WO2023078574A1

WO2023078574A1 - A pharmaceutical kit for oncolytic virotherapy of breast cancer, its preparation and use

Info

Publication number: WO2023078574A1
Application number: PCT/EP2021/080992
Authority: WO
Inventors: Beata HALASSY; Dubravko FORCIC; Tihana KURTOVIC
Original assignee: Sveuciliste U Zagrebu
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2023-05-11

Abstract

The present invention discloses a pharmaceutical kit consisting of composition I and composition II. Composition I comprises a viable Measles virus (MeV), Edmonston Zagreb vaccine strain with one or more pharmaceutical excipients. Composition II comprises a viable, Vesicular stomatitis virus (VSV) Indiana strain according to ATCC, with one or more pharmaceutical excipients. The final dosage forms are a suspension or a lyophilized powder for reconstitution of injectable suspension. The compositions I and II from the said kit are administered by a specific dosage regimen as a therapy complementing surgical excision. The dosage regimen in combination with surgery enables effective therapeutical treatment of carcinomas, in particularly the treatment of early-stage breast cancers, including locally recurrent breast cancer.

Description

A PHARMACEUTICAL KIT FOR ONCOLYTIC VIROTHERAPY OF BREAST CANCER, ITS PREPARATION AND USE

DESCRIPTION

Technical Field

The present invention relates to pharmaceutical product in the form of a kit consisting of two separate compositions containing oncolytic viruses for parenteral administration, to pharmaceutical technology of its manufacturing, and its medical use.

Technical Problem

The technical problem solved by the present invention includes an effective and safe intratumoral (i.t.) treatment of cancer with a combination of two different viruses of oncolytic properties, to avoid all obstacles known in the field of oncovirus-based therapies:

(i) maintenance of adequately high infective oncovirus concentration in tumour environment constantly, to prevent or minimize local virus-specific antibody response; with,

(ii) as few as possible adverse effects.

The present invention effectively solved such defined technical problem in a manner as described in the section Detailed Description of the invention.

Previous State of Art

Oncolytic virotherapy (OV therapy) is a promising therapy in development for many cancer types, that has been investigated for quite a long time, almost 100 years, without the success to enter into clinical practice, until recently. The whole field has speeded up in the last 20 years due to the development of technologies enabling detailed virology studies at a molecular level as well as evaluation of virus safety and anti-tumour efficacy, but also enabling high-titre virus production, e.g, see literature reference 1:

1) S. J. Russell, K. W. Peng, J. C. Bell: Oncolytic Virotherapy, Nat. Biotechnol. 30 (2014) 658-670.

Such speeding up resulted in the first (outside China) approval of OV therapy for metastatic melanoma, based on herpes simplex virus coding for GM-CSF and named Talimogene Laherparepvec or T-Vec, see literature reference 2:

2) S. L. Greig: Talimogene Laherparepvec: First Global Approval, Drugs 76 (2016) 147-154.

Although breast cancer (BC) was not originally targeted for OV therapy development, nowadays the OV therapy has being developed for this type of cancer, also, with several products in different phases of clinical development, see literature references 3-5:

3) S. Chaurasiya, Y. Fong: Viroimmunotherapy for breast cancer: promises, problems and future directions, Cancer Gene Ther. 28 (2021) 757-768.

4) P. Msaouel, M. Opyrchal, A. Dispenzieri, K. W. Peng, M. J. Federspiel, S. J. Russell, E. Galanis: Clinical Trials with Oncolytic Mealses Virus: Current Status and Future Prospects, Curr. Cancer Drug Targets 18 (2018) 177-187.

5) V. Martini, F. D'Avanzo, P. M. Maggiora, F. M. Varughese, A. Sica, A. Gennari: Oncolytic virotherapy: New weapon for breast cancer treatment, Ecancermedicalscience 14 (2020) 1-19.

The slow entrance of OV therapy in clinical practice is partially due to inappropriateness of preclinical models to fully predict effectiveness and safety in humans, see literature reference 1. Another reason might lay in the fact that OV therapy in clinical trials is mostly evaluated in metastatic patients that have already passed many rounds of chemotherapy, radiotherapy and/or immunotherapy (if available), and that have from that reason sub-optimally active, and often impaired activity of immune system.

Thorne disclosed a method for treating cancer with two different recombinant microorganisms that could be viruses, where the first recombinant virus replicates in a tumour cell and does not replicate in a non-tumour cell or displays attenuated replication in a nontumour cell, that is additionally characterized by at least one additional feature:

(i) enhancing or eliciting an immune response to a tumour protein that is not coded for or is not expressed by the first and second virus;

(ii) enhancing or eliciting an immune response to a protein expressed by a tumour associated cell that is not coded for or expressed by the first or second recombinant virus; see literature reference 6.

This document teaches that cancer could be treated with, among other microorganisms, with two different recombinant viruses which enable decreasing of the level expression of one or more tumour proteins, a change in population of immune cells in the subject, an enhancement in B-cell, CD4+ T cells, CD8+ T cells, cytokine production, or antigen presenting cell proliferation in the subject. The therapy regimen includes the administration of the first (prime) virus formulation, while the second (boost) virus formulation could be administered one or more times, 1, 2, 3, 4, 5, 6, 7 days, weeks, or even months after the primary administration, see literature reference 6.

The formulation of the second virus could be administered at the virus concentration between 10³-10¹⁵ viral particles/dose or 10³-10¹⁵ plaque forming units (PFU)/mL in various ways, including by intratumoral (i.t.) application via injection. Finally, this document discloses that the viruses that could be used either in the first (prime) formulation is recombinant Vesicular stomatitis virus (VSV) or recombinant Measles virus (MeV), see literature reference 6: 6) W02020146411A1; S. H. Thorne: Methods of treating cancer;

Applicant: Western Oncolytics Ltd, Pittsburg, PA, US; priority date: 07.01.2019.

The therapeutic method from this document is based on recombinant viruses, while the technical solution from the present invention is based on a native viruses' strains, MeV and VSV, what represents a key difference and brings an unexpected characteristic of the present invention.

Bell and Stojdl disclosed a therapeutic method for cancer treatment based on the use of an attenuated rhabdovirus with modified G protein, which is able to kill hyperproliferative cells like cancer cells and metastatic cancer cells, as well as the method for preforming this therapeutic procedure. This also includes the application of the second oncolytic virus which, among others, could be Measles virus. The administration of the viral composition is realized by a direct intratumoral injection in one or more dosages at the virus concentration from 10³-10¹⁵ PFU or VP per dose. Additionally, this document includes both very short time in-between the administration of the formulation I and formulation II, as well as much longer time interval, up to several (8) weeks. The document teaches that the cancer could be treated with two subsequent treatments with oncolytic viruses. In the first treatment, a modified VSV is used, while in the second treatment, MeV could be employed. It is important to outline that this invention excludes possible vice versa treatment: first with MeV formulation and then with modified VSV formulation, see literature reference 7:

7) W02011/070440A2; J. C. Bell, D. F. Stojdl: Oncolytic Rhabdovirus;

Applicant: Ottawa Hospital Research Institute, Ottawa, ON, CA; priority date: 10.12.2009.

