CN116421734A

CN116421734A - Freeze-drying protective agent applicable to oncolytic viruses and application thereof

Info

Publication number: CN116421734A
Application number: CN202111659603.9A
Authority: CN
Inventors: 赵海波; 林康; 刘小虎; 马燕彬; 王俊杰; 贾为国; 赵荣华
Original assignee: Shenzhen Double Promise Biological Technology Co ltd
Current assignee: Shenzhen Double Promise Biological Technology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-07-14
Also published as: WO2023125765A1

Abstract

The invention discloses a freeze-drying protective agent suitable for oncolytic viruses, which is a solution with functional components, wherein the functional components comprise the following components in percentage by mass under working concentration: 3 to 10 percent of sucrose, 0.3 to 1.0 percent of gelatin, 0.1 to 1.0 percent of Human Serum Albumin (HSA) and 0.1 to 1.0 percent of sodium glutamate. Compared with the traditional oncolytic virus liquid preparation, the freeze-dried preparation is easier to store and has good stability, and especially has good titer and tumor killing capability after freeze-drying.

Description

Freeze-drying protective agent applicable to oncolytic viruses and application thereof

Technical Field

The invention relates to the technical field of oncolytic viruses, in particular to a freeze-drying protective agent suitable for oncolytic viruses and application thereof.

Background

With the improvement of life research technology, a plurality of novel hot therapies are presented for treating tumor and cancer, and besides single/double antibodies, ADC (antibody coupled drugs) and PD-1/L1, cell therapies, exosomes, oncolytic viruses and the like are also used as therapeutic means in the field. Among them, oncolytic viruses (Oncolytic viruses, OVs) are receiving increasing attention from scientific research and industry because of their ability to replicate specifically within tumor cells and cause tumor cell lysis without affecting normal cells. Oncolytic viruses are a class of natural or genetically engineered viruses that selectively replicate within tumor tissue, thereby infecting or causing lysis of tumor cells, but not killing normal tissue. According to whether it has been genetically modified, it can be classified into two main categories: one class is wild-type strains and naturally attenuated strains, such as reoviruses, newcastle disease viruses, etc.; the other type is virus which can only proliferate in tumor cells after genetic modification, mainly adenovirus, herpes simplex virus, vaccinia virus, measles virus and the like.

Oncolytic virus therapy (Oncolytic virotherapy) is a novel tumor targeting therapy, which is an anti-tumor preparation using oncolytic viruses with tumor selectivity and multiple killing ways as main components, can directly kill tumor cells to achieve oncolytic effect, and releases tumor specific antigens in the process of tumor cell lysis to activate the anti-tumor immune response of an organism. Oncolytic viruses can kill cancer cells using a variety of mechanisms of action including cytolysis, apoptosis, antiangiogenesis, and cell necrosis. The virus infects tumor cells and then begins to replicate. The virus continues to replicate until finally the host cell membrane is "lysed" and the tumor cells can no longer contain the virus. Tumor cells are destroyed and the newly produced virus spreads to neighboring cancer cells to continue the cycle. It is important to note that all oncolytic viruses replicate only in cancer cells, which do not cause damage when they pass through normal tissues. Thus, once all tumor cells are eradicated, the oncolytic virus is no longer capable of replication and the immune system is able to clear it from the body.

The freeze-dried preparation can effectively protect the product, so that the original biological structure and physical properties of the product can be maintained, and the freeze-dried preparation has been widely applied to vaccine biological products. However, at present, few lyoprotectants suitable for oncolytic viruses are studied, most lyoprotectants are suitable for traditional attenuated or inactivated vaccines, the toxicity of oncolytic viruses is poor after being lyophilized by the traditional lyoprotectants, the titer is poor compared with that before being lyophilized, the thermal stability after being lyophilized is poor, the titer of oncolytic viruses is gradually poor when the oncolytic viruses exist in vivo for a long time when the oncolytic viruses are used for in vivo treatment, and the killing effect of oncolytic viruses on tumors is further affected. Therefore, the oncolytic virus preparations on the market at present are all liquid preparations, and compared with freeze-dried preparations, the liquid preparations have a plurality of defects, such as: the cost of preservation and transportation is high, and the preservation at the ultralow temperature of-80 ℃ is usually required. The risk is also high, if freezer or refrigerator trouble, the rising of temperature leads to the goods to melt, and the titer can drop about half, makes the goods scrap.

