Modified oncolytic parvovirus H-1PV with enhanced fitness and superior anticancer activity

Abstract

The present invention relates to a rodent H-1 parvovirus mutant that can propagate and spread through human tumor cells. In particular, the present invention relates to a parvovirus mutant (H-1PV DR) based on wild-type H-1PV but containing a 55-nucleotide repeat motif toward the right end in the presence of a 114-nucleotide internal deletion and the full length of the right-terminal ITR. This mutant exhibits improved anticancer activity. The present invention also relates to pharmaceutical compositions containing such parvovirus mutants and their use for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to a rodent H-1 parvovirus mutant that can propagate and spread through human tumor cells. In particular, the present invention relates to a parvovirus mutant (H-1PV DR) based on wild-type H-1PV but containing a 55-nucleotide repeat motif toward the right end in the presence of a 114-nucleotide internal deletion and the full length of the right-terminal ITR. This mutant exhibits improved anticancer activity. The present invention also relates to antibodies, pharmaceutical compositions, and kits containing such parvovirus mutants, as well as their use for the treatment of cancer. [Background technology]

[0002] H-1 parvovirus (H-1PV) is a widespread, physically minute, and genetically compact virus containing a linear, single-stranded DNA genome of approximately 5 kb encoding only two genes: the nonstructural proteins (NS) involved in replication and viral cytotoxicity, and the structural proteins (VP) that form the capsid (Figure 1A). Viral gene expression is controlled by two promoters, P4 and P38. At its terminus, the viral genome contains a palindromic sequence that forms a hairpin structure, the size of which varies between the left (approximately 120 nucleotides) and right (approximately 248 nucleotides) inverted terminal repeats (ITRs) [1].

[0003] ITRs are essential elements of the parvoviridae family, including adeno-associated virus (AAV), minute virus of mice (MVM), and H-1PV, and are involved in the initiation of viral replication, genome encapsidation, excision from viral DNA replication intermediates, and long-term maintenance of viral genome stability and transgene expression [2-4]. Rolling circle replication is initiated by a replicon-encoded endonuclease that introduces a single-stranded nick at a specific origin sequence, covalently attaches to the 5' end of the DNA at the nick, and provides a 3' hydroxyl group to initiate unidirectional leading-strand synthesis [5]. In heterotelomeric parvoviruses, such as MVM and H-1PV, the left end of the equivalent folded structure cannot be nicked, and terminal resolution is limited to the right end of the genome. Studies in MVM have demonstrated that the virus can amplify its linear single-stranded genome by using the right ITR, which sequentially unfolds and refolds to shuttle replication forks back and forth along the genome, creating continuous multimeric DNA strands. The viral initiator protein NS1 then excises individual genomes from this continuum by nicking specific origins present within the hairpin sequence and resuming synthesis. For nicking to occur, DNA bending proteins from the HMGB family must coordinate interactions between the NS1 complexes bound to each end of the hairpin stem, creating a roughly 30-bp double-helical loop centered on the intervening G-rich region of DNA [6]. Mutant ITRs failed to form loops and nicked even in the presence of NS1 and HMGB, suggesting that they are important for the activity of this hyperactive origin [7]. Because H-1PV shares significant similarity with MVM in its right-most inverted terminal repeat, H-1PV likely adopted the same mechanism as MVM by using the same functional elements during viral replication (Figure 1B) [4, 8, 9].

[0004] Wild-type H-1PV (wt H-1PV) was derived from the pSR19 molecular clone. The right-terminal ITR of the wild-type H-1PV (wt H-1PV) plasmid currently used for virus production in clinical trials is missing and incomplete due to the original vector design for H-1PV production [9]. Nevertheless, the H-1PV genome can replicate even with the missing ITR sequence, leading to the production of H-1PV vectors in transfection experiments and further propagation in NB-324K cells.

[0005] The oncolytic virus (OV) agent ParvOryx® utilizes the wild-type parvovirus H-1PV, which belongs to the Parvoviridae family, Parvovirinae subfamily, and Protoparvovirus genus [1]. Preclinical proof-of-concept studies in various cultured cell lines, animals

[20] , and xenograft models of several human tumor types have demonstrated its oncolytic and tumor-suppressive properties [9]. Recently, a Phase I / IIa clinical trial of ParvOryx® in patients with recurrent glioblastoma or metastatic pancreatic cancer demonstrated that wt H-1PV treatment was safe, well-tolerated, and exhibited anticancer activity [22, 23]. Of note, there are functional elements absent from the clinical trial wt H-1PV construct, which may have adverse effects on the construct's viral growth in either the virus-producing NB-324K cell line or human tumor cells. Therefore, wt H-1PV may be improved to circumvent these shortcomings. Summary of the Invention [Problem to be solved by the invention]

[0006] It is therefore an object of the present invention to provide H-1PV constructs with enhanced viral fitness and anti-cancer efficacy. [Means for solving the problem]

[0007] This technical problem is solved by providing the embodiments characterized in the independent claims. Preferred embodiments are the subject of the dependent claims.

[0008] The present invention relates to a protoparvovirus (PV) H-1PV DR that is based on wild-type H-1PV but contains a 55-nucleotide repeat motif towards the right end in the presence of an internal deletion of 114 nucleotides and the full length of the right-end ITR, which exhibits improved anticancer activity.

[0009] Animal viruses have a remarkable ability to adapt to new hosts and environments

[14] . Propagation of natural rat parvoviruses, such as standard H-1PV (st H-1PV), in human cells often results in the production of mutant particles with altered genomes [15-18]. Among the H-1PV mutant viruses analyzed to date, H-1 dr [with an internal deletion (d) and terminal repeats (r)] is fully viable. H-1 dr exhibits an in-frame 114-nucleotide deletion (nt 2022-2135) in the open reading frame encoding the nonstructural proteins NS1 and NS2, and a 55-nucleotide duplication (nt 4828-4883) toward the right terminal end of the viral genome (Figure 1D).

[0010] We found that the H-1 dr exhibited enhanced fitness after natural adaptation in human cells, resulting in significant anticancer activity. Therefore, we constructed a novel infectious recombinant molecular clone based on the characteristic genetic alterations in H-1 dr, which we named H-1PV DR.

[0011] Thus, the present invention describes an innovative proparvovirus H-1PV DR characterized by an internal deletion of 114 nucleotides and a 55 nucleotide repeat motif towards the right terminal end in the presence of the full length of the right terminal ITR, which shows improved anticancer activity in different cell lines derived from various tumor entities without altering the safety profile of the virus, which guarantees the clinical application of the novel virus to cancer patients.

[0012] Those skilled in the art can arrive at a parvovirus mutant according to the present invention by subjecting a starting strain to the introduction of the aforementioned modifications, resulting in the desired modification of biological properties, i.e., the ability of the parvovirus mutant to propagate and spread, and thereby kill, human tumor cells, starting from the known nucleic acid and amino acid sequences of the nonstructural proteins of parvoviruses, for example, from parvovirus H-1 [9]. Those skilled in the art can also easily test whether a particular mutant exhibits the desired biological properties listed by using the assays described in the examples below.

[0013] In a proof-of-concept study, a molecular clone of pst H-1PV (Figure 1A) was generated by inserting the full-length right-terminal ITR based on the H1-PV derived from the pSR19 isolate [9], and the pH-1PV DR (Figure 1D) was generated by deleting 114 nucleotides within the NS region and inserting a 55-nucleotide repeat motif toward the right terminal based on the pst H-1PV plasmid. To investigate whether the above modifications to the viral genome were compatible with the viral life cycle and fitness, virus production was performed as described in Example 2 below.

[0014] We investigated the production and infectivity of the H-1PV DR in comparison with that of wild-type (wt) H-1PV and standard (st) H-1PV. To this end, virus was produced and harvested by infection of NB-324K cells, as described in detail in Example 3 below. In these experiments, the H-1PV DR exhibited the best virus fitness in NB-324K cells, consistent with previous observations [9].

[0015] The enhanced fitness and increased infectivity of the H-1PV DR relative to wt H-1PV virions motivated us to investigate the efficiency of viral spread in NB-324K cells during viral propagation, as described in Example 3 below. The results demonstrated that the H-1PV DR promoted more efficient viral spread during the propagation process than wt H-1PV, suggesting that the novel features of the H-1PV DR, including its internal deletion and terminal repeats, may play a crucial role in stimulating the viral life cycle. The morphologies of the various viruses were examined by electron microscopy. Consistent with wt H-1PV, the H-1PV DR and st H-1PV viruses exhibited the characteristic parvoviral diameter of 25 nm and the same morphology at high resolution.