In contrast to the technical solution from the present invention, this patent is based on a combination of attenuated rhabdovirus, with modified G protein and not a native strain of the same. This detail brings a substantial difference to the solution from the present invention since such type of modified rhabdovirus is of completely different immunological response than specific native strains of MeV and VSV from the present invention.

Van den Pol and Wollmann described a method for treating cancer with chimeric VSV Indiana strain at the dosage of 10²-10¹² PFU by an injection into, or adjacent to, a tumour in the subject, see literature reference 8:

8) WO2015/077714A1; A. N. Van den Pol, G. Wollmann: Chimeric VSV virus compositions and methods of use thereof for treatment of cancer; Applicant: Yale University, New Haven, CT, US; priority date: 22.11.2013.

The solution from this document is also based on a modified, chimeric VSV, while the solution from the present invention is based on specific strains of native MeV and VSV.

Additionally, Russell and coworkers disclosed the method for treating cancer by a direct intratumoral injection of attenuated MeV, including its Edmonston Zagreb strain, in the dosage of 10³-10¹² PFU, see literature reference 9:

9) WO02/23994A1; S. J. Russell, A. Fielding, K.-W. Peng, D. Grote:

A Method for limiting the growth of cancer cells using an attenuated measles virus; Applicant: Mayo Foundation for Medical Education and Research, Rochester, MN, US; priority date: 22.09.2000.

The solution from this document is also based on attenuated MeV and did not recognize a synergistic effect of native Indiana strain VSV on integral oncolytic effect of such combination like in the present invention. According to our best knowledge, the solution from the present invention brings a new and inventive solution to the defined technical problem as disclosed in Detailed description of the invention.

Summary of the Invention

The present invention discloses a pharmaceutical kit for oncolytic virotherapy, consisting of:

(i) composition I, which is composed of:

- a viable Measles virus (MeV), Edmonston Zagreb measles vaccine strain, and,

- one or more pharmaceutical excipients, and,

(ii) composition II, which is composed of:

- a viable Vesicular stomatitis virus (VSV) Indiana strain, according to American Type Culture Collection (ATCC), and,

- one or more pharmaceutical excipients, and, where said excipients enable formation of the final dosage forms of a suspension or a lyophilized powder for reconstitution of injectable suspension.

The concentrations of viruses MeV and VSV in the composition I and II within the kit is as follows:

(i) MeV concentration in composition I is between 1^,10⁵-1^,10¹⁰ fifty- percent cell culture infective dose (CCID50) mL^-1; and,

(ii) VSV concentration in composition II, is between 1^,10⁷-1^,10¹¹ CCID50 mLA

Preferably, the concentration of viruses are as follows:

(i) MeV concentration in composition I is between 1^,10⁵-1^,10⁸ CCID₅₀ mL^-1, and,

(ii) VSV concentration in composition II is between 1^,10⁷-1^,10¹⁰ CCID50 mLA

Pharmaceutical excipients are selected from the group consisting of: pharmaceutical water for injections, stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives. The invention discloses the process for the preparation of the pharmaceutical kit in both dosage forms, of a suspension and a lyophilized powder for reconstitution of injectable suspension.

The pharmaceutical kit from the present invention is used for the cancer treatment by the new method which involves the administration of composition I and composition IT directly into the treated tumour by injection at several sites in a dose of 0.1-5 mL of each composition by the following dosage regimen:

(i) composition I is administered on days: 1, 4, 8, 12, 15, 19, and 22; followed by,

(ii) composition II, which is administered on day 29, and additionally, 2-4x, each 7-15 days; and,

(iii) composition I, 1x more, not later than two months after the surgical excision of the remaining tumour.

Preferably, the dose of the composition I and II is 1-2 mL in each administration stage.

The kit according to the present invention is use for the treatment of any kind of carcinomas.

Specifically, said kit is used for the treatment of cancers selected from the group consisting of: a carcinoma within the breast tissue, including: invasive ductal carcinoma and invasive lobular carcinoma, irrespective of the expression status of hormone receptors (ER/PR) or receptor for epidermal growth factor receptor (HER2) on tumour cells, Paget disease and inflammatory breast cancer.

Preferably, the pharmaceutical kit according to the present invention is used for the treatment of locally recurrent breast cancer.

Brief Description of Drawings

Figure 1 - Details of the oncolytic virotherapy regimen (arrows) with the pharmaceutical kit according to the present invention. The therapy resulted in tumour size decrease (circles), and virus-specific antibody increase (rectangles). Size of tumour was calculated from the three largest dimensions (l*w*h), like it was oval. MR - magnetic resonance; US - ultrasound; OncoMeV - measles virus from the composition I, and OncoVSV - vesicular stomatitis virus from the composition II, both used for the oncolytic virotherapy according to the present invention; NT - neutralising antibody titre.

Detailed Description

The present invention involves a pharmaceutical kit for oncolytic virotherapy, consisting of:

(i) composition I, which is composed of:

- a viable Measles virus (MeV), Edmonston Zagreb measles vaccine strain, and,

- one or more pharmaceutical excipients, and,

(ii) composition II, which is composed of:

(ii) VSV concentration in composition II, is between 1^,10⁷-1^,10¹¹ CCID50 mb^-1

Preferably, the concentration of viruses are as follows:

(i) MeV concentration in composition I is between 1^,10⁵-1^,10⁸ CCID₅₀ mL^-1, and, (ii) VSV concentration in composition II is between 1^,10⁷-1^,10¹⁰ CCID₅₀ mL^-1.

Pharmaceutical excipients are selected from the group consisting of: pharmaceutical water for injections, stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives.

Specifically, excipients are selected from the group consisting of: pharmaceutical water for injections, glycine, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-glutamine, L-histidine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L— phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L— tyrosine disodium salt dihydrate, L-valine, ascorbic acid, biotin, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, cyanocobalamin, inositol, D-glucose, gelatine, hydrolysed gelatine, sorbitol, mannitol, xylitol, erythritol, lactitol, maltitol, lipoic acid, sodium pyruvate, glycerol, 1,2-propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, calcium chloride (CaCl₂), magnesium sulfate (MgSO₄), potassium chloride (KC1), sodium bicarbonate (NaHCO₃), sodium chloride (NaCl), sodium dihydrogenphosphate (NaH₂PO₄ ) or its monohydrate (NaH₂PO₄ H₂O), potassium dihydrogenphosphate (KH₂PO₄), disodium hydrogenphosphate dihydrate (Na₂HPO₄·2H₂O), dipotassium hydrogenphosphate (K₂HPO₄) or its trihydrate (Na₂HPO₄·3H₂O), disodium citrate dihydrate (Na₃C₆H₅O₇·H₂O ), tripotassium citrate monohydrate (K ₃C₆H₅O₇·H₂O), citric acid, disodium edetate dihydrate (Na₂EDTA·2H₂O), benzyl alcohol, methyl 4-hydroxy benzoate, propyl 4-hydroxybenzoate, and 2-phenoxyethanol.

