d) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0155] (e) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, and Arg 185→Trp;

[0156] (f) Phe 122→Leu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0157] (g) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0158] (h) Ile 181→Met, Arg 185→Trp, Glu 255→Gly, and Lys 492→Ile;

[0159] (i) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Arg 358→Lys;

[0160] (j) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, Leu 260→Phe, and Lys 492→Ile;

[0161] (k) Ser 153→Phe, Arg 185→Trp, Glu 255→Gly and Leu 260→Phe;

[0162] (l) Ser 153→Phe, Glu 255→Gly, and Leu 260→Phe;

[0163] (m) Lys 156→Glu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0164] (n) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Leu 260→Phe;

[0165] (o) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Lys 492→Ile; or

[0166] (p) Ile 181→Met, Arg 185→Trp, Glu 379→Asn, and Lys 492→Ile.

[0167] In the viral particles of this disclosure, preferably, the glycoprotein contains a limited number of mutated amino acid residues compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (where SEQ ID NO: 4 is preferred). Preferably, the glycoprotein contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid residues, preferably mutated amino acid residues as defined herein. More preferably, the glycoprotein preferably contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid residues, preferably mutated amino acid residues as defined herein.

[0168] In the viral particles of this disclosure, preferably, the glycoprotein has at least about 80%, preferably at least about 85%, 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 99.5%, preferably at least about 99.7% sequence identity, or preferably is the same as.

[0169] In the viral particles of this disclosure, preferably, the viral particles comprise nucleic acids encoding glycoproteins, wherein the nucleic acids comprise a sequence having at least about 80%, preferably at least about 85%, 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 preferably identical to.

[0170] In the viral particles of this disclosure, preferably, the viral particles comprise an LCMV L-protein (LP) and / or nucleic acid encoding the LP, wherein the LP has at least about 80%, preferably at least about 85%, 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 with the L-protein sequence shown in SEQ ID NO: 10 or 23.

[0171] In the viral particles of this disclosure, preferably, the viral particles comprise nucleic acids encoding LP, wherein the nucleic acids comprise a sequence having at least about 80%, preferably at least about 85%, 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 preferably identical to.

[0172] In the viral particles of this disclosure, preferably, the viral particles comprise an LCMV nucleoprotein (NP) and / or nucleic acid encoding the NP, wherein the NP has at least about 80%, preferably at least about 85%, 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 preferably is the same as it.

[0173] In the viral particles of this disclosure, preferably, the viral particles comprise a nucleic acid encoding an NP, wherein the nucleic acid comprises a sequence having at least about 80%, preferably at least about 85%, 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 preferably identical to.

[0174] In the viral particles of this disclosure, preferably, the viral particles comprise an LCMV Z-protein (ZP) and / or nucleic acid encoding the ZP, wherein the ZP has at least about 80%, preferably at least about 85%, 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 preferably identical to it.

[0175] In the viral particles of this disclosure, preferably, the viral particles contain nucleic acid encoding ZP, wherein the nucleic acid contains a sequence having at least about 80%, preferably at least about 85%, 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 identical to the sequence shown in SEQ ID NO: 7 or 20 or the corresponding complementary sequence.

[0176] In the viral particles of this disclosure, preferably, the viral particles comprise LCMV GP and LCMV NP, and / or one or more nucleic acids encoding the LCMV GP and LCMV NP, wherein the LCMV GP and LCMV NP have at least about 80%, preferably at least about 85%, 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 preferably identical to, the sequences of: (a) SEQ ID NO: 30 and 6; (b) SEQ ID NO: (c) SEQ ID NO: 32 and 6; (d) SEQ ID NO: 33 and 6; (e) SEQ ID NO: 34 and 6; (f) SEQ ID NO: 35 and 6; (g) SEQ ID NO: 36 and 6; (h) SEQ ID NO: 37 and 6; (i) SEQ ID NO: 38 and 6; (j) SEQ ID NO: 39 and 6; (k) SEQ ID NO: 40 and 6; (l) SEQ ID NO: 41 and 6; (m) SEQ ID NO: 42 and 6; (n) SEQ ID NO: 43 and 6; (o) SEQ ID NO: 44 and 6; (p) SEQ ID NO: 45 and 6; (q) SEQ ID NO: 46 and 6; (r) SEQ ID NO: 47 and 6; (s) SEQ ID NO: 48 and 6; (t) SEQ ID NO: 49 and 6; (u) SEQ ID NO: 50 and 6; (v) SEQ ID NO: 51 and 6; (w) SEQ ID NO: 52 and 6; (x) SEQ ID NO: 53 and 6; (y) SEQ ID NO: 54 and 6; (z) SEQ ID NO: 55 and 6; (aa) SEQ ID NO: 56 and 6; or (bb) SEQ ID NO: 57 and 6.

[0177] In the viral particles of this disclosure, preferably, the viral particles comprise LCMV GP, LCMV LP, LCMV NP, and LCMV ZP, and / or one or more nucleic acids encoding said LCMV GP, LCMV LP, LCMV NP, and LCMV ZP, wherein said LCMV GP, LCMV LP, LCMV NP, and LCMV ZP have at least about 80%, preferably at least about 85%, 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 preferably identical: (a) SEQ ID NO: 30, 23, 6 and 21; (b) SEQ ID NO: 31, 23, 6 and 21; (c) SEQ ID NO: 32, 23, 6 and 21; (d) SEQ ID NO: 33, 23, 6 and 21; (e) SEQ ID NO: 34, 23, 6 and 21; (f) SEQ ID NO: 35, 23, 6 and 21; (g) SEQ ID NO: 36, 23, 6 and 21; (h) SEQ ID NO: 37, 23, 6 and 21; (i) SEQ ID NO: 38, 23, 6 and 21; (j) SEQ ID NO: 39, 23, 6 and 21; (k) SEQ ID NO: 40, 23, 6 and 21; (l) SEQ ID NO: 41, 23, 6 and 21; (m) SEQ ID NO: 42, 23, 6 and 21; (n) SEQ ID NO: 43, 23, 6 and 21; (o) SEQ ID NO: 44, 23, 6 and 21; (p) SEQ ID NO: 45, 23, 6 and 21; (q) SEQ ID NO: 46, 23, 6 and 21; (r) SEQ ID NO: 47, 23, 6 and 21; (s) SEQ ID NO: 48, 23, 6 and 21; (t) SEQ ID NO: 49, 23, 6 and 21; (u) SEQ ID NO: 50, 23, 6 and 21; (v) SEQ ID NO: 51, 23, 6 and 21; (w) SEQ ID NO: 52, 23, 6 and 21; (x) SEQ ID NO: 53, 23, 6, and 21; (y) SEQ ID NO: 54, 23, 6, and 21; (z) SEQ ID NO: 55, 23, 6, and 21; (aa) SEQ ID NO: 56, 23, 6, and 21; or (bb) SEQ ID NO: 57, 23, 6, and 21.

[0178] In the viral particles of this disclosure, preferably, the viral particles comprise an S segment containing a 5' untranslated region (UTR) that has at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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 identical to the sequence shown in SEQ ID NO: 24 or its corresponding complementary sequence.

[0179] In the viral particles of this disclosure, preferably, the viral particles comprise an L segment containing a 5' untranslated region (UTR) that has at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 70%, preferably at least about 75%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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 identical to, the sequence shown in SEQ ID NO: 26 or 28, or the corresponding complementary sequence.

[0180] In the preferred viral particles of this disclosure, the viral particles comprise an L segment containing a 3' untranslated region (UTR) that has at least about 60%, preferably at least about 65%, preferably at least about 70%, preferably at least about 70%, preferably at least about 75%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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 identical to, the sequence shown in SEQ ID NO: 27 or 29 or the corresponding complementary sequence.

[0181] In the viral particles of this disclosure, preferably, the viral particles comprise an S segment containing a nucleic acid sequence having at least about 80%, preferably at least about 85%, 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 with the sequence shown in SEQ ID NO: 1 or 11.

[0182] In the viral particles of this disclosure, preferably, the viral particles comprise an L segment, the L segment comprising a nucleic acid sequence having at least about 80%, preferably at least about 85%, 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 preferably the same as the sequence shown in SEQ ID NO: 2 or 15.

[0183] In the viral particles of this disclosure, preferably, the viral particles comprise an S segment derived from the WE strain (or a derivative thereof, such as P52) and an L segment derived from clone 13. As described in WO 2022 / 180203, such chimeric viral particles exhibit attenuated replication. Despite this attenuation of replication, such viruses generally retain or even enhance their potent antitumor activity compared to strains containing an L segment from the WE strain (or one of its derivatives, such as P52).

[0184] In the viral particles of this disclosure, preferably, the viral particles comprise an S segment containing a nucleic acid sequence having at least about 80%, preferably at least about 85%, 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 with the sequence shown in SEQ ID NO: 1 or 11. More preferably, the viral particle comprises an L segment containing 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 preferably the same nucleic acid sequence as the sequence shown in SEQ ID NO: 2 or 15 (preferably SEQ ID NO: 15).

[0185] Several LCMV strains are part of this disclosure. These LCMVs include the strains shown in Table A, which have the S segment derived from LCMV WE, including mutations in GP. Mutations compared to the glycoprotein shown in SEQ ID NO: 4 are shown in Table A. Additionally, the strains have the L segment of clone 13 shown in SEQ ID NO: 15. Preferred viral particles of this disclosure are the LCMV strains shown in Table A.

[0186] Table A

[0187]

[0188] Compared with reference viral particles, the viral particles of this disclosure preferably have an enhanced ability to enter tumor cells, preferably mouse or human tumor cells, and more preferably human tumor cells. The tumor cells are preferably cells from one of the following tumors: lung tumor cells, such as H1975 or A549, LLC, TC-1; gastrointestinal tumor cells, such as Gist-T1; melanoma cells, such as MaMel86a, MaMel51, A375, B16F10, or RPMI-7951; pancreatic tumor cells, such as 511950, 60590, 511950R, or 60590R; thyroid tumor cells, such as 8305C or C643; sarcoma cells, such as Gist-T1; breast tumor cells, such as HCC1954; cervical tumor cells, such as HeLa; liver tumor cells, such as HepG2; colon tumor cells, such as MC38, Sw620, or Sw480; neuroblastoma cells, such as SK-N-BE(2); and / or prostate tumor cells, such as TrampC2. The ability to enter the tumor cells can be determined using tests substantially as described in Example 10B. The assay preferably includes the following steps: pre-incubating target cells with virus (multiple of infection 0.1) at 4°C. After 1 hour of incubation, the cells are heated to 37°C. After heating the cells to 37°C, monensin is added to the culture at different time points (i.e., 0 min, 20 min, 60 min, or 180 min). The cells are then incubated for another 16 hours. After this incubation period, the viral protein in each cell is determined by staining the cells with anti-LCMV-NP antibody and analyzing the results in a flow cytometer. The ability to enter the cells is directly related to the percentage of cells infected in this assay. The reference viral particle is preferably a reference viral particle containing LCMV GP (SEQ ID NO: 4) of strain WE or LCMV GP (SEQ ID NO: 13) of strain P52, wherein GP of strain WE is preferred. Apart from GP, the reference particle is preferably the same as a preferred viral particle with enhanced ability to enter tumor cells.

[0189] Compared with reference viral particles, the viral particles of this disclosure preferably have enhanced infectivity against tumor cells, preferably mouse or human tumor cells, and more preferably human tumor cells. The tumor cells are preferably cells from one of the following tumors: lung tumor cells, such as H1975 or A549, LLC, TC-1; gastrointestinal tumor cells, such as Gist-T1; melanoma cells, such as MaMel86a, MaMel51, A375, B16F10, or RPMI-7951; pancreatic tumor cells, such as 511950, 60590, 511950R, or 60590R; thyroid tumor cells, such as 8305C or C643; sarcoma cells, such as Gist-T1; breast tumor cells, such as HCC1954; cervical tumor cells, such as HeLa; liver tumor cells, such as HepG2; colon tumor cells, such as MC38, Sw620, or Sw480; neuroblastoma cells, such as SK-N-BE(2); and / or prostate tumor cells, such as TrampC2. Infectivity to the tumor cells can be determined using a test substantially as described in Example 10C. The assay preferably includes the following steps: incubating target cells with virus (multiple of infection of 0.01 to 0.1) at 37°C for 16 hours. After this incubation period, the viral protein in each cell is determined by staining the cells with anti-LCMV-NP antibody and analyzing the results in flow cytometry. The ability of the virus to proliferate in a specific target cell culture is directly related to the percentage of cells infected in this assay. The reference viral particle is preferably an LCMV GP (SEQ ID NO: 4) or LCMV GP (SEQ ID NO: 13) containing strain WE, wherein strain WE GP is preferred. Apart from GP, the reference particle is preferably the same as a preferred viral particle with enhanced ability to enter tumor cells.

