CN112294948A

CN112294948A - Use of a semi-fluid comprising blood, a vaccine comprising the semi-fluid and a method for the preparation of the vaccine

Info

Publication number: CN112294948A
Application number: CN201910706670.8A
Authority: CN
Inventors: 邹方霖; 邹礼常; 王建霞; 王艺羲
Original assignee: Chengdu Kuachang Aopu Medical Technology Co ltd
Current assignee: Chengdu Kuachang Aopu Medical Technology Co ltd
Priority date: 2019-08-01
Filing date: 2019-08-01
Publication date: 2021-02-02

Abstract

The present invention relates to the use of a semi-fluid comprising blood, a vaccine comprising the same, a method for the preparation of the vaccine, and a method for combating pathogenic disease.

Description

Use of a semi-fluid comprising blood, a vaccine comprising the semi-fluid and a method for the preparation of the vaccine

Technical Field

Background

Due to the intense research, solid tumors are often used as a pathogen disease model for many serious pathogen diseases, especially intractable pathogen diseases. Under the pressure of organism immunoselection, cancer cells usually rely on the high mutation characteristic of the cancer cells to gradually establish an immunosuppressive microenvironment to resist and inhibit the immune recognition and reaction of the organism, and finally show the immune tolerance of the tumor body to the organism. The core of solid tumor vaccine development is the development of safe and effective antigens. The substances with various structural layers can be used as vaccine antigens, and a wide space is provided for vaccine development.

Microorganisms (e.g., bacteria, viruses, parasites, etc.) were first used as antigens in the development of solid tumor vaccines. Although some live or inactivated vaccines of microorganisms show some effectiveness, they are considered to have a higher safety risk. In addition to the use of BCG in the treatment of bladder cancer, such microbial vaccines have found little use clinically.

Certain tumor cells are subsequently used as antigens in the development of solid tumor vaccines. After the autologous or allogeneic tumor cells are treated by eliminating tumorigenicity, the immunogenicity of the autologous or allogeneic tumor cells shows a certain anti-tumor curative effect. The whole-cell vaccine has more comprehensive expression, but has complex components, weak specificity and low sexual (effectiveness)/price (safety) ratio. Pathogens often carry multiple specific antigenic determinants. Researchers are then looking to screen individual useful harmless antigenic sites (e.g., certain surface proteins, DNA, etc.) in tumor cells to make subunit vaccines. The discovery of so-called tumor-specific antigens and tumor-associated antigens has been encouraging. However, although these subunit vaccines are relatively unique in composition, highly specific, and highly safe, their immunogenicity is low, and the ratio of sex (efficacy)/price (safety) is still not high. The combination of the accuracy of subunit antigens and the powerful immunogenicity of attenuated live antigens of microorganisms derives the vector vaccine. And autologous dendritic cells (DC cells) loaded with subunit antigens in vitro are used for deriving the DC vaccine. However, these subunit antigen-derived vaccines still do not show efficacy against most solid tumors. With the development of the current genetic technology, tumor neoantigens (Neoantigen) are making people a lot again.

Tumor neoantigens refer to a class of proteins/epitope polypeptides that are caused by mutations in cancer cell genes, are absent from normal cells, and are recognized by immune cells. The number of mutant genes carried by a cancer cell is often huge (hundreds to thousands), the number of abnormal proteins produced by the cancer cell is not small (tens to thousands), and the number of tumor neoantigens is only a small part of the abnormal proteins. The development of new technologies (e.g., one's deceased mother gene sequencing technology, computer modeling, and protein mass spectrometry) has enabled the identification of these mutant genes and aberrant proteins, prediction of proteins with neoantigen potential, and the in vitro screening and synthesis of these neoantigens. However, the polypeptide sequence generally consists of several or tens of amino acids, and has a small molecular weight, a simple chemical structure, and high safety but weak immunogenicity, and it is still difficult to induce immune response of sufficient strength, and only few successful cases have been reported.

In addition, some allogeneic lymphocytes have also been used as antigens in the development of non-solid tumor vaccines. It has been found that after allogeneic hematopoietic stem cell transplantation (allo-HSCT), allogeneic lymphocytes migrate and proliferate in the recipient, and further initiate cytotoxic attack targeting the recipient target cells, mediating graft-versus-host disease (GVHD). The more mismatched sites that the donor and recipient HLA match, the greater the likelihood of severe GVHD occurring. Allogenic lymphocytes often also mediate graft-versus-leukemia (CVL) responses when GVHD occurs. In other non-hematologic malignancies, there is also a GVHD and CVL-like response (extensionally termed Graft Versus Tumor (GVT)). However, further studies have found that the dominant antigenicity of the allogeneic lymphocyte antigen is GVHD, showing only a lower GVT response. Thus, the predominant indications are mainly some slow-growing hematological tumors (e.g., chronic myelogenous leukemia), rather than rapidly progressing hematological tumors (e.g., acute leukemia, chronic leukemia that has progressed to the acute stage).

Attempts have also been made to use certain fluids containing allogeneic lymphocytes as antigens in the development of solid tumor vaccines. However, solid tumors often require a much more robust GVT response. And an increase in their GVT response also increases the morbidity and mortality of GVHD in patients. Such allogeneic lymphocyte vaccines thus limit their advantageous indications to solid tumors that are as close as possible to hematological tumors, for example, to the smallest tumor mass possible, or as soon as possible within 3-5 days after inoculation of the mice with the transplanted tumor. How to reduce GVHD while increasing the GVT response is a key difficulty. In the present clinical application, there are few attempts to treat solid tumors with such allogeneic lymphocyte vaccines.

Although microbial (antigen) vaccines, tumor cell subunit (antigen) vaccines, tumor neo (antigen) vaccines, allogeneic lymphocyte (antigen) vaccines are flawless in variety, the development of solid tumor vaccines seems to be little advanced from a clinical point of view. Big problems are also in old places: how can a solid tumor vaccine antigen be developed that has the desired (anti-tumor) antigenicity clinically effective, while not requiring antigenicity (e.g., GVHD) and being within clinically safe tolerances? Several other serious pathogenic diseases, especially intractable pathogenic diseases (e.g. hepatitis b), also have similar problems. Thus, there remains a need to develop new immunization strategies to meet the urgent clinical needs that the prior art has not yet met.

Disclosure of Invention

The object of the present patent application is to provide a vaccine antigen that mediates an effective immune effect against pathogenic diseases, without the need for antigenicity (e.g., GVHD) and with minimal size, as well as a vaccine comprising the antigen, a method of making the vaccine, and a method of combating pathogenic diseases with the vaccine.