Disclosure of Invention

Based on this, it is necessary to provide a lyoprotectant suitable for oncolytic viruses and its use.

The first object of the present invention is to provide a lyoprotectant suitable for oncolytic viruses, which is a solution having functional components, comprising, in mass percent at working concentration:

3 to 10 percent of sucrose, 0.3 to 1.0 percent of gelatin, 0.1 to 1.0 percent of Human Serum Albumin (HSA) and 0.1 to 1.0 percent of sodium glutamate

The second object of the present invention is to provide a method for preparing the lyoprotectant, comprising:

and dissolving the sterilized components in a solvent, and uniformly mixing.

The third object of the present invention is to provide a method for preparing the oncolytic virus lyophilized powder, comprising:

and mixing the oncolytic virus with the freeze-drying protective agent and then freeze-drying.

The fourth object of the invention is to provide the oncolytic virus freeze-dried powder prepared by the preparation method of the oncolytic virus freeze-dried powder.

The fifth purpose of the invention is to provide the application of the oncolytic virus freeze-dried powder in preparing medicaments for killing tumors.

The invention provides a freeze-drying protective agent for preparing freeze-drying powder of oncolytic viruses, which is easier to store and has good stability compared with the traditional liquid preparation of oncolytic viruses. The freeze-drying protective agent comprises the functional components of sucrose, gelatin, HSA and sodium glutamate, or also comprises arginine, urea and glycerol. The components are matched with each other in a proper proportion, so that the activity loss of the oncolytic virus is smaller in the freeze-drying process, the titer loss before and after freeze-drying is small, and in addition, the difference of killing capacity of the oncolytic virus on tumor cells before and after freeze-drying is within +40% to-20% (40% indicates that the killing capacity of the tumor cells after freeze-drying is improved by 40%, and-20% indicates that the killing capacity of the tumor cells after freeze-drying is reduced by 20%) when the freeze-drying protective agent is used. . In addition, the freeze-dried preparation can maintain stability for a long time at the temperature of the organism (37 ℃), is favorable for being applied to the organism as a tumor killing medicament and improves the time of tumor killing.

Drawings

FIG. 1 is a graph showing the expression of cytokine IL-12 in various samples according to example 2 of the present invention;

FIG. 2 is a graph showing the expression of cytokine IL15 in different samples according to example 2 of the present invention;

FIG. 3 is a graph showing the expression of hIgG4 from various samples of example 2 of the present invention;

FIG. 4 is a graph showing the killing ability of hepatoma cell Hep 3B prior to lyophilization of batch 1 samples using the a3 lyoprotectant of example 3 of the present invention;

FIG. 5 is a graph showing the killing ability of hepatoma cell Hep 3B prior to lyophilization of batch 2 samples using the a3 lyoprotectant of example 3 of the present invention;

FIG. 6 is a graph showing killing ability of hepatoma cell Hep 3B after lyophilization of batch 1 samples using the a3 lyoprotectant of example 3 of the present invention;

FIG. 7 is a graph showing killing ability of hepatoma cell Hep 3B after lyophilization of batch 2 samples using the a3 lyoprotectant of example 3 of the present invention;

fig. 8 shows the titer change before and after lyophilization and in the accelerated stability test for two batches of samples using the a3 lyoprotectant of example 2 of the present invention.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, and the endpoints recited.

When describing a measurable value, such as a parameter, amount, time period, etc., the term "about" as used herein is intended to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/-5% or less, more preferably +/-1% or less, more preferably +/-0.1% or less, from the specified value, such variations being suitable for use in the disclosed invention.

In a first aspect, an embodiment of the present invention provides a lyoprotectant suitable for oncolytic viruses, which is a solution having functional components, including, in mass percent at a working concentration:

sucrose about 3% to about 10%, gelatin about 0.3% to about 1.0%, HSA about 0.1% to about 1.0%, and sodium glutamate about 0.1% to about 1.0%.

In this application, the lyoprotectant may further comprise arginine, the arginine being present in an amount of about 0.1% to about 1.0% by mass at the working concentration.