[0190] Compared to the reference virus particles, the virus particles of this disclosure preferably have enhanced tropism for tumor cells, which are preferably tumors as described herein, preferably mouse or human tumor cells, and more preferably human tumor cells. The preferred viral particles of this disclosure may have enhanced tropism for tumor types selected from tumor cells as disclosed herein, preferably lung tumor cells, such as H1975 or A549, LLC, TC-1; gastrointestinal tumor cells, such as Gist-T1; melanoma cells, such as MaMel86a, MaMel51, A375, B16F10 or RPMI-7951; pancreatic tumor cells, such as 511950, 60590, 511950R or 60590R; thyroid tumor cells, such as 8305C or C643; sarcoma cells, such as Gist-T1; breast tumor cells, such as HCC1954; cervical tumor cells, such as HeLa; liver tumor cells, such as HepG2; colon tumor cells, such as MC38, Sw620 or Sw480; neuroblastoma cells, such as SK-N-BE(2); and / or prostate tumor cells, such as TrampC2. The preferred viral particles of this disclosure can exhibit enhanced tropism for more than one tumor type, including all tumor types selected from the above group. The reference viral particle is preferably a reference viral particle containing LCMV GP (SEQ ID NO: 4) of strain WE or LCMV GP (SEQ ID NO: 13) of strain P52, wherein strain GP of strain WE is preferred. Apart from GP, the reference particle is preferably the same as the preferred viral particles exhibiting enhanced tropism for human tumor cells.

[0191] The viral particles of this disclosure are preferably capable of entering cells in a manner independent of α-dystrophin. Therefore, the viral particles of this disclosure are preferably capable of entering cells via mechanisms other than α-dystrophin. The viral particles of this disclosure are preferably capable of entering cells that do not express or substantially do not express α-dystrophin. Preferably, the viral particles are capable of entering cells that do not express or substantially do not express α-dystrophin with approximately the same efficiency as entering cells that express α-dystrophin. The ability to enter cells in an α-dystrophin-independent or α-dystrophin-dependent manner can be measured substantially as described in Example 6.

[0192] The viral particles disclosed herein can be arenavirus particles other than LCMV particles pseudotyped with LCMV GP. "Pseudotyped" or "pseudotyped" refers to a virus, viral vector, or viral particle combined with an exogenous envelope protein, such as a non-LCMV viral particle combined with LCMV GP. In this case, the exogenous viral envelope protein can be used to alter host tropism or increase or decrease the stability of the viral particle. The viral particles can be vesicular stomatitis virus (VSV) particles pseudotyped with LCMV GP. The viral particles can be Pichinde virus particles pseudotyped with LCMV GP. The viral particles can be viral particles other than arenaviruses, preferably pseudotyped with LCMV GP as defined herein. The viral particles can be particles of viral vaccination vectors, preferably pseudotyped with LCMV GP as defined herein.

[0193] The viral particles disclosed herein preferably do not contain heterologous ORF. In this context, "heterologous ORF" refers to an ORF derived from an organism other than LCMV and / or an ORF encoding an artificial or synthetic protein.

[0194] The viral particle disclosed herein can be a viral particle comprising a glycoprotein (GP) of a sand virus and / or a nucleic acid encoding said GP, wherein said glycoprotein comprises at least one of the following amino acid residues:

[0195] (a) A mutated amino acid residue, or preferably an Asn residue, at the position corresponding to Ser 102 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at that position.

[0196] (b) A mutated amino acid residue, or preferably a Leu residue, at the position corresponding to Phe 122 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0197] (c) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Phe 129 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0198] (d) A mutated amino acid residue, or preferably a Gln residue, at the position corresponding to His 136 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a His residue at that position.

[0199] (e) A mutated amino acid residue at the position corresponding to Asn 152 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Tyr residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0200] (f) A mutated amino acid residue at the position corresponding to Ser 153 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Pro, Phe or Tyr residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at that position.

[0201] (g) A mutated amino acid residue at the position corresponding to Asn 154 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably an Asp residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0202] (h) A mutated amino acid residue at the His 155 position of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Leu or Tyr residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0203] (i) A mutated amino acid residue at the position corresponding to Lys 156 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Glu residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at that position.

[0204] (j) A mutated amino acid residue, or preferably a Thr residue, at the position corresponding to Ala 211 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at that position.

[0205] (k) A mutated amino acid residue at the position corresponding to Thr 217 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably an Ile residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0206] (l) A mutated amino acid residue at the position corresponding to Thr 218 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably an Ile residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0207] (m) A mutated amino acid residue, or preferably an Arg residue, at the position corresponding to Lys 256 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at that position.

[0208] (n) A mutated amino acid residue at the position corresponding to Leu 260 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Phe or Ile residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Leu residue at that position.

[0209] (o) A mutated amino acid residue at the position corresponding to Tyr 284 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a His residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Tyr residue at that position.

[0210] (p) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Ala 328 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at that position.

[0211] (q) A mutated amino acid residue at the position corresponding to Phe 332 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Leu residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at that position.

[0212] (r) A mutated amino acid residue at the position corresponding to Val 342 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably an Ala residue, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Val residue at that position.

[0213] (s) A mutated amino acid residue, or preferably a Lys residue, at the position corresponding to Arg 358 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not a wild-type residue of the GP at that position, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Arg residue at that position; and

[0214] (t) The Ile residue at the position corresponding to Lys 492 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, preferably not the wild-type residue of the GP at that position, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at that position.

[0215] The virus particle is preferably a sand virus particle, more preferably a sand virus other than LCMV, such as lunk virus (LUNV). Preferably, the virus particle is a sand virus particle, and the GP is the GP of the sand virus. Preferably, the at least one amino acid residue is at least one substitution of a natural amino acid residue of the sand virus GP. The GP is preferably a GP different from the LCMV GP.

[0216] The present invention also relates to an LCMV GP as described herein. Compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4), the glycoprotein preferably contains at least one mutated amino acid residue as defined herein in the context of a viral particle. The LCMV GP can be a separate GP. The LCMV GP can also be contained in a larger structure, for example, contained within a viral particle.

[0217] The glycoprotein disclosed herein is preferably a mutant LCMV strain WE glycoprotein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), wherein the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions included in the following regions: (a) the N-terminus of chain β1 defined by positions 59-89, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) helical α1 and / or α2 defined by positions 114-147, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) (e) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (f) Helix α3 defined by positions 187-199; (g) Ring 3 defined by positions 200-226; (h) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) H1 and H2 domains defined by positions 1-58; and / or (j) TM cytoplasmic domain defined by positions 439-498.

[0218] The glycoprotein disclosed herein is preferably a mutant LCMV strain WE glycoprotein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), wherein the mutant LCMV strain WE glycoprotein is selected from 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152, 153, 154, 155, 156, 163, 188, 1 One or more of the following positions contain at least one mutated amino acid residue: 98, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492.

[0219] Compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions selected from the following regions: (a) the N-terminus of chain β1 defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) helical α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) (e) a ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (f) a helix α3 defined by positions 187-199; (g) a ring 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; and / or (h) an N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365.

[0220] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions of helices α1 and / or α2 defined by positions 114-147 in (a), wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0221] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in the N-helix defined by positions 313-373 in (b), wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0222] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in the chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0223] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in (d) of the ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0224] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions of chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0225] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in (f) of the ring 3 defined by positions 200-226;

[0226] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in the H1, H2 domains defined by positions 1-58.

[0227] Compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more positions in the TM cytoplasmic domain defined by positions 439-498 in (h); and / or

[0228] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue at one or more of the α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more of the positions selected from 245-251 and 253-258.

[0229] Compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein of this disclosure preferably contains at least one mutated amino acid residue in any one of and any combination of regions (a), (b), (c), (d), (e), (f), (g), (h) and / or (i) as defined above.

[0230] The glycoprotein disclosed herein preferably has at least about 80%, preferably at least about 85%, 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 99.5%, preferably at least about 99.7% sequence identity with the glycoprotein sequence shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4).

[0231] In the glycoproteins disclosed herein, preferably, one or more positions containing at least one mutated amino acid residue are selected from positions 102, 122, 129, 132, 136, 152, 153, 154, 155, 156, 211, 217, 218, 256, 260, 284, 328, 332, 342, 358, and 492. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence (preferably SEQ ID NO: 4) of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13.

[0232] In the glycoproteins disclosed herein, preferably, the at least one mutated amino acid residue is selected from Ser 102→Asn, Phe 122→Leu, Phe 129→Ser, Lys 132→Arg, His 136→Gln, Asn 152→Tyr, Ser 153→Pro, Phe, or Tyr, Asn 154→Asp, His 155→Leu or Tyr, Lys 156→Glu, Ala 211→Thr, Thr 217→Ile, Thr 218→Ile, Lys 256→Arg, Leu 260→Phe or Ile, Tyr 284→His, Ala 328→Ser, Phe 332→Leu, Val 342→Ala, Arg 358→Lys and Lys 492→ Ile, including combinations thereof. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4).

[0233] In the glycoproteins disclosed herein, preferably, compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the GP contains the following mutated amino acid residue groups:

[0234] (a) Lys 492→Ile;

[0235] (b) Ala 211→Thr;

[0236] (c) Lys 260→Phe and Lys 492→Ile;

[0237] (d) Ser 153 → Pro and Lys 492 → Ile;

[0238] (e) Phe 122→Leu, His 136→Gln and Ser 153→Pro;

[0239] (f) Phe 122→Leu and Lys 492→Ile;

[0240] (g) Phe 122→Leu, His 136→Gln, Ser 153→Pro and Lys 492→Ile;

[0241] (h) Glu 255→Gly and Lys 492→Ile;

[0242] (i) Ser 153→Pro and Arg 358→Lys;

[0243] (j) Ser 153→Pro, Lys 256→Arg, Leu 260→Phe and Lys 492→Ile;

[0244] (k) Ser 153→Phe, Glu 255→Gly and Leu 260→Phe;

[0245] (l) Ser 153→Phe, Glu 255→Gly and Leu 260→Phe;

[0246] (m) Lys 156→Glu and Lys 492→Ile;

[0247] (n) Ser 153→Pro, Lys 256→Arg and Leu 260→Phe;

[0248] (o) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Lys 256→Arg, and Lys 492→Ile; or

[0249] (p) Glu 379→Asn and Lys 492→Ile.

[0250] In the glycoproteins disclosed herein, preferably, the at least one mutated amino acid residue is selected from Ser 102→Asn, Phe 122→Leu, His 136→Gln, Ser 153→Pro or Phe, Lys 156→Glu, Ala 211→Thr, Lys256→Arg, Leu 260→Phe, Tyr 284→His, Ala 328→Ser, Phe 332→Leu, Val 342→Ala, Arg358→Lys, and Lys 492→Ile, including combinations thereof. The at least one mutated amino acid residue is preferably compared to the linear polypeptide sequence (preferably SEQ ID NO: 4) of the wild-type LCMV strain WE GP shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13.

[0251] The glycoprotein disclosed herein may be equipped with additional modifications. Such modifications may include mutations at positions 181 and / or 185 of the GP, particularly Arg 185→Trp and / or Ile 181→Met. Viral particles containing such GPs exhibit stronger antitumor activity, such as a stronger early LCMV-guided T-cell response, compared to strains containing wild-type amino acids (Ile 181 and Arg 185) at positions 181 and 185. Preferred glycoproteins may have at least one mutated amino acid residue at positions 181 and / or 185, compared to the glycoprotein sequence shown in SEQ ID NO: 4. However, it is preferred that the glycoprotein contains mutations at both positions, compared to SEQ ID NO: 4. Preferred mutations at these positions are selected from Arg 185→Trp and Ile 181→Met or Val, more preferably from Arg 185→Trp and Ile 181→Met. Such mutations may be combined with other mutations disclosed herein, such as mutations that improve entry into tumor cells.