[0016] For biosafety reasons, especially for future clinical applications of the H-1PV DR, it is worthwhile to examine the stability of either the internal deletion or the terminal repeat of the H-1PV DR through successive rounds of infection in permissive NB-324K cells. To this end, seven successive rounds of infection were performed. At the end of each round of infection, viral genomic DNA was isolated, and genome stability was examined by PCR, as described in Example 4 below. No degradation or alteration of the H-1PV DR within the viral genome was observed after seven rounds of infection, indicating that both modified alterations were stably integrated into the viral genome. Further sequencing of the viral genome covering the modified regions confirmed the same observation. These results are consistent with the notion that the H-1PV DR is characterized by long-term maintenance of genome stability in human cells.

[0017] The more stable viral genome stability of the H-1PV DR in permissive transformed human cells provides insight into its immune and cytotoxic effects on normal human cells, peripheral blood mononuclear cells (PBMCs) from healthy donors. These experiments are described in detail in Example 5 below. It was shown that the virus does not affect immune cell populations or the viability of PBMCs, suggesting that PBMCs are not susceptible to the cytotoxic effects of the H-1PV DR and st H-1PV, which behaves similarly to wt H-1PV, which demonstrated complete safety and well-tolerated therapy in two recently completed clinical trials [22, 23].

[0018] Although the H-1PV DR showed identical capsid assembly patterns by electron microscopy, we examined whether the viral genome modifications altered neutralizing antibody recognition of the H-1PV DR at the molecular biological level. To this end, we performed a neutralization assay using specific neutralizing antibodies against the H-1PV DR using a cytotoxicity protection assay, as described in Example 6 below. The results showed similar profiles of neutralizing antibody recognition of the H-1PV DR on the capsid surface compared with wild-type H-1PV and st H-1PV, suggesting that the novel features of the internal deletions and terminal repeats of the H-1PV DR do not affect the viral capsid assembly pattern.

[0019] Since phase I / IIa clinical trials in patients were conducted using wt H-1PV (ParvOryx®) in both recurrent glioblastoma and metastatic pancreatic cancer, the applicant compared the anticancer effects of wt H-1PV and H-1PV DR to demonstrate the clinical significance of this novel virus for a new cancer treatment strategy. As mentioned above, the H-1PV DR was shown to have enhanced fitness and increased infectivity in NB-324K cells compared with wt H-1PV virions. This raised the question of whether the remarkable potency of the novel H-1PV DR due to the artificial expression of SV40 antigens in NB-324K cells is cell line dependent

[25] . Therefore, as described in Example 7 below, the production of H-1PV DR and wt H-1PV was examined in different permissive human cell lines derived from various tumor entities. Based on these experiments, the improved fitness of the H-1PV DR was found to be independent of SV40 antigen expression in transformed cells, indicating that the H-1PV DR possesses advantages for viral growth in cancer cells derived from various tumor entities, resulting in superior anticancer efficacy compared with wt H-1PV.

[0020] The enhanced viral production of the H-1PV DR, which has potent oncolytic activity in various permissive cancer cell lines, motivated us to explore the oncotoxic efficacy of the H-1PV DR in cancer cells with different permissiveness. To this end, we infected low-permissive, semi-resistant, and resistant cell lines with increasing amounts of virus, as described in Example 8 below. The enhanced oncotoxicity of the H-1PV DR was confirmed. However, it is noteworthy that viral entry is a prerequisite for the production of progeny viruses that cause oncotoxicity in cancer cells. This may explain the decrease in oncolytic activity from low-permissive cells to resistant cells. Importantly, the H-1PV DR not only inhibited cell proliferation in 2D cell cultures, but also in 3D spheroids, a highly translationally relevant model characterized by the heterogeneity of cancer cells and highly relevant to tumor biology. See Example 9 below. It can be observed that spheroids treated with the H-1PV DR exhibited slower increases in cancer cell size compared to spheroids treated with wild-type H-1PV. Interestingly, cancer cells treated with H-1PV DR showed almost stagnant spheroid growth over 14 days, suggesting that in addition to growth inhibition, there may also be an element of cell death. Altogether, these results provide proof-of-concept that H-1PV DR has superior anticancer activity to wild-type H-1PV, warranting the clinical application of this novel virus to cancer patients.

[0021] In summary, the present invention improved the viral fitness of H-1PV, including more efficient production and increased infectivity, thereby enhancing its anticancer efficacy. Notably, the virus with internal deletions and terminal repeats (H-1PV DR) of the present invention showed improved fitness, along with increased progeny production and enhanced infectivity, compared to st H-1PV and wt H-1PV. Importantly, the H-1PV DR exhibited superior oncolytic activity in different cell lines derived from various tumor entities. Verification of the therapeutic efficacy of the H-1PV DR in a highly translationally relevant 3D spheroid model confirmed the improved anticancer activity of the H-1PV DR and suggested future clinical studies of the novel virus. In conclusion, this is the first time that the H-1PV DR has been demonstrated to enhance the oncolytic activity of the virus without altering its safety profile, ensuring the clinical application of the novel virus to cancer patients. Furthermore, in order to achieve a personalized medical approach for H-1PV DR viral therapy, the feasibility of using cancer patient-derived organoids as a platform for predicting the response of individual patients to the tumor toxicity of H-1PV DR was investigated in this invention.This is the first time that the establishment of a platform using patient-derived organoids can predict the response of specific cancers to H-1PV DR treatment.

[0022] The present invention further relates to antibodies that specifically recognize the polypeptide regions of the parvovirus mutants described above, i.e., parvovirus mutants with deletions at positions that characterize the parvovirus mutants. The antibodies can be monoclonal, polyclonal, or synthetic antibodies or fragments thereof, such as Fab, Fv, or scFV fragments. Monoclonal antibodies are preferred. For production, animals, particularly rabbits or chickens for polyclonal antibodies and mice for monoclonal antibodies, are preferably immunized with the parvovirus mutants or fragments thereof. Further boosting of the animals can be performed with the same parvovirus mutants or fragments thereof. Polyclonal antibodies can then be obtained from the animal serum and egg yolk, respectively. Monoclonal antibodies can be obtained according to standard methods, see in particular the methods of Kohler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Enzymol. 73 (1981), 3). In this case, mouse myeloma cells are fused with spleen cells from the immunized animal. The antibody according to the present invention can be used in many ways, for example, for immunoprecipitation or isolation of the above-mentioned parvovirus mutants. The antibody can be used in a liquid-phase immunoassay or bound to a solid support. In this regard, the antibody can be labeled in various ways. Those skilled in the art are familiar with suitable markers and labeling methods. Examples of immunoassays are ELISA and RIA.

[0023] A kit for application of the present invention is also provided, which comprises: (a) a parvovirus mutant according to the present invention; (b) an antibody according to the invention; and / or optionally, (c) conventional auxiliary agents such as solvents, buffers, carriers, markers and controls;

[0024] The present invention also relates to a pharmaceutical composition comprising an effective amount of an antibody against a parvovirus (H-1PV DR) and / or a variant thereof in combination with a pharmaceutically acceptable carrier. The present invention also provides the use of said pharmaceutical combination for treating or preventing cancer. The present invention also relates to (a) the use of H-1PV DR for the preparation of a pharmaceutical composition, combination, or kit for treating or preventing cancer.

[0025] The term "treat" and its derivatives, as used herein, refers to therapeutic therapy. With respect to a particular condition, treatment means: (1) ameliorating the disease state or one or more biological manifestations of the disease state; (2) interfering with (a) one or more points in the biological cascade that causes or contributes to the disease state or (b) one or more biological manifestations of the disease state; (3) alleviating one or more symptoms, effects, or side effects associated with the disease state; or (4) slowing the progression of the disease state or one or more biological manifestations of the disease state.

[0026] As used herein, "prevention" is understood to refer to the prophylactic administration of a drug to substantially reduce the likelihood or severity of a condition or its biological manifestations, or to delay the onset of such a condition or its biological manifestations. Those skilled in the art will understand that "prevention" is not an absolute term. Preventive treatment is appropriate, for example, when a subject is considered to be at high risk of developing cancer, for example, when the subject has a strong family history of cancer, or when the subject has been exposed to a carcinogen.

[0027] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or medical response in a tissue, system, animal, or human that is desired, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" refers to any amount that results in the treatment, cure, prevention, or amelioration of a disease, disorder, or side effect compared to a corresponding subject who has not received such amount. This term also includes within its scope an amount effective to enhance normal physiological function. "Effective doses" useful for treating and / or preventing these diseases or disorders can be determined using methods known to those skilled in the art.