More specifically if the dosage form of the compositions I and II within said pharmaceutical kit is a suspension, then the excipients are selected from the group consisting of: pharmaceutical water for injections, L-arginine hydrochloride, L-cystine dihydrochloride, L— glutamine, L-histidine hydrochloride monohydrate, L-isoleucine, L— leucine, L-lysine hydrochloride, L—methionine, L-phenylalanine, L— threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, inositol, D-glucose, calcium chloride (CaCl₂), magnesium sulfate (MgSO₄), potassium chloride (KC1), sodium bicarbonate (NaHCO₃), sodium chloride (NaCl), sodium dihydrogenphosphate (NaH₂PO₄) or its monohydrate (NaH₂PO₄·H₂O), disodium edetate dihydrate (Na₂EDTA·2H₂O), and 2-phenoxyethanol.

Study of oncolytic effect of the pharmaceutical kit from the present invention

Here we disclose the results of OV therapy case study in humans with the pharmaceutical kit according to the present invention, in the treatment of early stage locally recurrent breast cancer. Although the therapy was not the part of approved and registered clinical study, and was conducted with natural, parental strains of otherwise engineered oncolytic viruses from the prior art, it demonstrates the effectiveness in quick reduction of tumour mass and offers some simple solutions for overcoming the major challenges in OV therapy development: virus exchange for OV immunogenicity and repeated intratumoral (i.t.) administration in short time periods for reaching permanent high doses of infective OVs.

Patient and methods

Patient and case history. The patient was 50-years old women with a history of local recurrence of triple negative breast cancer (TNCB). The tumour was firstly diagnosed in 2016 after sudden (overnight) swelling of the whole breast and treated with mastectomy followed by adjuvant chemotherapy (AC protocol). Pathohistological analysis of excised breast revealed several foci of invasive ductal cancer (largest 0.7 cm) of triple negative phenotype, while the 80% of ducts were with in situ cancer of the same phenotype. In 2018 the small local recurrence at the site of previous surgery (bellow the suture) was detected and surgically removed, followed with no adjuvant therapy due to the wish of the patient. This recurrence was pathohistologically diagnosed as TNBC again. However, at the site of excision a small seroma was left (bellow 1 cm) which was monitored at periodic controls. Magnetic resonance (MR) in August 2020 (as part of regular control protocol) diagnosed that the structure so far described as "seroma" has turned into a neoplastic formation, that increased in size to 2 cm (in January 2020 the size of "seroma" was estimated to 1.2 cm by ultrasound and mammography). The patient, who is also a scientist in the field of virology, and expecting that the tumour is of TNBC phenotype again for which no specific and effective therapy exists, informed her oncologists that she is going to try to treat this tumour with viruses similar to the oncoviruses that are in clinical development for breast cancer, before any other treatment. Her oncologists accepted to monitor the progress of the treatment, mainly with the aim to stop this before the possible huge damage. The oncolytic virotherapy started immediately after all necessary diagnostic methods were done (PET-CT scan) and core needle biopsy samples were taken, so the proper characterization and diagnosis of disease status could be done. At the moment of OV therapy start the results of performed analyses were not known yet. Upon the arrival of histopathological analysis of core biopsy material indicating that tumour evolved from TNBC to HER2+++, the oncologists suggested to the patient a neoadjuvant therapy combining chemotherapy with trastuzumab and pertuzumab before the surgery, and continuation with monoclonal antibodies up to one year, a therapy with a significant success in treating HER2+++ tumours. However, a patient, experiencing already some clinical benefits of the therapy with the compositions I and II from the kit, decided to continue to progress according to here described OV therapy protocol until the worsening of symptoms (which didn't arrive).

In the present study the pharmaceutical kit from the present invention from Example 1 was employed. Composition I and composition II from the kit were intratumorally and multifocally administered in a total volume of 1 to 2 mL, as indicated in Table 1. Dynamics of OV application is indicated in the Results section. There were in total 7 applications of the composition I and 3 applications of the composition II within the period of two months, see Table 1.

Table 1. The details of the treatment regimen of cancer with the pharmaceutical kit according to the present invention.

^a In this study, the composition I and composition II from the pharmaceutical kit of the present invention were prepared always freshly, immediately before the administration stage, just to avoid any eventual decrease of the viral titre due to the eventual virus's instability. ^b Type of cells on which the corresponding viruses, MeV or VSV, were cultivated for the preparation of the composition I and composition II of the pharmaceutical kit from the present invention.

Assessment of response to oncolytic virotherapy with the pharmaceutical kit according to the present invention

The changes in visual clinical presentation were described and photographed. The size of tumour was before the OV therapy estimated by PET-CT scan, phase contrast magnetic resonance imaging and two ultrasound imagings performed at two independent instruments by two independent radiologists. During the therapy, changes in tumour size and appearance were monitored regularly and often using ultrasound imaging. Phase contrast MR was performed once during the course of therapy, and the size of the tumour mass was at the end of therapy determined by pathohistologists on excised tumour. The tumour size was estimated by multiplying the largest dimensions in three directions like it was a rectangular (actual size was lesser, because the shape was oval) and expressed in cm³.

PHD analysis was performed on core biopsy tumour material before the OV therapy, and on excised tumour at the end of therapy, including ICH staining for HER, ER, PR within the regular diagnostic protocol, as well as additional staining for CD3, CD4, CD8, CD20, CD56 and PD1- L.

Antivirus antibody titres

Anti-MeV and anti-VSV antibody neutralizing titres were determined using a standard effective dose 50 (ED50) assay in a 96-well format according to the procedure described in literature references 10 and 11:

10) M. Brgles, T. Kurtovic, M. Lang Balija, A. Hecimovic, T. Muslin, B. Halassy: Impact of complement and difference of cell-based assay and ELISA in determination of neutralization capacity against mumps and measles virus, Journal of Immunological Methods, 490 (2021) 112957.

11) B. Halassy, T. Kurtovic, M. Brgles, M. L. Balija, D. Forcic: (2015). Factors influencing preclinical in vivo evaluation of mumps vaccine strain immunogenicity, Human Vaccines and Immunotherapeutics, 11 (2015) 2446-2454.

Briefly, two-fold serial dilutions of patient's serum samples taken at different time points during the therapy, each in octaplicate (50 μ/Lwell) were preincubated with approximately 20 CCID50 (50 μ/Lwell) of the respective virus at 37 °C and 5% V/V CO₂ for 90 minutes. After the addition of Vero cells (1.2*10⁵/mL for anti-MeV and 2·10⁵/mL for anti-VSV assay; 100 μ/Lwell), the plates were incubated at 37 °C and 5% V/V CO₂ . The wells with pure cell suspension served as cell growth control. After incubation period (6 days-long for anti-MeV and 4 days- long for anti-VSV assay), the wells with cytopathic changes visible under inverted optical microscope were counted, and the ED₅₀ (the amount of undiluted serum that inhibits the cytopathic effect in 50% of infected wells) calculated according to the Spearman-Karber method. Neutralization titre (NT) was expressed as the number of ED₅₀S in 1 mL of serum.