[0252] In the glycoproteins disclosed herein, preferably, GP contains the following mutated amino acid residue groups compared to the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4:

[0253] (a) Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0254] (b) Ile 181→Met, Arg 185→Trp, and Ala 211→Thr;

[0255] (c) Ile 181→Met, Arg 185→Trp, Lys 260→Phe, and Lys 492→Ile;

[0256] (d) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0257] (e) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, and Arg 185→Trp;

[0258] (f) Phe 122→Leu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0259] (g) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0260] (h) Ile 181→Met, Arg 185→Trp, Glu 255→Gly, and Lys 492→Ile;

[0261] (i) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Arg 358→Lys;

[0262] (j) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, Leu 260→Phe, and Lys 492→Ile;

[0263] (k) Ser 153→Phe, Arg 185→Trp, Glu 255→Gly and Leu 260→Phe;

[0264] (l) Ser 153→Phe, Glu 255→Gly, and Leu 260→Phe;

[0265] (m) Lys 156→Glu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0266] (n) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Leu 260→Phe;

[0267] (o) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Lys 492→Ile; or

[0268] (p) Ile 181→Met, Arg 185→Trp, Glu 379→Asn, and Lys 492→Ile.

[0269] In the glycoproteins disclosed herein, preferably, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or the LCMV strain P52 glycoprotein shown in SEQ ID NO: 13 (preferably SEQ ID NO: 4), the glycoprotein contains a limited number of mutant amino acid residues. Preferably, the glycoprotein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutant amino acid residues, preferably mutant amino acid residues as defined herein. More preferably, the glycoprotein preferably contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutant amino acid residues, preferably mutant amino acid residues as defined herein.

[0270] The glycoprotein disclosed herein preferably has at least about 80%, preferably at least about 85%, 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 99.5%, preferably at least about 99.7% sequence identity, or preferably is the same as.

[0271] This invention also relates to a nucleic acid molecule encoding the GP disclosed herein, preferably LCMV GP. Because the degeneracy of the genetic code allows certain codons to be substituted by other codons defining the same amino acids, this disclosure is not limited to the specific nucleic acid molecule encoding the GP described herein, but covers all nucleic acid molecules including the nucleotide sequence encoding said GP.

[0272] Nucleic acid molecules can be DNA or RNA (e.g., mRNA, viral RNA). Nucleic acid molecules can be single-stranded or double-stranded. Nucleic acid molecules can be linear or covalently closed to form a ring.

[0273] If a nucleic acid molecule (e.g., DNA) includes sequence elements containing information related to transcription and / or translation regulation, and said sequence is “operably linked” to a nucleotide sequence encoding a protein, it is called “capable of expressing the nucleic acid molecule” or “capable of allowing the expression of the nucleotide sequence.” Operable linking is a link in which the regulatory sequence element and the sequence to be expressed are linked in a manner that enables gene expression. The precise nature of the regulatory regions necessary for gene expression can vary between species, but typically these regions include promoters, which in prokaryotes contain the promoter itself (i.e., the DNA element that directs transcription initiation) and DNA elements that will emit translation initiation signals when transcribed into RNA. Such promoter regions typically include 5'-noncoding sequences involved in transcription and translation initiation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, or the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also include enhancers or repressors, as well as translation signals and leader sequences for targeting specific compartments of the host cell to the native protein.

[0274] Furthermore, the 3' non-coding sequence may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they can be replaced by signals that function in that cell.

[0275] Therefore, the nucleic acid molecules of this disclosure can be "operably linked" to one or more regulatory sequences, such as promoter sequences, to allow the expression of the nucleic acid molecule. In some embodiments, the nucleic acid molecules of this disclosure include a promoter sequence and a transcription termination sequence.

[0276] The nucleic acid disclosed herein preferably comprises a nucleic acid sequence having at least about 80%, preferably at least about 85%, 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 preferably being identical to the sequence shown in SEQ ID NO:58-85 or the corresponding complementary sequence.

[0277] The provided nucleic acid molecule may also be part of a vector or any other type of cloning vector, such as a plasmid, phage particle, bacteriophage, baculovirus, granulosome, or artificial chromosome. The provided nucleic acid molecule may be contained within an expression vector. In addition to the regulatory sequences and nucleotide sequences encoding the glycoproteins described herein, the cloning vector may include replication and control sequences derived from a species compatible with the host cell used for expression, as well as selection markers that confer an optional phenotype to the transformed or transfected cells. A large number of suitable cloning vectors are known in the art and are commercially available.

[0278] This invention also relates to a host cell comprising the nucleic acid molecules disclosed herein, and / or nucleic acid molecules comprising nucleic acid sequences encoding proteins of the viral particles described herein. The host cell of this disclosure may comprise cDNA of the viral particle genome. The cDNA of the genome may be contained in one or more nucleic acid molecules. The host cell of this disclosure is preferably an isolated host cell. The host cell of this disclosure is preferably not part of a human or animal body. The host cell of this disclosure is preferably not part of a whole plant. The host cell of this disclosure is preferably a non-human host cell.

[0279] The present invention also relates to a host cell comprising cDNA encoding an ORF encoding a glycoprotein as described herein, an ORF encoding an L protein as described in the viral particles of the present invention, an ORF encoding a nucleoprotein as described in the viral particles of the present invention, and an ORF encoding a Z protein as described in the viral particles of the present invention.

[0280] The present invention also relates to a host cell comprising cDNA of the LCMV S and LCMV L regions described in relation to the viral particles of this disclosure.

[0281] The techniques used to produce cDNA are common and routine techniques in molecular biology and DNA manipulation and production. Any cloning technique known to those skilled in the art can be used. These techniques are well-known and are available to technicians in laboratory manuals such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, ColdSpring Harbor Laboratory NY (2001).

[0282] The cDNA described herein can be introduced into plasmids. The cDNA described herein may be part of a DNA expression vector, or may be introduced into a DNA expression vector and optionally introduced into a host cell. The cDNA described herein, or the plasmid or vector containing that cDNA, preferably contains a promoter. Specific examples of promoters include RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 promoter, SP6 promoter, or T3 promoter.

[0283] The host cell can be any host cell suitable for cloning, expression, proliferation, or production of viral particles. The host cell can be prokaryotic or eukaryotic, such as *Escherichia 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, particularly HEK293 cells, such as HEK293T (CVCL_0063) or FreeStyle 293-F (CVCL_D603). FreeStyle293-F cells are commercially available, for example, from Thermo Fisher Scientific Inc. (catalog 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).

[0284] The present invention also relates to a method for generating viral particles of the present disclosure. The method includes culturing the host cells of the present disclosure under conditions suitable for viral particle formation.

[0285] Methods for generating viral particles may further include introducing the cDNA described herein into a host cell. Methods for generating viral particles may also include recovering and / or purifying the viral particles. Such recovery and / or purification methods are well known to those skilled in the art.

[0286] This invention also relates to a pharmaceutical composition comprising viral particles disclosed herein, such as arenavirus particles. The viral particles preferably comprise a lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acid encoding the glycoprotein described herein for the viral particles. This invention also relates to a pharmaceutical composition comprising a glycoprotein disclosed herein, a nucleic acid molecule disclosed herein, and / or a host cell disclosed herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient or carrier. The carrier may, for example, be selected from water, aqueous saline solutions, aqueous buffers, cell culture media, and combinations of at least two of the aforementioned carriers.

[0287] This invention also relates to viral particles (such as isoplasmic virus particles), glycoproteins, nucleic acid molecules, and / or host cells disclosed herein, for therapeutic purposes. The viral particles preferably comprise a lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acids encoding the glycoproteins described herein for the viral particles. The viral particles disclosed herein can be used to treat cancer or tumors.

[0288] This invention also relates to the use of the viral particles (such as arenavirus particles), glycoproteins, nucleic acid molecules, and / or host cells disclosed herein for the preparation of medicaments. The viral particles preferably comprise lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acids encoding the glycoproteins described herein for the viral particles. The medicaments are preferably used for the treatment of cancer or tumors.

[0289] This invention also relates to a method of treating a disease, the method comprising administering to a subject the viral particles (sand-like virus particles) disclosed herein, the glycoproteins disclosed herein, the nucleic acid molecules disclosed herein, and / or the host cells disclosed herein. The subject is preferably a subject in need of treatment. The viral particles, glycoproteins, nucleic acid molecules, and / or host cells are preferably administered in an effective amount. The viral particles preferably comprise lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and / or nucleic acids encoding the glycoproteins described herein for the viral particles. The disease is preferably cancer or a tumor.

[0290] The “subject” is a vertebrate, preferably a mammal, and more preferably a human. As used herein, the term “mammal” means any animal classified as a mammal, including but not limited to humans, livestock and farm animals, as well as zoo animals, sporting animals, or pet animals, such as mice, sheep, dogs, horses, cats, cattle, rats, pigs, apes (e.g., cynomolgus monkeys), to name just a few illustrative examples. Preferably, the mammal referred to herein is a human. The cancer or tumor can be a human or mouse cancer or tumor, preferably a human cancer or tumor.

[0291] An "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more applications.

[0292] Cancer or tumor can be any cancer or tumor disclosed in this article. Typically, cancer or tumor can be selected from carcinoma, melanoma, germ cell tumor, lymphoma, and sarcoma.

[0293] In the context of this disclosure, the term "carcinoma" should be understood as referring to a malignant tumor of epithelial origin. Preferably, the carcinoma is selected from anal cancer, bronchial cancer, lung cancer, endometrial cancer, gallbladder cancer, bladder cancer, hepatocellular carcinoma, testicular cancer, colon cancer, colorectal cancer, rectal cancer, laryngeal cancer, esophageal cancer, gastric cancer, breast cancer, kidney cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, oropharyngeal cancer, prostate cancer, thyroid cancer, and cervical cancer.

[0294] In the context of this disclosure, the term "sarcoma" should be understood to mean a malignant tumor of mesodermal origin. Sarcoma can be selected from angiosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, Kaposi's sarcoma, liposarcoma, leiomyosarcoma, malignant fibrous histiocytoma, neurogenic sarcoma, osteosarcoma, and rhabdomyosarcoma.

[0295] In the context of this disclosure, the term “melanoma” should be understood to mean a malignant tumor of melanocyte origin.

[0296] In the context of this disclosure, the term "lymphoma" should be understood to mean a malignant tumor of lymphoid origin.

[0297] In the context of this disclosure, the term “germ cell tumor” should be understood to mean a malignant tumor of embryonic origin.

[0298] The cancer or tumor to be treated can be a cold tumor. Preferred cold tumors include breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and glioblastoma.

[0299] The cancer or tumor to be treated can be a thermal tumor. Preferred thermal tumors include melanoma, bladder cancer, kidney cancer, head and neck cancer, and non-small cell lung cancer.

[0300] Preferred cancers or tumors to be treated include melanoma, colon cancer, fibrosarcoma, pancreatic cancer, thyroid cancer, lung cancer, adenocarcinoma, and gastrointestinal cancer.

[0301] Generally, the viral particles, glycoproteins, nucleic acid molecules, and / or host cells disclosed herein can be administered via any suitable route known to those skilled in the art. Administration typically 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, particularly intravenous administration, such as by injection or infusion.

[0302] The present invention also relates to a method for generating viral particles with enhanced ability to enter tumor cells, comprising:

[0303] a. Provide nucleic acid encoding the WE glycoprotein of the mutated LCMV strain described herein.

[0304] b. Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles containing the mutated LCMV strain WE glycoprotein; and

[0305] c. Optionally, assess the ability of viral particles containing the WE glycoprotein of the LCMV strain with the aforementioned mutation to enter and / or infect tumor cells.

[0306] The nucleic acid preferably encodes a mutant LCMV strain WE glycoprotein, which, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4), contains at least one mutant amino acid residue at one or more positions selected from the following regions: (a) the N-terminus of chain β1 defined by positions 59-89, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) helical α1 and / or α2 defined by positions 114-147, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) (e) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (f) Helix α3 defined by positions 187-199; (g) Ring 3 defined by positions 200-226; (h) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) H1 and H2 domains defined by positions 1-58; and / or (j) The TM cytoplasmic domain defined by positions 439-498; or containing at least one mutated amino acid residue at one or more of the following positions: 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152. 153, 154, 155, 156, 163, 188, 198, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492. The mutant LCMV strain WE glycoprotein can be any mutant LCMV strain WE glycoprotein disclosed herein.