One aspect of the present application provides the use of a semifluid comprising or formed from blood as an antigen in the manufacture of a vaccine for the treatment or inhibition of a pathogenic disease.

Another aspect of the application provides a vaccine for treating or inhibiting a pathogenic disease comprising blood or a semi-fluid formed from blood as an antigen.

A further aspect of the present application provides a method of treating a pathogenic disease comprising topically administering, preferably by local injection, to a subject individual in need thereof a vaccine comprising as an antigen blood or a semi-fluid formed from blood.

A further aspect of the present application provides a method of preparing a vaccine for treating or inhibiting a pathogenic disease, the vaccine comprising as an antigen a blood-containing or blood-forming semifluid, the method comprising the steps of:

a. providing blood;

b. subjecting the blood to a semifluidification treatment resulting in a semifluid, wherein the semifluidification is selected from one or more of: thickening of blood, coagulation of blood, and mechanical disruption of blood coagulation.

A further aspect of the disclosure provides a vaccine prepared according to the method disclosed herein.

The antigens according to the invention have the following advantages compared to microbial antigens of the prior art: higher immunogenicity against pathogen diseases, lower biosafety risks; compared with the tumor cells and subunit antigens thereof in the prior art, the method has the following advantages: higher immunogenicity against pathogen diseases, a broader spectrum of pathogen disease indications for immunogenicity; compared with the prior art, the allogeneic lymphocyte antigen has the following advantages: higher immunogenicity against pathogenic diseases, lower application rates, lower unwanted immune activity (e.g. GVHD).

The blood semi-fluid vaccine according to the invention has the following advantages compared to the microbial vaccines of the prior art: higher anti-pathogen disease effectiveness, higher biological safety, wider indication spectrum and more and better synergistic action selectivity; compared with the pathogen subunit vaccine in the prior art, the vaccine has the following advantages: higher effectiveness against pathogenic diseases, wider indication spectrum and more and better synergistic action selectivity; compared with the prior art, the allogeneic lymphocyte vaccine has the following advantages: higher efficacy against pathogenic disease, lower graft risk (e.g., GVHD), broader spectrum of indications, more selective synergy.

The anti-pathogenic disease regimen according to the present invention is more easily combined with other related treatment regimens. In addition, the application and the vaccine preparation are controllable, the cost is low, the treatment scheme is simple and easy to implement, and the safe and effective treatment is particularly beneficial to the broad population who is difficult to bear high expense.

Detailed Description

Another aspect of the invention provides a vaccine for treating or inhibiting a pathogenic disease comprising blood or a semi-fluid formed from blood as an antigen.

A further aspect of the invention provides a method of treating or inhibiting a pathogenic disease comprising topically administering, preferably by local injection, to a subject individual in need thereof a vaccine comprising as an antigen blood or a semi-fluid formed from blood.

In the present disclosure, the term "semi-fluid" refers to an object that does not flow under external pressure within a limited time (e.g., 20 seconds) and does not flow visually under the clinically acceptable external pressure, and is distinguished from fluid (fluid also has fluidity under no external pressure), solid (solid is not flowable under clinically acceptable external pressure), and semi-solid (semi-solid is only reversibly deformable under clinically acceptable external pressure). For example, the tissues of certain animal organs (e.g., muscle mass, liver, stomach, intestine, heart, lung, pancreas, cartilage, joints, etc.), and certain gels that do not flow under pressure (e.g., fibrin glue) are semi-solid, rather than semi-fluid; whereas conventional liquids containing blood cells (e.g., natural blood, suspensions with hematocrit ≦ 55%) are fluids.

In the present disclosure, the term "pathogen" refers to a pathogenic organism, including, for example, parasites (e.g., protozoa, worms, etc.), bacteria, fungi, viruses, rickettsia, chlamydia, mycoplasma, spirochetes, or other biological agents, including, for example, microbial recombinants, diseased cells (e.g., solid tumor cells); the term "pathogenic disease" refers to a disease associated with a pathogen.

In the present disclosure, the term "vaccine" refers to a biological agent capable of inducing a safe and effective immune response in an organism against a disease of interest (e.g., a solid tumor), thereby safely and effectively preventing or/and treating the disease; the term "vaccine antigen" (sometimes abbreviated as antigen in this disclosure) refers to a biological substance that is contained in a vaccine and whose predominant immune response is a desired immune response against a disease of interest (e.g., a solid tumor).

Within the scope of the present invention, the semifluid comprises blood selected from one or more of the following groups: natural blood, blood cell-enriched components of natural blood (e.g., concentrated blood of blood cells having a plasma content of 30-80% of the plasma content of natural blood), and engineered blood comprising a blood cell preparation and plasma (e.g., a mixture of leukocytes or/and lymphocytes and plasma).

In one embodiment, the hemocyte concentrated blood is selected from one or more of the following: the blood treatment agent comprises serum-free blood obtained by removing 20-60% of serum from natural blood, blood cell-added blood obtained by adding blood cells to the natural blood, and artificial blood containing blood cells and plasma proteins.

Within the scope of the present invention, the blood cells are selected from the group comprising one or more of: red blood cells, white blood cells, platelets and derivatives thereof. Within the scope of the present invention, the leukocytes are selected from the group comprising one or more of: granulocytes, monocytes, lymphocytes and derivatives thereof. Within the scope of the present invention, the lymphocytes are selected from the group comprising one or more of: t cells, B cells, naked cells, and derivatives thereof.

In one embodiment, the blood-containing semifluid preferably comprises as an antigen an invasion-like tissue antigen.

In the context of the present invention, the requirements (or basic solutions) for the blood-containing semifluid as an antigen are: such that (e.g., by applying conditions) it forms in vivo tissue-like nodules that are similar in composition (e.g., comprise cells and intercellular stroma), in nature (e.g., softness), morphologically/structurally, and sufficiently severe in invasiveness to be able to elicit sufficient immune recognition for it, and elicit a sufficient immune response against it as well as against its analogous diseased body (e.g., tumor body).