The IVIg preparation (Institute of Immunology Inc., Zagreb, Croatia) was calibrated against 3^rd WHO International Standard for Anti-Measles (97/648), and used as in-house reference material for determination of anti-MeV antibody neutralizing titre.

Results of the study

The tumour diagnosis before the start of the therapy with the kit according to the present invention

The tumour appeared as a hard, palpable nodule on the chest wall, at the site of previous mastectomy, bellow the surgical suture from the previous operations. Different imaging techniques described it as a circled plate at the base (chest wall side), with a bulging hill at the skin side. Phase contrast MR, PET-CT scan, and two ultrasound estimations performed at two different machines and by two independent radiologists all estimated perfectly matching size estimations of 2.47 ± 0.06 cm³. Phase contrast MR described spreading of the tumour into the pectoral muscle, while PET-CT analysis only raised suspicion of possible pectoral muscle infiltration. Skin infiltration was described by all three diagnostic imaging methods. Under ultrasound the tumour was presented as a hypoechoic and spiculated mass. The tumour was easily accessible since it was protruding from the chest wall, being bright red, inflamed, with a thin skin above it. PET-CET revealed that the illness is still localized, and the regional lymph nodes were calm.

The therapy according to the present invention with the pharmaceutical kit in neoadjuvant setting

Composition I therapy period

After performing all initial diagnostic imaging (ultrasound, phase contrast MR and PET-CT scanning) and one week after sampling tumour tissue by core biopsy for PHD diagnosis, the therapy started with i.t. composition I application. Composition I was applied 7 times in three- to four-day intervals, within the total period of three weeks. Each time composition I in the dosage form of the suspension was administered. The volumes and infective virus quantities applied each time are listed in Table 1, while the cumulative quantity of applied MeV through the composition I was 10⁷'⁹. There were no systemic side effects. First three applications were extremely hard to perform due to extreme hardness of the tumour mass, making the needle insertion and virus suspension application difficult. Small amounts of suspension were applied at multiple sites, and the administration was moderately painful. It was accompanied with prolonged bleeding from the injection site (dark, capillary venomous blood). A weak after the first composition I administration (before the third dose) mass seemed larger, more bulged, with extremely thin skin covering the mass and looking like it might break. Under ultrasound the mass looked more hypoechoic and more spiculated, a bit larger and more inflated. The first sign of improvement was observed on the day of 4^th application, 11 days after the therapy with the pharmaceutical kit according to the present invention start. The clinical picture was improved with less redness and less bulging. Under ultrasound the mass was less spiculated and better circumscribed. The majority of the mass was soft, when needle was injected, application was easier, no painful any more, and there was no bleeding from the injection site. It was the first time the 2 mL of suspension were easily administered. Until the last composition I administration on day 21, the mass was constantly improving clinically becoming palpatory smaller, with very little redness (it looked more like a healing bruises), the administration was getting easier, the mass was less spiculated and less hypoechoic, and was getting better circumscribed.

The therapeutic method for the treatment of cancer by the use of the pharmaceutical kit according to the present invention

Three days after the 7^th composition I, the administration of the composition IT from the kit of the present invention started. The tumour was not dense any more, the needle was easily inserted, and higher volume of virus suspension was easily administered, in comparison to the beginning of composition I treatment period. We applied i.t., at multiple foci, 2 mL of freshly prepared composition II. In the evening, the tumour was bulging, and very red. Twelve hours after the administration, at midnight, the patient was facing a strong fever and shivering that lasted for 2 hours. After it stopped, the patient had 40 °C, but without the feel of fatigue, or the overall body pain, usually connected with such a high body temperature. The patient took antipyretic (ibuprofen). The whole day the body temperature was between 39 and 40 °C, and was reduced by ibuprofen usage. The second day the temperature was the whole day around 38 °C, while on the third day there was no temperature any more. The tumour was palpatory very soft, like a sponge. This was the moment when tumour is of the highest softness during the whole OV therapy period. Due to strong side reaction, the composition II administration was temporarily ceased and control phase-contrast MR was performed two weeks after the first composition II dose. Although the size estimation was not substantially smaller in comparison to the size at the beginning of the therapy, the MR diagnosis was much improved, since there was no visible infiltration of the pectoral muscle. The increase in axillar lymph node size was described in both axillas. The patient was forwarded to surgeon that diagnosed also that the tumour mass is freely movable, and could be easily and safely removed by surgery. Until the date of surgery, the composition II administration was continued in one-week intervals. In the second administration only 1 mL was applied as a precautionary measure to reduce potential systemic side effects, but this time, there was no any side effect, just as after the last administration of composition II a week later, when 2 mL was applied again. The tumour looked at that time under ultrasound like a sac that was easily filled with a virus suspension which led to tumour inflation, like the air balloon. In total, there were three composition II administrations, separated by two and one weeks, respectively, within the total period of 24 days. Cumulative dose of administered composition II was 10⁹'¹ CCID50, and volumes and infective virus quantities in separated doses are listed in Table 1.

Composition therapy in an adjuvant setting

Two months after the tumour excision, the composition I was applied subcutaneously once around the surgical suture, as a preventive adjuvant treatment.

The tumour size changes during the cancer therapy with the pharmaceutical kit according to the present invention

At the beginning of the therapy with the pharmaceutical kit from the present invention the tumour size was estimated to 2.4710.06 cm³, representing the mean size and standard deviation from four measurements in different and independently performed imaging techniques. Tumour size was estimated from ultrasound measurements immediately before each administration. On day 41 phase-contrast MR size measurement was performed, and the final tumour size, et the end of therapy, is based on pathology measurement after tumour excision, on day 112. At the beginning, after administration of first two doses of the composition I, the tumour size was increasing, reaching maximum of 4.28 cm³ estimated on day 8. After that the tumour was constantly being reduced till the end of the therapy with the composition I, when the size was estimated to 1.61 cm³. After application of the first composition II dose, the size increased again to 2.17 cm³ on day 41 (14^th day after the composition II application), but then it started to reduce till the end of the composition II therapy, with the size of the excised tumour of 0.91 cm³.

Antiviral antibody responses

The patient had low, but measurable neutralizing antibody titres to both viruses before the therapy start. The rise in anti-MeV NT was registered 10 days after the composition I therapy started, with slight rise till the day 48, when the titre was close to 2 logs higher than before the therapy. Neutralising titre after composition II application was on day 3 after virus administration already increased for more than one log CCID50, reaching the maximum already after 9 days, and being more than 2 log CCID50 higher than before VSV administration. Induced high neutralizing antibody titres to both viruses stayed high till the end of OV therapy protocol.

Pathohistological and immunohistochemistry characterization of excised tumour

Besides demonstrating a significant reduction of tumour mass, pathohistological analysis revealed that tumour was placed only in subcutis, with no infiltration of either skin or pectoral muscle. The tumour showed strong lymphocytic infiltration (45% in relation to tumour mass). Areas of tumour bed rich in lymphocytes and fibrous tissue, without tumour cells, similar to the effect of neoadjuvant chemotherapy, were observed.