[0028] "Pharmaceutically acceptable" is meant to encompass any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to a subject in an effective dose. Additional pharmaceutically compatible carriers can include gels, bioabsorbable matrix materials, implantable elements containing therapeutic agents, or any other suitable vehicle, delivery or distribution means or material.

[0029] As used herein, the term "cancer" refers to the abnormal growth of cells or tissues and is understood to include malignant neoplastic growth. The term "neoplastic" refers to a neoplasm or something related to a neoplasm. In some embodiments, the cancer is a solid tumor, i.e., brain tumor (particularly glioma: ependymoma, astrocytoma [e.g., glioblastoma multiforme], oligodendroglioma, brainstem glioma, oligoastrocytoma); colorectal cancer (particularly non-MSI CRC), bladder cancer, liver cancer, breast cancer (particularly double- or triple-negative breast cancer), kidney cancer, head and neck squamous cell carcinoma, lung cancer (particularly lung squamous cell carcinoma, non-small cell lung cancer (NSCLS), small cell lung cancer (SCLC)), malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, or gastric cancer. The term "cancer" also encompasses metastasis of the aforementioned tumors in various organs. In a more preferred embodiment, the tumors treated are recurrent tumors. A particular advantage of the pharmaceutical composition of the present invention is that even cancer-initiating stem cells can be effectively treated. This has a positive effect on avoiding tumor recurrence and metastasis formation.

[0030] In other embodiments, the cancer is a hemolytic malignancy, i.e., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary interferon-positive large B-cell lymphoma, T-cell (histiocytocyte)-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0031] Administration can be carried out by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intratumoral, or intradermal administration. The route of administration will, of course, depend on the type of treatment and the type of compound contained in the pharmaceutical composition. The viral dosing regimen can be easily determined by the attending physician within the skill of the art based on patient data, observations, and other clinical factors, including, for example, the patient's size, body surface area, age, sex, administration time and route, tumor type and characteristics, the patient's overall health, and other medications the patient is receiving. The selection of a dosing regimen (also referred to herein as a dosing regimen) for the treatment of the present invention depends on several factors, including the entity's serum or tissue turnover rate, the level of symptoms, the entity's immunogenicity, and the accessibility of target cells, tissues, or organs in the individual being treated. Thus, the dosage and frequency of administration depend, in part, on the severity of the cancer being treated and the characteristics of the patient.

[0032] Because the viruses of the present invention contain infectious viral particles capable of penetrating the blood system, treatment can be administered (or at least initiated) by intravenous injection of the virus. Because long-term intravenous treatment is likely to be ineffective as a result of the formation of neutralizing antibodies to the virus, different modes of administration can be employed after the initial plan of intravenous virus administration, or such different administration techniques, such as intratumoral virus administration, can be used alternatively throughout the course of virus treatment.

[0033] As another specific administration technique, the virus (virus, vector, and / or cellular agent) can be administered to the patient from a source implanted in the patient. For example, a catheter, such as a silicone or other biocompatible material catheter, can be connected to a small subcutaneous reservoir (Rickham reservoir) placed in the patient during tumor removal or by another procedure, allowing the parvovirus composition to be locally injected at various times without further surgical intervention. The virus or derived vector can also be injected into the tumor by stereotactic surgical techniques or navigation targeting techniques.

[0034] Administration of the virus can also be achieved by continuous infusion of the virus particles or a fluid containing the virus particles at a low flow rate through an implanted catheter using a suitable pump system, for example, a peristaltic infusion pump or a convection-enhanced delivery (CED) pump.

[0035] Another method of administering the virus is through an implantable device constructed and arranged to dispense the parvovirus into the desired cancer tissue. For example, a virus-impregnated wafer can be used, which is attached to the edge of the resection cavity at the end of surgical tumor removal. Multiple wafers can be used in such therapeutic interventions. Cells that actively produce the virus or virus-based vector can be injected into the tumor or tumor cavity after tumor removal.

[0036] In the case of the parvovirus mutants of the present invention, infection results in the death of tumor cells but spares normal cells, thereby achieving tumor-specific therapy without adverse neurological or other side effects.

[0037] The therapy of the present invention can be used before or after surgery to remove the tumor, and can be used before, during or after radiation therapy.

[0038] The pharmaceutical composition may also contain one or more additional therapeutic agents, such as chemotherapeutic agents, biological therapeutic agents (including, but not limited to, antibodies against VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD40L, CTLA-4, OX-404-1BB, and ICOS), checkpoint inhibitors (e.g., antibodies against PD-1 or PD-L1), immunogenic agents (e.g., attenuated cancer cells, tumor antigens, tumor-derived antigens, or antigen-presenting cells such as dendritic cells pulsed with nucleic acids), immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), and cells transfected with a gene encoding an immunostimulatory cytokine (e.g., but not limited to, GM-CSF). Examples of chemotherapeutic agents include: alkylating agents, such as cyclophosphamide, busulfan, camptothecin, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, antibiotics, bleomycin, caminomycin, dactinomycin, daunorubicin, idarubicin, 5-fluorouracil (5-F11), methotrexate, cytarabine, platinum analogs, such as cisplatin and carboplatin; vinblastine, platinum; toposide (VP-16); ifosfamide, mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin, xeloda; ibandronate; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoids, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, or aromatase inhibitors. [Brief explanation of the drawings]

[0039] The invention is further explained with respect to the following figures:

[0040] [Figure 1]Genomic structure of the H-1PV DR. (A) Schematic diagram of the H-1PV genome. The H-1PV genome is characterized by a single line ending in an inverted terminal repeat (ITR) structure. The ITRs are depicted schematically to show their predicted structure. The viral early P4 promoter regulates expression of the NS genes (yellow boxes with arrows), which encode the nonstructural NS1 and NS2, and the late P38 promoter regulates expression of the VP genes (light yellow boxes with arrows), which encode the VP1 and VP2 viral proteins. The right-most ITR is thought to function as a self-priming origin for viral genome replication and encapsidation. (B) Predicted structure of the right-most ITRs of MVM and H-1PV. The 248-nucleotide right-most telomere is indicated by a black line. A 36-bp palindrome containing a repeat of the NS1-binding motif 5'-TGGT-3' (small light orange box) folds into two axial arms. NS1 oligomer assembly binds to the boxed 5'-TGGT-3' motif in the hairpin stem, indicated by the larger, light orange box. The nick site is indicated by a yellow lightning bolt, and the G-rich region is indicated by a green oval. (C) Cleavage structure of the right-end hairpin of H-1PV. The 182-nucleotide right-end ITR of H-1PV is indicated by a black line. A 36-bp palindrome containing a repeat of the NS1-binding motif 5'-TGGT-3' folds into two axial arms and is indicated by a small, light orange box. (D) Schematic of the H-1PV DR genome organization. The gray triangle indicates the location of the 114-nucleotide internal deletion, and the pink diamond indicates the location of the 55-nucleotide repeat motif toward the right end. The remaining features of the viral genome are described as in (A). [Figure 2]Increased progeny production and infectivity of H-1PV DR with improved fitness in permissive human NB-324K cells. (A) Increased infectivity of H-1PV DR compared to wt H-1PV and st H-1PV. Cells infected with the indicated viruses at an MOI of 0.01 PFU / cell were harvested 5 days post-infection. Titers of infectious and genome-containing particles were measured by plaque assay (in PFU / ml) and qPCR (in vg / ml), respectively. Genome-containing particle to infectivity (P / I) ratios are shown in the table. (B) The fold reduction in P / I ratios between wt H-1PV vs. st H-1PV and wt H-1PV vs. H-1PV DR is shown. [Figure 3] Assessment of the genomic stability of the H-1PV DR. (A) Production of the H-1PV DR through successive rounds of infection. This production scheme was employed to check the stability of the new features of the H-1PV DR, the internal deletion and terminal repeats. Transfection of the H-1PV DR in HEK293T cells resulted in the production of fully infectious viral particles. NB-324K cells were infected with the indicated viruses at an MOI of 0.01 PFU / cell. Five days later, infected cells were harvested, and progeny viral particles were purified, titrated, and used to infect freshly prepared batches of cells. Each round of infection and harvest corresponds to one passage, or one round of virus production (P); seven rounds of production were performed (P1–P7). Viral DNA was extracted from each production round, and genomic fragments containing the H-1PV DR internal deletion and terminal repeats were amplified by PCR. (B) Illustration of the flanking regions of the primer pairs in the H-1PV DR genome configuration. (C) The stability of the in-frame 114-nucleotide deletion (upper panel) and the 58-nucleotide repeat motif (lower panel) in the H-1PV DR was assessed by PCR analysis. [Figure 4]Evaluation of the profile of neutralizing antibody recognition of the H-1PV DR on the capsid surface. Neutralization assays of the indicated viruses with specific neutralizing antibodies against the H-1PV DR, as described in the Materials and Methods section. The activity of virus-neutralizing antibodies was determined using cytotoxicity protection assessment with the Cell Counting Kit-8 assay in NB-324K cells at an MOI of 10 PFU / cell, scored 72 hours after inoculation (upper panel). The effect of neutralizing antibodies on NB-324K cells using the same conditions as above was independently demonstrated by crystal violet staining assay (lower panel). [Figure 5] The more efficient production of H-1PV DR was not limited to NB-324K cells. H-1PV DR progeny production was measured in various human cell lines by plaque assay. Infectious progeny production of H-1PV DR and wild-type H-1PV was measured in human NB-324K, BxPC-1, Cal33, Hela, and U251 cell lines after infection at an MOI of 0.25 PFU / cell. The yield of infectious particles (total number of PFU) recovered from both the cell supernatant and pellet was measured by plaque assay on day 3 postinfection for NB-324K and day 4 for the remaining cell lines. Results represent the average of two independent experiments. [Figure 6] H-1PV DR has potent oncolytic activity in various human cell lines derived from various cancer entities. Cell proliferation and viability were assessed by Cell Counting Kit-8 assay and crystal violet staining assay, respectively, following infection with H-1PV DR and wild-type H-1PV at an MOI of 12 to 100 PFU / cell at 96 hours postinfection. (A) The virus-low permissive cell lines MeWo and SiHa; (B) the virus-semiresistant cell lines Capan-1 and AsPC-1; and (C) the virus-resistant cell lines FaDu and ME180. [Figure 7]Validation of the therapeutic effect of H-1PV DR in a highly translationally relevant 3D spheroid model. Real-time cell proliferation using the Incucyte® single spheroid assay was demonstrated in infections with H-1PV DR and wild-type H-1PV at an MOI of 10 PFU / cell up to 13 or 14 days post-infection. (A) Virus-semiresistant cell line AsPC-1. (B) Virus-resistant cell line FaDu. [Figure 8] Parvovirus H1 sequence [Figure 9] Response of human intestinal stem cell quadruple mutant isogenic KAPS organoids to H-1PV DR. Real-time cell proliferation was monitored by Incucyte® over 14 days following infection with H-1PV and H-1PV DR at increasing MOIs as indicated. (A, B) Human intestinal stem cell wt organoids. (C, D) Quadruple mutant isogenic KAPS organoids. [Figure 10] Response of patient-derived organoids (PDO) to H-1PV DR in colorectal cancer patient #1. Real-time cell proliferation was assessed by Incucyte® over 24 days following infection with H-1PV and H-1PV DR at increasing MOIs as indicated. (A, B) Normal tissue. (C, D) Primary tumor. (E, F) Metastatic tumor. [Figure 11] Response of patient-derived organoids (PDO) to H-1PV DR in colorectal cancer patient #2. Real-time cell growth was monitored by Incucyte® over 20 days following infection with H-1PV and H-1PV DR at increasing MOIs as indicated. (A, B) Normal tissue. (C, D) Primary tumor. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention will be further illustrated in the following examples, which should be understood as preferred embodiments, but are not intended to limit the present invention.

[0042] Example 1: Materials and Methods Plasmid constructs. For the construction of pst H-1PV, a fragment of the viral genome obtained by digesting the pSR19 clone with Hpal-Ndel was first subcloned into the pUC19 Hpal-Ndel vector to generate an adapted vector with a modified polylinker. A DNA fragment containing the full-length right-end ITR flanked by PshAI and Ndel restriction sites, synthetically produced by Biocat (Heidelberg, Germany), was inserted into this vector to obtain the construct pst H-1PV(Hpal-Ndel). Using the same strategy, a DNA fragment containing a 55-nucleotide repeat motif (nt 4828 to 4883) flanked by HpaI and PshAI restriction sites was introduced into pst H-1PV(HpaI-Ndel). The modified construct was finally subcloned back into its parent pSR19 backbone to generate the molecular clones pst H-1PV and pH-1PVVR. To construct the H-1PV DR, a deletion representing 114 in-frame nucleotides (nt 2022–2135) within the NS region of the EcoRI-Mfel-digested pDelHI plasmid was introduced into the pH-1PVR vector

[19] . As a result, the pst H-1PV and pH-1PV DR plasmids were obtained, and the replacement fragments were further confirmed by sequencing (Genewiz, Takeley Sanger Sequencing Laboratory, UK).

[0043] Cell culture. Simian virus 40 (SV40)-transformed human neonatal kidney NB-324K cells and HEK293T cells were as previously described [25, 26]. The human glioblastoma (GBM) cell line U251 was kindly provided by Dr. Iris Augustin (DKFZ, Heidelberg, Germany). The HeLa and SiHa cervical carcinoma (CC) cell lines were kindly provided by Dr. Angel Alonso (DKFZ, Heidelberg, Germany). The ME180 cervical carcinoma (CC) cell line was kindly provided by Dr. Elisabeth Schwarz (DKFZ, Heidelberg, Germany). The pancreatic ductal adenocarcinoma (PDAC)-derived cell lines AsPC-1, BxPC-1, and Capan-1 were kindly provided by Dr. Stephan Herzig (DKFZ, Heidelberg, Germany). The head and neck squamous cell carcinoma (HNSCC) cell lines FaDu and Cal33 were kindly provided by Dr. Ina Kurth (DKFZ, Heidelberg, Germany). The melanoma MeWo cell line was kindly provided by Dr. Jochen Utikal (DKFZ, Heidelberg, Germany). Cells were cultured in medium according to Table 1, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Gibco), and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin sulfate, Gibco), except for NB-324K cells, which were 5% FBS (Sigma-Aldrich). All cells were grown at 37°C, 5% CO2, and 95% humidity and were routinely checked for mycoplasma contamination using a mycoplasma detection kit (Venor GeM, Minerva Biolabs, Berlin, Germany) according to the manufacturer's instructions. [Table 1]

[0044] For experiments, cells were counted using trypan blue and a Countess™ automated cell counter (Invitrogen, USA) and seeded at the cell numbers shown in Table 2 unless otherwise stated. 3 ml of medium was used for 6-well plates, 5 ml of medium was used for 6-cm dishes, and 100 μl / well of medium was used for 96-well plates. [Table 2]

[0045] Transfection assay. HEK293T cells were seeded in 6-well plates containing 3 ml of Opti-MEM reduced serum medium (Invitrogen, catalog no. 31985). The next day, cells were transiently transfected with 6 μg of a plasmid carrying the viral genome (pwt H-1PV, pst H-1PV, or pH-1PV DR) using the transfection reagent Metafectene (Biontex Laboratories GmbH, catalog no. T020-1.0, Munich, Germany) at a 1:2 ratio (μg DNA:μl reagent) according to the manufacturer's instructions.

[0046] Viral infection and production. The viruses were primarily produced by transfection of 293T cells and subsequently amplified by infection of NB-324K cells at a multiplicity of infection (MOI) of 0.01 PFU per cell. Cell harvesting and virus purification were performed by iodixanol step gradient centrifugation as previously described

[27] .

[0047] Titration of infectious and complete particles. Infectious viral particles were titrated by plaque assay as previously described

[25] . Complete viral particles were quantified by quantitative real-time PCR as previously described

[28] . Viral titers were expressed as the number of viral genomes (vg) per milliliter of virus stock.

[0048] Electron microscopy. Viruses from the dialyzed preparations were adsorbed onto glow-discharged carbon-coated grids, washed with water (Braun, Ampuwa), and negatively stained with 1% aqueous uranyl acetate. Micrographs were acquired at 80 kV using an EM912 (Carl Zeiss, Oberkochen, Germany) equipped with a slow-scan CCD camera (TRS, Molenweis, Germany).

