Improved oncolytic parvovirus H-1PV with enhanced adaptability and excellent anti-cancer activity

Abstract

The present invention relates to rodent H-1 parvovirus variants that can be propagated and propagated through human tumor cells. In particular, the present invention relates to parvoviral variants (H-1PV DR) based on wild-type H-1PV, but containing internal deletions of 114 nucleotides and 55 nucleotide repeat motifs towards the right end in the presence of the full length of the right-end ITR. Such variants exhibit improved anti-cancer activity. The invention also relates to pharmaceutical compositions comprising such parvovirus variants and their use for the treatment of cancer.

Description

[0001] The present invention relates to a rodent H-1 parvovirus variant capable of replicating and spreading in human tumor cells. In particular, the present invention relates to a parvovirus variant (H-1PV DR) which is based on wild-type H-1PV but contains an internal deletion of 114 nucleotides and a 55-nucleotide repeat motif toward the right-end terminus in the presence of the full-length right-end ITR. This variant shows improved anti-cancer activity. The present invention also relates to antibodies, pharmaceutical compositions and kits containing such parvovirus variants, and their use for the treatment of cancer.

[0002] H-1 parvovirus (H-1PV) is a ubiquitous, physically tiny, genetically compact virus that contains a linear single-stranded DNA genome of ∼5 kb, which encodes only two genes, encoding a non-structural protein (NS) involved in the replication process and a structural protein (VP) forming the capsid, respectively ( Figure 1 A). The expression of viral genes is regulated by two promoters, P4 and P38. At its termini, the viral genome contains palindromic sequences that form hairpin structures, the size of which varies between left (∼120 nucleotides) and right (∼248 nucleotides) inverted terminal repeats (ITRs) [1].

[0003] ITR is an important component of the Parvoviridae family, including adeno-associated virus (AAV), minute virus of mice (MVM), and H-1PV, and is involved in the initiation of virus replication, the encapsidation of the genome, the excision from viral DNA replication intermediates, the long-term maintenance of viral genome stability, and the expression of transgenes [2-4]. Rolling circle replication is initiated by a replicon-encoded endonuclease that introduces a single-strand nick into a specific origin sequence, covalently attaches to the 5' end of the DNA at the nick, and provides a 3' hydroxyl group to prime unidirectional leading-strand synthesis [5]. In heterotelomeric parvoviruses, such as MVM and H-1PV, the left end in the equivalent turnaround configuration cannot be nicked, and terminal resolution is restricted to the right end of the genome. A study of MVM showed that the virus can use the ITRs on the right to amplify its linear single-stranded genome, and these ITRs unfold and refold sequentially, allowing the replication fork to shuttle back and forth along the genome, generating a continuous poly-DNA strand. The viral initiator protein NS1 then excises individual genomes from this continuum by making a cut and restarting synthesis at specific origins within the hairpin sequence. For the cut to occur, DNA-bending proteins from the HMGB family must coordinate the interaction between NS1 complexes bound to each end of the hairpin stem, forming a ~30-bp double-helical loop centered on the intervening G-rich region of DNA [6]. Mutant ITRs that do not form loops result in failed cleavage, even in the presence of NS1 and HMGB, indicating that it is essential for the activity of this hyperactive origin [7]. Since H-1PV has a high similarity to MVM at the right-end inverted terminal repeat, H-1PV apparently employs the same mechanism as MVM and uses the same functional elements during virus replication ( Figure 1 B) [4,8,9].

[0004] Wild-type H-1PV (wt H-1PV) is derived from the pSR19 molecular clone. The right-end ITR of the wild-type H-1PV (wt H-1PV) plasmid currently used for virus production in clinical trials is a truncated and incomplete sequence because the original vector design was for the production of H-1PV [9]. Nevertheless, even with the truncated ITR sequence, the H-1PV genome can be replicated, producing H-1PV vectors in transfection experiments and further propagated in NB-324K cells.

[0005] Oncolytic virus (OV) drugs Using wild-type parvovirus H-1PV, which belongs to the genus Protoparvovirus of the subfamily Parvovirinae in the family Parvoviridae [1]. Oncolytic and tumor-suppressive properties have been demonstrated in preclinical proof-of-concept studies in various cultured cell lines, animals

[20] , and xenograft models of several human tumor types [9]. Recently, Phase I / Ila clinical trials conducted in patients with recurrent glioblastoma or metastatic pancreatic cancer have shown that wt H-1PV treatment is safe, well-tolerated, and has anti-cancer efficacy [22, 23]. Notably, some functional elements are missing in the wt H-1PV constructs in the clinical trials. This may have an adverse effect on the viral propagation of the construct in the NB-324K cell line for virus production or in human tumor cells. Therefore, wt H-1PV can be improved to avoid these drawbacks.

[0006] Accordingly, an object of the present invention is to provide an H-1PV construct with enhanced viral adaptability and anti-cancer efficacy.

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

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

[0009] Animal viruses have an outstanding ability to adapt to new hosts and environments

[14] . The natural rat parvovirus H-1PV, such as standard H-1PV (st H-1PV), often produces variant particles with altered genomes when growing in human cells [15 - 18]. Among the H-1PV variant viruses analyzed so far, H-1dr [internal deletion (d) and terminal repeat (r)] is fully viable. H-1dr has an in-frame deletion of 114 nucleotides (nt2022 to 2135) within the open reading frames encoding the non-structural proteins NS1 and NS2, and a 55-nucleotide repeat (nt 4828 to 4883) toward the right-end terminus of the viral genome ( Figure 1 D).

[0010] The inventors found that the above-mentioned H-1dr may have enhanced adaptability after natural adaptation in human cells, thereby producing significant anti-cancer activity. Therefore, they constructed a novel infectious recombinant molecular clone based on the characteristic genetic variations of H-1dr and named it H-1PV DR.

[0011] Accordingly, the present invention describes the innovative protoparvovirus H-1PV DR, which is characterized by an internal deletion of 114 nucleotides and a 55-nucleotide repeat motif toward the right-end terminus in the presence of the full-length right ITR, which shows improved anti-cancer activity in different cell lines derived from various tumor entities without altering the safety of the virus, thus ensuring the clinical translation of the new virus in cancer patients.

[0012] Those skilled in the art can start from the known nucleic acid and amino acid sequences of the non-structural proteins of parvoviruses such as parvovirus H-1 [8] and introduce the modifications mentioned above that lead to the desired changes in biological properties, which are the ability of the parvovirus variant to replicate and spread in human tumor cells and thus kill human tumor cells, to obtain the parvovirus variant of the present invention. Those skilled in the art can also easily detect whether a specific variant exhibits the desired biological properties by using the assays described in the following examples.

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

[0014] The inventors detected the production and infectivity of H-1PV DR and compared it with wild-type (wt) H-1PV and standard-type (st) H-1PV. For this purpose, the virus was produced by infecting NB-324K cells and harvested according to the method described in detail in Example 3 below. In these experiments, H-1PV DR showed the best viral adaptability in NB-324K cells, which was consistent with the previous observations [9].