In the present disclosure, the term "tissue" refers to a multicellular structure comprising morphologically similar animal cells and intercellular substance; the term "invading tissue antigen" refers to an antigen that invades the body with a tissue-based structure (e.g., a semifluid comprising blood tissue) and is immunologically recognized and immunoresponsive; the terms "microbial antigen", "tumor cell antigen", "allogeneic immune cell antigen", "subunit antigen" refer to an antigen that enters the body with the basic structure of a microbe, tumor cell, allogeneic immune cell, or biological/tumor cell subunit, respectively, and is recognized and immunoresponsively by immunization; the terms "microbial vaccine", "tumor cell vaccine", "allogeneic immune cell vaccine", "subunit vaccine", "blood tissue vaccine" mean a vaccine whose antigens are microbial antigens, tumor cell antigens, allogeneic immune cell antigens, subunit antigens, or tissue antigen-mediated, respectively. The more complex the structure, the more antigenic determinants it usually carries. Apart from the same part of the immune mechanisms activated by the exact same epitope, different epitopes (e.g. immunogenic molecular weight, structural complexity, combinability, etc.) of different structural levels of the substance entering the body may activate different immune mechanisms. As a result, many vaccines used clinically are microbial vaccines, although subunit antigens are safer and easier to prepare.

In one embodiment, the blood-containing or blood-derived semifluid as an antigen preferably comprises as a tumor-invading tissue antigen.

In the scope of the present invention, the requirements (or basic technical solution) for the blood-containing semifluid as the antigen of the tumor-like invading tissue are as follows: such that (e.g., by applying conditions) it forms in vivo a tumor-like nodule that is similar in composition (e.g., amount of contained cells), nature (e.g., softness), morphology/structure, and sufficiently severe in invasiveness to be able to elicit sufficient immune recognition for it, and elicit a sufficient immune response against it as well as against its similar tumor.

In the present disclosure, the term "tumor-like invading tissue antigen" refers to an invading tissue-like antigen that has similarities in immunological recognition with tumor tissue (and not necessarily tumor cells) and whose induced immune response can attack tumor tissue.

In one embodiment, the semifluid is comprised in a topical application of the vaccine, and wherein the composition and morphology of the semifluid is such that it forms a semifluid nodule at the application site. In this embodiment, the blood-containing semifluid is preferably an antigen that induces an immune response as a local implant.

In one embodiment, the semifluid is comprised in the intralesional administration and/or the extrapathological topical administration of the vaccine, and wherein the composition and morphology of the semifluid is such that it forms a semifluid nodule at the site of administration. In one embodiment, the semifluid is contained in an intralesional (e.g., intratumoral) administration of the drug. In one embodiment, the semi-fluid is contained in a topical application outside the lesion of the drug (e.g., a topical application outside a tumor). In one embodiment, the semi-fluid is contained in an intralesional administration and an intralesional topical administration of the drug.

In one embodiment, the topical application outside the lesion comprises one or more of: subcutaneous, intramuscular, mucosal administration.

In one embodiment, the blood-containing or blood-forming semifluid is preferably included as an antigen as an invaded tissue-like antigen in a state remote from the native state, preferably in a state readily recognized by the immune system as severely damaged.

In one embodiment, the morphological/structural conditions comprising blood or a semifluid formed from blood as an antigen comprise: the blood contained by the semifluid is highly deviated from the native state, preferably severely damaged tissue, wherein the deviation from the native state comprises viscosification; the severe injury is selected from one or more injuries including: solidification, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage. In this embodiment, the blood-containing semifluid is preferably an antigen that induces an immune response as a highly deviated native tissue, as a locally severely damaged tissue.

In the context of the present invention, the thickening means that the blood is added to the blood cells and/or the additives in such a high amount that the system is no longer fluid but becomes a non-fluid viscous substance. The blood changes from fluid to semi-fluid, significantly away from the natural state.

In the present disclosure, the term "severe injury" refers to an injury state that not only loses physiological function but is recognized by the body's immune system and is eliminated in response to major trauma.

Within the scope of the present invention, the solidification damage, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage may be obtained by the following processes, respectively: solidification treatment, mechanical crushing, ultrasonic treatment, heat treatment, freeze thawing treatment, irradiation treatment and chemical treatment. It is well known that these treatments can alter tissue structure, morphology, causing severe tissue damage. These treatments not only cause tissue damage, but often also alter cellular structure, morphology, causing cellular damage (e.g., mechanical damage to cells, ultrasonic damage, thermal damage, freeze-thaw damage, radiation damage, chemical damage). Following these insults, animal tissues lose physiological function (e.g., are no longer available for organ or tissue transplantation, and are weakened in cell proliferation) and are more readily recognized and responded to by the body's immune system as a major trauma (e.g., its dominant antigenicity is no longer a xenogenic antigenicity).

In the context of the present invention, the solidification is a process of converting a liquid into a solid or semi-solid, which includes solidification processes selected from any liquid tissue known in the art, such as: self-coagulating (e.g., self-coagulating blood), thermal coagulating (e.g., thermal coagulating blood), coagulant coagulating (e.g., coagulant coagulating blood). Among them, thermal coagulation can be performed by heat treatment, and coagulation by a coagulant is performed by adding a coagulant to a liquid (for example, thrombin and calcium chloride are added to blood).

In the context of the present invention, mechanical crushing includes mechanical segmentation and shear crushing. The shear crushing means that the object to be treated is subjected to shear at a rotational speed of 10 rpm or more, preferably 10 to 50000 rpm, wherein the shear crushing can be carried out by means of a stirrer, a grinder or a homogenizer. A solid or semi-solid (e.g., a blood gel) can become a particle upon mechanical disruption.

In the context of the present invention, ultrasonic treatment means that the object to be treated (for example blood) is placed in an ultrasonic apparatus and subjected to ultrasound (for example at an operating frequency of 2 to 60kHZ) in order to destroy its structure.

Within the scope of the present invention, the heat treatment is selected from the group comprising one or more of: direct heat treatment, steam heat treatment, freeze-drying heat treatment, microwave heat treatment, radio frequency heat treatment and laser heat treatment, wherein the heat treatment temperature is more than or equal to 40 ℃, and preferably 60-115 ℃. Such as heat treatment of blood.

Within the scope of the present invention, the freeze-thaw treatment includes a freezing treatment and a thawing treatment of the frozen matter, wherein the freezing treatment is selected from mechanical refrigeration or/and liquid nitrogen refrigeration, and the freezing temperature is ≦ 60 ℃ and preferably-60 ℃ to-160 ℃. Such as freeze-thaw processing of blood.

Within the scope of the present invention, the irradiation treatment (e.g. with an intensity of 20-100Gy) is selected from one or more of the following: x-ray irradiation treatment, gamma-ray irradiation treatment, photosensitive drug + ultraviolet irradiation treatment. Such as irradiation treatment of blood, tissue particles.

In the context of the present invention, chemical treatment refers to the addition of a chemical breaker (e.g. a hardening agent such as an acid, base, alcohol, etc.) to the object to be treated to break its structure. Such as chemical treatment of blood.