Using routine immunohistochemical staining we stained residual tumour sample after the therapy with the pharmaceutical kit according to the present invention (11/2020) and compared it with core needle biopsy sample taken before the therapy (09/2020). Differentiation of infiltrating lymphocytes revealed stronger presence of T-lymphocytes (CD3 positive) than B lymphocytes (CD20+) and lack of NK and helper T lymphocytes (negative staining for CD4 and CD56) in both samples. The therapy of the cancer with pharmaceutical kit according to the present invention induced increase in both CD20-positive cells (from 10% to 70%) and CD8-positive cells (from 30% to 60%), indicating activation of adaptive immune response. Infiltration of macrophages (CD68- positive cells) was also increased. In addition, after OV therapy PDL1 expression was detected in contrast to the PDL1 negative phenotype before the treatment with viruses.

All experimental details of the study are described in Example 3 and schematically presented in Figure 1.

Discussion of the study results

We report the first case of oncolytic virotherapy in the treatment of the early stage, recurrent breast cancer, specifically with a combination of MeV (composition I) and VSV (composition II). Oncolytic viruses have slow entrance into the clinical practice due to different reasons, among which one important might be the intention and willingness of researchers to develop therapy for advanced cancer stages that cannot be effectively cured by currently existing and approved therapies. This means that OV therapy is mostly investigated in patients with metastatic diseases of different cancer types, that have undergone many cycles of different chemotherapy and radiotherapy regimes and from such a history being of weak immune and overall health status. In such situations OV therapy works only by direct virus killing of cancer cells, which is only a part of full potential of OV therapy. Induction of cancer-specific immune response of OV therapy cannot be fully activated. Early-stage breast cancer is not the current target in development of OV therapy. However, OV therapy has being clinically developed for advanced stages of different types of breast cancer, by the usage of measles virus-based virotherapeutics, see literature reference 4.

Here described study was feasible only due to unique situation in which a patient facing breast cancer local recurrence was also a scientist working in the field of virology, skilled to grow and characterize human viruses, see literature reference 12: 12) EP3408383B1; M. Brgles, B. Halassy, D. Forcic: An elution mobile phase and process for immunoaffinity chromatography of viruses; applicant:University of Zagreb (HR), Bia Separations d.o.o. (SI); priority date: 27. 01. 2016.

The tumour was of epithelial origin so viruses that are known to successfully infect epithelial cells were selected. Breast carcinoma has been already described as being abundant with CD46 and nectin-4, both utilized by the vaccine strain of measles for the entrance, particularly viruses of Edmonston lineage, so the usage of Edmonston- Zagreb vaccine strain was found appropriate, see literature reference 13:

13) O. V. Matveeva, S. A. Shabalina: Prospects for Using Expression Patterns of Paramyxovirus Receptors as Biomarkers for Oncolytic Virotherapy, Cancers 12 (2020) 3659.

Being aware that high virus titres are needed for successful OV therapy, and that infective viruses are quickly sequestered and/or neutralised after i.v. application, the composition I and composition II were administered often, in short intervals, directly into the tumour, with the aim to keep high quantity of infective viruses in tumour environment constantly. Further, knowing that virus-specific antibody response will be raised fast, and that viruses will be neutralised also locally, we exploited virus exchange strategy, by starting with MeV and switching to another oncolytic virus - VSV, after three weeks of therapy.

Although not using some genetically engineered viruses with improved oncolytic properties, but common natural (VSV) or vaccine (MeV) virus strains, the OV therapy according to the protocol disclosed by the present invention was very effective. After only two months of therapy, in which a total of 7.89 log CCID50 of the composition I and 9.07 log CCID50 of the composition II was applied, the following was achieved. The tumour was significantly reduced in size - from 2.47 cm³ estimated by four independent imaging techniques and analysis, to 0.91 cm³, which was the pathologic size of the excised tumour. The tumour was described to infiltrate skin above, and the muscle bellow at the beginning of the therapy, based on phase contrast MR and PET-CT imaging, while PHD analysis of excised material after OV therapy revealed that it was situated only in subcutis, with no infiltration of either skin or pectoral muscle. The clinical picture was significantly improved from hard bulged nodule at the beginning, with redness of the skin above it, to smaller nodule on palpation without redness of the skin any more (it was more brownish, like in a process of bruises healing). At the beginning it was extremely difficult to insert the needle and to release microquantities of the virus suspension, while at the end it was a soft tissue, the needle went through it easily, and up to 2 mL of virus suspension was easily administered. Under ultrasound imaging it looked like a hypoechoic, spiculated mass, which become less hypoechoic, less spiculated, better circumscribed, and significantly flattened at the end of the therapy. It looked like a sack that can be easily filled.

It is important to stress that all these signs of OV therapy effectiveness according to the present invention were achieved in only eight weeks of OV therapy, during which a patient did not experienced any serious side effects. The needle application and suspension administration were tolerably painful at the beginning of OV therapy. The only systemic side effect was experienced after the first application of VSV, manifested in 2 hours long period of fever with strong shivering started twelve hours after VSV application, since it was a first encounter of the patient with this virus. A patient experienced a high temperature the rest of the night and the following days, which gradually disappeared three days later.

Short-term outcome was undoubtedly beneficial in the here described case study of the therapy with the pharmaceutical kit according to the present invention of locally recurrent breast cancer. Although this is a representation of only one case, this study provides an obvious and clear evidence that the pharmaceutical kit from the present invention deserves to be considered for further clinical development. Preparation of the pharmaceutical kit according to the present invention

The pharmaceutical kit according to the present invention, in which the composition I and II are in the dosage form of suspension, is manufactured by the following manufacturing steps:

(i) the preparation of MeV is carried out by propagation either in MRC-5 at a cell density of 75,000 cells/cm² or in Vero cells culture at a cell density of 100,000 cells/cm², in both cases with Multiplicity of Infection (MOI) of 0.01 in Minimal Essential Medium (MEM) with 10% Fetal Bovine Serum (FBS) for 24 h at 36 °C,

(ii) then, the medium is replaced with fresh MEM, and cultivated at 32 °C under 5% V/V CO₂ atmosphere until the appearance of cytopathic changes,

(iii) then, the cell culture supernatant is collected and clarified by centrifugation for 10 minutes at 1400 g,

(iv) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and,

(v) the suspension is sterilized by 0.45 μm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions, while,

(vi) VSV is propagated in Vero cell cultures at a cell density of 100,000 cells/cm² with MOI of 0.5 in MEM with 10% FBS, at 37 °C for 4 h,

(vii) then, the medium is replaced with fresh MEM, and the cells are cultivated at 37 °C for 16-24 h under 5% V/V CO₂ atmosphere, until the appearance of cytopathic changes,

(viii)then, the cell culture supernatant is collected and clarified by centrifugation for 10 minutes at 1400 g,

(ix) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and, (x) the suspension is sterilized by 0.45 pm-filtration, yielding sterile suspension of the composition II, which is subsequently packed into vials under sterile conditions, and,

(xi) the filled vials with the composition I are packed together with the filled vials of the composition II, together with the product instruction, into boxes, giving final pharmaceutical kit whose compositions I and II are in the dosage form of suspension.