[0049] Evaluation of the stability of internal deletions and terminal repeats by PCR. NB-324K cells were infected with H-1PV DR through successive rounds. At the end of each infection round, viral genomic DNA was isolated as previously described

[26] . PCR was performed using CloneAmp HiFi PCR Premix (Takara BioClontech, catalog number 639298, Japan) with primer pairs (deletion: 5'-TCAATGCGCTCACCATCTCTG-3' vs. deletion: 5'-TTAGTCCAAGGTCAGCTCCTC-3' or repeat: 5'-TAATATGGTATTGGTTAACTGTAAAAAAT-3' vs. repeat: 5'-CAACCACCCAACCACCCTTT-3'). The PCR mixture was loaded onto a 2% or 3% agarose gel for electrophoresis. Images were recorded by INTAS (Intas Pharmaceuticals Limited, India).

[0050] Spectral flow cytometry data analysis. PBMCs were stained in PBS with 1:1000 diluted ZombieNIR as a live / dead stain for 30 minutes at room temperature in the dark. FcX and monocyte blocker (Biolegend) were then added for 10 minutes at room temperature in the dark, followed by the addition of fluorochrome-conjugated antibodies (Table 1). After a 60-minute incubation at room temperature in the dark, cells were washed and measured on an AURORA Spectral flow cytometer (Cytek Biosciences). Unsupervised data analysis, detailed below, was performed using OMIQ data analysis software (www.omiq.ai). Data were first manually gated to remove aggregates, dead cells, and debris, and then analyzed using 2.5 × 10 5CD45 + The data were subsampled to include leukocytes / groups. Next, FlowAI was run to check the files for any abnormal regions

[29] . FlowAI settings were as follows: all used files, all selected fluorescence channels and time periods, all used methods, and default settings. Subsequently, a dimensionality reduction analysis was performed using Uniform Manifold Approximation and Projection (UMAP) to visualize different subpopulations within the groups

[30] . UMAP settings were as follows: all used files, all fluorescence parameters except CD45 and Live / Dead, neighbors = 80, minimum distance = 0.7, components = 2, metric = Euclidean, learning rate = 1, epochs = 250, random seed = 9346, and embedding initialization = Spectral.

[0051] Monoclonal antibody development. Monoclonal antibody generation was performed according to technical principles

[31] . Mice were immunized with H-1PV virus particles via multiple injections. To enhance the immune response, 100 μl of Freund's complete adjuvant (Santa Cruz Biotechnology) was injected into the hind leg of each mouse. A booster injection was performed with Freund's incomplete adjuvant, followed by an injection of buffer alone. The fusion procedure to generate hybridoma cell clones was performed as follows: popliteal lymph nodes were surgically removed and placed in RPMI medium (Gibco). The lymph nodes were then triturated using a syringe plunger under a microscope. The cell mixture was centrifuged at 150 × g for 10 minutes at room temperature. The cells were resuspended in 1.5 ml of polyethylene glycol (PEG, Sigma-Aldrich), added for 1 minute, and mixed with a Pasteur pipette. Subsequently, 20 ml of RPMI medium was added over 4 minutes, and the cells were centrifuged at 150 x g for 10 minutes. They were then resuspended in HAT medium containing Hyper and cultured for 7 days. After 7 days, cell supernatants were screened for the presence of specific antibodies against the protein of interest by ELISA, followed by immunofluorescence. Effective mother clones were subcloned by limiting dilution to obtain monoclonal cell clones.

[0052] Neutralization assay. Virions (10 PFU / cell) were incubated with different clonal antibodies against H-1PV DR in a final volume of 60 μl of primary MEM medium for 30 min at 37°C. Residual infectivity was measured on NB-324K cells 72 h postinoculation using the Cell Counting Kit-8 assay as well as a crystal violet staining assay.

[0053] Cell Counting Kit-8 assay. Cells were seeded into 96-well plates containing 50 μl of medium per well. The culture media and cell numbers were as shown in Tables 1 and 2, namely, NB-324K, MeWo, SiHa, AsPC-1, Canpan-1, FaDu, and ME180. After 24 hours, 50 μl of FBS-free medium with or without st H-1PV or H-1PV DR was added per well. At 72 or 96 hours postinfection, cell proliferation was measured using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan) as previously described

[32] .

[0054] Crystal violet staining assay. Cell seeding and treatment were performed as described above. Cell viability was measured using a crystal violet assay as previously described

[33] .

[0055] Spheroid generation. 3D cell spheroids represented the heterogeneity of tumor models because cells in the outer layer of the spheroid had access to nutrients and oxygen, whereas hypoxic regions formed in the core of the spheroid due to the accumulation of cellular degradation products. Spheroids were generated from 20,000 AsPC-1 or Fadu cells using the hanging drop method in the presence of a 30% methylcellulose stock solution, as previously described

[34] . After 2–3 days, spheroids were transferred to low-attachment round-bottom 96-well plates. 50 μl / well of complete medium with or without H-1PV or H-1PV DR was added. Spheroid size was analyzed in real time using the Incucyte® 3D Single Spheroid Assay with the Spheroid Acquisition and Analysis tool. Typically, three images per well were acquired daily at 10x magnification. Analysis was performed using Incucyte® S3 2018A software.

[0056] Culture of wt and quadruple mutant isogenic KAPS organoids of human intestinal stem cells. Colon tumor organoids (KAPS) harboring combinations of mutations in KRAS, APC, TP53, and SMAD4 were generated as previously described

[35] . Quadruple mutant and wt organoids were cultured in advanced DMEM-F12 (Thermo, 12634010) in organoid medium containing 1x B27 supplement (Thermo, 12587010), 1x Glutamax (Gibco, 35050061), and 1x penicillin / streptomycin (Thermo, 15140122). Organoids were seeded in a 1:1 mixture of Matrigel and organoid medium. The medium was changed every other day. To evaluate viral therapy response, organoids were prepared as described in the next section on PDO culture.

[0057] Generation of colorectal cancer patient-derived organoids (PDOs). Organoids were established from patient-derived paired colon normal, primary, or metastatic tumor tissue collected at the time of surgical resection, according to a previous protocol

[36] . Briefly, after 2 weeks of Wnt-depleted selection to enrich for tumor and metastatic cells in each condition, all organoids were cultured in Cultrex Basement Membrane Extract Type 2 (BME; R&D Systems) domes covered with complete "hC" growth medium to ensure comparability between conditions. Complete organoid medium was prepared using Advanced DMEM / F12 (Gibco), 1x B27, 1x Glutamax, 10 mmol / L HEPES, 0.1 mg / ml primocin (all Thermo-Fisher), 1.25 mM N-acetylcysteine, 10 μM nicotinamide, 10 μM p38 inhibitor SB202190 (all Sigma-Aldrich), 0.5 nM Wnt surrogate-Fc fusion protein, 2% Noggin-conditioned medium, 2% Rspo3-conditioned medium (all U-ProteinExpress), 50 ng / ml EGF (Peprotech), 0.5 μM A83-01, and 1 μM PGE2 (both Tocris). To assess viral therapy response, organoids were seeded as single cells. Dissociation was achieved using TrypLE Express Enzyme (Gibco), and 8,000 single cells were seeded per well in 8 μl of 96-well plates. Within each experiment, triplicate wells were seeded and treated independently for each condition. After seeding, 100 μl of medium was added. Both H-1PV DR and H-1PV were dissolved in 50 μl of medium to achieve the desired MOI and added to the top 24 hours after initial seeding. Organoid growth was assessed by imaging every 3 days over 24 days using an Incucyte system (37°C, 5% CO2). Image analysis was performed using Incucyte 3D Spheroid Analysis software, allowing for measurement of PDO confluence over time.

[0058] Human material for organoid culture. Approval for this study was obtained by the Ethics Committee II of the Medical Faculty of Mannheim, University of Heidelberg. Written informed consent was obtained.

[0059] Example 2: Generation and production of H-1PV DR In a proof-of-concept study, a molecular clone of pst H-1PV (Figure 1A) was generated by inserting the full-length right-end ITR based on the previous pSR19 plasmid [9], and the pH-1PV DR (Figure 1D) was generated by deleting 114 nucleotides within the NS region and inserting a 55-nucleotide repeat motif toward the right end based on the pst H-1PV plasmid. To investigate whether the above modifications to the viral genome are compatible with the viral life cycle and fitness, virus production was performed. Typically, parvovirus production is first performed in HEK293T cells by transient transfection of pst H-1PV and pH-1PV DR constructs. Approximately 96 hours after transfection, cells are lysed, and newly assembled viral particles are purified and titrated by plaque assay and quantitative real-time PCR. Further amplification of the virus was performed in NB-324K cells. Cells are infected with the virus previously produced in the transfection at an MOI of 0.01 PFU (plaque-forming units / ml) per cell. When cytopathic effects, indicating viral replication and translocation, were detected in 80% of the cells, the cells were harvested, and the virus was purified and titrated. As expected, the virus was successfully purified from the cell lysate. For further study, wt H-1PV, st H-1PV, and H-1PV DR, designated pSR19, were transfected into HEK293T cells to generate master stocks, respectively. Each virus was produced in parallel by infecting NB-324K cells as described in Example 1 above.