[0015] The enhanced fitness and increased infectivity of H-1PV DR relative to the wt H-1PV virion facilitated the study of the virus propagation efficiency in NB-324K cells during the viral replication process, as described in Example 3 below. The results showed that the viral propagation efficiency of H-1PV DR during replication was higher than that of wt H-1PV, indicating that the novel features of internal deletion and terminal repeats of wt H-1PV DR might play a very important role in stimulating the viral life cycle. The morphology of various viruses was observed by electron microscopy. Consistent with the wt H1PV virus, the H-1PV DR and st H1PV viruses showed the characteristic parvovirus diameter of 25 nm and the same morphology at high resolution.

[0016] For biosafety, especially for the future clinical application of H-1PV DR, it was worthwhile to detect the stability of the internal deletion or terminal repeats of H-1PV DR by consecutive rounds of infection in permissive NB-324K cells. For this purpose, seven consecutive rounds of infection were performed. At the end of each round of infection, viral genomic DNA was isolated and the genomic stability was studied by PCR, as described in Example 4 below. After seven rounds of infection, no degradation or alteration was observed within the viral genome of H-1PV DR, indicating that both modifications were stably integrated into the viral genome. Further sequencing of the viral genome covering the modified region demonstrated the same observation. These results were consistent with the concept of H-1PV DR maintaining genomic stability in human cells over the long term.

[0017] H-1PV DR has higher viral genomic stability in permissive transformed human cells, which enabled the understanding of its immune effects and cytotoxicity on peripheral blood mononuclear cells (PBMCs) from healthy donors, which represent normal human cells. These experiments are described in detail in Example 5 below. The results showed that the virus had no effect on the immune cell population and the viability of PBMCs, indicating that PBMCs were insensitive to the cytotoxic effects of H-1PV DR and st H-1PV, and their performance was similar to that of wt H-1PV, which has been proven to have excellent safety and good treatment tolerance in two recently completed clinical trials [22, 23].

[0018] Although H-1PV DR showed the same capsid assembly pattern under the electron microscope, the recognition of neutralizing antibodies generated due to viral genome modification on H-1PV DR was tested at the molecular biology level. For this purpose, as described in Example 6 below, a neutralization assay of specific neutralizing antibodies against H-1PV DR was performed using cytotoxic protection assessment. The results showed that the recognition of neutralizing antibodies on the capsid surface of H-1PV DR was similar to that of wt H-1PV and st H-1PV, indicating that the new features of internal deletion and terminal repeats of H-1PV DR had no effect on the capsid assembly pattern of the virus.

[0019] Since the Phase I / IIa clinical trial of the patient used wt H-1PV Conducted in recurrent glioblastoma and metastatic pancreatic cancer, the applicant compared the anti-cancer effects of wt H-1PV and H-1PV DR to demonstrate the clinical translational significance of this novel virus in new strategies for cancer treatment. As mentioned above, the study showed that H-1PV DR had enhanced fitness and increased infectivity in NB-324K cells compared to the wt H-1PV virion. This raised the question of whether the superior ability of the novel H-1PV DR was cell line-dependent, as the SV40 antigen was artificially expressed in NB-324K cells

[25] . Therefore, as described in Example 7 below, the production of H-1PV DR and wt H-1PV was detected in different recipient human cell lines derived from various tumor entities. Based on these experiments, it could be excluded that the enhanced fitness of H-1PV DR was not dependent on the expression of the SV40 antigen in transformed cells, indicating that compared to wt H-1PV, H-1PV DR had the advantage of viral propagation in cancer cells derived from various tumor entities, resulting in excellent anti-cancer efficacy.

[0020] In various cancer cell lines receptive to it, the increased production of H-1PV DR virus with potent oncolytic activity prompted us to explore the oncolytic toxicity efficacy of H-1PV DR in cancer cells with different receptivities. For this purpose, as described in Example 8 below, cell lines with low virus receptivity, semi-virus-resistant cell lines, and virus-resistant cell lines were infected with increasing amounts of the virus. The results confirmed the enhanced oncolytic toxicity of H-1PV DR. However, it is worth noting that the entry of the virus is a prerequisite for the production of progeny viruses that cause oncolytic toxicity in cancer cells. This may explain why the oncolytic activity decreases from cells with low receptivity to resistant cells. Importantly, H-1PV DR can not only inhibit cell growth in 2D cell cultures but also in 3D cell spheroids in a highly transformation-related model, which is more relevant to tumor biology and represents the characteristics of cancer cell heterogeneity. Refer to Example 9 below. It can be observed that compared with the cell spheroids treated with wt H-1PV, the size of cancer cells in the cell spheroids treated with H-1PV DR increased more slowly. Interestingly, in the cancer cells treated with H-1PV DR, the growth of the cell spheroid size was almost blocked within 14 days, indicating that in addition to inhibiting proliferation, there may also be a component of cell death. In summary, these results demonstrate that H-1PV DR has more excellent anti-cancer activity than wt H-1PV, thus ensuring the clinical translation of the novel virus in cancer patients.

[0021] In summary, the present invention can improve the adaptability of the H-1PV virus, including more efficient production and increased infectivity, thereby enhancing the anti-cancer efficacy. Notably, compared with st H-1PV and wt H-1PV, the virus of the present invention containing internal deletions and terminal repeats (H-1PV DR) shows enhanced adaptability, increased progeny production, and enhanced infectivity. Importantly, H-1PV DR shows excellent oncolytic activity in different cell lines derived from various tumor entities. The verification of the therapeutic effect of H-1PV DR in a highly transformation-related model of 3D cell spheroids confirms the improved anti-cancer activity of H-1PV DR, which suggests future research on the novel virus at the clinical level. In summary, this is the first time to demonstrate that H-1PV DR enhances its oncolytic activity without changing the virus safety, thus ensuring the clinical translation of the novel virus in cancer patients. In addition, the present invention also explores the use of patient-derived organoids as a platform to predict the response of individual patients to the oncolytic toxicity of H-1PV DR to meet the feasibility of personalized medical approaches for H-1PV DR virus therapy. This is the first time to demonstrate that by establishing a platform using patient-derived organoids, the response of specific cancers to H-1PV DR treatment can be predicted.

[0022] In addition, the present invention also relates to antibodies that specifically recognize the above parvovirus variants, namely the polypeptide regions where the deletions in the parvovirus variants are located, and these polypeptide regions characterize the parvovirus variants. The antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof, such as Fab, Fv or scFV fragments. Preferably, monoclonal antibodies are considered. For production, it is advantageous to immunize animals with the above parvovirus variants or their fragments - especially immunizing rabbits or chickens to obtain polyclonal antibodies and immunizing mice to obtain monoclonal antibodies. The immunized animals can be further boosted with the same parvovirus variants or their fragments. Then polyclonal antibodies can be obtained from animal sera and egg yolks respectively. Monoclonal antibodies can be obtained according to standard methods, especially referring to 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 animals. The antibodies according to the present invention can be used in various ways, for example, for immunoprecipitation or isolation of the above parvovirus variants. The antibodies can be conjugated in immunoassays in liquid phase or on solid supports. In this regard, the antibodies can be labeled in various ways. Those skilled in the art are familiar with suitable labels and labeling methods. Examples of immunoassays are ELISA and RIA.

[0023] A kit for use in the applications of the present invention is also provided. The kit includes the following:

[0024] (a) A parvovirus variant according to the present invention;

[0025] (b) An antibody according to the present invention; and / or, optionally,

[0026] (c) Conventional auxiliaries such as solvents, buffers, carriers, labels and controls.