In this application, the lyoprotectant may further comprise glycerin, the glycerin being about 0.01% to about 0.15% by mass percent at the working concentration.

In this application, the lyoprotectant may further comprise urea, the urea being about 0.1% to about 1.0% by mass percent at the working concentration.

Compared with the traditional oncolytic virus liquid preparation, the freeze-drying protective agent for preparing the freeze-drying powder of the oncolytic virus is easier to store and has good stability. The freeze-drying protective agent comprises the functional components of sucrose, gelatin, HSA and sodium glutamate, or also comprises arginine, urea and glycerol. The components are matched with each other in a proper proportion, so that the activity loss of the oncolytic virus is smaller in the freeze-drying process, the titer loss before and after freeze-drying is small, and in addition, the difference of killing capacity of the oncolytic virus on tumor cells before and after freeze-drying is within +40% to-20% (40% indicates that the killing capacity of the tumor cells after freeze-drying is improved by 40%, and-20% indicates that the killing capacity of the tumor cells after freeze-drying is reduced by 20%) when the freeze-drying protective agent is used. In addition, the freeze-dried preparation can maintain stability for a long time at the temperature of the organism (37 ℃), is favorable for being applied to the organism as a tumor killing medicament and improves the time of tumor killing.

The freeze-dried preparation is the biggest difference from the liquid preparation in that: the freeze-dried preparation can remove most of water, and has the advantages of basically anaerobic storage condition, difficult oxidization of the product, better stability and longer shelf life. The lyophilized preparation has reduced requirement for storage conditions, reduced transportation and storage costs, and reduced risk. The fault tolerance of medication is reduced, patients do not worry about emergency before medication, and medication is delayed or not performed.

In the present invention, the terms "lyophilization" and "freeze-drying" are used interchangeably herein and refer to a substance that is dehydrated by first freezing and then reducing the pressure of the surrounding to allow the frozen water in the substance to sublimate. The material may be frozen in a freezer and then dried. But can also be frozen directly in the drying chamber by rapid vacuum.

In the preparation of the oncolytic virus freeze-dried preparation by the freeze-dried protective agent, the freeze-drying method can be a conventional virus or attenuated virus or vaccine freeze-drying method. In some embodiments, for example, the steps may be included: pre-freezing: the minimum temperature is less than or equal to minus 40 ℃, and the temperature is maintained for 2 to 3 hours after reaching the minimum temperature; sublimation drying stage: the final temperature is-15 to-10 ℃, the time for reaching the final temperature is 8-14 hours, and the vacuum pressure is controlled at 8-10 Pa; and (3) desorption drying stage: the final temperature is 34-37 ℃, the vacuum pressure is controlled to be 0.5-10 Pa, and the operation is carried out for 15-22 hours.

In the present invention, the term "working concentration" is used to define the proportion of the main active ingredient of the lyoprotectant at the time of treatment of the microorganism, and the concentration of the active ingredient in the lyoprotectant may be the working concentration or may be a mother liquor (e.g., a

mother liquor

2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50-fold concentrated) that can be diluted to that concentration.

In some embodiments, the functional ingredient consists of the following components in mass percent at the working concentration:

sucrose about 3% -10%, gelatin about 0.3% -1.0%, HSA about 0.1% -1.0%, sodium glutamate about 0.1% -1.0%, urea about 0.1% -1.0% and arginine about 0.1% -1.0%.

In other embodiments, the functional ingredient consists of the following components in mass percent at the working concentration: sucrose about 3% -9%, gelatin about 0.3% -0.9%, HSA about 0.1% -0.9%, sodium glutamate about 0.1% -0.9%, urea about 0.1% -0.9% and arginine about 0.1% -0.9%. In some embodiments, the solvent of the solution is water, such as deionized water, sterile water.

In particular, the sucrose content may also be 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5% or 9.0%. The gelatin may also be present in an amount of 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90% or 0.95%. The HSA content may also be 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.24%, 0.26%, 0.28% or 0.30%. The sodium glutamate may also be present in an amount of 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.16%, 0.18% or 0.20%. The urea content may also be 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75% or 0.80%.

Specifically, the arginine may also be present in an amount of 0, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.14%, 0.16%, 0.18% or 0.20%.