In one embodiment, the blood-containing semifluid is preferably included as an antigen as an invasive tissue-like semifluid antigen in a state remote from the native state, preferably in a state readily recognized by the immune system as a severe lesion.

In one embodiment, the blood-containing semifluid comprises the semifluid product of the blood, wherein the semifluid is selected from the group consisting of one or more of: viscosity thickening of the semi-fluid, solidification of the semi-fluid and breaking of the solidified substance. The thickening, coagulation and mechanical disruption all cause severe abnormalities or damage to blood tissue.

In the present disclosure, the term "semi-fluid viscosified" refers to a viscosification of a non-fluid dope to a semi-fluid dope. For example: when the hematocrit of the white blood cells/plasma or red blood cells/plasma of the engineering tissue reaches more than 70 percent, the tissue is converted from fluid to semi-fluid; the term "semi-fluid coagulation" refers to a coagulation that converts a liquid into a semi-fluid, such as self-coagulation of blood (e.g., coagulation without heat, without a coagulant), thermal coagulation (e.g., medium-low temperature heat coagulation), and the like; the term "comminution" means a process of fragmenting non-liquid or solidified matter by mechanical partitioning or shear-crushing so as to form a semi-fluid, preferably injectable semi-fluid, wherein the shear-crushing can be carried out by means of a stirrer, a mill or a homogenizer (e.g.at a rotation speed of > 10 rpm, preferably 10-50000 rpm).

In one embodiment, the blood-containing semifluid is a blood-containing semifluid dope. In one embodiment, the semi-fluid dope comprises a tackifier. In one embodiment, the tackifier is selected from the group comprising one or more of: polyethylene glycol, starch, sodium carboxymethyl cellulose, polyvinyl pyrrolidone.

In one embodiment, the morphological/structural conditions of the blood-containing semifluid as an antigen include: the semifluid is selected from one or more of the following groups: a semi-fluid dope comprising said blood, a semi-fluid coagulum comprising said blood, a disruption of a coagulum comprising said blood, preferably one or more selected from the group consisting of: a semi-fluid coagulum containing the blood, a disrupted coagulum containing the blood.

In one embodiment, the morphological/structural conditions of the blood-containing semifluid as an antigen include: the semifluid is selected from one or more of the following groups: a semi-fluid coagulum comprising the blood, a disruption of a coagulum comprising the blood, preferably one or more selected from the group consisting of: a semi-fluid coagulum containing the blood, a disrupted coagulum containing the blood.

In one embodiment, the coagulum includes a self-coagulum, a coagulant-coagulum, a thermal coagulum of the blood.

In the present disclosure, the term "blood self-coagulation" refers to a coagulation formed by the natural coagulation of blood. The term "thermal coagulation of blood" refers to the coagulation of blood formed by heat treatment. The term "blood/coagulant semi-fluid coagulum" refers to a semi-fluid coagulum (rather than a solid or semi-solid coagulum) formed by the addition of a coagulant to blood.

In one embodiment, the semi-fluid comprises blood self-coagulates.

In one embodiment, the semi-fluid comprises a blood/coagulant coagulum. In one embodiment, the clotting agent comprises a blood clotting agent, wherein the blood clotting agent is selected from the group consisting of one or more of: thrombin such as bovine thrombin, porcine thrombin, recombinant human thrombin, autologous thrombin, other blood coagulation proteins such as coagulation factors, prothrombin complexes, and their activated forms, calcium ion providers such as calcium chloride, calcium hydroxide, calcium carbonate.

In one embodiment, the blood-containing semifluid is a disruption of a coagulation comprising blood. In one embodiment, the disruptions of blood coagulation comprise blood coagulation particles. In one embodiment, the disruptant comprises a population of particles. In one embodiment, the particles are preferably macroscopic particles distinguishable to the naked eye. In one embodiment, the average diameter of the cross-section of the longest end of the particle is ≥ 100nm, preferably 500nm-1mm or 1 μm-1 mm.

In one embodiment, the blood contained in the semifluid has a hematocrit of>22% (or cell concentration of>5.6×10⁹One/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml) or 45% -86% (or cell concentration 11.5X 10)⁹-22×10⁹Individual cells/ml). For example, natural blood typically has a large hematocritAt 30% (e.g., the normal range of hematocrit of human blood is 37-50%), the hematocrit of the concentrated blood after removing part of the serum can reach 45-85%; the hematocrit in blood cells/plasma can reach 45-85%. The hematocrit compositionally ensures that the semifluid forms a semifluid nodule, particularly an invasive tissue-like semifluid nodule, in vivo.

In one embodiment, the semi-fluid comprises blood having a hematocrit of 55% to 86% (or a cell concentration of 14.0 x10)⁹-22×10⁹Individual cells/ml).

In one embodiment, the blood-containing or blood-derived semifluid is preferably included as an antigen as a far from native state, preferably in a larger sized invasion-like tissue semifluid antigen that is readily recognized by the immune system as a severely damaged state.

In one embodiment, the composition, properties, morphology/structure and application conditions of the semifluid are such that it forms a volume at the site of application>100mm³Preferably ≥ 200mm³The semifluid nodule.

In the present disclosure, the term "larger sized invasive tissue-like semifluid antigen" refers to an antigen that is specific for an immunogen due to the larger size of the invasive tissue-like semifluid antigen, and is distinguished from antigens in molecular form (e.g., microbial antigens, tumor antigens, allogeneic immune cell antigens, etc.), as well as antigens in semi-solid form (e.g., semi-solid graft antigens, etc.). The terms "microbial antigen", "tumor antigen", "allogeneic immune cell antigen", "semi-solid graft antigen" refer to an antigen for which a species-specific molecule of a microorganism, a pathogen-specific molecule of a tumor cell, a xenogeneic immune cell, a species of a semi-solid graft or a xenogeneic specific molecule, respectively, is a specific immunogen. When these antigens are used as vaccine antigens, the corresponding vaccines are referred to as "semifluid nodule antigen vaccines", "microbial vaccines", "tumor antigen vaccines", "allogeneic immune cell vaccines", and the like, respectively. The more complex the structure, the more antigenic determinants (e.g., epitopes) it usually carries. For example, many vaccines used clinically are still primarily live microbial vaccines, although microbial subunit antigens are safer and easier to prepare.

In one embodiment, the semi-fluid is preferably a squeezable semi-fluid. Within the scope of the present invention, the extrudable semi-fluid is defined as a semi-fluid that flows through a syringe at a clinically acceptable pressure.