[0027] The present invention also relates to a pharmaceutical composition comprising an effective amount of parvovirus (H-1PV DR) and / or an antibody against the variant, and a pharmaceutically acceptable carrier. The present invention also provides the use of the said pharmaceutical composition for the treatment or prevention of cancer. The present invention also relates to the use of H-1PV DR for the preparation of (a) a pharmaceutical composition, a combination or a kit for the treatment or prevention of cancer.

[0028] As used herein, the term "treatment" and its derivatives refer to therapeutic regimens. With respect to a particular condition, treatment means: (1) ameliorating the condition or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that causes or contributes to the condition, or (b) one or more biological manifestations of the condition, (3) alleviating one or more symptoms, effects, or side effects associated with the condition, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0029] 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 appreciate that "prevention" is not an absolute term. Prophylactic treatment is appropriate, for example, when a subject is considered to be at high risk of developing cancer, such as when the subject has a strong family history of cancer or the subject has been exposed to a carcinogen.

[0030] The term "effective amount" as used herein refers to the amount of a drug or agent that is capable of eliciting a biological or medical response in the tissue, system, animal, or human being sought, such as that sought by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to any amount that, compared to a corresponding subject not receiving that amount, is capable of effecting an improvement in the treatment, cure, prevention, or alleviation of a disease, disorder, or side effect. The scope of the term also includes amounts that effectively enhance normal physiological functions. The "effective dose" that can be used to treat and / or prevent these diseases or disorders can be determined by methods known to those skilled in the art.

[0031] "Pharmaceutically acceptable" refers to any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the patient to whom it is administered. Suitable pharmaceutical carriers are well known and include phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. These carriers can be formulated by conventional methods and administered to the subject in an effective dose. Other pharmaceutically compatible carriers can include gels, bioabsorbable matrix materials, implantable elements containing a therapeutic agent, or any other suitable vehicle, delivery or dispensing means, or material.

[0032] 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 related to a neoplasm. In some embodiments, the cancer is a solid tumor, namely brain cancer (especially gliomas: ependymomas, astrocytomas [such as glioblastoma multiforme], oligodendrogliomas, brainstem gliomas, oligoastrocytomas); colon cancer (especially non-multiple sclerosis-related colon cancer (non-MSICRC)), bladder cancer, liver cancer, breast cancer (especially triple-negative or double-negative breast cancer), kidney cancer, head and neck squamous cell carcinoma, lung cancer (especially squamous cell lung cancer, 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 includes metastases of the above tumors in various organs. In a more preferred embodiment, these tumors to be treated are recurrent tumors. A particular advantage of the pharmaceutical composition of the present invention is that even cancer-initiating stem cells can be successfully treated. This has a positive effect on avoiding tumor recurrence and metastasis formation.

[0033] In other embodiments, the cancer is a hematopoietic malignancy, namely 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 lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL) or small lymphocytic lymphoma (SLL).

[0034] Administration can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intratumoral or intradermal administration. Of course, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. Within the scope of the art, the dosage regimen of the virus can be easily determined by those skilled in the art and can be determined by the attending physician based on patient data, observations and other clinical factors, which include, for example, the patient's body size, body surface area, age, gender, time and route of administration, tumor type and characteristics, the patient's general health status, and other drug treatments the patient is receiving. The choice of the dosage regimen (also referred to herein as the administration regimen) of the therapy of the present invention depends on multiple factors, including the serum or tissue turnover rate of the entity, the symptom level, the immunogenicity of the entity, and the accessibility of the target cells, tissues or organs in the individual being treated. Thus, the dose and frequency of administration depend in part on the severity of the cancer being treated and the patient characteristics.

[0035] Since the virus according to the present invention contains infectious virus particles capable of penetrating the blood system, the treatment can be carried out (or at least initiated) by intravenous injection of the virus. Since long-term intravenous treatment is prone to become ineffective due to the formation of virus-neutralizing antibodies, after the initial intravenous virus administration regimen, different administration methods can be employed, or these different administration techniques, such as intratumoral virus administration, can be alternated throughout the virus treatment process.

[0036] As another specific administration technique, the virus (virus, vector and / or cell agent) can be administered to the patient from a source implanted in the patient's body. For example, a catheter, such as a catheter made of silicone or other biocompatible material, can be connected to a small subcutaneous reservoir (Rickham reservoir) installed in the patient's body during tumor resection or through a separate procedure to allow local injection of the parvovirus composition at different times without further surgical intervention. The virus or derived vector can also be injected into the tumor by stereotactic surgical techniques or navigational positioning techniques.

[0037] The administration of the virus can also be carried out by using a suitable pump system, such as a peristaltic infusion pump or a convection-enhanced delivery (CED) pump, to continuously infuse virus particles or a liquid containing virus particles at a low flow rate through an implanted catheter.

[0038] Another method of administering the virus is from an implant that is constructed and positioned to distribute the parvovirus to the desired cancer tissue. For example, a sheet impregnated with the virus can be used and attached to the edge of the resection cavity at the end of tumor resection. Multiple sheets can be used in such therapeutic interventions. After tumor resection, cells that actively produce the virus or virus-based vectors can be injected into the tumor or the tumor cavity.

[0039] In the case of the parvovirus variant of the present invention, infection results in killing of tumor cells without harming normal cells, thereby enabling tumor-specific treatment without adverse neurological or other side effects.

[0040] The therapy of the present invention can be used before or after surgical removal of the tumor, and can also be used before, during or after radiotherapy.

[0041] The pharmaceutical composition may further comprise one or more additional therapeutic agents. The additional therapeutic agents may be, for example, chemotherapeutic agents, biotherapeutic agents (including but not limited to antibodies against VEGF, EGFR, Her2 / neu, VEGF receptor, other growth factor receptors, CD20, CD40, CD40L, CTLA-4, OX-40 4-1BB, and ICOS), checkpoint inhibitors (e.g., antibodies against PD-1 or PD-L1), immunogenic reagents (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines (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-FU), methotrexate, cytarabine, platinum analogs such as cisplatin and carboplatin; vinblastine, platinum; etoposide (VP-16); ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone; teniposide; edatrexate; daunomycin; aminopterin, xeloda; ibandronate; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoids, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene or aromatase inhibitors.

[0042] The present invention is further described with reference to the accompanying drawings, which show:

[0043] Figure 1.Genomic structure of H-1PV DR. (A) Schematic representation of the H-1PV genome. The H-1PV genome is characterized by a single-stranded molecule terminated by inverted terminal repeat (ITR) structures. The ITRs are drawn to illustrate the predicted structure. The viral early P4 promoter regulates the expression of the NS genes (yellow arrow boxes) encoding the non-structural NS1 and NS2 proteins, and the late P38 promoter regulates the expression of the VP genes (light yellow arrow boxes) encoding the viral proteins VP1 and VP2. The right-end ITR is thought to be the self-priming origins for viral genome replication and encapsidation. (B) Predicted structures of the right-end ITRs of MVM and H-1PV. The 248-nucleotide right-end telomere is shown as a black line. The 36-bp palindromic structure containing the repeated NS1-binding motif, 5′-TGGT-3′ (light orange small boxes), folds into two axial arms. Assembly of NS1 oligomers binds to the 5′-TGGT-3′ motifs within the boxes in the hairpin stem, as shown by the larger light orange boxes. The nicking positions are indicated by yellow lightning bolts, and the G-rich regions are indicated by green ellipses. (C) Truncated structure of the right-end hairpin of H-1PV. The 182-nucleotide right-end ITR of H-1PV is shown as a black line. The 36-bp palindromic structure containing the repeated NS1-binding motif, 5′-TGGT-3′, folds into two axial arms, as indicated by the light orange small boxes. (D) Schematic representation of the genomic structure of H-1PV DR. The positions of the 114-nucleotide internal deletion are indicated by grey triangles, and the positions of the 55-nucleotide repeat motifs toward the right-end terminus are indicated by pink diamonds. Other features of the viral genome are as described in (A).

