MEASLES VIRUS ENCODING A TUMOR ANTIGEN

Abstract

The present invention relates to a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii). The present invention further relates to a polynucleotide encoding said recombinant virus of the family Paramyxoviridae and to a host cell comprising said recombinant virus of the family Paramyxoviridae and/or said polynucleotide encoding said recombinant virus of the family Paramyxoviridae. Moreover, the present invention relates to a method for activating immune cells with antitumor activity in a sample comprising cancer cells and to further means, methods, and uses related to the present invention.

Claims

1-42. (canceled)

43. A replication competent recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (a) the tumor antigen L-dopachrome-tautomerase (TRP2), (b) a fragment of the tumor antigen TRP2 comprising at least one antigenic epitope of TRP2, and (c) a variant of (a) or (b), wherein the variant is 90% identical to the sequence of TRP2 depicted in SEQ ID NO: 2.

44. The recombinant virus of the family Paramyxoviridae of claim 43, wherein said fragment of the tumor antigen TRP2 comprising at least one antigenic epitope of TRP2 comprises a sequence of from 7 to 15 contiguous amino acids.

45. The recombinant virus of the family Paramyxoviridae of claim 43, wherein TRP2 is human TRP2.

46. The recombinant virus of the family Paramyxoviridae of claim 43, wherein said recombinant virus is a recombinant Morbillivirus.

47. The recombinant virus of the family Paramyxoviridae of claim 46, wherein said recombinant Morbillivirus is a recombinant measles virus (MV).

48. The recombinant virus of the family Paramyxoviridae of claim 43, further comprising at least one expressible polynucleotide encoding a further activator of the immune response.

49. The recombinant virus of the family Paramyxoviridae of claim 48, wherein said at least one expressible polynucleotide encoding a further activator of the immune response is comprised in a polynucleotide encoding the recombinant virus of the family Paramyxoviridae.

50. A polynucleotide encoding the replication competent recombinant virus of claim 43.

51. A host cell comprising the replication competent recombinant virus of claim 43.

52. A method for activating immune cells with antitumor activity in a sample comprising cancer cells and immune cells, comprising: a) contacting said sample comprising cancer cells and immune cells with (i) the replication competent recombinant virus of the family Paramyxoviridae of claim 43; (ii) a polynucleotide encoding the recombinant virus of the family Paramyxoviridae of a)(i); (iii) a host cell comprising the recombinant virus of the family Paramyxoviridae of a)(i); (iv) a host cell comprising the polynucleotide of a)(ii); or (v) any combination of a)(i) to a)(iv); and thereby, b) activating immune cells with antitumor activity comprised in said sample.

53. A preparation of activated immune cells with antitumor activity obtained or obtainable by the method of claim 52.

54. A method for treating inappropriate cell proliferation comprising administering to a subject in need thereof: (i) the replication competent recombinant virus of the family Paramyxoviridae of claim 43; (ii) a polynucleotide encoding the replication competent recombinant virus of the family Paramyxoviridae of (i); (iii) a host cell comprising the replication competent recombinant virus of the family Paramyxoviridae of (i); (iv) a host cell comprising the polynucleotide of (ii); or (v) any combination of (i) to (iv).

55. The method of claim 54, wherein said inappropriate cell proliferation is cancer.

56. A kit comprising (i) the replication competent recombinant virus of the family Paramyxoviridae of claim 43; (ii) a polynucleotide encoding the replication competent recombinant virus of the family Paramyxoviridae of (i); (iii) a host cell comprising the replication competent recombinant virus of the family Paramyxoviridae of (i); (iv) a host cell comprising the polynucleotide of (ii); or (v) any combination of (i) to (iv); housed in a container.

57. A host cell comprising the polynucleotide according to claim 51.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0163] FIGS. 1A-1C: FIG. 1A) Schematic representation of the recombinant MV genome. cDNA encoding trp2 (1667 bp) was inserted into the MeV Schwarz genome into an additional transcription unit downstream of the hemagglutinin open reading frame. FIG. 1B) Upper panel: growth of measles virus constructs (MV-trp2 or MV (control)) on Vero cells; x-axis: time (hours), y-axis: titer (cell infectious units (ciu)/ml); lower panel: viability of host cells over the course of MV-trp2 and MV (control) replication, as compared to a mock-infected culture; x-axis: Time (hours), y-axis: relative viability compared to mock infected culture. FIG. 1C) Expression of trp2 by MV-trp2, but not by MV; upper panel: RT-PCR with trp2-specific primers; lower panel: immunoblot with anti-trp2 antibodies.