In one embodiment, the semifluid is an implant, preferably an injection, and its single animal dose is >0.1ml, preferably ≧ 0.2ml or 0.2-25 ml. The requirement for the semifluid to act as an antigen provides multiple oncosomal characteristics, while larger injection volumes make the resulting semifluid nodule more readily immunologically recognizable as a semifluid nodule antigen.

Within the scope of the present invention, the semi-fluid injection is a semi-fluid that can be administered directly by conventional injection systems. Semi-solid implants, in turn, are often surgically implanted or administered in a fluid (liquid) form a semi-solid (e.g., in situ gelling) nodule at the site of administration by conventional injection systems.

In one embodiment, the semi-fluid has a subcutaneous half-disappearance of ≧ 0.1 day, preferably 0.1-30 days.

Within the scope of the present invention, the blood contained in the semifluid is derived from a mammal. The mammal is selected from one or more of the following: human, pig, horse, sheep, cattle, rabbit, mouse. These animals may be either fully naturally evolved animals or animals modified by biotechnology (e.g., gene editing technology) (e.g., GAL antigen knockout pigs).

In one embodiment, the blood-containing semifluid preferably includes as its dominant antigenicity an antigen antigenic against a pathogenic disease rather than the host.

The semifluid according to the invention, when used as an antigen under the above conditions, is preferably a semifluid whose dominant antigenicity is the antigenicity against said pathogenic disease and not against the host.

In one embodiment, the semifluid is preferably a semifluid with a tumor inhibition rate ≥ host resistance rate, preferably a tumor inhibition rate/host resistance rate ≥ 150%.

The complexity of immune recognition and immune response caused by substances as antigens far outweighs the consequences caused by their use as chemotherapeutic drugs. In a multi-level and diversified network pattern of an immune system, one substance may cause various immune reactions and show various antigenicities. In the present disclosure, the term "immunodominant response" refers to the primary immune response that is activated; the term "dominant antigenicity" refers to the antigenicity exhibited in a dominant immune response. For example, allogeneic lymphocytes can attack normal cells (graft versus host response) and cancer cells (graft versus tumor response) of the recipient, and can also activate lymphocytes of the recipient to attack cancer cells (host versus tumor response) and allogeneic cells (host versus graft response). Since the predominant immune response is graft-versus-host reaction, followed by graft-versus-tumor reaction, which is weak, the allogeneic lymphocytes in the liquid drug are essentially an anti-host antigen, which can be used as an anti-tumor cell (e.g., leukemia) antigen, but are difficult to be used as solid tumor vaccine antigens. Therefore, the blood contained therein can be optimized by the following means.

Within the scope of the present invention, the blood contained by the semifluid is preferably selected from blood with minimized xenogeneic or xenogeneic antigenic activity.

In the present disclosure, the term "xenoantigen" refers to an antigen derived from a different species of a subject and representative of its species specificity; the term "allogenic antigen" refers to antigens derived from alloallelic differences in a subject, such as major histocompatibility antigens (MHC antigens, e.g., Human Leukocyte Antigens (HLA)), minor histocompatibility antigens (mH antigens), and other histocompatibility antigens (e.g., human blood group antigens, tissue-specific antigens, etc.).

In one embodiment, the blood is preferably one or more selected from the group consisting of: allogeneic blood, allogeneic blood and autologous blood, which have the same ABO blood type or similar HLA. In one embodiment, the blood comprises autologous blood. In one embodiment, the blood may be autologous blood and/or allogeneic blood.

In one embodiment, the vaccine comprising blood or a semifluid formed from blood as an antigen may further comprise an active ingredient against a pathogenic disease. In the case where the vaccine comprises an active ingredient of the pathogenic disease, the blood-containing or blood-derived semifluid may be used as an immunopotentiating antigen, wherein the immunopotentiation means that the vaccine of the semifluid and active ingredient produces a greater therapeutic immune response than either of the individual doses of the vaccine. The immune enhancement comprises a synergistic effect of the semi-fluid with a vaccine of the active ingredient.

Within the scope of the present invention, said synergistic effect comprises one or more aspects of a synergistic effect against pathogenic diseases selected from the group consisting of: immune synergy and/or chemotherapy synergy. For example, the inventive semi-fluid intratumoral drug may be involved in the release of intratumoral home antigens as a tissue-damaging component, while the synergistic effect with chemotherapeutic drugs enhances tissue destruction and activates and releases intratumoral home antigens, facilitating immune enhancement.

In one embodiment, the active ingredient against the pathogenic disease is selected from one or more of chemotherapeutic drugs or/and biologicals against the pathogenic disease.

In one embodiment, the ratio of the amounts of the active ingredients in the vaccine (w/w or v/v) is (0.1-30)/100. In one embodiment, the amount ratio (w/w or v/v) of the chemotherapeutic agent in the vaccine is (0.1-30)/100. In one embodiment, the amount ratio (w/w or v/v) of said biological product in said vaccine is (0.1-30)/100.

In one embodiment, the biological product is selected from one or more of the group comprising: a cell preparation, an immunomodulatory antibody, a cytokine, a pathogen, or a pathogen subunit.

In one embodiment, the cell preparation has a cell content of greater than 10⁵Cell/mm³Preferably 10⁵～10⁹Cell/mm³。

In the present disclosure, the term "cell preparation" refers to cells obtained by engineered production methods (e.g., cell purification, cell culture, etc.), such as lymphocytes obtained by the purification of a cell mixture from the spleen after lysis of erythrocytes. In one embodiment, the semi-fluid comprises autologous blood and a cell preparation. In one embodiment, the ratio of the amount of said cell preparation to the amount of blood cells (v/v) is between 10% and 90%. In one embodiment, the cell preparation is preferably derived from allogeneic, syngeneic and autologous cells, more preferably autologous cells.

In one embodiment, the cell preparation is a preparation selected from natural or engineered cells comprising one or more of the following: tissue-rich cells such as muscle cells; immune cells such as peripheral blood mononuclear cells, T cells, B cells, NK cells, lymphocytes; stem cells such as mesenchymal stem cells, hematopoietic stem cells.

In the scope of the present invention, the immunomodulatory antibody-like drugs are selected from one or more of the following groups: antibody blockers against inhibitory receptors (e.g., blocking antibodies against CTLA-4 molecules and PD-1 molecules), antibody blockers against ligands of inhibitory receptors, activating antibodies against immune response cell surface stimulatory molecules (e.g., OX40, CD137, 4-1BB, etc.), neutralizing antibodies against immunosuppressive molecules in the solid tumor microenvironment, such as TGF-p 1.