[0060] Example 3: Increased progeny production and infectivity of fitness-enhanced H-1PV DR in permissive human NB-324K cells. We investigated the production and infectivity of H-1PV DR compared with those of wild-type (wt) H-1PV and standard (st) H-1PV. To this end, viruses were simultaneously produced by infection of NB-324K cells and harvested 72 hours postinfection. The amounts of genome-containing (i.e., complete) and plaque-forming (i.e., infectious) particles were measured by quantitative real-time PCR and plaque assay, respectively. The amounts of complete (vg / ml) virus particles and infectious titers (given in PFU / ml) from two independent experiments are listed in Figure 2A. The amount of complete particles produced by infection with wt H-1PV was significantly higher than that of st H-1PV and H-1PV DR, but the latter two produced higher titers of infectious virions than wt H-1PV. Interestingly, as shown in the right column of Figure 2A, the corresponding complete particle-to-infectivity (P / I) ratio was dramatically reduced, resulting in a significant (8- to 10-fold) reduction in the P / I ratio with st H-1PV and H-1PV DR (Figure 2B). These results suggest that the full-length right-end ITR is a critical component of parvoviruses involved in viral genome encapsidation, excision from viral DNA replication intermediates, and high yields of infectious virions. Last but not least, H-1PV DR demonstrated the best viral fitness in NB-324K cells, consistent with previous observations [9].

[0061] The improved fitness and increased infectivity of the H-1PV DR relative to wild-type H-1PV virions motivated us to investigate the efficiency of virus spread in NB-324K cells during virus propagation. To this end, plaque assays were performed. All viruses produced a mixture of small and large plaques. Clearly, large plaques were more frequent in cells infected with the H-1PV DR, whereas small plaques were more frequent in cells infected with the wild-type H-1PV DR. These results indicated that the H-1PV DR facilitated more efficient virus spread during the propagation process than wild-type H-1PV, suggesting that the internal deletion and terminal repeats of the H-1PV DR may play a crucial role in stimulating the viral life cycle. The morphology of the various viruses was examined by electron microscopy. Consistent with wild-type H-1PV, the H-1PV DR and stH1PV viruses exhibited the characteristic parvoviral diameter of 25 nm and the same morphology at high resolution.

[0062] Example 4: Evaluation of the stability of internal deletions and terminal repeats of the H-1PV DR For biosafety reasons, especially for future clinical applications of the H-1PV DR, it is worthwhile to examine the stability of either the internal deletion or the terminal repeat of the H-1PV DR through successive rounds of infection in permissive NB-324K cells. To this end, seven successive rounds of infection were performed, as shown in Figure 3A. At the end of each round of infection, viral genomic DNA was isolated, and genome stability was examined by PCR using primers flanking the region between nucleotides 2022 and 2135 or between nucleotides 4828 and 4883, as shown in Figure 3B.

[0063] No degradation or alteration of the H-1PV DR within the viral genome was observed after seven rounds of infection, indicating that both modified mutations were stably integrated into the viral genome, as shown in lanes 1 to 4 in Figure 3C, which correspondingly represent 4 to 7 viral passages. Unmodified wt H-1PV was used as a control (Figure 3C, lane 5). Further sequencing of the viral genome covering the modified region substantiated the same observation. These results are consistent with the notion that the H-1PV DR is characterized by long-term maintenance of genome stability in human cells.

[0064] Example 5: Insensitivity of normal human cells to the cytotoxic effects of H-1PV DR The greater viral genome stability of H-1PV DR in permissively transformed human cells provided insight into its immune and cytotoxic effects on peripheral blood mononuclear cells (PBMCs) from healthy donors, which represent normal human cells. To this end, spectral flow cytometry assays were performed on ex vivo cultured PBMCs infected with wt H-1PV, st H-1PV, and H-1PV DR at an MOI of up to 10 PFU per cell, respectively. Data were analyzed using a 36-color immunophenotyping panel. UMAP plots showed highly overlapping immune cell populations in all conditions analyzed, including mock-infected PBMCs, demonstrating the lack of impact of viral infection on any immune cell population under viral challenge. The virus did not affect the viability of immune cell populations or PBMCs, suggesting that PBMCs were not sensitive to the cytotoxic effects of H-1PV DR or st H-1PV, but behaved similarly to wt H-1PV.

[0065] Example 6: Evaluation of the profile of neutralizing antibody recognition of H-1PV DR on the capsid surface Although the H-1PV DR showed identical capsid assembly patterns by electron microscopy, we examined the occurrence of neutralizing antibody recognition of the H-1PV DR at the molecular biological level due to viral genome modifications. To this end, a neutralization assay using specific neutralizing antibodies against the H-1PV DR was performed using a cytotoxicity protection assessment. The activity of virus-neutralizing antibodies was measured by a Cell Counting Kit-8 assay scored 72 hours after inoculation in NB-324K cells at an MOI of 10 PFU per cell (Figure 4, upper panel). As a control, mouse IgG showed no virus-neutralizing activity and resulted in a failure of cytotoxicity protection after incubation with the virus. In contrast, a different monoclonal antibody against the H-1PV DR showed a remarkable ability to neutralize virus infectivity and produced potent cytotoxicity protection as mock-infected NB-324K cells. Interestingly, the same observation was found when experiments were performed using wild-type and st-type H-1PV under the same experimental settings. Using the same conditions, we independently demonstrated similar effects of neutralizing antibodies against various viruses in NB-324K cells by crystal violet staining assay (Fig. 4, lower panel). These results indicated similar profiles of neutralizing antibody recognition of the H-1PV DR on the capsid surface compared with wild-type H-1PV and st H-1PV, suggesting that the novel features of the internal deletion and terminal repeats of the H-1PV DR do not affect the viral capsid assembly pattern.

[0066] Example 7: Efficient production of H-1PV DR in different permissive human cancer cell lines As mentioned above, the H-1PV DR demonstrated enhanced fitness and increased infectivity in NB-324K cells compared to wild-type H-1PV virions. This raised the question of whether the remarkable potency of the novel H-1PV DR due to artificially expressed SV40 antigens in NB-324K cells was cell line dependent

[25] . Therefore, production of the H-1PV DR and wild-type H-1PV was examined in different permissive human cell lines derived from various tumor entities. NB-324K, pancreatic ductal adenocarcinoma (PDAC) BxPC-1, head and neck squamous cell carcinoma (HNSCC) Cal33, cervical carcinoma HeLa, and glioblastoma (GBM) U251 cells were infected with the H-1PV DR and wild-type H-1PV at an MOI of 0.25 PFU per cell. Infectious virions were harvested on day 3 postinfection for NB-324K cells and day 4 for the remaining cell lines and quantified by plaque assay. The total amount of infectious particles (cell pellet and medium combined) was not only higher in NB-324K cells upon infection with H-1PV DR than with wt H-1PV, but also increased in the remaining four cell lines derived from different tumor entities (Figure 5). Based on these observations, the improved fitness of H-1PV DR was found to be independent of SV40 antigen expression in transformed cells, indicating that H-1PV DR has a viral growth advantage in cancer cells derived from various tumor entities, resulting in superior anticancer efficacy compared to wt H-1PV.