Typical procedure for the preparation of the pharmaceutical kit from the present invention in the dosage form of a suspension is described in Example 1.

Alternatively, the pharmaceutical kit according to the present invention, in which the composition I and II are in the dosage form of lyophilized powder for reconstitution of injectable suspension, is manufactured by the following manufacturing steps:

(v) the suspension is sterilized by 0.45 μm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions,

(vi) the vials are subjected to lyophilization and closing with suitable sterile cap, while,

(vii) VSV is propagated in Vero cell cultures at a cell density of 100,000 cells/cm² with MOI of 0.5 in MEM + 10% FSB, at 37 °C for 4 h,

(viii)then, the medium is replaced with fresh MEM, and the cells are cultivated at 37 °C for 16-24 h under 5% V/V CO₂ atmosphere, until the appearance of cytopathic changes,

(ix) then the cell culture supernatant was collected and clarified by centrifugation for 10 minutes at 1400 g,

(x) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and,

(xi) the suspension is sterilized by 0.45 μm-filtration, yielding sterile suspension of the composition II, which is subsequently packed into vials under sterile conditions, and,

(xii) the vials are subjected to lyophilization and closing with suitable sterile cap, while,

(xiii)the filled vials with the composition I are packed together with the filled vials of the composition II, together with the product instruction, into boxes, giving final pharmaceutical kit whose compositions I and II are in the dosage form of a lyophilized powder for reconstitution of injectable suspension.

Typical procedure for the preparation of the pharmaceutical kit from the present invention in the dosage form of a lyophilized powder for reconstitution of injectable suspension is described in Example 2.

Use of the pharmaceutical kit according to the present invention

The pharmaceutical kit from the present invention is used for the cancer treatment by the new method which involves the administration of composition I and composition II directly into the treated tumour by injection at several sites in a dose of 0.1-5 mL of each composition by the following dosage regimen: (i) composition I is administered on days: 1, 4, 8, 12, 15, 19, and 22; followed by,

(ii) composition IT, which is administered on day 29, and additionally, 2-4x, each 7-15 days; and,

(iii) composition I, lx more, not later than two months after the surgical excision of the remaining tumour.

The typical dosage regimen for administration of the pharmaceutical kit from the present invention is described in Table 1.

The kit according to the present invention is used for the treatment of any kind of carcinomas.

In conclusion, the pharmaceutical kit from the present invention provides an effective and safe treatment of cancers, especially breast cancers, and more particularly, when combined with surgical cancer excision.

Examples

Example 1. Preparation of the pharmaceutical kit according to the present invention in the dosage form of suspension The pharmaceutical kit according to the present invention, in which the composition I and II are in the dosage form of suspension, is manufactured by the following manufacturing steps:

(v) the suspension is sterilized by 0.45 pm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions, while,

(ix) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and,

(x) the suspension is sterilized by 0.45 pm-filtration, yielding sterile suspension of the composition II, which is subsequently packed into vials under sterile conditions, and, (xi) the filled vials with the composition I are packed together with the filled vials of the composition II, together with the product instruction, into boxes, giving final pharmaceutical kit whose compositions I and II are in the dosage form of suspension.

The standardization of the composition I against the content of MeV within the formulation via the determination of the CCID₅₀ was performed according to the procedure described in Example 3.

Example 2. Preparation of the pharmaceutical kit according to the present invention in the dosage form of a lyophilized powder for reconstitution of injectable suspension

(v) the suspension is sterilized by 0.45 μm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions, (vi) the vials are subjected to lyophilization and closing with suitable sterile cap, while,

The standardization of the composition II against the content of VSV within the formulation via the determination of the CCID₅₀ was performed according to the procedure described in Example 3.

Example 3. Study of oncolytic effect of the pharmaceutical kit from the present invention

In the present study the pharmaceutical kit from the present invention from Example 1 was employed. Composition I and composition II from the kit were intratumorally and multifocally administered in a total volume of 1 to 2 mL, as indicated in Table 1. Dynamics of OV application is indicated in the Results section. There were in total 7 applications of the composition I and 3 applications of the composition II within the period of two months month, see Table 1.

The changes in visual clinical presentation were described and photographed. The size of tumour was before the OV therapy estimated by PET-CT scan, phase contrast magnetic resonance imaging and two ultrasound imaging performed at two independent instruments by two independent radiologists. During the therapy, changes in tumour size and appearance were monitored regularly and often using ultrasound imaging. Phase contrast MR was performed once during the course of therapy, and the size of the tumour mass was at the end of therapy determined by pathohistologists on excised tumour. The tumour size was estimated by multiplying the largest dimensions in three directions like it was a rectangular (actual size was lesser, because the shape was oval) and expressed in cm³.

Antivirus antibody titres Anti-MeV and anti-VSV antibody neutralizing titres were determined using a standard effective dose 50 (ED50) assay in a 96-well format according to the procedure described in literature references 6 and 7. Briefly, two-fold serial dilutions of patient's serum samples taken at different time points during the therapy, each in octaplicate (50 μL/well) were preincubated with approximately 20 CCID₅₀ (50 μL/well) of the respective virus at 37 °C and 5% V/V CO₂ for 90 minutes. After the addition of Vero cells (1.2*10⁵/mL for anti-MeV and 2 x 10⁵/mL for anti-VSV assay; 100 μ/Lwell), the plates were incubated at 37 °C and 5% V/V CO₂ . The wells with pure cell suspension served as cell growth control. After incubation period (6 days-long for anti-MeV and 4 days- long for anti-VSV assay), the wells with cytopathic changes visible under inverted optical microscope were counted, and the ED50 (the amount of undiluted serum that inhibits the cytopathic effect in 50% of infected wells) calculated according to the Spearman-Karber method. Neutralization titre (NT) was expressed as the number of ED₅₀S in 1 mL of serum.

