RNA viruses for immunovirotherapy

Abstract

The present invention relates to a recombinant virus of the family Paramyxoviridae, comprising at least one expressible polynucleotide encoding a secreted activator of the immune response, to a polynucleotide encoding the same, and to a kit comprising the same. Moreover, the present invention relates to a method for treating cancer in a subject afflicted with cancer, comprising contacting said subject with a recombinant virus of the family Paramyxoviridae of the invention, and thereby, treating cancer in a subject afflicted with cancer.

Claims

1. A polynucleotide encoding a recombinant virus of the family Paramyxoviridae, the polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 6, 7, 8, and/or 9, wherein the recombinant virus comprises at least one expressible polynucleotide encoding a secreted activator of the immune response.

2. The polynucleotide of claim 1, wherein said recombinant virus is a recombinant Morbillivirus.

3. The polynucleotide of claim 2, wherein said recombinant Morbillivirus is a recombinant measles virus (MV).

4. The polynucleotide of claim 3, wherein the recombinant MV is derived from vaccine strain Schwarz (Edmonston A).

5. The polynucleotide of claim 1, wherein the secreted activator of the immune response is a ligand for an immune checkpoint blockade protein.

6. The polynucleotide of claim 1, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against CTLA 4.

7. The polynucleotide of claim 6, wherein the secreted antagonistic single-chain antibody against CTLA-4 comprises the amino acid sequence of SEQ ID NO:1.

8. The polynucleotide of claim 1, wherein the secreted activator of the immune response is a secreted antagonistic single-chain antibody against PD-L1.

9. The polynucleotide of claim 8, wherein the secreted antagonistic single-chain antibody against PD-L1 comprises the amino acid sequence of SEQ ID NO:3.

10. The polynucleotide of claim 1, further comprising a second expressible polynucleotide encoding a second secreted activator of the immune response.

11. The polynucleotide of claim 10, wherein said second expressible polynucleotide encoding a secreted activator of the immune response is a cytokine or a second antagonist of an inhibitory factor of a T-cell or an antagonist of a negative immune regulator of the tumor-immune microenvironment.

12. A method for treating cancer in a subject afflicted with cancer, comprising a) contacting said subject with the polynucleotide according to claim 1, and b) thereby, treating cancer in a subject afflicted with cancer.

13. The method of claim 12, wherein said cancer is a solid cancer, a metastasis, or a relapse thereof.

14. The method of claim 12, wherein treating cancer is reducing tumor burden.

15. The method of claim 12, wherein said cancer is malignant melanoma, head and neck cancer, hepatocellular carcinoma, pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastric carcinoma, colorectal carcinoma, lymphomas or leukemias.

16. A kit comprising at least the polynucleotide of claim 1 housed in a container.

17. A medicament comprising the polynucleotide of claim 1, and at least one pharmacologically acceptable excipient.

18. An in vitro method for activating immune cells in a sample comprising cancer cells and immune cells, comprising a) contacting said sample comprising cancer cells and immune cells with the polynucleotide according to claim 1, and b) thereby, activating immune cells comprised in said sample.

Description

FIGURE LEGENDS

(1) FIG. 1 shows a schematic representation of recombinant Measles Virus (MV) genomes. Top panel: MV encoding a secretable antibody (sY) for immune checkpoint modulation bottom panel: MV encoding a secretable antibody for immune checkpoint modulation and a second additional immunomodulatory transgene (X).