[0044] Figure 2 .Increased progeny yield and infectivity of H-1PV DR with enhanced fitness in the recipient human NB-324K cells. (A) Increased infectivity of H-1PV DR compared to wt H-1PV and st H-1PV. Cells were infected with the indicated viruses at an MOI of 0.01 PFU per cell and harvested on day 5 post-infection. Titers of infectious particles and genome-containing particles were determined by plaque assay (in PFU / ml) and qPCR (in vg / ml), respectively. The ratio of genome-containing particles to infectivity (P / I) is shown in the table. (B) Demonstration of a multiplicative decrease in the P / I ratio between wt H-1PV and st H-1PV and between wt H-1PV and H-1PV DR.

[0045] Figure 3.Evaluation of the genomic stability of H-1PV DR. (A) H-1PV DR was produced by serial infection for several rounds. This production protocol was adopted to detect the stability of the novel features of internal deletions and terminal repeats in H-1PV DR. Complete infectious virus particles are generated after transfection of H-1PV DR into HEK293T cells. NB-324K cells were infected with the indicated virus at an MOI of 0.01 PFU per cell. Five days later, the infected cells were collected, and the progeny virus particles were purified, titrated, and used to infect a newly prepared batch of cells. In the figure, each round of infection and harvest corresponds to one passage, i.e., one round of virus production (P); seven rounds of production were performed (from P1 to P7). Viral DNA was extracted from each round of production, and genomic fragments encompassing the internal deletion and terminal repeats of H-1PV DR were amplified by PCR. (B) Schematic representation of the flanking regions of the primer pairs in the genomic structure of H-1PV DR. (C) The stability of the in-frame 114-nucleotide deletion (upper panel) and 58-nucleotide repeat motif (lower panel) in H-1PV DR was evaluated by PCR analysis.

[0046] Figure 4 .Evaluation of the neutralizing antibody recognition profile of H-1PV DR on the capsid surface. As described in the Materials and Methods section, neutralization assays were performed on the indicated viruses with specific neutralizing antibodies against H-1PV DR. The activity of the virus-neutralizing antibodies was determined on NB-324K cells by cytotoxicity protection using the Cell Counting Kit-8, at an MOI of 10 PFU per cell and scored 72 hours after inoculation (upper panel). The effect of the neutralizing antibodies on NB-324K cells was independently verified by crystal violet staining assay under the same conditions as described above (lower panel).

[0047] Figure 5 .High-yield production of H-1PV DR is not limited to NB-324K cells. The progeny yields of H-1PV DR in different human cell lines were measured by plaque assay. In human NB-324K, BxPC-1, Cal33, Hela, and U251 cell lines, the infectious progeny yields of H-1PV DR and wt H-1PV were determined after infection at an MOI of 0.25 PFU per cell. The yields of infectious particles (total PFU) recovered from the cell supernatant and pellet were determined by plaque assay on day 3 post-infection for the NB-324K cell line and on day 4 post-infection for the remaining cell lines. The results represent the mean of two independent experiments.

[0048] Figure 6.H-1PV DR has excellent oncolytic activity in various human cell lines derived from different cancer entities. At 96 hours post-infection with H-1PV DR and wt H-1PV, the amount of MOI increased from 12 to 100 PFU per cell, and cell proliferation and viability were evaluated using the Cell Counting Kit-8 and crystal violet staining assays, respectively. (A) Cell lines MeWo and SiHa with low virus receptivity. (B) Virus semi-resistant cell lines Capan-1 and AsPC-1. (C) Virus-resistant cell lines FaDu and ME180.

[0049] Figure 7 .Verification of the therapeutic effect of H-1PV DR in a 3D cell spheroid highly transformed-related model. H-1PV DR and wt H-1PV were infected at an MOI of 10 PFU per cell until 13 or 14 days post-infection, and cell real-time proliferation was recorded by a 3D single spheroid assay system ( 3D Single Spheroid Assays). (A) Virus semi-resistant cell line AsPC-1. (B) Virus-resistant cell line FaDu.

[0050] Figure 8. Sequence of parvovirus H1

[0051] Figure 9 : Response of quadruple mutant isogenic KAPS organoids of human intestinal stem cells to H-1PV DR. As shown, H-1PV and H-1PV DR were infected at increasing MOIs for 14 days, and cell real-time proliferation was monitored by . (A, B) wt human intestinal stem cell organoids. (C, D) Quadruple mutant isogenic KAPS organoids.

[0052] Figure 10 : Response of patient-derived organoids (PDOs) from colorectal cancer patient #1 to H-1PV DR. As shown, H-1PV and H-1PV DR were infected at increasing MOIs for 24 days, and cell real-time proliferation was evaluated by . (A, B) Normal tissue. (C, D) Primary tumor. (E, F) Metastatic tumor.

[0053] Figure 11 : Response of patient-derived organoids (PDOs) from colorectal cancer patient #2 to H-1PV DR. As shown, after infecting H-1PV and H-1PV DR at increasing MOIs for 20 days, cell real-time proliferation was detected by . (A, B) Normal tissue. (C, D) Primary tumor.

[0054] The present invention is further described in the following examples. These examples should be understood as preferred embodiments, but are not intended to limit the present invention.

[0055] Example 1: Materials and Methods

[0056] Plasmid construction. To construct pst H-1PV, first, the viral genomic fragment obtained by digesting pSR19 clone with HpaI-NdeI was subcloned into the pUC 19HpaI-NdeI vector to generate an adapter vector with a modified polylinker. A DNA fragment containing the full-length right ITR and flanked by PshAI and NdeI restriction sites, synthesized and manufactured by Biocat GmbH (Heidelberg, Germany), was shuttled into this vector to construct pst H-1PV (HpaI-NdeI). Using the same strategy, a DNA fragment containing a 55-nucleotide repeat motif (nt4828 to 4883) and flanked by HpaI and PshAI restriction sites was introduced into pstH-1PV (HpaI-NdeI). Finally, the modified construct was subcloned back into its parental pSR19 backbone to generate the molecular clones pst H-1PV and pH-1PV R. To construct H-1PV DR, a 114-nucleotide (nt2022 to 2135) in-frame deletion within the NS region of the EcoRI-MfeI digested pDelH1 plasmid was introduced into the pH-1PV R vector

[19] . The resulting pst H-1PV and pH-1PV DR plasmids were further verified by sequencing (Genewiz, Takeley Sanger Sequencing Laboratory, UK) of the replaced fragments.