[0067] Example 8: Superior tumor toxicity of H-1PV DR over wt H-1PV The enhanced viral production of H-1PV DR, which has potent oncolytic activity in various permissive cancer cell lines, motivated us to explore the oncotoxic efficacy of H-1PV DR in cancer cells with different permissiveness. To this end, virus-low permissive cell lines, including melanoma MeWo and cervical cancer SiHa (Figure 6A), virus-semiresistant cell lines, including pancreatic ductal adenocarcinoma (PDAC) Capan-1 and AsPC-1 (Figure 6B), and virus-resistant cell lines, including head and neck squamous cell carcinoma (HNSCC) FaDu and cervical cancer ME180 (Figure 6C), were infected with increasing amounts of H-1PV DR virus (MOI of 12 to 100 PFU per cell). Cells were treated with either wt H-1PV or virus dilution buffer as a mock treatment. Four days after infection, virus-induced cytotoxicity was measured by analyzing cell proliferation using the Cell Counting Kit-8 assay. As shown in the upper panel of Figure 6A, dramatically reduced cell proliferation was observed in both cell lines treated with H-1PV DR compared to wt H-1PV in a virus dose-dependent manner. The enhanced tumor toxicity of H-1PV DR was also confirmed by analyzing cell viability using a crystal violet staining assay (lower panel of Figure 6A). However, reduced cell proliferation and viability in either semi-resistant cell lines, Capan-1 or AsPC-1, was only observed at higher virus titers, and stronger inhibition of cell proliferation was observed in cells treated with H-1PV DR (Figure 6B). Furthermore, even at the highest virus titers, wt H-1PV had no effect on either virus-resistant cell lines, FaDu or ME180 cells. In FaDu cells treated with H-1PV DR, only a slight decrease in cell proliferation was observed at the highest virus titer (Figure 6C). It is noteworthy that viral entry is a prerequisite for the production of progeny viruses that result in tumor toxicity in cancer cells. This may explain the reason for the decrease in oncolytic activity from less permissive to resistant cells.

[0068] Importantly, H-1PV DR inhibited cell proliferation not only in 2D cell culture but also in 3D spheroids, a translationally relevant model that represents the heterogeneity of cancer cells and is more relevant to tumor biology. For spheroid generation, we established a protocol involving forming spheroids by hanging drop culture using medium supplemented with methylcellulose before transferring to ultra-low attachment microplates. Spheroid size upon treatment with H-1PV DR and wt H-1PV at an MOI of 10 PFU per cell was measured using the Incucyte® 3D Single Spheroid Assays spheroid acquisition and analysis tool. This device can monitor cell growth in real time. Figures 8A and 8B show the increase in spheroid size upon treatment. It can be observed that spheroids treated with H-1PV DR showed a slower increase in size compared to wt H-1PV in AsPC-1 cells (Figure 7A). Interestingly, treatment with H-1PV DR significantly delayed growth in FaDu cells over 14 days (Figure 7B), suggesting that in addition to growth inhibition, a cell death component may also be present. Altogether, these results provide proof-of-concept that H-1PV DR possesses superior anticancer activity to wild-type H-1PV, warranting clinical application of this novel virus to cancer patients.

[0069] Example 9: Response of quadruple mutant isogenic KAPS organoids of human intestinal stem cells to H-1PV DR. Oncolytic viruses (OVs) are powerful novel therapeutic agents in cancer treatment, but not all patients receiving oncolytic virotherapy for solid tumors demonstrate durable responses. Recently, the idea of ​​using human organoids to individually screen drugs for a given patient has been proposed. The ability of patient-derived tumor organoids to recapitulate key features of the original cancer tissue makes them useful as preclinical models for cancer research and precision medicine. A representative colorectal cancer organoid model, mutant isogenic KAPS organoids, is developed by introducing a quadruple mutant (KRAS, APC, P53, and SMAD4) into cultured human intestinal stem cells using CRISPR / Cas9 technology, allowing them to grow as tumor cells with invasive cancer characteristics in vivo and in vitro

[35] . To investigate whether H-1PV DR exhibits superior anticancer properties in human organoid cultures, wild-type (wt) and mutant isogenic KAPS organoids were infected with either H-1PV or H-1PV DR, as shown in Figure 9. Organoid growth was assessed by imaging every 3 days over 14 days using IncuCyte 3D spheroid analysis software. As expected, normal human intestinal stem cells were not susceptible to either virus, even at increasing viral doses, compared with mock treatment, suggesting that H-1PV DR is safe and not pathogenic to normal cells (Figures 9A and 9B). Both viruses were tumor-toxic in mutant isogenic KAPS organoids under treatment. Interestingly, organoids treated with H-1PV DR increased in size much more slowly than those treated with H-1PV (Figures 9D and 9C). Even at the lowest MOI of 25, organoid growth was largely inhibited over 14 days under treatment with H-1PV DR, suggesting that H-1PV DR had a more potent tumor toxicity than H-1PV, coupled with a component of cell death in mutant isogenic KAPS organoids. In contrast, treatment with H-1PV only showed tumor toxicity at the highest MOI of 100.In conclusion, these results provide proof-of-concept that H-1PV DR has superior anticancer activity to H-1PV in the human quadruple mutant isogenic KAPS organoid model.

[0070] Example 10: Response of patient-derived organoids (PDO) to H-1PV DR in colorectal cancer patient #1 and patient #2 Colorectal cancer patient-derived organoids (PODs) have recently been used to study carcinogenesis and potential prediction of clinical response to chemotherapy. The enhanced and superior anticancer activity of H-1PV DR in engineered human colorectal cancer organoids prompted us to explore the feasibility of using PDOs as a platform for predicting response to H-1PV DR tumor toxicity in colorectal cancer patients. To this end, PDOs were established from normal, primary, or metastatic tumor tissues collected at the time of surgical resection according to the protocol described in the methods section

[36] . Histological evaluation of patient specimens was routinely performed after surgery. To evaluate virotherapy response, colorectal cancer PDOs were exposed to either H-1PV or H-1PV DR with increasing viral doses. Measurement of the confluence area of ​​PDOs over time was performed as previously described. Consistent with the results in organoids derived from human intestinal stem cells, normal tissues were not sensitive to either H-1PV or H-1PV DR, even with increasing viral doses, compared with mock treatment, suggesting that H-1PV DR was safe and not harmful to normal cells in the two patients (Figures 10A, 10B, and 11A). Conversely, both viruses exhibited tumor toxicity in primary tumor cells upon infection in patient #1 (Figures 10C and 10D). Interestingly, organoids treated with H-1PV DR (Figure 10D) increased in size much more slowly in a viral dose-dependent manner compared with organoids treated with H-1PV (Figure 10C). However, only H-1PV DR showed anticancer activity in primary tumor cells from patient #2 (Figure 11D), whereas H-1PV did not, even at the highest MOI of 100, suggesting that primary tumor cells from patient #2 were highly resistant to H-1PV and responded poorly to it (Figure 11C). Although metastatic tumor cells proliferated much slower than primary tumors, infection with H-1PV DR at the highest MOI significantly delayed organoid growth in metastatic tumor cells over a 24-day period (Figure 11F), suggesting that H-1PV DR has potent tumor toxicity that suppresses tumor cell proliferation once infected.In summary, these results provide proof-of-concept that H-1PV DR has superior anticancer activity to H-1PV, warranting the clinical application of novel viruses to colorectal cancer patients. Importantly, the feasibility of using PDO to establish a platform for predicting colorectal cancer (CRC) response to H-1PV DR was demonstrated for the first time, demonstrating the feasibility of targeting cancer cells in specific individual patients to fulfill a personalized medicine approach, resulting in improved therapeutic efficacy with better clinical outcomes. (References)