(2) FIG. 2 shows a Western Blot of culture supernatants of cells infected with MV-sY variants. These data demonstrate that the encoded antibodies against CTLA-4 and PD-L1, respectively, are synthesized in full-length and secreted. Lane 1: MV-EGFP (control virus expressing EGFP); lane 2: MV H-sCTLA-4; lane 3: MV H-sPD-L1; lane 4: MV H-IgG Fc (control, expressing only the antibody constant region)

(3) FIG. 3 shows an ELISA for the binding of the MV-encoded secretable antibody to its respective antigen. Optical density is given as adsorption values at 450 nm vs. dilutions of culture supernatants of cells infected with the MV-sY variants. These data demonstrate specific recognition of and binding to the cognate antigen (sCTLA-4 to CTLA-4 and sPD-L1 to PD-L1, respectively) without cross-reactions (circle: control). Triangle up: sCTLA-4 to CTLA-4; triangle down: sPD-L1 to PD-L1, respectively, open circle: negative control.

(4) FIG. 4 shows in vitro growth kinetics of recombinant MV-sY variants in an infected human melanoma cell line. Titers of progeny particles are given as infectious units per ml at the indicated time points for each group. These data demonstrate equal kinetics of both variants which are comparable to the control. Triangle up: MV H-sCTLA-4; triangle down: MV H-sPD-L1; diamond: MV H-IgG Fc (control, expressing only the antibody constant region).

(5) FIG. 5 shows an in vitro cytotoxicity assay (XTT) of human melanoma cell line recombinant MV-sY variants. Medium cell viability and standard deviations are given as percentage at the indicated time points for each group (mock treated cells defining 100% viability). These data demonstrate equal potential of both variants to lyse tumor cells. Triangle up: MV H-sCTLA-4; triangle down: MV H-sPD-L1; diamond: MV H-IgG Fc (control, expressing only the antibody constant region).

(6) FIG. 6 shows in vivo anti-tumor activity of the recombinant MV-sY variants against human melanoma in a subcutaneous murine xenograft model. Top panel: tumor volume growth curve (mm.sup.3) of treated animal vs. time after implantation (medium volume and standard deviation per group). Square: MV H-EGFP, triangle up: MV H-sCTLA-4, triangle down: MV H-sPD-L1. Bottom panel: Kaplan-Meier plot showing the fraction of treated animals surviving vs. time after implantation of melanoma cells. In this immunodeficient model, MV encoding sCTLA-4 or sPD-L1 were as efficient as a parental control virus for oncolysis of human melanoma.

(7) FIG. 7 shows therapeutic effects of the recombinant MV-sY variants in an immunocompetent model of murine melanoma. Control virus, diamond; MV HCD20-sCTLA-4, triangle up; MV HCD20-sPD-L1, triangle down. Top panel: tumor volume growth curve (mm.sup.3) of treated animal vs. time after implantation (medium volume and standard deviation per group), bottom panel: Kaplan-Meier plot showing the fraction of treated animals surviving vs. time after implantation of melanoma cells. Treatment with MV-sCTLA-4 as well as with MV-sPD-L1 led to a significant delay of tumor progression, treatment with MV-sPD-L1 led to a significant prolongation of median overall survival.

(8) FIG. 8 shows FACS analyses of murine lymphocytes reflecting in vivo effects of the recombinant MV-sY variants on tumor-infiltrating lymphocytes after treatment of immunocompetent mice bearing syngeneic melanoma tumors. The FACS analyses demonstrate equal downregulation of regulatory T cells (FOXP3.sup.+, left panel) for both variants and a differentiated regulation of cytotoxic T cells (CD8.sup.+, right panel). MV HCD20-IgG Fc (control virus), diamond; MV HCD20-sCTLA-4, triangle up; MV HCD20-sPD-L1, triangle down. Mock treated animals received carrier fluid only (circle).

