Method for producing an antitumoral arenavirus as well as arenavirus mutants
AssigneeAbalos Therapeutics GmbH
InventorsKarl LANG, Cornelia HARDT, Michael BERGERHAUSEN, Hilal BHAT
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Abstract
The invention relates to a mutant of an arenavirus having improved antitumoral properties. The invention also relates to a method of generating such an arenavirus mutant, related pharmaceutical compositions, medical uses, methods of treatment, and isolated proteins and nucleic acids.
Claims29
A mutant of lymphocytic choriomeningitis virus, wherein the mutant is capable of undergoing a stronger propagation in a tumor cell as compared to the wild type lymphocytic choriomeningitis virus strain WE, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said glycoprotein comprises at least one of the mutations Arg 185→Trp and Ile 181→Met as compared to the wild type glycoprotein sequence set forth in SEQ ID NO:
- 10.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said glycoprotein 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% sequence identity, or is preferably identical, to a sequence set forth in any one of SEQ ID NOs:
- 18, 26, 34, 42, 50, and 58.
The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a glycoprotein, wherein said nucleic acid comprises a sequence that 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 is preferably identical, to a sequence set forth in SEQ ID NOs:
- 17, 25, 33, 41, 49, and 57.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein 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%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or is preferably identical, to a sequence set forth in any one of SEQ ID NOs:
- 24, 32, 40, 48, 56, and 64.
The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the following mutations:
- Lys 253→Arg;
- Lys 1512→Met;
- Lys 1513→Glu;
- Ser 1758→Phe;
- Phe 1995→Ser;
- Ile 2094→Val;
- Lys 2115→Glu;
- Thr 2141→Ala;
- Arg 2175→Lys;
- Thr 2185→Ala as compared to the wild type L-protein sequence set forth in SEQ ID NO:
- 16.
The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a L-protein, wherein said L-protein comprises one of the following sets of mutations:
- (a) Ser 1758→Phe;
- (b) Phe 1995→Ser;
- optionally Ile 2094→Val;
- and optionally Thr 2141→Ala;
- (c) Lys 1513→Glu;
- Phe 1995→Ser;
- and optionally Arg 2175→Lys;
- (d) Lys 253→Arg;
- Lys 1512→Met;
- Lys 2115→Glu;
- optionally Thr 2141→Ala;
- Thr 2185→Ala
- (e) Phe 1995→Ser;
- or
- (f) Lys 2115→Glu,
- as compared to the wild type L-protein sequence set forth in SEQ ID NO:
- 16.
The mutant of any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an L-protein, wherein said nucleic acid is complementary to a sequence that 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%, preferably at least about 99.8%, preferably at least about 99.9% sequence identity, or that is preferably identical, to a sequence set forth in SEQ ID NOs:
- 17, 25, 33, 41, 49, and 57.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an nucleoprotein, wherein said nucleoprotein 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 that is preferably identical, to a nucleoprotein set forth in any one of SEQ ID NOs:
- 12, 20, 28, 36, 44, 52, and 60.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding an nucleoprotein, wherein said nucleic acid is complementary to a sequence that 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 that is preferably identical, to a nucleoprotein set forth in any one of SEQ ID NOs:
- 11, 19, 27, 35, 43, 51, and 59.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a Z-protein, wherein said Z-protein 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 that is preferably identical, to a sequence set forth in any one of SEQ ID NOs:
- 14, 22, 30, 38, 46, 54, and 62.
The mutant any one of the preceding claims, wherein the mutant comprises a nucleic acid encoding a Z-protein, wherein said nucleic acid comprises a sequence that 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 that is identical, to a sequence set forth in any one of SEQ ID NOs:
- 13, 21, 29, 37, 45, 53, and 61.
