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 multispecific binding polypeptide, said multispecific binding polypeptide comprising a first binding domain binding to a surface molecule of an immune cell with antitumor activity, preferably a lymphocyte, more preferably a T cell or a dendritic cell, and a second binding domain binding to a tumor-associated antigen; 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 recombinant virus of the family Paramyxoviridae, comprising at least one expressible polynucleotide encoding a multispecific binding polypeptide, said multispecific binding polypeptide comprising: a) a first binding domain binding to a surface molecule of an immune cell with antitumor activity, and b) a second binding domain binding to a tumor-associated antigen.

2. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said immune cell with antitumor activity is selected from the group consisting of lymphocyte, T cell or and dendritic cell.

3. The recombinant virus of the family Paramyxoviridae of claim 2, wherein said surface molecule of a T cell is selected from the group consisting of CD3, CD2, CD5, CD6, CD9, CD11A, CD25 (IL-2 receptor alpha-chain), CD26, CD28, CD29, CD40L, CD43, CD44, CD45RO, CD45RA, CD45RB, CD47, CD58 (LFA-3), CD69, CD70, CXCR4, CD107a, CD122 (IL-2 receptor beta-chain), CD132 (IL-2 receptor gamma-chain), CD134, CD137 and CD247.

4. The recombinant virus of the family Paramyxoviridae of claim 2, wherein said surface molecule of a T cell is CD3.

5. The recombinant virus of the family Paramyxoviridae of claim 2, wherein said surface molecule of a dendritic cell is selected from the group consisting of CD1a/b, CD11c, CD16a, CD40, CD68, CD80, CD83, CD86, IFNAR1 (interferon-alpha/beta receptor), CD119 (interferon-gamma receptor 1), CDB197 (CCR7), CD205 (DEL-205), CD209 (DC-SIGN) and CD227 (MUC1).

6. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said tumor-associated antigen is selected from the group consisting of androgen receptor (AR), BCL-1, calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial cell adhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogen receptors (mER), FAP, HER2/neu, human high molecular weight melanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52, melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesterone receptor (PGR), prostate specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33.

7. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said tumor-associated antigen is CEA or CD20

8. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said multispecific binding polypeptide is a bispecific binding polypeptide.

9. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said recombinant virus is a recombinant Morbillivirus.

10. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said recombinant virus is a recombinant measles virus (MV).

11. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said first binding domain comprises a single-chain antibody against CD3.

12. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said second binding domain comprises a single-chain antibody against CEA.

13. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said second binding domain comprises a single-chain antibody against CD20.

14. The recombinant virus of the family Paramyxoviridae of claim 1, wherein the at least one expressible polynucleotide encoding a multispecific binding polypeptide is comprised in a polynucleotide encoding the recombinant virus of the family Paramyxoviridae.

15. The recombinant virus of the family Paramyxoviridae of claim 1, wherein said multispecific binding polypeptide further comprises a cytokine.

16. A polynucleotide encoding the recombinant virus of the family Paramyxoviridae according to claim 1.

17. A combined preparation for simultaneous, separate or sequential use comprising at least one virus of the family Paramyxoviridae and at least one multispecific binding polypeptide.

18. An in vitro method for treating 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 a recombinant virus of the family Paramyxoviridae of claim 1, and b) thereby, activating immune cells with antitumor activity comprised in said sample.

19. A method for treating cancer in a subject afflicted with cancer, comprising a) contacting said subject with a recombinant virus of the family Paramyxoviridae according to claim 1, with a polynucleotide encoding said recombinant virus of family Paramyxoviridae or a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:6-9, and b) thereby, treating cancer in a subject afflicted with cancer.

20. The method of claim 19, wherein step a further comprises contacting said subject with a multispecific binding polypeptide.

21. A kit comprising at least the recombinant virus of the family Paramyxoviridae according to claim 1 housed in a container.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1: Schematic of a viral genome of a multispecific binding polypeptide-encoding measles virus (MV-MBP). The 16 kb genomic RNA comprises 6 genes, encoding 8 proteins. This MeV-MBP additionally encodes eGFP upstream of N open reading frame (ORF). The transgene encoding the multispecific binding polypeptide was inserted downstream of the H ORF. The construct comprises approximately 1,600 bp. The exact number of nucleotides is a multiple of six, which is a requirement for measles viruses. Convenient restriction sites were introduced at the indicated positions to easily transfer the construct to different vectors and to exchange the TAA-targeting domain. Both, the variable domains and the scFvs were connected via glycine-serine peptide linkers. The N-terminal HA-tag and C-terminal 6His-tag are useful for detection and purification purposes. The actual multispecific binding polypeptide sequence is preceded by a Kozak sequence to enhance translation of the transgene RNA transcript. In addition, an Ig-chain leader sequence is fused to the N-terminus of the multispecific binding polypeptide, directing its expression to the secretory pathway.