In this application, the lyoprotectant may comprise sucrose 3% -8%, gelatin 0.3% -1.0%, HSA0.1% -1.0% and sodium glutamate 0.1% -1.0% (in mass% at working concentration). In certain embodiments, the lyoprotectant further comprises arginine 0.1% -1.0% (in mass percent at working concentration).

In this application, the lyoprotectant may comprise sucrose 4% -7%, gelatin 0.3% -0.8%, HSA0.2% -0.6% and sodium glutamate 0.1% -0.5% (in mass% at working concentration). In certain embodiments, the lyoprotectant further comprises arginine, glycerol, and/or urea.

In this application, the lyoprotectant may comprise sucrose 4% -7%, gelatin 0.3% -0.8%, HSA0.2% -0.6% and sodium glutamate 0.1% -0.5% (in mass% at working concentration). In certain embodiments, the lyoprotectant further comprises arginine in an amount of 0.1% to 0.5% (by mass at the working concentration). In certain embodiments, the lyoprotectant further comprises 0.1% urea (in mass percent at the working concentration). In certain embodiments, the lyoprotectant further comprises 0.1% (in mass percent at the working concentration) glycerol.

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2% and sodium glutamate 0.1% -0.3% (in mass% at working concentration). In certain embodiments, the lyoprotectant further comprises arginine, glycerol, and/or urea.

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2% and sodium glutamate 0.1% -0.3% (in mass% at working concentration). In certain embodiments, the lyoprotectant further comprises 0.1% arginine (in mass percent at the working concentration). In certain embodiments, the lyoprotectant further comprises 0.5% urea (in mass percent at the working concentration). In certain embodiments, the lyoprotectant further comprises 0.05% glycerol (in mass percent at the working concentration).

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3%, arginine 0.1% -0.15% and urea 0.5% -0.6% (in mass% at working concentration).

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3%, arginine 0.1% -0.15% and glycerol 0.05% -0.06% (in mass% at working concentration).

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3% and urea 0.5% -0.6% (in mass% at working concentration).

In this application, the lyoprotectant may comprise sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3% and glycerol 0.05% -0.06% (in mass% at working concentration).

Optionally, the lyoprotectant may also contain a buffer substance, as the term is used herein, refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffer nature of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffered solution. Buffers generally do not have an infinite ability to maintain the pH of a solution or composition. Instead, they are generally able to maintain a pH within a specific range, for example, pH 7 to pH 9. Generally, buffers are capable of maintaining a pH at its pKa and within the next logarithm (see, e.g., mohan, buffers, A guide for the preparation and useof Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffer salts or preferably non-ionic buffer components such as Tris and HEPES. The buffer which can be used in the method of the present invention is preferably selected from the group consisting of phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2-hydroxymethyl-1, 3-propanediol (Tris) buffer, TRIS buffered saline solution (TBS), TRIS/EDTA (TE), earle's Balanced Salt Solution (EBSS).

For example, lyoprotectants described herein may comprise EBSS as a buffer. For another example, lyoprotectants described herein may comprise Tris-HCl as a buffer. For another example, lyoprotectants described herein may comprise Tris as a buffer. The lyoprotectants of the present application may comprise one or more (e.g., two) buffers.

In some embodiments, the lyoprotectant has a working ph of 6.8 to 7.4.

In a second aspect, an embodiment of the present invention provides a method for preparing a lyoprotectant according to any one of the preceding embodiments, including:

and dissolving the sterilized components in a solvent, and uniformly mixing.

In a third aspect, an embodiment of the present invention provides a method for preparing the oncolytic virus lyophilized powder according to any one of the above embodiments, comprising:

oncolytic viruses are mixed with the lyoprotectant described in any of the above examples and lyophilized.

Factors affecting the protective properties of lyoprotectants come mainly from two aspects: firstly, microorganism factors, microorganism types and structure differences, selection of a culture method, microorganism types (such as oncolytic viruses, and the requirement of still retaining tumor killing effect after freeze-drying) and the like; secondly, the formula of the protective agent is that the raw materials of the protective agent, the properties of the protective agent, the proportion of the raw materials, the freeze-drying curve suitable for the vaccine and the like are selected. When the formulation of the lyoprotectant is selected, the above-mentioned several main factors are considered, a proper formulation and matching are selected, and the lyophilization curve is formulated through repeated verification.