[0071] 1. Cotmore, SF and Tattersall, P. (2014) Parvoviruses: small does not mean simple. Annu. Rev. Virol. 1, 517-537 2. Savy A, Dickx Y, Nauwynck L, Bonnin D, Merten OW, Galibert L. (2017) Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus / Sf9 Cells System. Hum Gene Ther Methods. 28, 277-289 3. Willwand K, Baldauf AQ, Deleu L, Mumtsidu E, Costello E, Beard P, Rommelaere J. (1997) The minute virus of mice (MVM) nonstructural protein NS1 induces nicking of MVM DNA at a unique site of the right-end telomere in both hairpin and duplex conformations in vitro. J Gen Virol. 78, 2647-55 4. Rhode SL 3rd, Klaassen B. (1982) DNA sequence of the 5' terminus containing the replication origin of parvovirus replicative form DNA. J Virol. 41, 990-999 5. Tattersall P, Ward DC. (1976) Rolling hairpin model for replication of parvovirus and linear chromosomal DNA. Nature 263, 106-109 6. Cotmore SF, Tattersall P. (1998) High-mobility group 1 / 2 proteins are essential for initiating rollingcircle- type DNA replication at a parvovirus hairpin origin. J. Virol. 72, 8477-8484 7. Cotmore SF, Christensen J, Tattersall P. (2000) Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin. J Virol. 74, 1332-1341. 8. Rhode SL 3rd, Paradiso PR. (1983) Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation. J Virol. 45, 173-184 9. Faisst S, Faisst SR, Dupressoir T, Plaza S, Pujol A, Jauniaux JC, Rhode SL, Rommelaere J. (1995) Isolation of a fully infectious variant of parvovirus H-1 supplanting the standard strain in human cells. J Virol. 69, 4538-4543 10. Yan Z, Zak R, Zhang Y, Engelhardt JF. (2005) Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol. 79, 364-379 11. Zhou Q, Tian W, Liu C, Lian Z, Dong X, Wu X. (2017) Deletion of the B-B' and C-C' regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression. Sci Rep. 7, 5432. 12. Colella P, Ronzitti G, Mingozzi F. (2017) Emerging Issues in AAV-Mediated In Vivo Gene Therapy. Mol Ther Methods Clin Dev. 1, 87-104 13. Willwand K, Mumtsidu E, Kuntz-Simon G, Rommelaere J. (1998) Initiation of DNA replication at palindromic telomeres is mediated by a duplex-to-hairpin transition induced by the minute virus of mice nonstructural protein NS1. J Biol Chem. 273, 1165-1174 14. Simmonds P, Aiewsakun P, Katzourakis A. (2019) Prisoners of war - host adaptation and its constraints on virus evolution. Nat Rev Microbiol 17, 321-328 15. Carter, B. J. (1984) Variant and defective interfering parvoviruses. In K. I. Berns (ed.). The parvoviruses. p209-258 16. Faust, E. A., and A. Hogan. (1990) Defective interfering particles. In P. Tijssen (ed.). Handbook of parvoviruses. p91-107 17. Rhode, S. L. (1978) Defective interfering particles of parvovirus H-1. J. Virol. 27, 347-356 18. Rhode SL 3rd. (1978) Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication. J Virol. 25, 215-223 19. Weiss N, Stroh-Dege A, Rommelaere J, Dinsart C, Salome N. (2012) An in-frame deletion in the NS protein-coding sequence of parvovirus H-1PV efficiently stimulates export and infectivity of progeny virions. J. Virol. 86, 7554-7564 20. Nuesch J, Thomas N, Plotzky C, Rommelaere J (2011) Modified rodent parvovirus capable of propagating and spreading through human gliomas. PCT / EP201 1 / 002306 21. Thomas, N. (2011) Therapy of brain tumors (gliomas) with the oncolytic parvovirus H-1 (H-1PV): Preclinical studies to optimize virotherapy in vitro and in vivo. [Doctoral dissertation, University of Kaiserslautern, Germany]. ProQuest Dissertations Publishing 22. Geletneky, K., Hajda J, Angelova AL, Leuchs B, Capper D, Bartsch AJ, Neumann JO, Schoening T, Huesing J, Beelte B, Kiprianova I, Roscher M, Bhat R, von Deimling A, Brueck W, Just A, Frehtman V, Loebhard S, Terletskaia-Ladwig E, Fry J, Jochims K, Daniel V, Krebs O, Dahm M, Huber B, Unterberg A, Rommelaere J. (2017) Oncolytic H-1 Parvovirus shows safety and signs of immunogenic activity in a first phase I / IIa glioblastoma trial. Mol Ther. 25, 2620-2634 23. Hajda J, Leuchs B, Angelova AL, Frehtman V, Rommelaere J, Mertens M, Pilz M, Kieser M, Krebs O, Dahm M, Huber B, Engeland CE, Mavratzas A, Hohmann N, Schreiber J, Jaeger D, Halama N, Sedlaczek O, Gaida MM, Daniel V, Springfeld C, Ungerechts G. (2021) Phase 2 Trial of Oncolytic H-1 Parvovirus Therapy Shows Safety and Signs of Immune System Activation in Patients With Metastatic Pancreatic Ductal Adenocarcinoma. Clin Cancer Res. 27, 5546-5556 24. Willemsen A, Zwart MP. (2019) On the stability of sequences inserted into viral genomes. Virus Evol. 5, vez045 25. Tattersall P, Bratton J. (1983) Reciprocal productive and restrictive virus cell interactions of immunosuppressive and prototype strains of minute virus of mice. J. Virol. 46, 944-955 26. Kestler, J., Neeb, B., Struyf, S., Van Damme, J., Cotmore, S. F., D'Abramo, A., Tattersall, P., Rommelaere, J., Dinsart, C. and Cornelis, J. J. (1999) cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses. Hum Gene Ther. 10, 1619-1632 27. Leuchs B, Roscher M, Mueller M, Kuerschner K, Rommelaere J. (2016) Standardized large-scale H-1PV production process with efficient quality and quantity monitoring. J Virol Methods. 229, 48-59 28. Li J, Bonifati S, Hristov G, Marttila T, Valmary-Degano S, Stanzel S, Schnoelzer M, Mougin C, Aprahamian M, Grekova SP, Raykov Z, Rommelaere J, Marchini A. (2013) Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas. EMBO Mol Med. 5, 1537-1555 29. Monaco G, Chen H, Poidinger M, Chen J, de Magalhaes JP, Larbi A. (2016) flowAI: automatic and interactive anomaly discerning tools for flow cytometry data. Bioinformatics. 32, 2473-2480 30. McInnes L, Healy J, Melville J. (2018) UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. ArXiv e-prints 31. Koehler, G., Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256, 495-497 32. T. Masuda, M. Endo, Y. Yamamoto, H. Odagiri, T. Kadomatsu, T. Nakamura, H. Tanoue, H. Ito, M. Yugami, K. Miyata, J. Morinaga, H. Horiguchi, I. Motokawa, K. Terada, M. Morioka, I. Manabe. Iwase , H. , Mizuta , H. , & Oike , Y. (2015). ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling. Scientific reports , 5 , 9170 [ PMC free article ] [ PubMed ] 33. Feoktistova M, Geserick P, Leverkus M. (2016) Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harb Protoc. 2016 , pdb.prot087379 [ PMC free article ] [ PubMed ] 34. Cavo, M., Delle Cave, D., D'Amone, E. Giuseppe Gigli, E. Enza Lonardo, Loretta L. del Mercato, L. (2020). Sci Rep 10 , 10192 35. Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, Sachs N, Overmeer RM, Offerhaus GJ, Begthel H, Korving J, van de Wetering M, Schwank G, Logtenberg M, Cuppen E, Snippert HJ, Medema JP, Kops GJ, Clevers H. (2015) Sequential cancer mutations in cultured human intestinal stem cells. Nature 7, 43-47. 36. Pleguezuelos-Manzano C, Puschhof J, van den Brink S, Geurts V, Beumer J, Clevers H. (2020) Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells. Curr Protoc Immunol. 130, e106.

Claims

1. An H-1 parvovirus mutant containing an in-frame 114-nucleotide deletion from nucleotides 2022 to 2135 in the open reading frame encoding the nonstructural proteins NS1 and NS2, and a 55-nucleotide duplication from nucleotides 4828 to 4883 toward the right terminal end in the wild-type H-1PV genome of FIG.

2. An antibody against the NS1 and / or NS2 protein of the parvovirus mutant according to claim 1, characterized in that it binds only to the mutant protein having the deletion and not to the wild-type protein.

3. (a) the parvovirus of claim 1; (b) an antibody according to claim 2; and / or optionally (c) conventional auxiliary agents, such as solvents, buffers, carriers, markers and controls; wherein one or more representatives of components (a) to (c) may each be present.

4. A pharmaceutical composition comprising: (a) the parvovirus mutant of claim 1 or the antibody of claim 2; and (b) a pharmaceutically acceptable carrier.

5. Use of the parvovirus mutant of claim 1 or the antibody of claim 2 for the preparation of a pharmaceutical composition for the treatment of cancer.

6. The use according to claim 5, characterized in that the cancer is a solid or hematological tumor.

7. 7. The use according to claim 6, characterized in that the solid tumor is brain cancer, colon cancer, bladder cancer, liver cancer, breast cancer, kidney cancer, squamous cell carcinoma of the head and neck, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma or gastric cancer and / or tumor metastasis.

8. 8. The use according to claim 7, wherein the hematological tumor is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell (histiocyte)-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL) and / or metastases thereof.

9. The parvovirus mutant of claim 1 for use in a method for treating cancer.

10. The parvovirus mutant for use according to claim 9, characterized in that the cancer is a solid or hematological tumor.

11. The parvovirus mutant for use according to claim 10, characterized in that the solid tumor is brain cancer, colon cancer, bladder cancer, liver cancer, breast cancer, kidney cancer, head and neck squamous cell carcinoma, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma or gastric cancer and / or tumor metastasis.

12. 12. The parvovirus mutant for use according to claim 11, characterized in that the hematological tumor is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell (histiocyte)-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL) and / or metastases thereof.

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