[0103] FIGS. 2a-2c show specific binding of the indicated multispecific binding polypeptides to their respective binding domain targets.

[0104] FIG. 2a shows specific binding of the indicated multispecific binding polypeptides to their respective TAA targets in terms of recombinant human protein in a sandwich ELISA format. Polypeptides with the second binding domain directed to CEA bind the recombinant CEA full length protein (rCEA). The negative controls mock and non-relevant protein (NRP) (recombinant PD-L1 protein) indicate a specific binding to rCEA. Polypeptides with the second binding domain directed to CD20 show a similar binding specificity to rCD20. Multispecific binding polypeptides were detected via anti-HA-tag antibodies.

[0105] FIG. 2b shows FACS analyses demonstrating specific binding of the indicated multispecific binding polypeptides with the first binding domain directed to human CD3 (hCD3) to peripheral blood mononuclear cells (PBMC) isolated from donor blood.

[0106] FIG. 2c shows FACS analyses demonstrating specific binding of the indicated multispecific binding polypeptides with the first binding domain directed to murine CD3 (mCD3) on murine splenocytes. Polypeptides with the first binding domain directed to mCD3 were not found to recognize hCD3 on PBMCs and vice versa, polypeptides with the first binding domain directed to hCD3 were not found to recognize mCD3 on murine splenocytes.

[0107] FIGS. 3a-3c show multispecific binding polypeptides-directed cytotoxicity to target cells mediated by PBMCs.

[0108] FIG. 3a shows specific killing of MC38 cells expressing the TAA-target CEA in the presence of PBMCs and multispecific binding polypeptides with the first binding domain directed to hCD3 and the second binding domain directed to CEA. Controls with a non-target cell line or a multispecific binding polypeptide with the second binding domain directed to an irrelevant TAA show no specific tumor cell lysis.

[0109] FIG. 3b shows cytotoxicity for multispecific binding polypeptides with the second binding domain directed to CD20 in a PBMC concentration-dependent manner.

[0110] FIG. 3c shows cytotoxicity for multispecific binding polypeptides with the second binding domain directed to CD20 in a MBP concentration-dependent manner.

EXAMPLES

[0111] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1: Production of Virus Encoded Recombinant Multispecific Binding Polypeptides

[0112] The amplification of the said pcpNSe-multispecific-binding-polypeptide (pcpNSe-MBP) MV (Edmonston B vaccine strain anti-genome with multispecific binding polypeptide gene downstream of H open reading frame (ORF) FIG. 1) was performed in NEB 10-beta bacteria, grown in LB medium (Carl Roth) containing 100 g/ml ampicillin (Carl Roth). Viral particles were rescued from pcpNSe plasmids and subsequently propagated three times on Vero cells to maximize viral titers. The term rescue of negative-strand RNA viruses is known to the skilled person. Transfection of the plasmids into Vero cells was carried out with FuGENE HD (Promega) according to a standard protocol and cells were incubated at 37 C. for approximately 65 h. When syncytia had formed, virus particles were harvested according to the standard procedure. In brief: supernatant was discarded and cells were scraped into fresh medium. Medium was frozen in liquid nitrogen and thawed once, vortexed and centrifuged. Supernatant containing the viral particles was aliquoted and stored at 80 C. For the production of the multispecific binding polypeptides, 510.sup.6 Vero cells were seeded in 15 cm dishes and infected with an MOI of 0.03 in 10 ml OptiPRO SFM serum-free medium (Gibco, Invitrogen). Cells were kept at 37 C. for approximately 40 h and then transferred to 32 C. for additional 20 to 25 h. Supernatants were transferred to 50 ml tubes and centrifuged at 2,000g, 4 C. for 10 min. Supernatants were passed through a 0.22 m filter (Merck) and multispecific binding polypeptides were purified via the C-terminal 6His-tag by affinity chromatography according to standard protocol (Qiagen). His-tagged multispecific binding polypeptides were eluted with 500 mM imidazole and subsequently desalted using centrifugal filters (Amicon, Merck).

Example 2: Characterization of Binding Specificity of Recombinant Multispecific Binding Polypeptides

[0113] Specific binding of the multispecific binding polypeptide to human and murine CD3 and their respective TAA-targets was assessed via FACS analysis and sandwich ELISA, respectively.