In some embodiments, the method of lyophilization comprises:

pre-freezing, primary drying and secondary drying.

In a fourth aspect, an embodiment of the present invention provides an oncolytic virus lyophilized powder prepared by the method for preparing an oncolytic virus lyophilized powder according to any one of the embodiments above.

In the present application, the oncolytic virus used may be selected from ssDNA-like viruses, dsDNA-like viruses, ssRNA-like viruses or dsRNA-like viruses; and/or the oncolytic virus used may be selected from wild-type or naturally attenuated strains, genetically engineered selectively attenuated strains, genetically loaded strains or genetically transcription targeted strains.

The wild-type or naturally attenuated strain may be selected from newcastle disease virus, reovirus, mumps virus, west nile virus, adenovirus, vaccinia virus, etc.

The genetically engineered selective attenuated strain, e.g., ONYX-015, G207, may be artificially deleted for tumor selectivity of viral replication.

The gene-loaded virus strain may be loaded with an exogenous gene such as granulocyte macrophage colony-stimulating factor (GM-CSF), such as JX-594 or T-VEC.

The gene transcription targeted virus strain, i.e. the insertion of tissue or tumor specific promoters in front of the virus essential genes, controls oncolytic virus replication in tumor cells, is e.g. G92A.

The ssDNA viroid may be selected from parvoviruses (parvoviruses), such as H-1PV virus.

The dsDNA virus may be selected from herpes simplex virus (herpes simplex virus), adenovirus (adeno virus), poxvirus; preferably, the adenovirus is selected from Enadenotucirev, DNX-2401, C-REV, NG-348, prosapak, CG0070, ADV-TK, EDS01, KH901, H101, H103, VCN-01, telomelysin (OBP-301), preferably the herpes simplex virus is type I herpes simplex virus HSV-1; the herpes simplex virus is selected from R3616, T-VEC, HF10, G207, NV1020 and OrientX 010, and the poxvirus is selected from Pexa-Vec (vaccinia virus), JX-594 (vaccinia virus), GL-ONC1 and Myxoma.

The ssRNA viroid may be selected from Picornavirus, alphavirus, retroviruses, paramyxoviruses, rhabdoviruses; preferably, the Picornavirus is selected from CAVATAK, PVS-RIPO, CVA21 (enterovirus), RIGVIR, the alphaviruses are selected from M1, sindbis AR339, semliki Forest virus, the Retroviruses are selected from Toca511, the Paramyxoviruses are selected from MV-NIS, PV701 (Newcastle disease virus), and the Rhabdoviruses are selected from VSV-IFN beta, MG1-MAGEA3, VSV-GP. The ssRNA viroid may also be selected from reovirus (reovirus), coxsackievirus (coxsackievirus), polio virus (polio virus), sesamum indicum valley virus (seneca valley virus), measles virus (measles virus), newcastle disease virus (newcastle disease virus), vesicular stomatitis virus (vesicular stomatitis virus, VSV), influenza virus.

The dsRNA virus may be selected from Reoviruses; preferably, the Reoviruses are selected from the group consisting of pelaroep, reovirus (reoysin), vaccinia virus (vaccinia virus), mumps virus, human immunodeficiency virus (human immunodeficiency virus, HIV). In this application, the oncolytic virus may comprise endogenous or exogenous sequences encoding therapeutic substances for cancer treatment. For example, the therapeutic substance may be a cytokine and/or an immune checkpoint inhibitor.

Cytokines may include interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20; interferon (IFN) - α, ifnβ, or IFN- γ -, tumor necrosis factor α (tnfα), CD40L, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte colony-stimulating factor (G-CSF), chemokines (such as Neutrophil Activating Protein (NAP), macrophage Chemoattractant and Activating Factor (MCAF), RANTES, and macrophage inflammatory peptides (MIP-1 a and MIP-1B), complement components and their receptors, immune system accessory molecules (e.g., B7.1 and B7.2), adhesion molecules (e.g., ICAM-1, 2 and 3), and adhesion receptor molecules.