[0307] In step b, the mutated LCMV strain WE glycoprotein is preferably expressed together with other viral proteins required for the production of viral particles. These other viral proteins may include NPs, LPs, and / or ZPs disclosed herein, preferably NPs, LPs, and / or ZPs disclosed in the context of viral particles of this invention.

[0308] Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells can be performed, for example, by signal transduction assays, such as assays measuring NF-κB and / or IRF3 / 7 activation in tumor cells infected with the viral particles. Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells preferably includes comparing the viral particles containing the mutated LCMV strain WE glycoprotein to a reference viral particle or a reference value. The reference viral particle preferably contains the wild-type glycoprotein shown in SEQ ID NO: 4 or 13, wherein SEQ ID NO: 4 is preferred. Preferably, the reference viral particle differs only in the sequence of the glycoprotein. The reference value is preferably a reference value derived from the reference viral particle. Compared to a value obtained from a viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4), the reference value is preferably at least as high as or higher. Preferably, the reference viral particle differs only in the sequence of the glycoprotein. The tumor cells are preferably tumor cell lines, such as lung cancer cell lines.

[0309] Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include isolating viral particles. Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include providing viral particles. Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include selecting viral particles.

[0310] The present invention also relates to a method for generating viral particles with enhanced tropism for tumor cells, comprising:

[0311] a. Provide nucleic acid encoding the WE glycoprotein of the mutated LCMV strain described herein.

[0312] b. Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles containing the mutated LCMV strain WE glycoprotein; and

[0313] c. Optionally, assess the ability of viral particles containing the WE glycoprotein of the LCMV strain with the aforementioned mutation to enter and / or infect tumor cells.

[0314] Preferably, the viral particles have enhanced tropism for at least one of the following tumors and / or tumor (cells): lung tumors (cells), such as H1975 or A549, LLC, TC-1; gastrointestinal tumors (cells), such as Gist-T1; melanoma (cells), such as MaMel86a, MaMel51, A375, B16F10 or RPMI-7951; pancreatic tumors (cells), such as 511950, 60590, 511950R or 60 590R; thyroid tumors (cells), such as 8305C or C643; sarcomas (cells), such as Gist-T1; breast tumors (cells), such as HCC1954; cervical tumors (cells), such as HeLa; liver tumors (cells), such as HepG2; colon tumors (cells), such as MC38, Sw620 or Sw480; neuroblastomas (cells), such as SK-N-BE(2); and / or prostate tumors (cells), such as TrampC2.

[0315] The nucleic acid preferably encodes a mutant LCMV strain WE glycoprotein, which, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4), contains at least one mutant amino acid residue at one or more positions selected from the following regions: (a) the N-terminus of chain β1 defined by positions 59-89, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) helical α1 and / or α2 defined by positions 114-147, wherein the at least one mutant amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) (e) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (f) Helix α3 defined by positions 187-199; (g) Ring 3 defined by positions 200-226; (h) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) H1 and H2 domains defined by positions 1-58; and / or (j) The TM cytoplasmic domain defined by positions 439-498; or containing at least one mutated amino acid residue at one or more of the following positions: 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152. 153, 154, 155, 156, 163, 188, 198, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492. The mutant LCMV strain WE glycoprotein can be any mutant LCMV strain WE glycoprotein disclosed herein.

[0316] In step b, the mutated LCMV strain WE glycoprotein is preferably expressed together with other viral proteins required for the production of viral particles. These other viral proteins may include NPs, LPs, and / or ZPs disclosed herein, preferably NPs, LPs, and / or ZPs disclosed in the context of viral particles of this invention.

[0317] Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells can be performed, for example, by signal transduction assays, such as assays measuring NF-κB and / or IRF3 / 7 activation in tumor cells infected with the viral particles. Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells preferably includes comparing the viral particles containing the mutated LCMV strain WE glycoprotein to a reference viral particle or a reference value. The reference viral particle preferably contains the wild-type glycoprotein shown in SEQ ID NO: 4 or 13, wherein SEQ ID NO: 4 is preferred. Preferably, the reference viral particle differs only in the sequence of the glycoprotein. The reference value is preferably a reference value derived from the reference viral particle. Compared to a value obtained from a viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4 or 13 (preferably SEQ ID NO: 4), the reference value is preferably at least as high as or higher. Preferably, the reference viral particle differs only in the sequence of the glycoprotein. The tumor cells are preferably tumor cell lines, such as lung cancer cell lines.

[0318] Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include isolating viral particles. Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include providing viral particles. Preferably, if the ability of viral particles to enter tumor cells is higher than that of reference viral particles and / or a reference value, the method may further include selecting viral particles.

[0319] The present invention also relates to viral particles having enhanced ability to enter tumor cells or enhanced tropism for tumor cells, which can be obtained by the methods disclosed herein for generating viral particles having enhanced ability to enter tumor cells and / or by the methods disclosed herein including generating viral particles having enhanced tropism for tumor cells.

[0320] It must be noted that, as used herein, the singular forms “a,” “an,” and “the” include plural references, and vice versa, unless the context clearly indicates otherwise.

[0321] Unless otherwise stated, the term “at least” preceding a series of elements should be understood to mean each element in the series.

[0322] Those skilled in the art will recognize or be able to determine, using no more than conventional experiments, a number of equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be covered in this invention.

[0323] The term “and / or” as used anywhere in this document includes the meaning of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0324] As used herein, the term "about" or "approximately" means within 20% of a given value or range, preferably within 10%, and more preferably within 5%. However, it also includes the specific number stated, for example, about 20 includes 20.

[0325] Unless the context otherwise requires, throughout this specification and claims, the word “comprise” and its variations, such as “comprises” and “comprising”, shall be understood to mean covering the said integer or step or set of integers or steps, but not excluding any other integer or step or set of integers or steps. When used herein, the term “comprising” may be replaced by the terms “containing” or “including”, or sometimes by the term “having” when used herein.

[0326] When used herein, "consisting of" excludes any element, step, or component not specifically mentioned in the claimed elements. When used herein, "consisting substantially of" does not exclude materials or steps that do not materially influence the essential and novel features of the claims.

[0327] In each instance of this document, any of the terms “comprising,” “consisting essentially of,” and “consisting of” can be replaced by any of the other two terms. For example, the term “comprising” implies that it also provides explicit support for “consisting essentially of” and “consisting of,” the term “consisting essentially of” implies that it also provides explicit support for “comprising” and “consisting of,” and the term “consisting of” implies that it also provides explicit support for “consisting essentially of” and “comprising.” The possibility that these terms are interchangeable should not be construed as meaning that these terms are synonymous.

[0328] The invention is further characterized by the following items.

[0329] Project 1. A viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0330] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0331] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0332] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0333] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0334] (e) Spiral α3 defined by positions 187-199;

[0335] (f) Ring 3 defined by positions 200-226;

[0336] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0337] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0338] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0339] (j) The TM cytoplasmic domain defined at positions 439-498;

[0340] Compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4, the viral particle has an enhanced ability to enter tumor cells.

[0341] Project 2. The viral particle of Project 1, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein contains at least one mutated amino acid residue at one or more locations comprising the following regions:

[0342] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0343] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0344] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0345] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0346] (e) Spiral α3 defined by positions 187-199;

[0347] (f) Ring 3 defined by positions 200-226;

[0348] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; and / or

[0349] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365.

[0350] Project 3. The viral particle described in Project 1, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein contains at least one mutated amino acid residue at one or more locations comprising the following regions:

[0351] (a) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0352] (b) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0353] (c) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0354] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0355] (e) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0356] (f) Ring 3 defined by positions 200-226;

[0357] (g) H1 and H2 structural domains defined by positions 1-58;

[0358] (h) The TM cytoplasmic domain defined at positions 439-498; and / or

[0359] (i) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258.

[0360] Project 4. A viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said glycoprotein, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, is selected from 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152, 153, 154, 155, 156, 163, 18 One or more of the following positions contain at least one mutated amino acid residue: 8, 198, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492, wherein the viral particle has an enhanced ability to enter tumor cells compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4.

[0361] Item 5. The viral particle of any one of the preceding items, wherein the glycoprotein has at least 95% sequence identity with SEQ ID NO: 4.

[0362] Item 6. The viral particle of any one of the preceding items, wherein the one or more positions are selected from positions 102, 122, 129, 132, 136, 152, 153, 154, 155, 156, 211, 217, 218, 256, 260, 284, 328, 332, 342, 358 and 492.

[0363] Item 7. The viral particle of any one of the preceding items, wherein the at least one mutated amino acid residue is selected from Ser 102 → Asn, Phe 122 → Leu, Phe 129 → Ser, Lys 132 → Arg, His 136 → Gln, Asn 152 → Tyr, Ser 153 → Pro, Phe or Tyr, Asn 154 → Asp, His 155 → Leu or Tyr, Lys 156 → Glu, Ala 211 → Thr, Thr 217 → Ile, Thr 218 → Ile, Lys 256 → Arg, Leu 260 → Phe or Ile, Tyr 284 → His, Ala 328 → Ser, Phe 332 → Leu, Val 342 → Ala, Arg 358 → Lys, and Lys 492 → Ile.

[0364] Item 8. The viral particle of any one of the preceding items, wherein, compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the GP comprises the following mutated amino acid residue group:

[0365] (a) Lys 492→ Ile;

[0366] (b) Ala 211→ Thr;

[0367] (c) Lys 260→ Phe and Lys 492→ Ile;

[0368] (d) Ser 153 → Pro and Lys 492 → Ile;

[0369] (e) Phe 122 → Leu, His 136 → Gln and Ser 153 → Pro;

[0370] (f) Phe 122 → Leu and Lys 492 → Ile;

[0371] (g) Phe 122 → Leu, His 136 → Gln, Ser 153 → Pro and Lys 492 → Ile;

[0372] (h) Glu 255 → Gly and Lys 492 → Ile;

[0373] (i) Ser 153 → Pro and Arg 358 → Lys;

[0374] (j) Ser 153 → Pro, Lys 256 → Arg, Leu 260 → Phe and Lys 492 → Ile;

[0375] (k) Ser 153 → Phe, Glu 255 → Gly and Leu 260 → Phe;

[0376] (l) Lys 156 → Glu and Lys 492 → Ile;

[0377] (m) Ser 153 → Pro, Lys 256 → Arg and Leu 260 → Phe;

[0378] (n) Phe 122 → Leu, His 136 → Gln, Ser 153 → Pro, Lys 256 → Arg and Lys492 → Ile; or

[0379] (o) Glu 379 → Asn and Lys 492 → Ile.

[0380] Item 9. The viral particle of any one of the preceding items, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein further includes a mutation at position 181 and / or 185, wherein the mutation is preferably selected from Ile 181→Met or Val and Arg 185→Trp.

[0381] Item 10. The viral particle of any one of the preceding items, wherein, compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the GP comprises the following mutated amino acid residue group:

[0382] (a) Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0383] (b) Ile 181→Met, Arg 185→Trp, and Ala 211→Thr;

[0384] (c) Ile 181→Met, Arg 185→Trp, Lys 260→Phe, and Lys 492→Ile;

[0385] (d) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0386] (e) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, and Arg 185→Trp;

[0387] (f) Phe 122→Leu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0388] (g) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0389] (h) Ile 181→Met, Arg 185→Trp, Glu 255→Gly, and Lys 492→Ile;

[0390] (i) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, and Arg 358→Lys;

[0391] (j) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, Leu 260→Phe, and Lys 492→Ile;

[0392] (k) Ser 153→Phe, Arg 185→Trp, Glu 255→Gly and Leu 260→Phe;

[0393] (l) Ser 153→Phe, Glu 255→Gly, and Leu 260→Phe;

[0394] (m) Lys 156→Glu, Ile 181→Met, Arg 185→Trp, and Lys 492→Ile;

[0395] (n) Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Leu 260→Phe;

[0396] (o) Phe 122→Leu, His 136→Gln, Ser 153→Pro, Ile 181→Met, Arg 185→Trp, Lys 256→Arg, and Lys 492→Ile; or

[0397] (p) Ile 181→Met, Arg 185→Trp, Glu 379→Asn, and Lys 492→Ile.