[0114] (A) ELISA: 96-well plates (Nunc Maxisorp, Thermo Fisher) were coated with 100 l recombinant human full length CEA (AbD Serotec) or CD20 (Abnova) in PBS [1 g/ml] and kept at 4 C. for at least 16 h. Wells were washed twice with 200 l PBS and blocked with 200 L blocking buffer (PBS supplemented with 5% FCS and 0.05% Tween20 (Biotium)) for 2 h at room temperature. Subsequently wells were washed three times with PBS and incubated with 100 l sample per well for 2 h at room temperature. Wells were washed four times with PBS-T (PBS supplemented with 0.05% Tween20) and twice with PBS. High affinity anti-HA-biotin antibody (clone BMG-3F10, Roche) was diluted in blocking buffer (1:500). 100 L antibody solution was added to each well and incubated for 45 min at room temperature. Wells were washed five times with PBS-T and twice with PBS. Streptavidin-horseradish peroxidase (Dianova) was diluted in blocking buffer (1:500) and 100 L streptavidin solution was added to each well and incubated for 10 min at room temperature. Wells were washed seven times with PBS-T and twice with PBS. 100 l substrate (1-Step Ultra TMB-ELISA, Thermo Fisher) was added to each well and incubated for 3 to 30 min at room temperature. The reaction was stopped with 100 l 2N sulfuric acid per well. Absorbance was measured at 450 nm using a microplate reader (Infinite M200 Pro).

[0115] With the described ELISA procedure, said multispecific binding polypeptides with the second binding domain directed to either CEA or CD20 showed specific binding to their respective antigens (FIG. 2a). The negative controls mock and non-relevant protein (NRP) (recombinant PD-L1 protein) indicate that binding to the respective antigens occurs in a specific manner.

[0116] (B) FACS: Using flow cytometry, cells were discriminated based on their size, structure and surface-expression of particular molecules. We detected cell-bound multispecific binding polypeptides via the C-terminal 6His-tag and anti-His-tag-FITC antibody (DIA920, Dianova, 10% in 50 l). Therefore, we labeled 510.sup.5 human PBMCs from donor blood or murine splenocytes with multispecific binding polypeptides in FACS buffer (PBS supplemented with 1 FCS and 0.05% sodium azide, 10% in 50 l, 30 min on ice). To reduce unspecific antibody binding during the following staining procedure, Fc receptors present on cells were blocked using Kiovig (Baxter) (for human cells) or mouse BD Fc Block (for mouse CD16/CD32) (5% in 50 l FACS buffer, 5 min on ice). Cells were washed and resuspended in FACS buffer containing antibodies specific for His-tag, CD3, CD4 and CD8 (BD Biosciences, 5% in 50 l). After 30 min on ice, cells were washed with FACS buffer and resuspended in 500 l DAPI [0.2 g/ml]. Cells were washed and resuspended in 200 to 300 l FACS buffer and analyzed using an LSR II system (BD Biosciences).

[0117] FACS analyses demonstrated specific binding of the multispecific binding polypeptides with the first binding domain directed to human CD3 (hCD3) to peripheral blood mononuclear cells (PBMC) isolated from donor blood (FIG. 2b) and to murine CD3 (mCD3) on murine splenocytes (FIG. 2c). Polypeptides with the first binding domain directed to mCD3 were not found to recognize hCD3 on PBMCs and vice versa, polypeptides with the first binding domain directed to hCD3 were not found to recognize mCD3 on murine splenocytes.

Example 3: Induction of T Cell Effector Function in Resting Human and Murine T Cells by Recombinant Multispecific Binding Polypeptides

[0118] We performed lactate dehydrogenase (LDH) release assays to assess the potential of the multispecific binding polypeptides to induce T cell effector functions, directed to specific tumor cells. 510.sup.3 target cells were cocultured with effector T cells at an effector to target cell ratio (E:T ratio) of 50:1 or various E:T ratios of 50:1, 25:1, 12:1, 6:1, 3:1 and 1:1 on 96-well round-bottom plates in 100 l RPMI/well in triplicates. Multispecific binding polypeptides were added to each well at a final concentration of 100 ng/ml or at various concentrations of 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml and 0 pg/ml. Spontaneous release of LDH from target and effector cells were measured separately, as well as maximum LDH release from target cells only using the provided lysis solution. Cells were cocultured for 24 h at 37 C. Subsequently plates were centrifuged for 4 min at 250g and 50 l supernatant was transferred to a 96-well flat-bottom plate. LDH concentration was measured according to the manufacturer's protocol. Tumor-specific T cell-mediated lysis in percent was calculated as:

(experimental releasespontaneous release target cellsspontaneous release effector cells)/(maximum release target cellsspontaneous release target cells)100

[0119] FIGS. 3a to c demonstrate increased target cell-specific T cell effector function in the presence of the respective multispecific binding polypeptides. Tumor cell killing occurred in a T cell- and multispecific binding polypeptide concentration-dependent manner. The specificity controls in FIGS. 3a and 3b demonstrate that neither the binding of the multispecific binding polypeptides to the effector cell alone, nor the coculture with the respective target cell line in the presence of a CD3 binding multispecific binding polypeptide were sufficient to induce T cell effector functions.