In one embodiment, the cytokine is selected from one or more of the following: tumor necrosis factor, interferon, interleukin.

In one embodiment, the pathogen in the pathogen or subunit of pathogens is selected from one or more of the following groups: tumors, microorganisms.

In the present disclosure, the term "pathogen antigen" refers to an antigen derived from a pathogen, including, for example, pathogens, pathogen subunits, and the like; the term "pathogen subunit" refers to immunologically active components of pathogens and artificial analogs thereof, such as parasites, bacteria, viruses, tumor cells, their immunologically active components (DNA, proteins, polypeptide fragments, etc.) (e.g., tumor subunit antigens, unmethylated cytosine guanine dinucleotide-deoxyoligonucleotides (CpG ODNs), etc.).

In one embodiment, the pathogen antigen is selected from one or more of the following groups: microbial antigens, tumor antigens.

In one embodiment, the microbial antigen is selected from antigens derived from one or more of the following groups of microorganisms: bacteria such as one or more of the following: streptococcus pyogenes, Serratia marcescens, Bacillus Calmette-Guerin, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus, and Bacillus bifidus; viruses are, for example, one or more of the following: hepatitis B virus, adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, West Nick valley virus, Coxsackie virus, reovirus; parasites such as plasmodium.

In one embodiment, the tumor antigen comprises an antigen of a tumor selected from one or more of the following groups: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma.

In one embodiment, the pathogen antigen further comprises an in situ antigen.

In the context of the present invention, the term "in situ antigen" refers to a substance contained in a diseased variant in vivo that is likely to induce a specific immune response in the body (e.g., intratumoral solid tumor cell material, exosomes, polypeptides and nucleic acid sequences, etc. containing information on the antigens of a solid tumor), which actually contains a large amount of antigenic information that is different from the normal body, except that this information is masked by certain pathological factors (e.g., tumor microenvironment) and thus cannot be recognized and reacted by the conventional immune system. The chemotherapeutic agent may then release the shielded in situ antigen. For example, the semi-fluid intratumoral drug of the invention may be involved in the release of intratumoral in situ antigens as a tissue damaging component, and the chemotherapeutic drug may also be involved in the release of intratumoral in situ antigens.

In one embodiment, the chemotherapeutic agent is selected from one or more of cytotoxic agents and/or conventional ineffective but topically effective compounds.

In the present disclosure, the term "cytotoxic drug" refers to a drug (e.g., an anti-tumor chemotherapeutic) that is effective by absorption at a safe dose, selected from any pharmaceutically acceptable cytotoxic drug, preferably selected from those known in the art, and more preferably selected from those approved or to be approved by, or loaded or to be loaded in, the chinese, U.S. or european official pharmacopoeia (e.g., FDA or chinese drug administration).

In one embodiment, the cytotoxic drug may be one or more selected from the group consisting of: uracil derivatives, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complex, and taxanes; preferably one or more selected from the following drugs and their analogous derivatives: 5-fluorouracil, gemcitabine, epirubicin, an anti-solid tumor antibiotic, teniposide, a metal platinum complex and paclitaxel.

In one embodiment, the concentration of the chemotherapeutic agent in the semi-fluid is greater than 50% of its saturation concentration, preferably 50-500% of its saturation concentration, wherein the saturation concentration refers to the saturation concentration of the anti-solid tumor chemotherapeutic agent in its pharmaceutical vehicle.

In one embodiment, the conventionally ineffective but topically effective compound is selected from one or more of the following groups: amino acid nutrient, ineffective aromatic compound, and bioactive component.

In the present disclosure, the term "conventional ineffective but topically effective compound" refers to a drug (e.g., an anti-tumor chemotherapeutic) that is clinically ineffective by absorption at a safe dose, selected from drugs other than the anti-tumor chemotherapeutic already loaded in, for example, the chinese, us, or european official pharmacopoeia.

In the context of the present invention, the amino acids, amino acid salts, oligopeptides as the amino acid nutrient are preferably amino acids or salts thereof selected from the group consisting of: alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, tyrosine, serine, cysteine, methionine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, beta-alanine, taurine, gamma-aminobutyric acid (GABA), theanine, citrulline, ornithine; more preferably an amino acid or a salt thereof selected from the group or an oligopeptide comprising or consisting of: arginine, lysine, glycine, cysteine, alanine, serine, aspartic acid, glutamic acid. In one embodiment, the concentration (w/w) of the amino acid based nutrient in the vaccine is greater than or equal to 5%, preferably 10-35% or 18-35%, more preferably 15% -35% or 20% -35%.

In the present disclosure, the term "ineffective aromatic compound" refers to an aromatic compound that is not effective in inhibiting tumors by absorption at a safe dose. The ineffective aromatic compound includes any ineffective aromatic compound which is pharmaceutically acceptable. Within the scope of the present invention, the ineffective aromatic compound is one or more selected from the group consisting of: pigment aromatic compounds, salicylic acid compounds and quinoline compounds. In one embodiment, the concentration (w/v) of the ineffective aromatic compound in the vaccine is not less than 0.35%, preferably 0.35-20%.

In the present disclosure, the term "chromoaromatic compound" refers to a pharmaceutically acceptable aromatic compound capable of selectively absorbing or reflecting light of a specific wavelength at a target region, which may include, for example, vital dyes, photosensitizers, and colored chemotherapeutic agents.

In one embodiment, the pigment aromatic compound may be one or more selected from the group consisting of: methylene blue (including its hydrates), patent blue, isothio blue, bengal red, mixed porphyrin-based photosensitizers, porphyrin-based compounds (e.g., porphyrins, porphins, purpurins, endogenous porphyrins), nitrophenol compounds. In one embodiment, the concentration (w/v) of the pigment aromatic compound in the vaccine is not less than 0.35%, preferably 0.5-10%.

In one embodiment, the salicylic acid-based compound is one or more selected from salicylic acid and one or more of the following compounds and derivatives thereof: acetylsalicylic acid, difluorosalicylic acid, disalicylate, dicumarol and aspirin lysine. In one embodiment, the concentration (w/v) of the salicylate compound in the vaccine is 0.5% or more, preferably 0.5-2.0%.

In one embodiment, the quinolines are selected from water-soluble quinolines, preferably from one or more of the following: quinine, quinine hydrochloride, quinine dihydrochloride, chloroquine. In one embodiment, the concentration (w/v) of said quinolinic compound in said animal semisolid is ≥ 3%, preferably 3-6%.