Immune checkpoints are proteins that regulate certain types of cells of the immune system, such as T cells, which play a central role in cell-mediated immunity. While immune checkpoints help to control immune responses in the examination, they can also prevent T cells from killing cancer cells. Immune checkpoint inhibitors (or simply "checkpoint inhibitors") can block immune checkpoint protein activity, releasing the "brake" on the immune system and allowing T cells to better kill cancer cells.

As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or modulates one or more checkpoint proteins, either entirely or in part. Checkpoint proteins regulate T cell activation or function.

Checkpoint inhibitors may include small molecule inhibitors, or may include antibodies or antigen binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed on all NK, γδ and memory cd8+ (αβ) T cells), CD160 (also known as BY 55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR and various B-7 family ligands. Ligands of the B7 family include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies or antigen binding fragments thereof, other binding proteins, biotherapeutic agents, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049.

In a fifth aspect, an embodiment of the present invention provides an application of the oncolytic virus lyophilized powder according to any one of the embodiments above in preparing a medicament for killing a tumor.

In some embodiments, the tumor against which the oncolytic virus is directed is selected from at least one of lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphatic cancer, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, or leukemia.

The oncolytic virus performs at least one of the following killing mechanisms on tumor cells: direct lysis of tumor cells, in situ vaccine and distal effect, induction of innate immunity, stimulation of adaptive immune responses, destruction of tumor vasculature and/or improvement of inhibitory microenvironment.

The following are specific examples.

Example 1

Oncolytic viruses in embodiments of the present application comprise exogenous IL-12, IL-15 and Fc fusion protein expression cassettes. The formulation of the lyoprotectant was sequentially adjusted in the following order, and the changes in titer and stability of the lysozyme before and after lyophilization were measured by the plaque method described in pharmacopoeia, and the complex solvent for titer measurement was water for injection. The "%" in each formula represents the mass percent of the component in the lyoprotectant.

(1) The formula of the selected 4 groups of freeze-drying protective agents is as follows:

6a 3% sucrose+1% gelatin+1.0% sodium glutamate+1.0% HSA+EBSS (Earle's Balanced salt solution)

6b 8% sucrose+0.3% gelatin+0.1% sodium glutamate+0.1% HSA+EBSS+Tris-HCl

7a 3% sucrose+1% gelatin+1.0% sodium glutamate+1.0% HSA+1.0% arginine+EBSS

7b 8% sucrose+0.3% gelatin+0.1% sodium glutamate+0.1% HSA+0.1% arginine+EBSS+Tris-HCl

The preparation method of the freeze-drying protective agent comprises the following steps:

the freeze-drying protective agent is dissolved in purified water according to a formula, and each component is stirred by a glass rod respectively and then the volume is fixed respectively. And then, the freeze-drying protective agents with fixed volume are filled into sterilization for sterilization. The pH value is adjusted to 6.8-7.4.

And adding the oncolytic virus into the prepared freeze-drying protective agent, uniformly mixing, and transferring to a freeze-drying tube for freeze-drying.

Oncolytic virus titers using the 4-group lyoprotectant formulations are shown in table 1.

TABLE 1

As can be seen from Table 1, the 4 formulations showed a decrease in titer after lyophilization, but all decreased within 1.0Log, indicating that they were substantially stable and met the expected requirements.

(2) And (3) formula adjustment:

a1 4% sucrose+0.8% gelatin+0.6% HSA+0.5% sodium glutamate+0.5% arginine+0.1% glycerol+EBSS

A2 7% sucrose+0.3% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% glycerol+EBSS

A3 4% sucrose+0.8% gelatin+0.6% HSA+0.5% sodium glutamate+0.5% arginine+1% urea+EBSS

A4 7% sucrose+0.3% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% urea+EBSS

B1 4% sucrose+0.8% gelatin+0.6% HSA+0.5% sodium glutamate+0.5% arginine+0.1% glycerol+EBSS+Tris

B2 7% sucrose+0.3% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% glycerol+EBSS+Tris

B3 4% sucrose+0.8% gelatin+0.6% HSA+0.5% sodium glutamate+0.5% arginine+1% urea+EBSS+Tris

B4 7% sucrose+0.3% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% urea+EBSS+Tris.

The titers of the 8-group lyoprotectant formulations are shown in table 2.