[0057] Cell culture. Simian virus 40 (SV40)-transformed human neonatal kidney NB-324K cells and HEK293T cells were cultured as described previously [25, 26]. The human glioblastoma (GBM) cell line U251 was a kind gift from Dr. Iris Augustin (DKFZ, Heidelberg, Germany). The HeLa and SiHa cervical cancer (CC) cell lines were provided by Dr. Angel Alonso (DKFZ, Heidelberg, Germany). The ME180 cervical cancer (CC) cell line was provided by Dr. Elisabeth Schwarz (DKFZ, Heidelberg, Germany). The pancreatic ductal adenocarcinoma (PDAC)-derived cell lines AsPC-1, BxPC-1, and Capan-1 were provided by Dr. Stephan Herzig (DKFZ, Heidelberg, Germany). The head and neck squamous cell carcinoma (HNSCC) cell lines FaDu and Cal33 were provided by Dr. Ina Kurth (DKFZ, Heidelberg, Germany). The melanoma cell line MeWo was a kind gift from Dr. Jochen Utikal (DKFZ, Heidelberg, Germany). Cells were cultured in the media listed in Table 1, supplemented with 10% fetal bovine serum (FBS), except for NB-324K cells which were supplemented with 5% FBS (Sigma Aldrich), 2 mM L-glutamine (Gibco), and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin sulfate, Gibco). All cells were grown at 37 °C, 5% CO2, and 95% humidity, and were routinely checked for mycoplasma contamination using a Mycoplasma Detection Kit according to the manufacturer's instructions (VenorGeM, Minerva Biolabs, Berlin, Germany).

[0058] Table 1: Cell lines and corresponding media and supplements (where applicable)

[0059] Cell line Culture medium Supplement 1 Supplement 2 Supplement 3 HEK293T DMEM NB-324K MEM U251 DMEM HeLa DMEM SiHa DMEM ME180 DMEM AsPC-1 DMEM BxPC-1 DMEM Capan-1 DMEM FaDu DMEM 100 mM Sodium pyruvate 1 M Hepes Non-essential amino acids Cal33 DMEM MeWo DMEM PBMC RPMI1640

[0060] For experiments, cells were counted using trypan blue and a Countess TM Automated Cell Counter (Invitrogen, USA), and were seeded at the cell numbers shown in Table 2 unless otherwise specified. 3 ml of media was used for 6-well plates, 5 ml of media was used for 6 cm dishes, and 100 μl / well was used for 96-well plates.

[0061] Table 2: Cell numbers seeded in experiments

[0062]

[0063]

[0064] Transfection assay. HEK293T cells were seeded into 6-well plates with 3 ml of Opti-MEM reduced serum medium (Invitrogen, Cat. No. 31985). The next day, 6 μg of plasmid carrying the viral genome (pwt H-1PV, pstH-1PV or pH-1PV DR) was transiently transfected into the cells using the transfection reagent Metafectene (Biontex Laboratories GmbH, Cat. No. T020-1.0, Munich, Germany) at a ratio of 1:2 (μg DNA: μl reagent) according to the manufacturer's instructions.

[0065] Virus infection and production. The above viruses were mainly produced by transfecting 293T cells and then amplified by infecting NB-324K cells at a multiplicity of infection (MOI) of 0.01 PFU per cell. Cells and purified viruses were harvested by iodixanol step gradient centrifugation according to the method described previously

[27] .

[0066] Titration of infectious and intact particles. The titration of infectious virus particles was performed using the plaque assay method as described previously

[25] . The quantification of intact virus particles was performed using the real-time quantitative PCR method as described previously

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

[0067] Electron microscopy. Viruses in the dialysis preparation were adsorbed onto glow discharged carbon coated grids, washed with water (Braun, Ampuwa), and negatively stained with 1% uranyl acetate aqueous solution. Micrographs were acquired at 80 kV using an EM 912 equipped with a slow-scan CCD camera (TRS, Moorenweis, Germany) (Carl Zeiss Company, Oberkochen, Germany).

[0068] The stability of internal deletions and terminal repeats was evaluated by PCR. NB-324K cells were infected with consecutive rounds of H-1PV DR. At the end of each round of infection, viral genomic DNA was isolated as described previously

[26] . PCR was performed using CloneAmp HiFi PCR Premix (Takara Bio Clontech. Cat. No. 639298, Japan) with primer pairs (deletion forward primer 5′-TCAATGCGCTCACCATCTCTG-3′ and deletion reverse primer 5′-TTAGTCCAAGGTCAGCTCCTC-3′ or repeat forward primer 5′-TAATATGGTATTGGTTAACTGTAAAAAAT-3′ and repeat reverse primer 5′-CAACCACCCAACCACCCTTT-3′). The PCR mixture was added to 2% or 3% agarose gels for electrophoresis. Images were recorded by INTAS (Intas Pharmaceuticals Limited, India).

[0069] Spectral flow cytometry data analysis. PBMCs were stained with 1:1000 diluted ZombieNIR as a live-dead stain in PBS for 30 minutes at room temperature in the dark. Then, after adding FcX and monocyte blocker (Biolegend) for 10 minutes at room temperature in the dark, fluorescently labeled antibodies (Table 1) were added. After incubation for 60 minutes at room temperature in the dark, the cells were washed and measured on an AURORA spectral flow cytometer (Cytek Biosciences). Unsupervised data analysis as detailed below was performed using OMIQ data analysis software (www.omiq.ai). First, the data were manually gated to remove aggregates, dead cells, debris, and then the data were subsampled to include 2.5x10 5 CD45 +Leukocyte group. Next, run flowAI to check for any abnormal regions of the files

[29] . The flowAI settings are as follows: use all files, select all fluorescence channels and time, use all methods and default settings. Subsequently, perform dimensionality reduction analysis using Uniform Manifold Approximation and Projection (UMAP) to visualize the different subpopulations in the group

[30] . The UMAP settings are as follows: use all files, use 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, Embedding Initialization = spectral.

[0070] Development of monoclonal antibodies. The generation of monoclonal antibodies was carried out according to the technical principle

[31] . For mouse immunization, multiple injections were performed using the viral particle H-1PV DR. To enhance the immune response, 100 μl of Freund's complete adjuvant (Santa Cruz Biotechnology) was injected into the hind legs of each mouse. Booster injections were carried out using Freund's complete adjuvant and then only buffer was injected. The operation of the fusion process to generate hybridoma cell clones is as follows: surgically excise the popliteal lymph nodes and place them in RPMI medium (Gibco). Subsequently, grind the lymph nodes with a syringe plunger under a microscope. The cell mixture was centrifuged at 150 xg for 10 minutes at room temperature. The cells were resuspended in 1.5 ml of polyethylene glycol (PEG, Sigma-Aldrich), added gradually within 1 minute, and mixed with a Pasteur pipette. 20 ml of RPMI medium was added gradually and continuously within 4 minutes, then centrifuged at 150 xg for 10 minutes. After that, the cells were resuspended in HAT medium containing Hyper and cultured for 7 days. After 7 days, the cell supernatant was screened for the presence of specific antibodies against the protein of interest by ELISA and subsequent immunofluorescence. The verified parental clone was subcloned by limiting dilution to obtain monoclonal cell clones.