In the present disclosure, the term "biologically active ingredient" refers to a biologically extract and its analogs having pharmaceutical activity. The term "biological extract" refers to a specific component (e.g., a purified product based on a specific structure) obtained by separation and other processes using biological materials such as plants and microorganisms as raw materials. The term "analog" refers to a natural product, derivative, semi-synthetic, or synthetic, although not identical, but similar in structure and/or nature.

Within the scope of the present invention, the biologically active ingredient is selected from the group consisting of biological extracts and analogues thereof having one or more of the following structures: glycosides, polyphenols, polysaccharides, terpenes, and flavones.

In one embodiment, the chemotherapeutic agent is selected from one or more of the following groups: anti-pathogenic chemotherapeutic drugs such as 5-fluorouracil, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complex, paclitaxel, amino acid nutrients such as arginine, lysine, glycine, cysteine, glutamic acid, or salts thereof, or oligopeptides comprising the same, ineffective aromatic compounds such as methylene blue, acetylsalicylic acid, quinine monohydrochloride, quinine dihydrochloride, bioactive ingredients such as algal polysaccharides, medicinal plant polysaccharides, fungal polysaccharides, artemisinin.

In one embodiment, the vaccine further optionally comprises an adjuvant selected from conventional adjuvants.

In the present disclosure, the term "adjuvant" refers to the additive disclosed in the present invention, which can enhance the body's ability to respond to the vaccine antigen or change the type of immune response after being mixed with the vaccine antigen. According to this definition, vaccine adjuvants differ from immunopotentiators in the general sense that their action often does not have to be mixed with vaccine antigens.

In the present disclosure, the term "conventional adjuvant" refers to any suitable adjuvant known to those skilled in the art, which may be, for example, an oil/milk adjuvant. In the present disclosure, the term "oil/milk adjuvant" refers to an oil or/and emulsion based adjuvant (e.g. freund's adjuvant, MF 59).

The additive in the semi-fluid according to the present disclosure may further optionally include an excipient. The excipient may be any suitable one known to those skilled in the art and may include, for example, one or more of the following: dispersion media, preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers and the like. Such as an antioxidant (e.g., ascorbic acid).

In the present disclosure, the vaccine is for use in treating or inhibiting a pathogenic disease. In one embodiment, the pathogen disease comprises one or more of the following groups: tumors, microbial infections.

In one embodiment, the pathogenic disease is a solid tumor. In the present disclosure, the term "solid tumor" is used to refer to any malignant tumor having a tumor body, while e.g., leukemia, malignant lymphoma, etc., are non-solid tumors. In one embodiment, the solid tumor is preferably selected from the group consisting of tumors with a volume of 400mm or more³More preferably, the tumor volume is more than or equal to 600mm³The malignant tumor of (2).

In one embodiment, the solid tumor comprises: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma.

In one embodiment, the pathogenic disease is selected from a microbial infection. In one embodiment, the microbial infection includes a viral infection and a bacterial infection. In one embodiment, the viral infection includes, for example, hepatitis b virus infection, hepatitis c virus infection, aids virus infection; the bacterial infections include, for example, leprosy, chronic mucocutaneous candidiasis.

The vaccine in the present application is a topical vaccine which, when used for the prevention and treatment of disease, may also be administered in combination with other therapies, such as interventional therapies, systemic chemotherapy, other immunotherapy (e.g. de-immune tolerant immunotherapy), photodynamic therapy, sonodynamic therapy, surgical intervention or a combination of such therapies to further enhance the therapeutic effect.

According to yet another aspect of the present disclosure, there is provided a method of preparing a vaccine for treating or inhibiting a pathogenic disease, the vaccine comprising as an antigen a blood-containing or blood-forming semifluid, the method comprising the steps of:

a. providing blood;

Where relevant, the definitions and descriptions of various pertinent terms in the foregoing aspects of the disclosure herein apply to this aspect as well.

In one embodiment, the semi-fluidized treatment comprises one or more of: semi-fluid visco-thickening, thermal solidification, solidification with a solidifying agent, and wherein the thermal solidification is selected from one or more of: direct heat treatment, steam heat treatment, microwave heat treatment, radio frequency heat treatment, laser heat treatment, preferably microwave heat treatment.

In the method of preparing a vaccine according to the present disclosure, after providing blood, the blood may be subjected to severe injury treatment and semi-fluid may be obtained. Alternatively, the provided blood can be prepared as a semi-fluid and then subjected to a severe injury treatment, wherein the severe injury treatment is selected from one or more of the following treatments disclosed herein: mechanical crushing, solidification treatment, heat treatment, freeze-thaw treatment, irradiation treatment and chemical treatment.

In one embodiment, the severe injury treatment comprises mechanical disruption. In one embodiment, the severe damage treatment comprises a heat treatment. In one embodiment, the severe insult treatment comprises a freeze-thaw treatment. In one embodiment, the severe damage treatment comprises an irradiation treatment. In one embodiment, the severe damage treatment comprises a chemical treatment.

In the method for preparing a vaccine according to the present disclosure, an active ingredient for anti-pathogenic disease may be further added to be mixed before or after the semi-fluidization treatment.

In the method for preparing a vaccine according to the present disclosure, the semi-fluid vaccine prepared as above may be optionally packaged, and the packaged product may be prepared for clinical use as a preparation (preferably, an implant, more preferably, an injection) or further prepared as a lyophilized preparation (e.g., a powder for injection) for clinical use.

In the present disclosure, the term "injection" refers to a drug that is administered through a needle cannula in compliance with the injection standards of the drug administration. The injection includes systemic injection (such as intravenous injection) and topical injection (such as subcutaneous injection, intramuscular injection, and blood injection).

Within the scope of the invention, the needle cannula comprises, for example: a conventional syringe needle tube, a conventional puncture needle, a conventional implantation needle, and a conventional infusion needle.

The process conditions of the freeze-drying treatment include, for example: the pre-freezing condition is that the temperature is kept at minus 45 ℃ for 4 hours; sublimation drying condition is that the heating rate is 0.1 ℃/min, and the heating is kept for at least 10 hours when the temperature is raised to-15 ℃; the desorption drying conditions were 30 ℃ for 6 hours. The lyophilized preparation can be mixed with a liquid medium (e.g., water or an aqueous solution containing a viscosity-enhancing agent) and then directly administered topically.

Based on the studies described in more detail below, the vaccine of the present invention shows an organic unity of rapid and follow-up treatment, short and long-lasting effects, and little damage to the normal blood of patients, although specific mechanisms remain to be further studied, thereby achieving a pharmaceutical effect of safely and effectively treating diseases.