[0164] FIG. 2: IFN-gamma ELISPOT analysis of co-cultures described in Example 2; upper panels: photographs of culture dishes after ELISPOT assay; lower graph: quantification of the spots in culture dishes as shown in the upper panels.

[0165] FIG. 3: Eliciting anti-tumor immunity in mice by intratumoral injection of MV-trp2 (Example 3); upper panel: Treatment schedule; lower left panel: quantification of results of an IFN-gamma ELISPOT analysis using splenocytes of mice from different treatment groups in co-culture with MC38-hCD46 cells infected with MV-trp2 or parental MV; lower right panel: quantification of results of an IFN-gamma ELISPOT analysis using splenocytes of mice from different treatment groups in co-culture with B16 cells.

[0166] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1

[0167] Recombinant measles viruses encoding the tumor-associated antigen (TAA) trp2 have been generated according to known methods (MV-trp2, FIG. 1A).

[0168] Vero cells were transduced with parental MV or MV encoding trp2 with a multiplicity of infection (MOI)=3. At designated time points, cells were harvested and progeny viral particles were determined in titration assays by serial dilution. Insertion of the tumor-associated antigen does not impair viral replication and cytotoxic effects (FIG. 1B, upper panel). At designated time points, cell viability was determined using a colorimetric XTT assay (FIG. 1B, lower panel). Mock treated cells were used as a reference for viability=1.0.

[0169] RNA was extracted from Vero cells infected with parental MV or MV encoding trp2. cDNA synthesis was performed using oligo-dT primers. PCR was performed with trp2-specific primers. Cells infected with parental MV or MV encoding trp2 were lysed in RIPA buffer for protein extraction. After SDS PAGE of cell lysates, Western blot analysis was performed with trp2-specific antisera. Beta-actin was detected as a loading control. A melanosome preparation was used as a positive control. Tumor-antigen expression in infected cells was confirmed both on the mRNA- and protein level (FIG. 1C).

Example 2

[0170] Trp2-specific T cells were co-cultured with MC38-hCD46 cells infected with MV-trp2 or parental MV or with B16 cells or stimulated with the immunodominant trp2 peptide. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. MV encoding the trp2 tumor antigen (TAA) activate TAA-specific T cells: Co-culture of trp2-specific T cells with murine cells infected with MV-trp2 leads to significantly higher IFN-gamma secretion than co-culture with parental MV (FIG. 2).

Example 3

[0171] 1×10.sup.6 B16ova hCD46 cells were implanted into the flank of C57BL/6J mice (day 0). Starting on day 10, mice received intratumoral injections of 1×10.sup.6 cell infectious units (ciu) of MV-trp2, 1×10.sup.6 ciu of parental MV in a total volume of 1004 or 1004 carrier fluid (mock). Mice were sacrificed on day 23 and spleens were extracted for ELISPOT analysis.

[0172] Splenocytes of mice from different treatment groups were co-cultured with MC38-hCD46 cells infected with MV-trp2 or parental MV. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. Splenocytes of mice from different treatment groups were co-cultured with B16 cells. After 16 hours of co-culture, IFN-gamma ELISPOT analysis was performed. It was found that intratumoral injection of MV-trp2 can enhance TAA-specific and tumor-specific immunity (FIG. 3).

[0173] Thus, in the present invention, an oncolytic MV vaccine was generated to elicit a tumour antigen-specific immune response. With this approach, the MV vector encoded tumour antigen is expressed in the context of viral danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) which serve as “natural adjuvants”. Furthermore, MV-mediated oncolysis functions as an in situ tumour vaccine: MV infection causes immunogenic cell death within the tumour accompanied by release of tumour-associated antigens, resulting in efficient antigen cross-presentation and epitope spread