Results of the study

Composition I therapy period

Three days after the 7^th composition I, the administration of the composition II from the kit of the present invention started. The tumour was not dense any more, the needle was easily inserted, and higher volume of virus suspension was easily administered, in comparison to the beginning of composition I treatment period. We applied i.t., at multiple foci, 2 mL of freshly prepared composition II. In the evening, the tumour was bulging, and very red. Twelve hours after the administration, at midnight, the patient was facing a strong fever and shivering that lasted for 2 hours. After it stopped, the patient had 40 °C, but without the feel of fatigue, or the overall body pain, usually connected with such a high body temperature. The patient took antipyretic (ibuprofen). The whole day the body temperature was between 39 and 40 °C, and was reduced by ibuprofen usage. The second day the temperature was the whole day around 38 °C, while on the third day there was no temperature any more. The tumour was palpatory very soft, like a sponge. This was the moment when tumour is of the highest softness during the whole OV therapy period. Due to strong side reaction, the composition II administration was temporarily ceased and control phase-contrast MR was performed two weeks after the first composition II dose. Although the size estimation was not substantially smaller in comparison to the size at the beginning of the therapy, the MR diagnosis was much improved, since there was no visible infiltration of the pectoral muscle. The increase in axillar lymph node size was described in both axillas. The patient was forwarded to surgeon that diagnosed also that the tumour mass is freely movable, and could be easily and safely removed by surgery. Until the date of surgery, the composition IT administration was continued in one-week intervals. In the second administration only 1 mL was applied as a precautionary measure to reduce potential systemic side effects, but this time, there was no any side effect, just as after the last administration of composition II a week later, when 2 mL was applied again. The tumour looked at that time under ultrasound like a sac that was easily filled with a virus suspension which led to tumour inflation, like the air balloon. In total, there were three composition II administrations, separated by two and one weeks, respectively, within the total period of 24 days. Cumulative dose of administered composition II was 10⁹'¹ CCID₅₀, and volumes and infective virus quantities in separated doses are listed in Table 1.

Composition therapy in an adjuvant setting

Antiviral antibody responses

The patient had low, but measurable neutralizing antibody titres to both viruses before the therapy start. The rise in anti-MeV NT was registered 10 days after the composition I therapy started, with slight rise till the day 48, when the titre was close to 2 logs higher than before the therapy. Neutralising titre after composition IT application was on day 3 after virus administration already increased for more than one log CCID₅₀, reaching the maximum already after 9 days, and being more than 2 log CCID₅₀ higher than before VSV administration. Induced high neutralizing antibody titres to both viruses stayed high till the end of OV therapy protocol.

Pathohistological and immunohistochemistry characterization of excised tumour

Besides demonstrating a significant reduction of tumour mass, pathohistological analysis revealed that tumour was placed only in subcutis, with no infiltration of either skin or pectoral muscle. The tumour shows strong lymphocytic infiltration (45% in relation to tumour mass). Areas of tumour bed rich in lymphocytes and fibrous tissue, without tumour cells, similar to the effect of neoadjuvant chemotherapy, were observed.

Using routine immunohistochemical staining we stained residual tumour sample after the therapy with the pharmaceutical kit according to the present invention (11/2020) and compared it with core needle biopsy sample taken before the therapy (09/2020). Differentiation of infiltrating lymphocytes revealed stronger presence of T-lymphocytes (CD3 positive) than B lymphocytes (CD20+) and lack of NK and helper T lymphocytes (negative staining for CD4 and CD56) in both samples. The therapy of the cancer with pharmaceutical kit according to the present invention induced increase in both CD20-positive cells (from 10% to 70%) and CD8-positive cells (from 30% to 60%), indicating activation for adaptive immune response. Infiltration of macrophages (CD68- positive cells) was also increased. In addition, after OV therapy PDL1 expression was detected in contrast to the PDL1 negative phenotype before the treatment with viruses.

The typical dosage regimen for administration of the pharmaceutical kit from the present invention is described in Table 1. The results of the study are schematically presented in Figure 1.

Conclusion

The present invention solves the technical problem of effective and safe intratumoral (i.t.) treatment of cancer with a specific combination of two different viruses of specific strains of MeV and VSV with oncolytic properties.

Being aware that high virus titres are needed for successful OV therapy, and that infective viruses are quickly sequestered and/or neutralised after i.v. application, the composition I and composition IT were administered often, in short intervals, directly into the tumour, with the aim to keep high quantity of infective viruses in tumour environment constantly. Further, knowing that virus-specific antibody response will be raised fast, and that viruses will be neutralised also locally, we exploited virus exchange strategy, by starting with MeV and switching to another oncolytic virus - VSV, after three weeks of therapy.

Additionally, the therapy with the pharmaceutical kit from the present invention is generally safe for human use. No serious adverse effects were observed. The needle application and suspension administration were tolerably painful at the beginning of OV therapy. The only systemic side effect was experienced after the first application of VSV, manifested in 2 hours long period of fever with strong shivering started twelve hours after VSV application, particularly if the subject is at a first encounter with this virus. A patient could experience a high temperature the rest of the night and the following days, which gradually disappeared three days later.

Industrial Applicability

The composition of a pharmaceutical kit according to the present invention is used in pharmaceutical industry for the production of a medicament intended for the treatment of several types of cancers. Therefore, the industrial applicability of the present invention is obvious.

The project leading to this disclosure has received funding from the European Regional Development Fund, grant number KK.01.1.1.01.0006, "Strengthening the capacity of CerVirVac for research in virus immunology and vaccinology".

List of Abbreviations

AC protocol - AC chemotherapy; a combination of two chemotherapy drugs used to treat breast cancer, doxorubicin (adriamycin) and cyclophosphamide anti-MeV NT - neutralising antibody titre for MeV ATCC - American Type Culture Collection

CCID₅₀ - fifty-percent cell culture infective dose CD3 - cluster of differentiation 3; a protein complex and T cell co-receptor that is involved in activating cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4 naive T cells) ED₅₀₅ - effective dose, a dose or concentration in serum that produces a biological response in 50% of the population

ER/PR - expression status of hormone receptors

FBS - Fetal Bovine Serum

HER2 - receptor for epidermal growth factor receptor i.t. - intratumoral (administration) i.v. - intravenous (administration)

MeV - Measles virus (MeV)

MEM - Minimal Essential Medium

MOI - Multiplicity of Infection

MR - magnetic resonance

MRC-5 - Medical Research Council cell strain 5

NK cells - natural killer cells

NT - neutralising antibody titre

PET-CT - positron emission tomography / computed tomography

PFU - plaque forming units

TNCB - triple negative breast cancer

VSV - Vesicular stomatitis virus (VSV)