[0071] Neutralization assay. The virus (10 PFU per cell) was incubated with different anti-H-1PV DR monoclonal antibodies in primary MEM medium with a final volume of 60 μl at 37 °C for 30 minutes. At 72 hours post-inoculation, residual infectivity assays were performed on NB-324K cells using the Cell Counting Kit-8 assay and crystal violet staining assay.

[0072] Cell Counting Kit-8 assay. Cells were seeded in 96-well plates containing 50 μl / well of medium. Their respective media and cell numbers are shown in Tables 1 and 2, namely NB-324K, MeWo, SiHa, AsPC-1, Canpan-1, FaDu, and ME180. After 24 hours, 50 μl / well of FBS-free medium containing or not containing st H-1PV or H-1PV DR was added. At 72 or 96 hours post-infection, cell proliferation was detected using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan) as described previously

[32] .

[0073] Crystal violet staining assay. Cells were seeded and treated as described above. Cell viability was determined using the crystal violet assay as described previously

[33] .

[0074] Generation of cell spheroids. 3D cell spheroids represent the heterogeneity of tumor models because cells on the outer layer of the cell spheroid can obtain nutrients and oxygen, while in the center of the cell spheroid, the accumulation of cell degradation products forms a hypoxic area. As described previously

[34] , cell 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. After 2-3 days, the formed cell spheroids were transferred to a low-adhesion round-bottom 96-well plate. 50 μl of complete medium containing or not containing st H-1PV or H-1PV DR was added to each well. Using the spheroid collection and analysis tool, 3D single spheroid assay system ( 3D Single Spheroid Assays), the size of the cell spheroids was analyzed in real time. Generally, 3 images per well were acquired at a magnification of 10 times per day. Analysis was performed using S3 2018A software.

[0075] Culture of human intestinal stem cell wild-type (wt) and quadruple mutant isogenic KAPS organoids. Colorectal tumor organoids carrying the KRAS, APC, TP53, and SMAD4 (KAPS) mutation combination were generated as previously described

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

[0076] Generation of patient-derived organoids (PDOs) in colorectal cancer. Organoids were established from patient-derived paired colon normal tissues, primary or metastatic tumor tissues collected at the time of surgical resection according to a previous protocol

[36] . Briefly, after 2 weeks of Wnt deprivation to enrich tumor and metastatic cells under the corresponding conditions, all organoids were cultured in dome-shaped structures of Cultrex Basement Membrane Extract Type 2 (BME; R&D Systems) and covered with complete "hC" expansion medium to ensure comparability under different conditions. The complete organoid medium consisted of Advanced DMEM / F12 (Gibco), 1×B27, 1×Glutamax, 10 mmol / l HEPES, 0.1 mg / ml primocin (all from Thermo-Fisher), 1.25 mM N-acetylcysteine, 10 μM nicotinamide, 10 μM p38 inhibitor SB202190 (all from Sigma-Aldrich), 0.5 nM Wnt replacement-Fc fusion protein, 2% noggin conditioned medium, 2% Rspo3 conditioned medium (all from U-Protein Express), 50 ng / ml EGF (Peprotech), 0.5 μM A83-01 and 1 μM PGE2 (all from Tocris). To evaluate the response to viral therapy, the organoids were single-cell seeded. Dissociation was performed using TrypLE Express enzyme (Gibco), and 8,000 single cells were plated in 1 dome of 8 μl each per well of a 96-well plate. In each experiment, the seeding of the wells was repeated three times and each condition was independently processed. After seeding, 100 μl of medium was added. H-1PV DR and H-1PV were both dissolved in 50 μl of medium to reach the desired MOI and added 24 hours after the first seeding. The growth of the organoids was evaluated using the IncuCyte system (37 °C, 5% CO2), and imaging was performed every 3 days for 24 days. Image analysis was performed using the Incucyte 3D cell spheroid analysis software, which allowed measurement of the confluent area of the PDOs over time.

[0077] Human materials for organoid culture. This study was approved by the Ethics Committee II of the Mannheim Medical Faculty of Heidelberg University. Written informed consent was obtained.

[0078] Example 2: Generation and production of H-1PV DR

[0079] In the proof-of-concept study, the molecular cloning of pst H-1PV ( Figure 1 A) was generated by inserting the full-length right ITR on the basis of the previous pSR19 plasmid [9], and pH-1PV DR ( Figure 1 D) was generated by deleting 114 nucleotides within the NS region and inserting a 55-nucleotide repeat motif to the right end on the basis of 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 carried out. Generally, parvovirus production is first carried out by transiently transfecting the constructs of pst H-1PV and pH-1PV DR in HEK293T cells. Approximately 96 hours after transfection, the cells were lysed, the newly assembled virus particles were purified, and titrated by plaque assay and real-time quantitative PCR. Further amplification of the virus was carried out in NB-324K cells. Cells were infected with a MOI of 0.01 PFU (plaque-forming units / ml) per cell using the virus previously produced in transfection. When cytopathic effects indicating virus replication and excretion 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 named pSR19 were transfected into HEK293T cells respectively to generate master stocks. Individual viruses were prepared in parallel by infecting NB-324K cells as described in Example 1 above.

[0080] Example 3: Increased progeny production and infectivity of H-1PV DR and enhanced fitness in recipient human NB-324K cells

[0081] The inventors examined the production and infectivity of H-1PV DR and compared them with wild-type (wt) H-1PV and standard-type (st) H-1PV. For this purpose, virus was produced while infecting NB-324K cells, and the virus was harvested 72 hours after infection. The numbers of genome-containing (i.e., complete) and plaque-forming (i.e., infectious) particles were determined by real-time quantitative PCR and plaque assay respectively. Figure 2 A lists the numbers of complete virus particles (in vg / ml) and infectious titers (in PFU / ml) in two independent experiments. Although the number of complete particles produced by infecting wt H-1PV was much higher than that produced by infecting st H-1PV and H-1PV DR, the latter two produced higher infectious virus titers than wt H-1PV. Interestingly, as Figure 2As shown in the right column, the corresponding intact particle-to-infectivity ratio (P / I) decreased sharply, resulting in a significant decrease (8 - 10-fold) in the P / I ratio of st H-1PV and H-1PV DR ( Figure 2 B). These results indicate that the full-length right ITR is a key element for parvovirus to participate in viral genome encapsidation, excision from viral DNA replication intermediates, and lead to high yields of infectious virions. Last but not least, H-1PV DR showed the best viral fitness in NB-324K cells, which is consistent with previous observations [9].

[0082] H-1PV DR has enhanced fitness and increased infectivity compared to wt H-1PV virions, which facilitated the study of the viral dissemination efficiency in NB-324K cells during the viral replication process. For this purpose, plaque assays were performed. All viruses produced a mixture of plaques of different sizes. Different from that, in cells infected with H-1PV DR, the frequency of large plaques was higher, while in cells infected with wt H-1PV, the frequency of small plaques was higher. These results indicate that H-1PV DR has a higher viral dissemination efficiency during replication compared to wt H-1PV, suggesting that the novel features of internal deletion and terminal repeats of H-1PV DR may play a very important role in stimulating the viral life cycle. The morphology of various viruses was examined by electron microscopy. Consistent with wt H1PV, H-1PV DR and st H1PV viruses showed the characteristic parvovirus diameter of 25 nm and the same morphology at high resolution.