[0398] Item 11. The viral particle of any one of the preceding items, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid residues as defined in any one of items 1-10.

[0399] Item 12. The viral particle of any one of the preceding items, wherein the GP has 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 99.5%, preferably at least about 99.7%, or is preferably identical to it.

[0400] Item 13. The viral particle of any one of the preceding items, wherein the viral particle comprises a nucleic acid encoding a glycoprotein, wherein the nucleic acid comprises a sequence having 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%, or preferably identical to the sequence shown in SEQ ID NO: 58-85 or the corresponding complementary sequence.

[0401] Item 14. The viral particle according to any one of the preceding items, said viral particle comprising an LCMV L-protein (LP) and / or nucleic acid encoding said LP, wherein said LP has 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 with the L-protein sequence shown in SEQ ID NO: 10 or 23.

[0402] Item 15. The viral particle of any one of the preceding items, wherein the viral particle comprises a nucleic acid encoding LP, wherein the nucleic acid comprises a sequence having 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 preferably identical to, the sequence shown in SEQ ID NO: 9 or 22 or the corresponding complementary sequence.

[0403] Item 16. The viral particle according to any one of the preceding items, said viral particle comprising an LCMV nucleoprotein (NP) and / or nucleic acid encoding said NP, wherein said NP has 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 preferably identical to, the nucleoprotein sequence shown in SEQ ID NO: 6.

[0404] Item 17. The viral particle according to any one of the preceding items, the viral particle comprising a nucleic acid encoding an NP, wherein the nucleic acid comprises a sequence having 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%, or preferably identical to it.

[0405] Item 18. The viral particle according to any one of the preceding items, said viral particle comprising an LCMV Z-protein (ZP) and / or nucleic acid encoding said ZP, wherein said ZP has 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 preferably identical to, the ZP sequence shown in SEQ ID NO: 8 or 21.

[0406] Item 19. The viral particle of any one of the preceding items, wherein the viral particle comprises a nucleic acid encoding ZP, wherein the nucleic acid comprises a sequence having 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 being identical to the sequence shown in SEQ ID NO: 7 or 20 or the corresponding complementary sequence.

[0407] Item 20. The viral particle of any one of the preceding items, wherein the viral particle comprises LCMV GP and LCMV NP, and / or one or more nucleic acids encoding said LCMV GP and LCMV NP, wherein said LCMV GP and LCMV NP have 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 preferably identical to, the following LCMV GP and LCMV NP sequences:

[0408] (a) SEQ ID NO: 30 and 6;

[0409] (b) SEQ ID NO: 31 and 6;

[0410] (c) SEQ ID NO: 32 and 6;

[0411] (d) SEQ ID NO: 33 and 6;

[0412] (e) SEQ ID NO: 34 and 6;

[0413] (f) SEQ ID NO: 35 and 6;

[0414] (g) SEQ ID NO: 36 and 6;

[0415] (h) SEQ ID NO: 37 and 6;

[0416] (i) SEQ ID NO: 38 and 6;

[0417] (j) SEQ ID NO: 39 and 6;

[0418] (k) SEQ ID NO: 40 and 6;

[0419] (l) SEQ ID NO: 41 and 6;

[0420] (m) SEQ ID NO: 42 and 6;

[0421] (n) SEQ ID NO: 43 and 6;

[0422] (o) SEQ ID NO: 44 and 6;

[0423] (p) SEQ ID NO: 45 and 6;

[0424] (q) SEQ ID NO: 46 and 6;

[0425] (r) SEQ ID NO: 47 and 6;

[0426] (s) SEQ ID NO: 48 and 6;

[0427] (t) SEQ ID NO: 49 and 6;

[0428] (u) SEQ ID NO: 50 and 6;

[0429] (v) SEQ ID NO: 51 and 6;

[0430] (w) SEQ ID NO: 52 and 6;

[0431] (x) SEQ ID NO: 53 and 6;

[0432] (y) SEQ ID NO: 54 and 6;

[0433] (z) SEQ ID NO: 55 and 6;

[0434] (aa) SEQ ID NO: 56 and 6; or

[0435] (bb) SEQ ID NO: 57 and 6.

[0436] Item 21. The viral particle of any one of the preceding items, wherein the viral particle comprises LCMV GP, LCMV LP, LCMV NP, and LCMV ZP, and / or one or more nucleic acids encoding said LCMV GP, LCMV LP, LCMV NP, and LCMV ZP, wherein said LCMV GP, LCMV LP, LCMV NP, and LCMV ZP have 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 preferably identical to, the following:

[0437] (a) SEQ ID NO: 30, 23, 6 and 21;

[0438] (b) SEQ ID NO: 31, 23, 6 and 21;

[0439] (c) SEQ ID NO: 32, 23, 6 and 21;

[0440] (d) SEQ ID NO: 33, 23, 6 and 21;

[0441] (e) SEQ ID NO: 34, 23, 6 and 21;

[0442] (f) SEQ ID NO: 35, 23, 6 and 21;

[0443] (g) SEQ ID NO: 36, 23, 6 and 21;

[0444] (h) SEQ ID NO: 37, 23, 6 and 21;

[0445] (i) SEQ ID NO: 38, 23, 6 and 21;

[0446] (j) SEQ ID NO: 39, 23, 6 and 21;

[0447] (k) SEQ ID NO: 40, 23, 6 and 21;

[0448] (l) SEQ ID NO: 41, 23, 6 and 21;

[0449] (m) SEQ ID NO: 42, 23, 6 and 21;

[0450] (n) SEQ ID NO: 43, 23, 6 and 21;

[0451] (o) SEQ ID NO: 44, 23, 6 and 21;

[0452] (p) SEQ ID NO: 45, 23, 6 and 21;

[0453] (q) SEQ ID NO: 46, 23, 6 and 21;

[0454] (r) SEQ ID NO: 47, 23, 6 and 21;

[0455] (s) SEQ ID NO: 48, 23, 6 and 21;

[0456] (t) SEQ ID NO: 49, 23, 6 and 21;

[0457] (u) SEQ ID NO: 50, 23, 6 and 21;

[0458] (v) SEQ ID NO: 51, 23, 6 and 21;

[0459] (w) SEQ ID NO: 52, 23, 6 and 21;

[0460] (x) SEQ ID NO: 53, 23, 6 and 21;

[0461] (y) SEQ ID NO: 54, 23, 6 and 21;

[0462] (z) SEQ ID NO: 55, 23, 6 and 21;

[0463] (aa) SEQ ID NO: 56, 23, 6 and 21; or

[0464] (bb) SEQ ID NO: 57, 23, 6 and 21.

[0465] Item 22. The viral particle of any one of the preceding items, wherein the viral particle comprises an S segment containing a 5' untranslated region (UTR) having at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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% sequence identity, or preferably identical to.

[0466] Item 23. The viral particle of any one of the preceding items, wherein the viral particle comprises an S segment containing a 3' untranslated region (UTR) having at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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% sequence identity, or preferably identical to it.

[0467] Item 24. The viral particle of any one of the preceding items, wherein the viral particle comprises an L segment containing a 5' untranslated region (UTR) having at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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 identical to, the sequence shown in SEQ ID NO: 26 or 28, or the corresponding complementary sequence.

[0468] Item 25. The viral particle of any one of the preceding items, wherein the viral particle comprises an L segment containing a 3' untranslated region (UTR) having at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, 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% sequence identity, or preferably identical to.

[0469] Item 26. The viral particle of any one of the preceding items, wherein the viral particle comprises an S segment, the S segment comprising a nucleic acid sequence having 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 with the sequence shown in SEQ ID NO: 1 or 11.

[0470] Item 27. The viral particle of any one of the preceding items, wherein the viral particle comprises an L segment, the L segment comprising a nucleic acid sequence having 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 preferably the same as the sequence shown in SEQ ID NO: 2 or 15.

[0471] Item 28. The viral particle of any one of the preceding items, wherein the viral particle has an enhanced ability to enter human tumor cells compared with the reference viral particle.

[0472] Item 29. The viral particle of any one of the preceding items, wherein the viral particle has enhanced tropism for human tumor cells compared with the reference viral particle.

[0473] Item 30. The viral particle of any one of the preceding items, wherein the viral particle is capable of entering cells in a manner independent of α-dystrophin.

[0474] Item 31. The viral particle of any one of the preceding items, wherein the viral particle is infectious.

[0475] Item 32. The viral particle of any one of the preceding items, wherein the viral particle is capable of replication.

[0476] Item 33. The viral particle of any one of the preceding items, wherein the viral particle has a dual-segment genome.

[0477] Item 34. The virus particle of any one of the preceding items, wherein the virus particle is a sand-like virus particle.

[0478] Item 35. The viral particle of any one of the preceding items, wherein the viral particle is an LCMV particle.

[0479] Item 36. Virus particles as described in any one of Items 1-34, wherein the virus particles are sand-like virus particles other than LCMV particles pseudotyped using LCMV GP.

[0480] Item 37. The viral particle of any one of Items 1-32, wherein the viral particle is a vesicular stomatitis virus (VSV) particle pseudotyped with LCMV GP.

[0481] Item 38. The viral particle of any one of Items 1-32, wherein the viral particle is a Piccind virus particle pseudotyped with LCMV GP.

[0482] Item 39. Virus particles of any one of Items 1-32, wherein the virus particles are particles of viruses other than sand-borne viruses.

[0483] Item 40. The viral particle of any one of Items 1-32, wherein the viral particle is a particle of a viral vaccine vector.

[0484] Item 41. The viral particle of any one of Items 1-34, wherein the viral particle does not contain ORF from an organism other than LCMV.

[0485] Item 42. An LCMV GP, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein contains at least one mutated amino acid residue as defined in any one of Items 1-12.

[0486] Item 43. A nucleic acid molecule encoding the LCMV GP described in Item 42.

[0487] Item 44. The nucleic acid molecule of Item 43, wherein the nucleic acid molecule comprises a sequence having 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%, or preferably identical to the sequence shown in SEQ ID NO: 58-85 or the corresponding complementary sequence.

[0488] Item 45. A viral particle comprising a glycoprotein (GP) of a sand virus and / or a nucleic acid encoding said GP, wherein said glycoprotein comprises at least one of the following amino acid residues:

[0489] (a) A mutated amino acid residue, or preferably an Asn residue, at the position corresponding to Ser 102 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position.

[0490] (b) A mutated amino acid residue, or preferably a Leu residue, at the position corresponding to Phe 122 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0491] (c) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Phe 129 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0492] (d) A mutated amino acid residue, or preferably a Gln residue, at the position corresponding to His 136 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a His residue at said position.

[0493] (e) A mutated amino acid residue, or preferably a Tyr residue, at the position corresponding to Asn 152 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0494] (f) A mutated amino acid residue at the position corresponding to Ser 153 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Pro, Phe or Tyr residue, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position.

[0495] (g) A mutated amino acid residue, or preferably an Asp residue, at the position corresponding to Asn 154 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0496] (h) A mutated amino acid residue at the His 155 position of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, or preferably a Leu or Tyr residue, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0497] (i) A mutated amino acid residue, or preferably a Glu residue, at the position corresponding to Lys 156 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0498] (j) A mutated amino acid residue, or preferably a Thr residue, at the position corresponding to Ala 211 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at said position.

[0499] (k) A mutated amino acid residue, or preferably an Ile residue, at the position corresponding to Thr 217 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0500] (l) A mutated amino acid residue, or preferably an Ile residue, at the position corresponding to Thr 218 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0501] (m) A mutated amino acid residue, or preferably an Arg residue, at the position corresponding to Lys 256 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0502] (n) A mutated amino acid residue, or preferably a Phe or Ile residue, at the position corresponding to Leu 260 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Leu residue at said position.

[0503] (o) A mutated amino acid residue, or preferably a His residue, at the position corresponding to Tyr 284 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Tyr residue at said position.

[0504] (p) A mutated amino acid residue, or preferably a Ser residue, at the position corresponding to Ala 328 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at said position.

[0505] (q) A mutated amino acid residue, or preferably a Leu residue, at the position corresponding to Phe 332 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position.

[0506] (r) A mutated amino acid residue, or preferably an Ala residue, at the position corresponding to Val 342 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Val residue at said position.

[0507] (s) A mutated amino acid residue, or preferably a Lys residue, at the position corresponding to Arg 358 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle preferably has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Arg residue at said position; and

[0508] (t) The Ile residue at the position corresponding to Lys 492 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position.