Claims

CLAIMS 1. A pharmaceutical kit for oncolytic virotherapy, consisting of: (i) composition I, which is composed of: - a viable Measles virus <MeV>, Edmonston Zagreb measles vaccine strain, and, - one or more pharmaceutical excipients, (ii) composition II, which is composed of: - a viable Vesicular stomatitis virus <VSV> Indiana strain, according to American Type Culture Collection <ATCC>, and, - one or more pharmaceutical excipients, and, where said excipients enable formation of the final dosage forms of a suspension or a lyophilized powder for reconstitution of injectable suspension. 2. The pharmaceutical kit for oncolytic virotherapy according to claim 1, wherein: (i) MeV concentration in composition I is between 1•10⁵-1•10¹⁰ fifty-percent cell culture infective dose <CCID₅₀> mL^-1; and, (ii) VSV concentration in composition II, is between 1•10⁷-1•10¹¹ CCID₅₀ mL^-1. 3. The pharmaceutical kit for oncolytic virotherapy according to claim 2, wherein: (i) MeV concentration in composition I is between 1•10⁵-1•10⁸ CCID50 mL^-1, and, (ii) VSV concentration in composition II is between 1•10⁷-1•10¹⁰ CCID₅₀ mL^-1. 4. The pharmaceutical kit for oncolytic virotherapy according to any of preceding claims, wherein the excipients are selected from the group consisting of: pharmaceutical water for injections, stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives. 5. The pharmaceutical kit for oncolytic virotherapy according to any of preceding claims, wherein the excipients are selected from the group consisting of: pharmaceutical water for injections, glycine, ^L-alanine, ^L-arginine hydrochloride, ^L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride monohydrate, L-cystine dihydrochloride, L-glutamic acid, L- glutamine, L-histidine, L-histidine hydrochloride monohydrate, L- isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- tyrosine disodium salt dihydrate, L-valine, ascorbic acid, biotin, choline chloride, calcium ^D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, cyanocobalamin, inositol, D-glucose, gelatine, hydrolysed gelatine, sorbitol, mannitol, xylitol, erythritol, lactitol, maltitol, lipoic acid, sodium pyruvate, glycerol, 1,2- propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, calcium chloride <CaCl2>, magnesium sulfate <MgSO₄>, potassium chloride <KCl>, sodium bicarbonate <NaHCO₃>, sodium chloride <NaCl>, sodium dihydrogenphosphate <NaH2PO4> or its monohydrate <NaH2PO4•H2O>, potassium dihydrogenphosphate <KH2PO4>, disodium hydrogenphosphate dihydrate <Na₂HPO₄•2H₂O>, dipotassium hydrogenphosphate <K₂HPO₄> or its trihydrate <K2HPO4•3H2O>, disodium citrate dihydrate <Na3C6H5O7•H2O>, tripotassium citrate monohydrate <K3C6H5O7•H2O>, citric acid, disodium edetate dihydrate <Na₂EDTA•2H₂O>, benzyl alcohol, methyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, and 2-phenoxyethanol. 6. The pharmaceutical kit for oncolytic virotherapy according to any of preceding claims, wherein: - the compositions I and II are in the dosage form of suspension, and, - excipients are selected from the group consisting of: pharmaceutical water for injections, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, L- histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L^{-lysine hydrochloride, L-methionine, L-phenylalanine, L-} threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, inositol, D-glucose, calcium chloride <CaCl₂>, magnesium sulfate <MgSO₄>, potassium chloride <KCl>, sodium bicarbonate <NaHCO3>, sodium chloride <NaCl>, sodium dihydrogenphosphate <NaH₂PO₄> or its monohydrate <NaH₂PO₄•H₂O>, disodium edetate dihydrate <Na₂EDTA•2H₂O>, and 2-phenoxyethanol. 7. A process for the preparation of pharmaceutical kit according to any of claims 1-6, characterised by the following manufacturing steps: (i) the preparation of MeV is carried out by propagation either in MRC-5 at a cell density of 75,000 cells/cm² or in Vero cells culture at a cell density of 100,000 cells/cm², in both cases with Multiplicity of Infection <MOI> of 0.01 in Minimal Essential Medium <MEM> with 10% Fetal Bovine Serum <FBS> for 24 h at 36 °C, (ii) then, the medium is replaced with fresh MEM, and cultivated at 32 °C under 5% V/V CO₂ atmosphere until the appearance of cytopathic changes, (iii) then, the cell culture supernatant is collected and clarified by centrifugation for 10 minutes at 1400 g, (iv) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and, (v) the suspension is sterilized by 0.45 µm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions, while, (vi) VSV is propagated in Vero cell cultures at a cell density o^{f 100,000 cells/cm2 with MOI of 0.5 in MEM with 10% FBS,} at 37 °C for 4 h, (vii) then, the medium is replaced with fresh MEM, and the cells are cultivated at 37 °C for 16-24 h under 5% V/V CO2 atmosphere, until the appearance of cytopathic changes, (viii) then, the cell culture supernatant is collected and clarified by centrifugation for 10 minutes at 1400 g, (ix) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and, (x) the suspension is sterilized by 0.45 µm-filtration, yielding sterile suspension of the composition II, which is subsequently packed into vials under sterile conditions, and, (xi) the filled vials with the composition I are packed together with the filled vials of the composition II, together with the product instruction, into boxes, giving final pharmaceutical kit whose compositions I and II are in the dosage form of suspension. 8. A process for the preparation of pharmaceutical kit according to any of claims 1-6, wherein it includes the following manufacturing steps: (i) the preparation of MeV is carried out by propagation either in MRC-5 at a cell density of 75,000 cells/cm² or in Vero cells culture at a cell density of 100,000 cells/cm², in both cases with Multiplicity of Infection <MOI> of 0.01 in Minimal Essential Medium <MEM> with 10% Fetal Bovine Serum <FBS> for 24 h at 36 °C, (ii) then, the medium is replaced with fresh MEM, and cultivated at 32 °C under 5% V/V CO₂ atmosphere until the appearance of cytopathic changes, (iii) then, the cell culture supernatant is collected and clarified by centrifugation for 10 minutes at 1400 g, (iv) optionally, one or more additional excipients, such as stabilizing agents, tonicity agents, buffers, chelating agents, antioxidants, and preservatives are added and dissolved, and,

(v) the suspension is sterilized by 0.45 pm-filtration, yielding a sterile suspension of the composition I, which is subsequently packed into vials under sterile conditions,

(viii) then, the medium is replaced with fresh MEM, and the cells are cultivated at 37 °C for 16-24 h under 5% V/V CO₂ atmosphere, until the appearance of cytopathic changes,

(xi) the suspension is sterilized by 0.45 pm-filtration, yielding sterile suspension of the composition II, which is subsequently packed into vials under sterile conditions, and,

(xiii) the filled vials with the composition I are packed together with the filled vials of the composition II, together with the product instruction, into boxes, giving final pharmaceutical kit whose compositions I and II are in the dosage form of a lyophilized powder for reconstitution of injectable suspension. 9. Use of pharmaceutical kit according to any of claims 1-6, wherein the method for treatment of cancers includes the administration of composition I and composition II directly into the treated tumour by injection at several sites in a dose of 0.1-5 mL of each composition by the following dosage regimen: (i) composition I is administered on days: 1, 4, 8, 12, 15, 19, and 22; followed by, (ii) composition II, which is administered on day 29, and additionally, 2-4x, each 7-15 days; and, (iii) composition I, 1x more, not later than two months after the surgical excision of the remaining tumour. 10. Use of pharmaceutical kit according to claim 9, wherein the dose is 1-2 mL. 11. Use of pharmaceutical kit according to claims 9 and 10, wherein the cancers are selected from any kind of carcinomas. 12. Use of pharmaceutical kit according to claim 11, wherein the cancer is a carcinoma within the breast tissue, including: invasive ductal carcinoma and invasive lobular carcinoma, irrespective of the expression status of hormone receptors <ER/PR> or receptor for epidermal growth factor receptor <HER2> on tumour cells, Paget disease and inflammatory breast cancer. 13. Use of pharmaceutical kit according to claim 12, wherein the cancer is locally recurrent breast cancer.