TABLE 2

Preliminary stability study:

the formulations of group 8 in Table 2 all showed better lyoprotection, formulations A1, A2, A3 and A4 were subjected to preliminary stability studies to determine the change in titer after 7 days and 14 days of storage at 37℃after lyophilization, respectively. Two freeze-dried batches were separately assayed. The results are shown in tables 3 and 4.

TABLE 3 Table 3

TABLE 4 Table 4

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As can be seen from tables 3 and 4, there was no significant difference between the 8 formulations, and the lyophilized samples had better stability at 37 ℃. Therefore, the freeze-dried preparation formula obtained through process development and optimization meets the expected requirements.

(3) Formula adjustment is carried out on the basis of A1, A2, A3 and A4:

a1 6% sucrose+0.6% gelatin+0.1% HSA+0.3% sodium glutamate+0.1% arginine+0.05% glycerol+EBSS

a2 6% sucrose+0.6% gelatin+0.1% HSA+0.3% sodium glutamate+0.05% glycerol+EBSS

a3 5% sucrose+0.5% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% arginine+0.5% urea+EBSS

a4 5% sucrose +0.5% gelatin +0.2% hsa +0.1% sodium glutamate +0.5% urea + EBSS.

And adding the prepared freeze-drying protective agent into the oncolytic virus, mixing, and transferring to a freeze-drying tube for freeze-drying.

The titers of the 4-group lyoprotectant formulations are shown in table 5.

TABLE 5

As can be seen from Table 5, after the formulation was modified, the difference in titer before and after lyophilization was 0.5.+ -. 0.1Log PFU/ml, and in the stability test at 37 ℃, the titers of the two groups a1 and a2 were 0.56Log PFU/ml and 0.86Log PFU/ml, and the titers of the two groups a3 and a4 were 0.25Log PFU/ml and 0.24Log PFU/ml, respectively, and although the stability test at 14d was 4, the titers of the two groups a3 and a4 were better, and the stability of the titers before and after lyophilization and the heat stability after lyophilization were better than the results of the respective groups of the lyoprotectants tried before.

The oncolytic virus lyophilized formulation of formulation a3 was further tested for titers before and after lyophilization and for accelerated stability testing as described above, and two batches were tested for titers after 7 days, 14 days, 21 days and 28 days of storage at 37 ℃ after lyophilization, respectively. As shown in Table 6 and FIG. 8, the lyophilized preparation of oncolytic virus of formulation a3 showed substantially no change in titer before and after lyophilization, and showed little change in titer after 28 days at 37℃and still remained stable. The freeze-dried formula provided by the invention has good stability after long-term storage.

TABLE 6

Titer (lgPFU/ml)	a3 batch 1	a3 batch 2
			Before lyophilization	7.80	7.92
After lyophilization	7.70	7.79
			37℃-7d	7.51	7.41
37℃-14d	7.31	7.33
			37℃-21d	7.29	7.16
37℃-28d	7.20	7.01

Example 2 detection of foreign protein expression before and after sample lyophilization

This example uses a3 formulation as an example, using samples before and after lyophilization to infect Vero cells, and ELISA method to detect the cytokine IL-12 and IL15, and the expression of hIgG4 before and after lyophilization for 3 days. The lyoprotectant formulation used was a two lot a3 formulation: 5% sucrose+0.5% gelatin+0.2% HSA+0.1% sodium glutamate+0.1% arginine+0.5% urea.

Vero cells were plated into 6-well plates, counted, and based on the count results (about 1x10 ⁶ cell/ml) was used to adjust the viral pfu concentration. Cells were digested with Trypsin, medium in wells was aspirated, washed with PBS, lyophilized virus solution diluted with DMEM medium was added, diluted in concentration gradient, and the well plate was placed on a shaker and incubated for 2h at room temperature. After incubation, the wells were aspirated, 2ml of 1% FBS MEM medium was added to each well, and the wells were incubated at 37℃with CO ₂ The incubator was cultured for 72 hours.

The amount of IL-15 and IL-12 secreted by Vero cells after infection with virus was measured using Duoset Ancitilary rengent kit (manufacturer: RD system, cat# DY 008), human IL-12p70 elisa SA kit (manufacturer: RD system, cat# DY 1270) and Human IL-15/IL-15Ra complex (manufacturer: RD system, cat# DY 6924) and read with Thermo Multiskan Go microplate reader at 450nm and 570 nm. Data analysis used Prism8 and Excel software, and curve fitting used a four-term fit.