[0509] Item 46. Viral particles according to Item 45, wherein the GP is a GP different from the LCMV GP.

[0510] Item 47. The virus particle according to Item 45, wherein the at least one amino acid residue is at least one substitution of a natural amino acid residue of the GP of the sand-like virus.

[0511] Item 48. A host cell comprising the cDNA of the genome of a nucleic acid molecule described in Item 43 or 44 or a viral particle described in any one of Items 1-41 and 45-47.

[0512] Item 49. A method for producing viral particles according to any one of items 1-41 and 45-47, comprising culturing the host cells described in item 48 under conditions suitable for viral particle formation.

[0513] Item 50. A pharmaceutical composition comprising any one of items 1-41, the GP of item 42, the nucleic acid molecule of item 43 or 44, or any one of items 45-47, sand-like virus particles.

[0514] Item 51. The virus particle described in any one of Items 1-41, the GP described in Item 42, the nucleic acid molecule described in Item 43 or 44, or the sand-like virus particle described in any one of Items 45-47, for use in treatment, preferably for use in cancer treatment.

[0515] Item 52. Use of the viral particles of any one of Items 1-41, the GP of Item 42, the nucleic acid molecule of Item 43 or 44, or the sand-like viral particles of any one of Items 45-47 in the preparation of a medicament, wherein the medicament is preferably used for cancer treatment.

[0516] Item 53. A method of treating a disease comprising administering to a subject in need an effective amount of any of the following: viral particles from items 1-41, GP from item 42, nucleic acid molecules from items 43 or 44, or sand-like viral particles from any of the following items 45-47, wherein the disease is preferably cancer.

[0517] Project 54. A method for generating viral particles with enhanced ability to enter tumor cells, comprising:

[0518] (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0519] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0520] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0521] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0522] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0523] (e) Spiral α3 defined by positions 187-199;

[0524] (f) Ring 3 defined by positions 200-226;

[0525] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0526] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0527] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0528] (j) The TM cytoplasmic domain defined at positions 439-498; and

[0529] (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and

[0530] (iii) Optionally assess the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO: 4.

[0531] Project 55. A method for generating viral particles with enhanced tropism for tumor cells, comprising:

[0532] (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions:

[0533] (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87;

[0534] (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113;

[0535] (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147;

[0536] (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157;

[0537] (e) Spiral α3 defined by positions 187-199;

[0538] (f) Ring 3 defined by positions 200-226;

[0539] (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258;

[0540] (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365;

[0541] (i) The H1 and H2 structural domains defined by positions 1-58; and / or

[0542] (j) The TM cytoplasmic domain defined at positions 439-498; and

[0543] (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and

[0544] (iii) Optionally assess the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells and / or non-tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a reference value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO: 4.

[0545] Item 56. The method of Item 54 or 55, wherein the viral particles have enhanced tropism for at least one of the following tumor cells: lung tumor cells, such as H1975 or A549, LLC, TC-1; gastrointestinal tumor cells, such as Gist-T1; melanoma cells, such as MaMel86a, MaMel51, A375, B16F10 or RPMI-7951; pancreatic tumor cells, such as 511950, 60590, 511950R or 60590R; thyroid tumor cells, such as 8305C or C643; sarcoma cells, such as Gist-T1; breast tumor cells, such as HCC1954; cervical tumor cells, such as HeLa; liver tumor cells, such as HepG2; colon tumor cells, such as MC38, Sw620 or Sw480; neuroblastoma cells, such as SK-N-BE(2); and / or prostate tumor cells, such as TrampC2.

[0546] Item 57. The method of any one of Items 54-56, wherein the mutant LCMV strain WE glycoprotein is GP as defined in any one of Items 1-12.

[0547] Item 58. A viral particle having enhanced ability to enter tumor cells or enhanced tropism for tumor cells, which can be obtained by any one of items 54-57.

[0548] Example

[0549] Example 1: Rapid Evolution Platform Identifies Missense Mutations that Promote Tumor Tropicality

[0550] The ambiguous RNA genome of arenavirus consists of the following: an approximately 3.5 kb S region encoding the proglycoprotein-polyprotein complex (GPC) and nucleoprotein (NP), and a 7.2 kb L region encoding RNA-dependent RNA polymerase (RdRP) and Z-protein. During replication, the viral polymerase will... -5 The error rate produces multiple genetically closely related progeny, called quasi-species. These quasi-species only gain an advantage when they are capable of replication and outperform their counterparts. Typically, the viral strain best suited to its environment will exhibit optimal replication (“survival of the fittest”). Therefore, we hypothesize that passage of arenavirus LCMV-WE in tumor cells will lead to the enrichment of mutations, thereby accelerating viral proliferation in cancer cells.

[0551] In the initial approach, LCMV-WE was proliferated for 7, 13, 42, or 52 generations on human or mouse cancer cells with or without 5-fluorouracil (5-FU). This process was then normalized by passage the virus 10–12 times in 7 human and 8 mouse tumor cell lines, each with or without the antiviral mutagen 5-FU, which limited viral replication by further enhancing the mutation rate. Four to five parallel cultures were set up for each combination to determine whether mutations occurred completely randomly with low selection bias (different mutants appearing in parallel cultures) or selection occurred, i.e., specific mutants preferentially appeared in one or more specific tumor cell lines (the same mutations appearing in parallel cultures). Viruses obtained from the so-called “rapid evolution platform (FEP)” were reverse transcribed, and cDNA of the S region was sequenced. We identified 6 missense mutations (5 AA positions) in the stable signal peptide (SSP), 44 missense mutations (40 AA positions) in GP1, and 19 missense mutations (18 AA positions) in GP2 (Table 1). The location of these 65 amino acid (AA) positions (74 missense mutations) in LCMV-GPC may affect viral proliferation in cancer cells. Some of these mutations occur more than once in the same or other tumor cell lines, indicating that missense mutations of certain amino acids are more critical for viral entry and proliferation than other amino acids. Figure 1 AC).

[0552] Next, we tested whether the acquired mutations were retained when the virus from the primary tumor cell culture was further passaged in other tumor cell lines. Therefore, the long-passaged (52-generation) mutant virus LCMV-WE subtype P52 (I181M, R185W) was further propagated for 23-55 generations in different tumor cell lines. Both mutations remained stable in the p52 viral subtypes. Figure 2 In one experiment, in the presence of 5-FU, the codon for amino acid 181 was changed to M181V via a single nucleotide polymorphism, which was a forward mutation rather than a reversion mutation (M181I). During passages 23 to 55, the p52 virus acquired additional mutations in five different tumor cell lines. Of the 16 viral mutations observed, 13 appear to be “private” because they were established only in one tumor cell line, while the virus acquired additional “shared” mutations in each of the five tumor cell lines, regardless of the presence of 5-FU. In the A375 tumor cell line, the p52 virus carrying a genetic background known for weak DAG binding (H136, S153, L260, H155) acquired the mutation H155Y, which transforms the p52 mutant into an isotype that binds α-DG with high affinity. When P52 virus proliferated on HCC1954, A549, and SW620 tumor cells, it acquired the mutation S153P, indicating that genotypes with weak DAG binding ability (H136, S153, L260, H155) could further reduce DAG binding through the conversion of S153 to P153. In the SW620 cell line, in the absence of 5-FU, P52 virus acquired two mutations—S153P and H155Y—resulting in genotypes (H136, P153, L260, Y155). Under these conditions, it is expected that Y155 (high DAG binding) cannot compensate for the loss of high affinity α-DG binding by S153. The mutant T217I in the tumor cell line RPMI-7951 and the T217S and S153P in the cell line SW620 are expected to have weak or weakened DAG binding, since it is not known that T217I is directly involved in DAG binding.

[0553] Next, we compared the accumulation of viral mutations in LCMV-WE (A211) and the YF13 subtype (T211). The mutant T211 obtained in YF13 was stably expressed in 4 / 4 parallel cultures, each with or without 5-FU ( Figure 3 After passage, YF13 acquired other mutations. Figure 3The mutation spectrum of 211T positive viruses is more selective than that of LCMV-WE 211A viruses, especially in the presence of 5-FU. When a mixture of six different viruses (WE, p42, p52, Y18, YF13, FP7) was passaged in the C643 cell line, the virus (211T carrier) outperformed the 211T non-carrier and acquired additional mutations. Figure 3 ).

[0554] Example 2: Rapid Evolution Platform Identifies Tumor-Preference Mutation Hotspots

[0555] Next, we evaluated which regions in LCMV-GPC showed the highest mutation frequencies and were therefore potentially particularly important for tumor tropism. We analyzed 15 domains in LCMV-GP (…). Figure 4 A). In the chain β7 domain, we identified only one mutation in 18 AS positions, in stark contrast to the loop 1, which showed 6 mutations in 10 AS positions. Figure 4 A and Figure 5 This clearly demonstrates that some regions within the LCMV-GP are more important than others in determining tumor tropism. To identify highly mutated regions, we compared the mutation frequencies of different domains and found that 10.3% and 15.4% were the most significant values ​​for the low-mutation domain group, indicating that the threshold between low and high mutation rates was 12.85%. Figure 4 B + C). Furthermore, the significance of mutation frequencies was analyzed ( Figure 5 Besides loops 1 and 3, the N-terminus of chain β1, chains β3, β4, β5, N-helices, helical α1, α2, α4, α5 domains, TM cytoplasmic domain, and H1 and H2 domains showed a significant enhancement of mutations. Figure 1 a). In summary, we identified several domains that exhibit significant mutation frequencies during tumor cell proliferation. It is believed that some of these identified regions interact with the host membrane, thereby determining LCMV entry.

[0556] Example 3: LCMV GP mutant triggers enhanced viral entry into tumor cells

[0557] Viral entry is triggered by the interaction of LCMV glycoproteins with host cell proteins and membranes. Although LCMV-GP utilizes glycosylated α-dystrophic glycan (DAG) as an entry receptor, other receptors have also been described. Some tumor cells (including those from FEP) do not necessarily express high levels of DAG. Therefore, attempts have been made to hypothesize that viral mutants selected from FEP improve viral entry into cancer cells. Consistent with this, the amino acid positions known to trigger DAG binding and LCMV-GP dimerization are severely mutated in viruses derived from FEP. Figure 6 The amino acids from the fusion peptide / ring region were not mutated. Figure 6 To test our hypothesis that the mutant region affects viral entry into tumor cells, we quantified the entry of LCMV into A549 cells. Given that recombinant chimeric viruses exhibit attenuated viral replication, we used a recombination system carrying the S segment of the WE strain and the L segment of LCMV clone 13. Fourteen sites in seven regions were selected to generate recombinant chimeric viruses carrying the mutated S segment (carrying LCMV-GP and NP) and the L segment from LCMV clone 13. A549 cells were exposed to the recLCMV mutant, and entry was terminated with monensin at different time points. All tested mutants showed accelerated entry into the human tumor cell line A549. Figure 7 (A & B). We then investigated whether other amino acids at the mutation site could similarly accelerate entry into tumor cells. In a rapid evolution platform, polar and neutral serine were mutated to hydrophobic proline. To further analyze this site, we generated mutations expressing either hydrophobic phenylalanine or neutral and polar tyrosine. Both amino acids accelerated entry into A459 cells (A & B). Figure 7 C). Exchanged with (and basic histidine), Figure 7 C) Similar to the hydrophobic leucine, the neutral and polar amino acid tyrosine at position 155 accelerates entry into A549 cells. From these data, we conclude that other amino acids besides the mutant amino acids identified in FEP can also accelerate entry into tumor cells. Next, we investigated whether other positions within the identified region, which were not detected in FEP, could similarly enhance tumor cell entry. To this end, we selected one mutation in the loop 1 region and analyzed the virus carrying this mutation in an entry assay. Indeed, this virus showed a significantly enhanced entry into A549 tumor cells. Figure 7 D). In summary, we have demonstrated that viral entry into cancer cells is a major mechanism leading to the selection of mutated viruses on a rapidly evolving platform.

[0558] Example 4: FEP recognizes mutations that promote the replication of specific tumor cells.