[0083] Example 4: Assessment of the stability of the internal deletion and terminal repeats of H-1PV DR

[0084] For biosafety, especially for the future clinical application of H-1PV DR, it is worth examining the stability of the internal deletion or terminal repeats of H-1PV DR by continuous infection for several rounds in the recipient NB-324K cells. For this purpose, as Figure 3 shown in A, seven consecutive rounds of infection were performed. As Figure 3 shown in B, at the end of each round of infection, viral genomic DNA was isolated and PCR was performed using primers flanking the region of nucleotides 2022 to 2135 or nucleotides 4828 to 4883 to detect genomic stability.

[0085] After seven rounds of infection, no degradation or alteration was observed within the viral genome of H-1PV DR, indicating that these two modified alterations were stably integrated into the viral genome. As Figure 3 shown in C, lanes 1 to 4 represent the 4th to 7th generations of virus passage, respectively. Unmodified wt H-1PV was used as a control ( Figure 3C, lane 5). Further sequencing of the viral genome covering the modified region demonstrated the same observations. These results are consistent with the concept that H-1PV DR maintains genomic stability in human cells over the long term.

[0086] Example 5: Normal human cells are insensitive to the cytotoxic effects of H-1PV DR

[0087] H-1PV DR has higher viral genomic stability in recipient transformed human cells, which enables an understanding of its immune effects and cytotoxic effects on healthy donor peripheral blood mononuclear cells (PBMCs) representing normal human cells. For this purpose, PBMCs were cultured and infected in vitro at a maximum MOI of 10 PFU per cell with wt H-1PV, st H-1PV, and H-1PV DR, respectively, and spectral flow cytometry analysis was performed. Data analysis was performed using a 36-color panel for immunophenotyping. UMAP plots showed that under any analysis conditions including mock-infected PBMCs, the immune cell populations highly overlapped, indicating that viral infection had no effect on any immune cell population under the assault of viral infection. These viruses had no effect on the immune cell population and the viability of PBMCs, indicating that PBMCs are insensitive to the cytotoxic effects of H-1PV DR and st H-1PV, and behaved similarly to wt H-1PV.

[0088] Example 6: Evaluation of the neutralizing antibody recognition profile of H-1PV DR on the capsid surface

[0089] Although H-1PV DR showed the same capsid assembly pattern by electron microscopy, the recognition of H-1PV DR at the molecular biology level by neutralizing antibodies generated due to viral genome modification was tested. For this purpose, a neutralization assay was performed by cytotoxic protection using specific neutralizing antibodies against H-1PV DR. At 72 hours post-inoculation, the activity of viral neutralizing antibodies was measured on NB-324K cells using a cell counting kit-8 at an MOI of 10 PFU per cell ( Figure 4 , upper figure). As a control, mouse IgG showed no viral neutralizing activity and thus no cytotoxic protection after incubation with the virus. In contrast, different monoclonal antibodies against H-1PV DR showed a distinct ability to neutralize viral infectivity and produced effective cytotoxic protection like mock-infected NB-324K cells. Interestingly, the same observations were also found when experiments were performed using wt H-1PV and st H-1PV under the same experimental settings. Under the same conditions, the similar effects of neutralizing antibodies on different viruses in NB-324K cells were independently demonstrated by the crystal violet staining assay ( Figure 4, (see the figure below). These results indicate that, compared with wt H-1PV and st H-1PV, the recognition of H-1PV DR on the capsid surface by neutralizing antibodies is similar, suggesting that the new features of internal deletion and terminal repeats of H-1PV DR have no effect on the virus capsid assembly pattern.

[0090] Example 7: Efficient production of H-1PV DR in different recipient human cancer cell lines

[0091] As described above, it has been demonstrated that H-1PV DR has enhanced fitness and increased infectivity in NB-324K cells compared to H-1PV wt virions. This raises the question of whether the superior ability of the novel H-1PV DR is cell line-dependent, as the SV40 antigen is artificially expressed in NB-324K cells

[25] . Therefore, the production of H-1PV DR and wt H-1PV was examined in different recipient human cell lines derived from various tumor entities. Cells of NB-324K, pancreatic ductal adenocarcinoma (PDAC) BxPC-1, head and neck squamous cell carcinoma (HNSCC) Cal33, cervical cancer HeLa, and glioblastoma (GBM) U251 were infected with H-1PV DR and wt H-1PV at an MOI of 0.25 PFU per cell. Infectious virions were harvested from NB-324K cells 3 days post-infection, and the remaining cell lines were harvested 4 days post-infection and quantified by plaque assay. Not only was the total amount of infectious particles (the sum of cell pellet and medium) higher in NB-324K cells after infection with H-1PV DR than with wt H-1PV, but it also increased in the remaining four cell lines from different tumor entities ( Figure 5 ). Based on these observations, it can be excluded that the enhanced fitness of H-1PV DR in transformed cells is not SV40 antigen expression-dependent, suggesting that H-1PV DR has an advantage in virus propagation in cancer cells derived from various tumor entities compared to wt H-1PV, resulting in excellent anti-cancer effects.

[0092] Example 8: The oncolytic toxicity of H-1PV DR is superior to that of wt H-1PV

[0093] The enhanced virus production of H-1PV DR with strong oncolytic activity in various recipient cancer cell lines prompted the inventors to explore the efficacy of the oncolytic toxicity of H-1PV DR in cancer cells with different susceptibilities. For this purpose, melanoma MeWo cells and cervical cancer SiHa cells were infected with increasing amounts (MOI from 12 to 100 PFU per cell) of H-1PV DR, wt H-1PV virus, or treated with virus dilution buffer as a mock ( Figure 6A) Cell lines with low virus receptivity, including pancreatic ductal adenocarcinoma (PDAC) Capan-1 cells and AsPC-1 cells ( Figure 6 B) Cell lines with semi-resistance to the virus, and including head and neck squamous cell carcinoma (HNSCC) FaDu cells and cervical cancer ME180 cells ( Figure 6 C) Cell lines with resistance to the virus,. Four days after infection, the cell proliferation was analyzed using the Cell Counting Kit-8 to determine the virus-induced cytotoxicity. As Figure 6 Shown in the upper panel of A, it was observed that compared with wt H-1PV, in the two cell lines treated with H-1PV DR, the cell proliferation decreased significantly in a virus dose-dependent manner, and. The analysis of cell viability using the crystal violet staining assay also confirmed the enhanced oncolytic toxicity of H-1PV DR ( Figure 6 A, lower panel). However, a decrease in cell proliferation and survival rate of the semi-resistant cell lines Capan-1 or AsPC-1 was only observed at a relatively high virus titer, and a stronger inhibition of cell growth was observed in the cells treated with H-1PV DR ( Figure 6 B). In addition, even at the highest virus titer, wtH-1PV had no effect on the virus-resistant cell lines FaDu or ME180 cells. At the highest virus titer, only a slight decrease in cell proliferation was observed in FaDu cells treated with H-1PV DR ( Figure 6 C). It is noteworthy that virus entry is a prerequisite for the production of progeny viruses that cause oncolytic toxicity in cancer cells. This may explain why the oncolytic activity decreases from cells with low receptivity to resistant cells.