(9) The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1. GENERATION OF RECOMBINANT MEASLES VIRUSES

(10) Construction of Recombinant MV Genomes in DNA Plasmids

(11) The genome of the measles vaccine strain Schwarz (Genbank Acc NO: AF266291.1 GI:9181912) was cloned into a pUC19-based plasmid. For later generation of viral particles from a DNA plasmid in a transfected mammalian host cell line, the 5-end of the MV leader was fused to the CMV minimal promoter, and the 3-end of the MV trailer is followed by the Hepatitis Delta virus ribozyme sequence and a eukaryotic polyA signal (note: with respect to the natural 3.fwdarw.5-orientation of negative-strand ()RNA viruses, the sequence of the DNA copy is annotated in the usual 5 .fwdarw.3-orientation; this corresponds to the viral sequence in antigenomic (+)RNA orientation; the same condition applies for the cloned viral genome with respect to the direction of the CMV promoter-driven transcription through RNA polymerase II). An additional MV-specific transcription unit (ATU) was inserted into the 3-untranslated region (UTR) of the H gene. The H-ATU consists of viral transcription control elementsa copy of gene end signal from the N gene and gene start signal of the P geneand the unique cloning site MauBI for insertion of transgenic open reading frames (ORF).

(12) The coding sequences for the claimed immunomodulatory transgenes were cloned into a mammalian expression vector, providing a secretion signal and a HA-tag at the N-terminus as well as a myc-tag at the C-terminus. The respective ORFs were excised as 5 -MluI 3-AscI fragments and inserted into the MV H-ATU plasmid via the compatible MauBI site, leading to the novel vectors (FIG. 1). Due to technical reasons, in later infection experiments of murine cells, the H protein was replaced by the fully re-targeted HCD20 (Ungerechts et al., Cancer Res. 2007, 67: 10939-10947). Thus, transgenic murine cells expressing CD20 can be infected via re-targeted MV.

(13) Generation and Propagation of Recombinant MV

(14) Recombinant MV particles were generated from cDNA constructs according to Martin et al. (J Virol. 2006; 80: 5708-5715) with slight modifications. Vero cells (510.sup.5 per 6-well) were transfected with 5 g of the recombinant MV plasmid, together with 500 ng N, 100 ng P and 500 ng L expression plasmids using FugeneHD at a ratio of 3:1. Four to six days after transfection, cell culture supernatants were transferred onto fresh cells. To prepare virus stocks, Vero cells (African green monkey, normal kidney) were infected at a MOI of 0.03 and incubated at 37 C. for 36 to 48 hours. Viral particles were harvested by one freeze/thaw cycle and centrifugation from their cellular substrate resuspended in Opti-MEM (Invitrogen). Virus preparations can be further purified by GMP-complying protocols for ultracentrifugation or tangential flow filtration. All following infection experiments were performed with viral stocks from the third passage. Titers were determined by 50% tissue culture infectious dose (TCID.sub.50) titration on Vero cells. For generation and propagation of fully re-targeted viruses, all procedures were done analogously using Vero-His cells (Nakamura et al., Nat Biotechnol 2005; 23: 209-214).

EXAMPLE 2. CHARACTERIZATION OF CLONED SECRETABLE ANTIBODIES

(15) MV-Mediated Expression of Secretable Antibodies

(16) Human melanoma cells Mel888 were seeded into a six-well plate (1.510.sup.5 per well) and infected with variant viruses at MOI of 1. Twenty-four hours after infection, supernatants were collected and passed through a 0.2 m filter. Antibodies were precipitated using Protein A Sepharose and detected by immunoblot with an anti-HA antibody (FIG. 2). Arrows indicate full-length antibodies against CTLA-4 and PD-L1 (60 kDa) and the IgG Fc (30 kDa) domain, respectively. These data demonstrate that the encoded antibodies against CTLA-4 and PD-L1, respectively, are synthesized in full-length and secreted.

(17) Binding of Secretable Antibodies to their Respective Cognate Antigens

(18) Vero cells were seeded in six-well plates (210.sup.5 cells per well) and infected at MOI of 3 with the indicated viruses. 36 hours after infection, cell culture supernatants were collected and passed through a 0.2 m filter. Nunc Maxisorp 96-well plates were coated with 100 ng recombinant protein each of CTLA-4 and PD-L1, respectively. Wells were blocked with FBS and a dilution series of equal volumes of supernatants of cells equally treated and infected with MV H-sY variants were added to the ELISA plates (FIG. 3). After 2 h incubation and washing, the secreted antibodies sCTLA-4 and sPD-L1 were detected with anti-HA-Biotin, HRP-Streptavidin and TMB as substrate. Supernatants from MV H-sCTLA-4 were used as a control for binding to PD-L1 and vice versa (circles). These data demonstrate specific recognition of and binding to the cognate antigen mediated by the respective secretable antibody without cross-reactions.