A method of producing an antitumoral arenavirus, in particular an arenavirus, which has improved antitumor properties over an original arenavirus, the process comprising the following steps:
- a) plating primary tumor cells or cells of the cell line H1975, C643 or Tramp-C2 onto and/or into a nutrient medium,
- b) inoculating the plated primary tumor cells or plated cells of the cell line H1975, C643 or Tramp-C2 with an original arenavirus,
- c) incubating the inoculated primary tumor cells or inoculated cells of the cell line H1975, C643 or Tramp-C2 under conditions which are suitable for infecting at least a portion of the inoculated primary tumor cells or the inoculated cells of the cell line H1975, C643 or Tramp-C2 with the original arenavirus,
- d) extracting an arenavirus-containing cell culture supernatant from a cell culture containing the incubated primary tumor cells or the incubated cells of the cell line H1975, C643 or Tramp-C2,
- wherein the step sequence a) to d) is repeated a plurality of times, wherein the primary tumor cells or the cells of the cell line H1975, C643 or Tramp-C2 when performing the first repetition of step sequence a) to d), for performing step b) is inoculated with the arenavirus-containing cell culture supernatant or a part thereof extracted when performing step d) before the first repetition of the step sequence a) to d), and wherein the primary tumor cells or the cells of cell line H1975, C643 or Tramp-C2 when performing each further repetition of the step sequence a) to d), for performing step b) is inoculated with the arenavirus-containing cell culture supernatant or a part thereof extracted when performing step d) of a previous repetition of the step sequence a) to d).
A lymphocytic choriomeningitis virus mutant comprising
- a glycoprotein with at least one mutation, wherein the at least one mutation is an amino acid substitution of the isoleucine at position 181 of the glycoprotein by another amino acid, preferably methionine, and/or an amino acid substitution of the arginine at position 185 of the glycoprotein by another amino acid, preferably tryptophan, and/or
- an L-protein having at least one mutation, wherein the at least one mutation is an amino acid substitution of the lysine at position 1513 of the L-protein by another amino acid, preferably glutamate, and/or an amino acid substitution of the phenylalanine at position 1995 of the L-protein by another amino acid, preferably serine, and/or an amino acid substitution of isoleucine at position 2094 of the L-protein by another amino acid, preferably valine, and/or an amino acid substitution of threonine at position 2141 of the L-protein by another amino acid, preferably alanine, and/or an amino acid substitution of arginine at position 2175 of the L-protein by another amino acid, preferably lysine.
An isolated protein or peptide, in particular glycoprotein, L-protein, nucleoprotein, or Z-protein, comprising or consisting of an amino acid sequence according to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 10, SEQ ID NO:
- 18, SEQ ID NO:
- 26, SEQ ID NO:
- 34, SEQ ID NO:
- 42, SEQ ID NO:
- 50, SEQ ID NO:
- 58, SEQ ID NO:
- 24, SEQ ID NO:
- 32, SEQ ID NO:
- 40, SEQ ID NO:
- 48, SEQ ID NO:
- 56, or SEQ ID NO:
- 64, SEQ ID NO:
- 12, SEQ ID NO:
- 20, SEQ ID NO:
- 28, SEQ ID NO:
- 36, SEQ ID NO:
- 44, SEQ ID NO:
- 52, SEQ ID NO:
- 60, SEQ ID NO:
- 14, SEQ ID NO:
- 22, SEQ ID NO:
- 30, SEQ ID NO:
- 38, SEQ ID NO:
- 46, SEQ ID NO:
- 54, or SEQ ID NO:
- 62.
An isolated nucleic acid, in particular encoding for a glycoprotein, L-protein, nucleoprotein, or Z-protein, wherein the nucleic acid has a nucleic acid sequence or consists of a nucleic acid sequence which is or is complementary to a nucleic acid sequence according to SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6 or SEQ ID NO:
- 8, SEQ ID NO:
- 9, SEQ ID NO:
- 17, SEQ ID NO:
- 25, SEQ ID NO:
- 33, SEQ ID NO:
- 41, SEQ ID NO:
- 49, SEQ ID NO:
- 57, SEQ ID NO:
- 23, SEQ ID NO:
- 31, SEQ ID NO:
- 39, SEQ ID NO:
- 47, SEQ ID NO:
- 55, SEQ ID NO:
- 63, SEQ ID NO:
- 11, SEQ ID NO:
- 19, SEQ ID NO:
- 27, SEQ ID NO:
- 35, SEQ ID NO:
- 43, SEQ ID NO:
- 51, SEQ ID NO:
- 59, SEQ ID NO:
- 13, SEQ ID NO:
- 21, SEQ ID NO:
- 29, SEQ ID NO:
- 37, SEQ ID NO:
- 45, SEQ ID NO:
- 53, or SEQ ID NO:
- 61.
Dependencies are inferred from the claim text.