[0559] Although we identified 19 mutations that accelerated viral entry into A549 cells, only mutations 102, 153, and 260 were initially found in A549 cell passages. The other mutations were obtained from passages of different cell lines. This suggests that some of the identified mutations accelerate viral entry into multiple cancer cells. To investigate whether these mutations exhibit tumor cell tropism, 20 recombinant LCMV strains carrying a single mutation were generated. Viral entry was tested on six highly diverse human cancer cell types and three healthy cell types. For cancer cells, we selected cell lines from sarcoma, lung cancer, and melanoma. For healthy cells, we selected neurons, myotubes, and human myoblasts. Almost all of the tested mutations showed accelerated proliferation in at least one tested cell line. Figure 8 a). Some tested mutations showed accelerated proliferation in a range of cancer cells ( Figure 8 a), while some mutations show enhanced replication only in one tumor cell line ( Figure 8 a). To gain a deeper understanding of the pervasiveness of viral replication, we plotted the infection rates of H1975 and MaMel86a cells against other tested tumor cell lines ( Figure 8 b). Interestingly, most mutations accelerated proliferation in multiple cell types. Figure 8 b).

[0560] As expected, both lung cell lines showed very similar mutational patterns that accelerated viral replication (A459 vs. H1975). Figure 8 b). However, unrelated tumor cell lines also showed similar sensitivity to mutations ( Figure 8 b). The spider diagram showing the acceleration fold reveals that most mutations in the tests are of a common type, meaning they proliferate at an accelerated rate in most tumor cell lines tested. Figure 8 c). Interestingly, some mutations tested reduced proliferation in both tumor and healthy cells. We consider two possible explanations. First, these mutations may still accelerate entry into tumor cells but subsequently limit viral replication. Second, these mutations may provide the virus with other selective advantages. In summary, our data suggest that mutations in different regions within LCMV-GP can modulate entry into tumor cells without affecting entry into healthy cells.

[0561] Example 5: Combinations of mutations can have synergistic effects

[0562] To investigate whether mutations from different regions could be combined, we generated recombinant viruses carrying multiple combined mutations. The combination of mutations I181M, R185W, and K492I exhibited strong infection activity against various tumor cell lines. Figure 9A). Next, we investigated whether other combinations of mutations identified by FEP or reported to affect cell tropism could improve tumor cell-specific tropism. We generated 15 recombinant LCMVs with combined mutations and tested them in a cohort of four human and four mouse cancer cell lines. As expected, most viruses showed accelerated replication in the human cancer cell line A549 ( Figure 9 (A and B). It is noteworthy that some viruses appear to have broad replication capabilities, while others exhibit specific tropism (A and B). Figure 9 A and B). Therefore, we identified the different mutations into four groups: generalized, human, murine, and cell-specific. The generalized virus showed accelerated replication in all eight tested cell types (A and B). Figure 9 B). 'Human cancer type' shows accelerated replication only in human cancer cells and not in mouse cancer cells. Figure 9 B). 'Mouse cancer cell type' showed stronger replication in mouse cancer cells than in human cancer cells. Figure 9 B), and cell type-specific viruses exhibit highly diverse proliferation in different cell types. Figure 9 (B) Although we did not perform site-by-site comparisons of different mutations, based on these data, we believe that mutations 256R and 358K are associated with higher human-specific tropism, while mutation 153F is associated with more mouse-specific expression. Mutation 156E appears to trigger cell-type-specific tropism. Mutations 122L, 129S, 136Q, 260F, and 490E enhance tumor cell-specific proliferation. In summary, we identified mutations that can be combined to obtain additional effects. Depending on the type of combination, the virus exhibits tropism for human cancer, mouse cancer, or specific cell types.

[0563] Example 6: Entry into tumor cells is independent of αDG

[0564] We found that the identified mutations accelerated viral entry into several tumor cell types, including A549 cells. Since LCMV-GP can bind to glycosylated αDG as an entry receptor, we then analyzed entry into WT and αDG knockout A549 cells. Recombinant chimeric viruses carrying (WT) or (MUT) mutations GP-F122L, GP-S153P, and GP-I181M-R185W-K492I were incubated with αDG-deficient or competent cells. As expected, all viruses showed accelerated entry into A549 cells. Figure 10 Although viral entry into A549 cells is partially dependent on αDG, GP-S153P and GP-I181M-R185W-K492I do not depend on αDG for entry. Figure 10It is noteworthy that the WT virus strain (155H) used did indeed show very little αDG-dependent entry into tumor cells, consistent with reports indicating that 155Y mediates a high affinity for αDG. Figure 10 This indicates that tumor cell-specific entry is not mediated by affinity for αDG.

[0565] Example 7: Viruses with combined mutations exhibit accelerated antitumor activity

[0566] Next, we aimed to analyze whether specific entry into tumor cells is beneficial for cancer treatment. First, we analyzed the viral distribution of different tumor-loving strains. These strains contained the following mutations in GP:

[0567] .

[0568] As expected, the new strain showed almost no replication in healthy mouse tissues. Replication was enhanced in tumor cells. Next, we analyzed whether this favorable viral distribution affected the antitumor activity of LCMV. To do this, we infected mice carrying MC38-OVA with different tumor-loving strains and then analyzed tumor growth. Indeed, all the tumor-loving strains tested showed strong antitumor activity. Figure 11 Mechanistically, we hypothesize the existence of an enhanced inflammatory response within the tumor, as we detected accelerated cytokine levels seven days after treatment. Figure 11 A). In summary, the mutant viruses are more specific than the WT strain, making them promising candidates for further development.

[0569] Example 8: Mutations identified by LCMV-WE exhibit similar activity in other viruses expressing LCMV-GP.

[0570] We found that LCMV-GP with the tumoritropic mutations we detected exhibited highly enhanced tumor cell entry and thus superior performance in tumor therapy. Next, we investigated whether these mutations could also be transferred to other arenaviruses. To this end, we introduced several identified mutations into the glycoproteins of Lunk virus (LUNV), and these glycoproteins were expressed in a chimeric LCMV background virus (containing the L segment of LCMV clone 13 and the S segment of GP from Lunk virus and NP from LCMV strain WE). Mutations at positions 155, 156, and 256 of LCMV-GP were introduced into the Lunk arenavirus glycoproteins. These correspond to positions 152, 153, and 253. The introduced mutations were Y152H, K153E, and K253R. The ability to replicate into different cell lines was analyzed in an infectivity assay. These viruses were analyzed in an entry assay in A549 cells. Indeed, mutations in these glycoproteins similarly enhanced entry into A549 cells. Figure 12 In summary, we found that the mutations can transfer to other arenavirus glycoproteins. Furthermore, if the mutated glycoprotein is expressed on other backbone viruses, it can also enhance tumor entry.

[0571] Example 9: Discussion

[0572] In this study, the inventors of this application demonstrated, using a rapid evolution platform, that mutations inducing tumor cell-specific tropism can be identified. Several point mutations and structural regions within LCMV-GP were identified that trigger tumor cell-specific entry. These mutations can be combined and act synergistically in tumor cell proliferation. Due to accelerated site-specific replication and immune activation, but limited proliferation in healthy tissues, the use of mutant viruses for tumor therapy is proprietary.

[0573] Although the increased entry into tumor cells was found to be independent of αDG, the exact molecular entry mechanism remains unclear. Interestingly, several mutations were found to accelerate proliferation not only in human cancer cells but also in mouse cancer cells. Viral proliferation was limited in healthy human cells or healthy mouse tissues (in vivo). This suggests that tumors in both species must overexpress this receptor.

[0574] In summary, the inventors of this application modified LCMV to target tumors. The resulting virus replicates rapidly in cancer cells, replicates to a limited extent in healthy organs, and exhibits enhanced anti-tumor activity.

[0575] Example 10: Materials and Methods

[0576] A. Mice: All mice used were kept on a C57BL / 6 background. All mice were housed in individual ventilated cages, authorized by Veterinäramt Nordrhein Westfalen (Düsseldorf, Germany), and in accordance with German animal protection laws or institutional guidelines of the Ontario Cancer Institute.

[0577] B. Entry Assay: An entry assay was performed to determine the ability of a specific virus to enter cells. In this assay, cells (i.e., A459 cells, directly after harvest) were pre-incubated with the virus (multiple of infection 0.1) at 4°C (in 96-well plates, in 200 μL of the appropriate cell culture medium) to allow the virus to bind to the cell membrane. Since LCMV entry is an active process, the virus could not enter the cells under these conditions. After one hour of incubation, it was expected that most of the virus had bound to the cells, and the cells were heated to 37°C. Monensin, which inhibits further viral entry, was added to the culture at different time points after heating the cells to 37°C (i.e., 0 min, 20 min, 60 min, or 180 min). The cells were then incubated for an additional 16 hours. This incubation time provided sufficient time for the virus to replicate its RNA and produce viral proteins. After this incubation period, the viral proteins in each cell were determined by staining the cells with anti-LCMV-NP antibody (clone VL4) and analyzing the results by flow cytometry. For this purpose, cells were harvested with trypsin / EDTA, fixed with 2% formalin for 10 minutes, and then washed twice with FACS buffer (PBS, 1% FCS, 5 mM EDTA, and 0.1% sodium azide) supplemented with 0.01% saponin. After washing, cells were stained with anti-LCMV-NP (homemade VL4) for 30 minutes. After two additional washes, the primary antibody was detected with fluorescently labeled anti-rat antibody. In this assay, replication and protein production will only occur in cells that were infected before the addition of monensin. Cells that did not undergo the entry process before the addition of monensin were uninfected and therefore did not produce viral RNA and proteins. Therefore, in this assay, the ability to enter cells is directly related to the percentage of infected cells.

[0578] C. Infectivity Assay: The infectivity assay measures the ability of the virus to enter cells, replicate within cells, and produce viral proteins. In this assay, cells (i.e., A459 cells, MaMel86a cells, and MaMel51 cells, directly after harvest) were incubated with the virus (multiple of infection ranging from 0.01 to 0.1) at 37°C for 16 hours (in 96-well plates, in 200 μL of the corresponding cell culture medium). During this incubation period, the virus proliferates in the cell culture. Viral particles produced in the cell culture can infect new cells. After this incubation period, viral proteins in each cell are determined by staining the cells with anti-LCMV-NP antibody (clone VL4) and analyzing them by flow cytometry. For this purpose, cells are harvested with trypsin / EDTA, fixed with 2% formalin for 10 minutes, and then washed twice with FACS buffer (PBS, 1% FCS, 5 mM EDTA, and 0.1% sodium azide) supplemented with 0.01% saponin. After washing, the cells were stained with anti-LCMV-NP (homemade VL4) for 30 minutes. After two additional washes, the primary antibody was detected using fluorescently labeled anti-rat antibody. In this assay, the percentage of infected cells was directly correlated with the virus's ability to proliferate in a given cell culture. It could also be correlated with the ability to enter the cell if we assume that entry into the cell is the rate-limiting step.

[0579] D. Tumor growth experiment: To facilitate tumor transplantation, 10 μL of PBS was injected subcutaneously. 6 MC38-OVA (MC38-OVA) cells of mouse colon cancer. Tumor growth was monitored every two days.

[0580] E. Statistical Analysis: Means were compared using unpaired Student's two-tailed t-test. Data are presented as mean ± SEM. Student's two-tailed t-test was used to detect significant differences between groups. Additionally, the Chi-Quadrat test was used. The Mantel-Cox test was used to compare survival. The statistical significance level was set at P < 0.05.

[0581] F. Cells: 511950 and 60590 are primary tumor cells isolated from transgenic mouse pancreatic cancer. 511950R and 60590R are derived from 511950 and 60590 cells treated with trametinib. 8305C (CVCL_1053) is derived from undifferentiated thyroid carcinoma. A549 (CVCL_0023) is a human lung adenocarcinoma cell line. A375 (CRL-1619) is a malignant melanoma cell line. B16F10 (CVCL_0159) is a mouse melanoma cell line. C643 (CVCL_5969) is a human undifferentiated thyroid cancer cell line. Gist-T1 cells are primary sarcoma cells. HCC1954 (CVCL_1259) is a breast ductal carcinoma cell line. H1975 (CVCL_1511, ATCC, CRL-5908) is a human lung cancer cell line. HeLa (CVCL_0030) is derived from papillomavirus-associated cervical adenocarcinoma. HepG2 (CVCL_0027) is derived from hepatoblastoma. HEK293FT is human embryonic kidney cells. LLC (CVCL_4358) is Lewis lung cancer (mouse). MC38 (CVCL_B288) is a mouse colon adenocarcinoma cell line. MOPC cells are mouse oropharyngeal cells. MC57 (CVCL_4985) is a mouse fibroblast cell line in which isavirus LCMV replicates well. RPMI-7951 cells (CVCL_1666) are human melanoma cells. SK-N-BE (2) (CRL-2271) is a neuroblastoma cell line. Sw620 (CVCL_0547) is a human colon adenocarcinoma cell line. Sw480 (CVCL_0546) is derived from colon adenocarcinoma. TC-1 (CVCL-4699) is mouse lung cancer. TrampC2 is a mouse adenocarcinoma cell line (CVCL_3615). UKE-Mel-51 (= MaMel51), Mel-86a (= MaMel86a), 118b, and 118c are primary tumor cells isolated from human melanoma metastases.