EXAMPLE 3. GROWTH KINETICS OF THE RECOMBINANT MV IN VITRO

(19) To determine viral growth kinetics in one-step growth curves, human melanoma cells Mel888 were seeded into a six-well plate (110.sup.5 per well) and infected with the indicated MV vectors at an MOI of 3. At designated time points, cells were harvested and progeny viral particles were determined by titration assays (FIG. 4). These data demonstrate equal kinetics of both variants in target melanoma cells and that encoding of secretable full-length antibodies by the MV vector does not impair viral replication.

EXAMPLE 4. CYTOTOXICITY OF THE RECOMBINANT MV IN VITRO

(20) To address the cytolytic effect of MV vectors encoding secretable anti-CTLA-4 and anti-PD-L1 antibodies against human melanoma cells, in vitro infection experiments were performed with Sk-Mel28 and Mel888 cells for qualitative evaluation via microscopic inspection. Syncytia formation on human cell lines was delayed compared to the simian producer cell line Vero. Nevertheless, by 48 hours after infection MV H-sCTLA-4 and MV H-sPD-L1 had spread across the entire cell layer. Cytopathic effects were as pronounced as those caused by the control virus MV-EGFP.

(21) Cytopathic effects of oncolytic MV on human and murine melanoma cell lines were quantified by cell viability assays. Human melanoma cells Mel888 were seeded into a six-well plate (110.sup.5 per well) and infected with the indicated MV vectors at an MOI of 1. At designated time points after infection, cell viability was determined using the colorimetric XTT assay (FIG. 5). Viability of mock treated cells was defined as 100%. Both recombinant MV H-sY variants rapidly lysed melanoma cells, leading to complete cell killing after 48 hours, demonstrating equal potential of both variants to lyse tumor cells.

EXAMPLE 5. IN VIVO ANTI-TUMOR ACTIVITY OF THE RECOMBINANT MV IN A XENOGRAFT MODEL

(22) Oncolytic efficacy of MV expressing secretable antibodies was assessed in a xenograft model of human melanoma (FIG. 6). 510.sup.6 Mel888 cells were implanted subcutaneously into the right flank region of NOD/SCID mice. When tumors reached an average volume of 50 mm.sup.3, mice received intratumoral injections of 210.sup.6 cell infectiuos units (ciu) per dose on five consecutive days applying MV H-EGFP (control virus), MV H-sCTLA-4 or MV H-sPD-L1. Mock treated animals received carrier fluid only. Tumor volumes were determined every third day using a caliper. Mice were sacrificed when tumor volumes exceeded 1500 mm.sup.3 or when tumor ulceration occurred.

(23) MV treatment led to a significant delay in tumor progression (FIG. 6, top panel). On day 19 after implantation (seven days after the last treatment), mock treated mice had a mean tumor volume of 115 l, while the mean tumor volume in mice treated with MV was 25 l (square: MV H-EGFP), 20 l (triangle up: MV H-sCTLA-4) and 21 l (triangle down: MV H-sPD-L1), respectively. Mean tumor volumes and standard error bars for each group are shown.

(24) MV treatment led to a significant survival benefit (FIG. 6, bottom panel). Median overall survival was 24 days for mock controls, whereas all but one of the MV-treated mice survived over 50 days after tumor implantation. Complete tumor remission and long-term survival was observed in 85% of treated animals.