[0094] Importantly, H-1PV DR not only inhibits cell growth in 2D cell cultures, but also inhibits cell growth in 3D cell spheroids of highly transformed-related models, which is more relevant to tumor biology and represents the heterogeneity of cancer cells. For the generation of cell spheroids, a protocol has been established that includes supplementing methylcellulose in the medium before transferring to ultra-low attachment microplates, and forming cell spheroids by the hanging drop method. Using the cell spheroid collection and analysis tool, using 3D single cell spheroid assay system ( 3D Single Spheroid Assays) to measure the size of cell spheroids treated with H-1PV DR and wt H-1PV at an MOI of 10 PFU per cell. This device can monitor cell proliferation in real time. Figures 8A and B depict the increase in the size of cell spheroids after treatment. It can be observed that in AsPC-1 cells, compared with the cell spheroids treated with wt H-1PV, the size of the cell spheroids treated with H-1PV DR increased more slowly ( Figure 7A). Interestingly, in FaDu cells, the growth of the size of spheroids treated with H-1PV DR was almost blocked over 14 days ( Figure 7 B), suggesting that in addition to inhibiting proliferation, there may be a component of cell death. Collectively, these results provide proof-of-concept that H-1PV DR has stronger anti-cancer activity than wt H-1PV, thus warranting clinical translation of the novel virus for cancer patients.

[0095] Example 9: Response of quadruple mutant isogenic KAPS organoids of human intestinal stem cells to H-1PV DR.

[0096] Oncolytic viruses (OVs) are powerful new therapeutic agents in cancer treatment. However, not all patients receiving oncolytic virus therapy for solid tumors can exhibit durable responses. Recently, the idea of using human organoids to individually screen drugs for specific patients has been proposed. The ability of patient-derived tumor organoids to recapitulate key features of the original cancer tissue makes them a preclinical model for cancer research and precision medicine. A representative colorectal cancer organoid model, the mutant isogenic KAPS organoids, was generated by introducing quadruple mutations (KRAS, APC, P53, and SMAD4) into cultured human intestinal stem cells using CRISPR / Cas9 technology, growing into tumor cells with invasive cancer features in vivo and in vitro

[35] . To test whether H-1PV DR has excellent anti-cancer activity in human organoid cultures, wild-type (wt) and mutant isogenic KAPS organoids were infected with H-1PV or H-1PV DR, as Figure 9 shown. The growth of the organoids was evaluated by imaging every 3 days for 14 days using IncuCyte 3D spheroid analysis software. As expected, compared with mock treatment, normal human intestinal stem cells were insensitive to both viruses with increasing virus amounts, indicating that H-1PV DR is safe and not pathogenic to normal cells ( Figure 9 A and 9B). Both viruses were oncolytic to the treated mutant isogenic KAPS organoids. Interestingly, compared with H-1PV, the increase in the size of the organoids treated with H-1PV DR was much slower ( Figure 9 D and 9C). Over 14 days of treatment with H-1PV DR, the growth of the organoids was almost inhibited even at the lowest MOI of 25, indicating that H-1PV DR has stronger oncolytic activity than H-1PV in mutant isogenic KAPS organoids, with a component of cell death. In contrast, treatment with H-1PV showed oncolytic activity only at the highest MOI of 100. Collectively, these results provide proof-of-concept that in the human quadruple mutant isogenic KAP organoid model, H-1PV DR has superior anti-cancer activity than H-1PV.

[0097] Example 10: Response of patient-derived organoids (PDOs) from colorectal cancer patients #1 and #2 to H-1PV DR

[0098] Recently, patient-derived organoids (PDOs) from colorectal cancer patients have been used to study cancer development and potential prediction of clinical response to chemotherapy. The excellent anti-cancer activity of H-1PV DR enhanced in engineered human colorectal cancer organoids prompted the inventors to explore the feasibility of using PDOs as a platform to predict the tumorotoxic response of colorectal cancer patients to H-1PV DR. To this end, PDOs were established from normal tissues, 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 performed routinely after surgery. To evaluate the response to virus therapy, colorectal cancer PDOs were exposed to increasing amounts of H-1PV or H-1PV DR. As previously described, the change in the confluent area of PDOs over time was measured. Consistent with the results of organoids derived from human intestinal stem cells, normal tissues were insensitive to both H-1PV or H-1PV DR with increasing virus amounts compared to mock treatment, indicating that H-1PV DR is safe and does no harm to normal cells of the two patients ( Figure 10 A, 10B, and Figure 11 A). In contrast, both viruses exhibited tumorotoxicity in the primary tumor cells infected in patient #1 ( Figure 10 C and 10D). Interestingly, compared to the organoids treated with H-1PV ( Figure 10 C), the size of the organoids treated with H-1PV DR ( Figure 10 D) increased much slower and in a virus dose-dependent manner. However, only H-1PV DR showed anti-cancer activity in the primary tumor cells of patient #2 ( Figure 11 D), while H-1PV showed no anti-cancer activity even at the highest MOI of 100, indicating a stronger resistance and poorer reactivity of the primary tumor cells of patient #2 to H-1PV ( Figure 11 C). Although the proliferation state of metastatic tumor cells was much lower than that of primary tumors, the growth of the size of organoids in metastatic tumor cells was almost inhibited 24 days after infection with H-1PV DR at the highest MOI ( Figure 11F), indicating that H-1PV DR has strong oncolytic toxicity after infection and can inhibit the proliferation of tumor cells. Collectively, these results provide proof-of-concept that H-1PV DR has superior anti-cancer activity compared to H-1PV, ensuring the clinical translation of this novel virus for patients with colorectal cancer. Importantly, it demonstrates for the first time the feasibility of using patient-derived organoids (PDOs) to establish a platform for predicting the response of colorectal cancer (CRC) to H-1PV DR, targeting the cancer cells of specific patients to meet the personalized medicine approach, thereby improving treatment efficacy and achieving better clinical outcomes.

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Claims

1. An H-1 parvovirus variant, which contains, in the wild-type H-1PV genome of Figure 8, a deletion of 114 nucleotides in-frame in the open reading frames encoding non-structural proteins NS1 and NS2 at nucleotides 2022 to 2135, and a duplication of 55 nucleotides at nucleotides 4828 to 4883 towards the right end.

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

3. A kit, which comprises: (a) The parvovirus variant of claim 1, and (b) the antibody according to claim 2; and / or, optionally, (c) conventional auxiliaries such as solvents, buffers, carriers, markers and controls, wherein each of components (a) to (c) may be present in one or more representatives.

4. A pharmaceutical composition, which comprises (a) the parvovirus variant according to claim 1 or the antibody according to claim 2 and (b) a pharmaceutically acceptable carrier.

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

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

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

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 lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL) or small lymphocytic lymphoma (SLL) and / or its metastasis.

9. Use of the parvovirus variant according to claim 1 in a method for the treatment of cancer.

10. The use of the parvovirus variant according to claim 9, wherein the cancer is a solid tumor or a hematological tumor.

11. Use of the parvovirus variant of claim 10, wherein the solid tumor is brain cancer, colon cancer, bladder cancer, liver cancer, breast cancer, kidney cancer, head / 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. Use of the parvovirus variant of claim 11, 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 lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL) or small lymphocytic lymphoma (SLL) and / or its metastasis.

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