The results are shown in the following tables and figures 1-3 and 7, and there was no significant change in the amounts of secreted IL-12 and IL15 after infection of Vero cells before and after lyophilization of the samples, indicating that lyophilization had substantially no effect on the activity of the viral samples. The carried protein can still be expressed normally.

TABLE 7

Example 3 detection of killing Effect on tumor cells before and after sample lyophilization

In this example, CCR-8 chromogenic method was used to examine the killing ability of samples to liver cancer cell Hep 3B before and after lyophilization. The lyoprotectant was the same as in example 2.

Resuscitating, passaging Hep 3B cells, digesting, counting, plating, and adjusting the cell suspension concentration to 4x10 with culture medium ⁵ cells/ml, then added to 96-well plates at 100 ul/well. Viral samples before and after lyophilization were serially diluted in DMEM medium and added to 96-well plates with cells spread to infect cells with MOI of 20, 4, 0.8, 0.16, 0.032, 0.006, 0.0128, 0.0003 and 0.0000512. Placing at 37deg.C, CO ₂ The incubator was cultured for 72 hours. The activity of the Hep 3B cells after infection was measured with CCK8 (manufacturer: YEASEN, cat. No. 40203ES 80) and read with a Thermo Multiskan Go microplate reader at 450nm and 650 nm. Data analysis used Prism8 and Excel software, and curve fitting used a four-term fit.

The results are shown in the following tables and figures 4-7 and 8,

TABLE 8

In addition, mycoplasma (EZ-PCR Mycoplasma Detection Kit, manufacturer: BI, product number: 20-700-20) and endotoxin (Toxin SensorTM Chromogenic LAL Endotoxin Assay Kit, manufacturer: kirsrui, product number: L00350C) were detected on samples before and after lyophilization, and mycoplasma and endotoxin were not detected within the minimum detection limit, indicating that the samples of the present application have higher safety.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.

Claims

1. A lyoprotectant suitable for oncolytic viruses, characterized in that it is a solution having functional components comprising, in mass percent at working concentration:

3 to 10 percent of sucrose, 0.3 to 1.0 percent of gelatin, 0.1 to 1.0 percent of Human Serum Albumin (HSA) and 0.1 to 1.0 percent of sodium glutamate.

2. The lyoprotectant of claim 1, further comprising arginine in the working concentration in mass percent of 0.1% -1.0%.

3. The lyoprotectant of claim 1, further comprising 0.1% -1.0% urea in mass% at the working concentration.

4. The lyoprotectant of claim 1, further comprising 0.01% -0.15% glycerol in mass percent at the working concentration.

5. The lyoprotectant of any one of claims 1-4, wherein the functional ingredients consist of, in mass percent at working concentration:

sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3%, urea 0.5% -0.6% and arginine 0.1% -0.15%; or alternatively

Sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3% and urea 0.5% -0.6%; or alternatively

Sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3% and glycerin 0.05% -0.06%; or alternatively

Sucrose 5% -6%, gelatin 0.5% -0.6%, HSA0.1% -0.2%, sodium glutamate 0.1% -0.3%, glycerin 0.05% -0.06% and arginine 0.1% -0.15%.

6. The lyoprotectant of any one of claims 1-4, wherein the solvent of the solution is water.

7. The lyoprotectant of any one of claims 1-4, further comprising a buffer substance, wherein the lyoprotectant has a working ph of 6.8-7.4.

8. The method for preparing a lyoprotectant according to any one of claims 1 to 7, comprising:

and dissolving the sterilized components in a solvent, and uniformly mixing.

9. The preparation method of the oncolytic virus freeze-dried powder is characterized by comprising the following steps:

the oncolytic virus is mixed with the lyoprotectant of any one of claims 1-7 and then lyophilized.

10. The oncolytic virus lyophilized powder prepared by the method of claim 9.

11. The oncolytic virus lyophilized powder of claim 10, wherein the oncolytic virus is selected from a DNA virus or an RNA virus.

12. Use of an oncolytic virus lyophilized powder according to any one of claims 10-11 in the manufacture of a medicament for killing a tumor.