(25) In this immunodeficient model, MV encoding sCTLA-4 or sPD-L1 were both as efficient as a parental control virus for oncolysis of human melanoma.

EXAMPLE 6. IN VIVO THERAPEUTIC EFFECTS IN AN IMMUNOCOMPETENT MURINE MELANOMA MODEL

(26) Therapeutic efficacy of immunovirotherapy in vivo was assessed in a syngeneic immunocompetent murine melanoma model (FIG. 7). 110.sup.6 B16-CD20 cells (transgenic mouse melanoma cell line expressing CD20 ectopically for CD20-targeted MV infection) were implanted subcutaneously into the right flank region of C57BL/6 mice. When tumors reached an average volume of 50 mm.sup.3, mice received intratumoral injections of 210.sup.6 ciu per dose on five consecutive days applying MV HCD20-IgG Fc (control virus, diamond, n=9), MV HCD20-sCTLA-4 (triangle up, n=11) or MV HCD20-sPD-L1 (triangle down, n=11). Mock treated animals received carrier fluid only (circle, n=10).

(27) Treatment with MV expressing the secretable antibody variants led to a delay in tumor progression in both cases (FIG. 7, top panel). Tumor volumes on day 15 after implantation revealed a significantly lower tumor volume in mice treated with MV-sCTLA-4 compared to mock and MV-IgG Fc controls (p<0.0001 and p=0.036 in paired students' t-test, respectively). In case of mice treated with MV-sPD-L1, a subgroup of mice experienced tumor remission.

(28) While reduced tumor volumes at early time points did not prolong overall survival (FIG. 7, bottom panel) in mice treated with MV-sCTLA-4, mice responding to MV-sPD-L1 also survived longer compared to mock and IgG Fc controls (p=0.0047 and p=0.031 in log rank test, respectively).

EXAMPLE 7. IN VIVO EFFECTS OF THE RECOMBINANT MV ON TUMOR-INFILTRATING LYMPHOCYTES

(29) To investigate possible mechanisms of immunomodulatory effects by MV-mediated checkpoint blockade, tumor-infiltrating lymphocytes were characterized by flow cytometry (FIG. 8). Immunomodulatory effects were assessed in a syngeneic immunocompetent murine melanoma model. 110.sup.6 B16-CD20 cells (transgenic mouse melanoma cell line expressing CD20 ectopically for CD20-targeted MV infection) were implanted subcutaneously into the right flank region of C57BL/6 mice. When tumors reached an average volume of 50 mm.sup.3, mice received intratumoral injections of 210.sup.6 ciu per dose on five consecutive days applying MV HCD20-IgG Fc (control virus, diamond), MV HCD20-sCTLA-4 (triangle up) or MV HCD20-sPD-L1 (triangle down). Mock treated animals received carrier fluid only (circle). Twenty-four hours after the last treatment, mice were sacrificed and tumors were explanted. Single-cell suspensions of tumors were stained with antibodies specific for CD45.2, CD3, CD8, CD4 and CD25. Cells were fixed, permeabilized and stained with a FoxP3-specific antibody. Tumor-infiltrating lymphocytes were analyzed by flow cytometry of the stained samples using AriaII and FACS DIVA Software. Left panel: The Abundance of CD3+ CD4+ CD25+ FoxP3+ regulatory T cells as percentage of all CD3+ T cells is shown for each treatment group. Right panel: The Abundance of CD3+ CD8+ cytotoxic T cells as percentage of all T cells is shown for each treatment group.

(30) Treatment with MV H-sCTLA-4 and MV H-sPD-L1 led to a favorable immune profile with lower abundance of negative regulatory T cells and, particularly in the case of MV H-sPD-L1, to a higher abundance of cytotoxic T cells in treated tumors. The data suggest that MV-mediated blockade of CTLA-4 as well as of PD-L1 leads to beneficial reduction of regulatory T cells, whereas only PD-L1 inhibition led to a statistically significant increase in cytotoxic T cells.