[0582] The embodiments exemplarily described herein may be practiced in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, the terminology and expressions used herein are used as descriptive rather than restrictive terms, and in using these terms and expressions, no equivalents of any portion of the features shown and described are intended to be excluded, but rather various modifications are recognized as being within the scope of the invention. Therefore, it should be understood that while these embodiments have been specifically disclosed by way of preferred embodiments and optional features, modifications and variations thereof may be sought by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. Each narrower group of species and subgenus falling within the scope of the general disclosure also forms part of the invention. This includes the general description of the invention, with conditions or negative limitations that remove any subject matter from that species, regardless of whether such removed material is specifically stated herein. Furthermore, when features are described in the form of a Markush group, those skilled in the art will recognize that this disclosure is therefore also described in the form of any individual member or subgroup within that Markush group.

[0583] Equivalents: Many equivalents of the invention described herein would be recognized or determined by a person skilled in the art using no more than conventional experimentation. The following claims are intended to cover these equivalents.

[0584] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents, and substances described herein, and therefore these can be varied. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0585] All publications referenced throughout this specification (including all patents, patent applications, scientific publications, manufacturers' instructions, user manuals, etc.) are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the invention was not prior to the disclosure described due to prior art. If any material incorporated by reference contradicts or is inconsistent with this specification, the specification shall supersede any such material.

[0586] Further embodiments will become apparent from the following claims.

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Claims

1. A viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, and compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, said mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue selected from the following: Lys 492 → Ile, Ser 102 → Asn, Phe 122 → Leu, Phe 129 → Ser, Lys 132 → Arg, His 136 → Gln, Asn 152 → Tyr, Ser 153 → Pro, Phe or Tyr, Asn 154 → Asp, His 155 → Leu or Tyr, Lys 156 → Glu, Thr 217 → Ile, Thr 218 → Ile, Lys 256 → Arg, Leu 260 → Ile, Tyr 284 → His, Ala 328 → Ser, Phe 332 → Leu, Val 342 → Ala and Arg 358 → Lys, wherein the viral particles have an enhanced ability to enter tumor cells compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO:

4.

2. The viral particle of claim 1, wherein the glycoprotein has at least 95% sequence identity with SEQ ID NO:

4.

3. The viral particle of claim 1 or 2, wherein the GP comprises the following mutated amino acid residue group compared with the linear polypeptide sequence of the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4: (a) Lys 492→ Ile; (b) Ala 211→ Thr; (c) Lys 260→ Phe and Lys 492→ Ile; (d) Ser 153 → Pro and Lys 492 → Ile; (e) Phe 122 → Leu, His 136 → Gln and Ser 153 → Pro; (f) Phe 122 → Leu and Lys 492 → Ile; (g) Phe 122 → Leu, His 136 → Gln, Ser 153 → Pro and Lys 492 → Ile; (h) Glu 255 → Gly and Lys 492 → Ile; (i) Ser 153 → Pro and Arg 358 → Lys; (j) Ser 153 → Pro, Lys 256 → Arg, Leu 260 → Phe and Lys 492 → Ile; (k) Ser 153 → Phe, Glu 255 → Gly and Leu 260 → Phe; (l) Lys 156 → Glu and Lys 492 → Ile; (m) Ser 153 → Pro, Lys 256 → Arg and Leu 260 → Phe; (n) Phe 122 → Leu, His 136 → Gln, Ser 153 → Pro, Lys 256 → Arg and Lys 492 → Ile; or (o) Glu 379 → Asn and Lys 492 → Ile.

4. The viral particle according to any one of the preceding claims, wherein the GP has 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 99.5%, preferably at least about 99.7%, or is preferably identical to it.

5. The viral particle according to any one of the preceding claims, wherein the viral particle has an enhanced ability to enter human tumor cells compared with the reference viral particle, or wherein the viral particle has an enhanced tropism for human tumor cells compared with the reference viral particle.

6. An LCMV GP, wherein, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the glycoprotein comprises at least one mutated amino acid residue as defined in any one of claims 1-5.

7. A nucleic acid molecule encoding the LCMV GP of claim 6, wherein the nucleic acid molecule preferably comprises a sequence having 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%, or preferably identical to the sequence shown in SEQ ID NO: 58-85 or the corresponding complementary sequence.

8. A viral particle comprising a glycoprotein (GP) of a sand virus and / or nucleic acid encoding said GP, wherein said glycoprotein comprises at least one of the following amino acid residues: (a) The Ile residue at the position corresponding to Lys 492 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position; (b) An Asn residue at the position corresponding to Ser 102 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position. (c) The Leu residue at the position corresponding to Phe 122 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position. (d) The Ser residue at the position corresponding to Phe 129 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position. (e) A Gln residue at the His 136 position of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a His residue at said position. (f) The Tyr residue at the position corresponding to Asn 152 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position; (g) Pro, Phe, or Tyr residues at the position corresponding to Ser 153 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Ser residue at said position. (h) An Asp residue at the position corresponding to Asn 154 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position. (i) A Leu or Tyr residue at the His 155 position of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position. (j) Glu residues at the position corresponding to Lys 156 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing Lys residues at said position. (l) The Ile residue at the position corresponding to Thr 217 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position; (m) The Ile residue at the position corresponding to Thr 218 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position; (n) An Arg residue at the position corresponding to Lys 256 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Lys residue at said position. (o) A Phe residue at the position corresponding to Leu 260 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Leu residue at said position; (p) His residue at the position corresponding to Tyr 284 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Tyr residue at said position; (q) The Ser residue at the position corresponding to Ala 328 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing an Ala residue at said position. (r) The Leu residue at the position corresponding to Phe 332 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Phe residue at said position. (s) An Ala residue at the position corresponding to Val 342 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing a Val residue at said position; and (t) Lys residues at the position corresponding to Arg 358 of the GP of the LCMV strain WE glycoprotein shown in SEQ ID NO: 4, wherein the viral particle has an enhanced ability to enter tumor cells compared with a reference viral particle containing Arg residues at said position.

9. A host cell comprising the cDNA of the genome of the nucleic acid molecule of claim 7 or the viral particle of any one of claims 1-5 and 8.

10. A method for producing viral particles according to any one of claims 1-5 and 8, comprising culturing the host cell of claim 9 under conditions suitable for viral particle formation.

11. A pharmaceutical composition comprising the virus particles of any one of claims 1-5, the GP of claim 6, the nucleic acid molecule of claim 7, or the sand-like virus particles of claim 8.

12. The virus particle of any one of claims 1-5, the GP of claim 6, the nucleic acid molecule of claim 7, or the sand-like virus particle of claim 8, for use in treatment.

13. The viral particles of claim 12, used in cancer treatment.

14. Use of the virus particle of any one of claims 1-5, the GP of claim 6, the nucleic acid molecule of claim 7, or the sand-like virus particle of claim 8 in the preparation of a medicament, wherein the medicament is preferably used for cancer treatment.

15. A method of treating a disease, comprising administering to a subject in need an effective amount of any one of claims 1-5, the GP of claim 6, the nucleic acid molecule of claim 7, or the sand-like virus particle of claim 8, wherein the disease is preferably cancer.

16. A method for generating viral particles with enhanced ability to enter tumor cells, comprising: (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions: (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (e) Spiral α3 defined by positions 187-199; (f) Ring 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) The H1 and H2 structural domains defined by positions 1-58; and / or (j) The TM cytoplasmic domain defined at positions 439-498; and (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and (iii) Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO:

4.

17. A method for generating viral particles with enhanced tropism for tumor cells, comprising: (i) Providing a nucleic acid encoding a mutant LCMV strain WE glycoprotein, wherein, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, the mutant LCMV strain WE glycoprotein contains at least one mutant amino acid residue at one or more positions comprising the following regions: (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (e) Spiral α3 defined by positions 187-199; (f) Ring 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) The H1 and H2 structural domains defined by positions 1-58; and / or (j) The TM cytoplasmic domain defined at positions 439-498; and (ii) Expressing the mutated LCMV strain WE glycoprotein and other viral proteins to produce viral particles comprising the mutated LCMV strain WE glycoprotein; and (iii) Assessing the ability of viral particles containing the mutated LCMV strain WE glycoprotein to enter and / or infect tumor cells and / or non-tumor cells, preferably including comparing viral particles containing the mutated LCMV strain WE glycoprotein with a reference value, wherein the reference value is preferably at least as high as or higher than the value obtained by viral particles containing the wild-type glycoprotein shown in SEQ ID NO:

4.

18. The method of claim 16 or 17, wherein the viral particles have enhanced entry ability or enhanced tropism for at least one of the following tumor cells: lung tumor cells, such as H1975 or A549, LLC, TC-1; gastrointestinal tumor cells, such as Gist-T1; melanoma cells, such as MaMel86a, MaMel51, A375, B16F10, or RPMI-7951; pancreatic tumor cells, such as 511950, 605. 90, 511950R or 60590R; thyroid tumor cells, such as 8305C or C643; sarcoma cells, such as Gist-T1; breast tumor cells, such as HCC1954; cervical tumor cells, such as HeLa; liver tumor cells, such as HepG2; colon tumor cells, such as MC38, Sw620 or Sw480; neuroblastoma cells, such as SK-N-BE(2); and / or prostate tumor cells, such as TrampC2.

19. A viral particle having an enhanced ability to enter tumor cells or an enhanced tropism for tumor cells, which can be obtained by the method of claim 15 or 16.

20. A viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said GP, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, which, compared to the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, contains at least one mutant amino acid residue at one or more positions comprising the following regions: (a) The chain β1 N-terminus defined by positions 59-89, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 59-78 and 81-87; (b) Chain β3, β4 and / or β5 defined by positions 90-113, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 90-93, 98-103 and 106-113; (c) Helices α1 and / or α2 defined by positions 114-147, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 126-130 and 136-147; (d) Ring 1 defined by positions 148-157, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 148-157; (e) Spiral α3 defined by positions 187-199; (f) Ring 3 defined by positions 200-226; (g) α4 and α5 domains defined by positions 245-265, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 245-251 and 253-258; (h) An N-helix defined by positions 313-373, wherein the at least one mutated amino acid residue is preferably located at one or more positions selected from 313-349 and 353-365; (i) The H1 and H2 structural domains defined by positions 1-58; and / or (j) The TM cytoplasmic domain defined at positions 439-498; Compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4, the viral particle has an enhanced ability to enter tumor cells.

21. A viral particle comprising a lymphocytic choriomeningovirus (LCMV) glycoprotein (GP) and / or nucleic acid encoding said glycoprotein, wherein said glycoprotein is a mutant LCMV strain WE glycoprotein, which, compared with the wild-type LCMV strain WE glycoprotein shown in SEQ ID NO: 4, is selected from 18, 28, 36, 39, 48, 51, 60, 61, 62, 63, 66, 71, 74, 88, 94, 102, 103, 105, 106, 112, 119, 120, 121, 122, 128, 129, 132, 133, 136, 141, 144, 149, 151, 152, 153, 154, 155, 156, 163, 18 One or more of the following positions contain at least one mutated amino acid residue: 8, 198, 203, 207, 211, 217, 218, 222, 236, 252, 255, 256, 260, 280, 284, 308, 327, 328, 332, 335, 339, 342, 343, 344, 357, 358, 369, 374, 382, ​​388, 406, 426, 451, 456, 471, 477, 491, and 492, wherein the viral particle has an enhanced ability to enter tumor cells compared to a reference viral particle containing the wild-type glycoprotein shown in SEQ ID NO: 4.

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