Examples

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto. In the following examples, all experimental animals were performed according to the relevant regulations and industry discipline. Unless otherwise specified, all tests were carried out according to the usual methods.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Some of the additives used in the following examples are listed in table 1.

TABLE 1

In the present invention, L-amino acids are abbreviated as amino acids (for example, L-arginine is abbreviated as arginine), and reduced glutathione is abbreviated as glutathione.

The experimental animals used in the following examples were all purchased from professional laboratory animals company and were all SPF (Specific Pathogen Free) grade animals. Taking mice as an example, there are 5 types: BALB/C mice, C57BL/6 mice, CB6F1 mice, CC3HF1 mice, nude mice, wherein: the CB6F1 mouse is a BALB/C X C57BL/6 hybrid F1 generation female mouse (the phenotype is H-2)^b/d) The CC3HF1 mouse is a BALB/C x C3H hybrid F1 generation female mouse (the phenotype is H-2)^d/k) The nude mice are mutant line (BALB/c-nu) mice obtained by introducing nude genes (nu) into BALB/c mice. The mice are healthy females with the age of 6-8 weeks and the body weight of 17.5-20.5 g. In the following examples, BALB/C mice are abbreviated as B mice and C57BL/6 mice are abbreviated as C mice, unless otherwise indicated. Experiment movementThe organ and blood are purchased by laboratory animal companies or prepared by routine laboratory animal preparation.

In the following examples, unless otherwise indicated, subcutaneous transplantation of tumor animals was performed according to the general practice of subcutaneous inoculation of solid tumor cells according to the guidelines issued by the drug administration. Unless otherwise indicated, solid tumors grow to the desired volume (e.g., mice carry tumors 30-500 mm)³) Then for successful modeling, the model was randomly divided into experimental groups of 6 animals each using PEMS 3.2 software (compiled by the national institutes of public health, western, university, Sichuan). Items for experimental observation, measurement and analysis include general state, body weight, food intake, animal graft versus host disease, solid tumor volume, tumor weight, survival time, and the like.

The solid tumor volume calculation formula is as follows:

solid tumor volume (V) ═ l/2 × a × b²Wherein a represents a solid tumor length and b represents a solid tumor width.

The solid tumor growth inhibition rate (abbreviated as tumor inhibition rate in the invention) is calculated by the following formula:

tumor inhibition rate Y (%) ═ (CW-TW)/CW × 100%, where TW is the average tumor weight of the study group; CW is the average tumor weight of the negative control group.

The observation items and scores of the animal graft versus host disease are shown in table 2 below.

TABLE 2 graft versus host disease score

Observation index	0 point (min)	1 minute (1)	2 is divided into
				Body mass reduction	<10％	10％～25％	>25％
Ability to move	Is normal	Attenuation of	Standing still
				Fur and fur	Is normal	Mild disorder	Color difference and disorder
Degree of skin integrity	Is normal	Mild molting of toe and tail	Severe hair loss
				Posture of a person	Is normal	Mild degree of bow back	Severe arch with upward

Each animal scored 5 points in total, with a maximum of 10 points and a minimum of 0 points.

The graft-versus-host ratio is calculated as follows:

(iii) anti-host rate Z (%) (TP-CP)/10 × 100%, wherein TP is the mean value of study group graft-versus-host disease scores; CP is the average value of the negative control group graft-versus-host disease score; 10 is the highest possible value of the graft versus host disease score for each animal.

In the following examples, experimental results (e.g. tumor weights) are expressed as means ± standard deviation (x ± s), and differences between two experimental animal groups and group means are compared by significance testing using statistical software SPSS 19.0, the testing being performed using statistic t, test level α being 0.05, P <0.05 indicating that the differences are statistically significant, otherwise they are not statistically significant.

Within the scope of the present invention, the combination of drug A and drug B is designated as B/A. In the following examples, unless otherwise indicated, agent a and agent B are the blood-containing semifluid and active components, respectively. Unless otherwise stated, the drug effects of A, B, A/B are tumor inhibition. Improving the drug effect of antitumor drugs is always the biggest medical problem in the world. Even a few percent of efficacy is difficult and difficult to improve, so that the theoretical expectation of drug combination is usually not high, and once realized, the significance is great. The drug effect generated by the combined use of the drugs can be theoretically determined according to the judgment of q:

q-the actual combined effect/theoretical purely additive expected effect.

When q is 1, the actual combination effect is in accordance with theoretical expectations, showing additive effects; when q is less than 1, the actual effect is weaker than the theoretical expectation, and antagonism is shown; when q >1, the actual combination effect is more than theoretically expected and shows a synergistic effect.

The q calculation used in the present disclosure is based on the modified just-in-gold method (just-in-gold, addition in combined medication, proceedings of Chinese pharmacology 1980; 1(2), 70-76) of the Burgi method (Burgi Y. Pharmacology; Drug actions and reactions. cancer res.1978, 38(2), 284-285):

q＝E_A+B/(E_A+E_B-E_A·E_B)，

wherein E_A+BFor the actual combined effect of the A and B drugs, E_AFor the single-use effect of A, E_BThe effect of the single medicine B is shown, (E)_A+E_B-E_A·E_B) The expected effect is simply added for the pharmacology theory of A drug and B drug. To better fit the practical error range in animal experiments, he further replaced q 1 with q 1 ± 15% as an additive decision formula.

Another method for determining the efficacy of anti-tumor combinations, which is common in the literature, is based on a significance test (e.g., p-test). The pharmacological effects (tumor inhibition rate) of the compositions in both the q-and p-judgment were judged as a clear relationship between the actual combined effect and the theoretical expectation in the following antitumor animal experiments:

1) when q is 0.85. ltoreq. 1.15, and E_A+BAnd E_ABetween or E_A+BAnd E_BIf the difference between the two is not statistically significant (p is more than or equal to 0.05), the result is judged to be additive effect or effect which is not over expected;

2) when q is<0.85, and E_A+BAnd E_AAnd E_A+BAnd E_BThe difference between them is statistically significant (p)<0.05), the antagonism is judged to be obvious;

3) when q is>1.15, and E_A+BAnd E_AAnd E_A+BAnd E_BThe difference between them is statistically significant (p)<0.05), the synergistic effect is judged to be obvious.

Example 1: preparation of vaccines

In the following examples, the blood used is selected from one or more of the following groups:

1) natural blood extracted from animals;

2) a cell-enriched natural blood component enriched to contain a fraction of blood cells (e.g., leukocytes or lymphocytes) and plasma. For example, blood cells concentrate blood;

3) engineered blood comprising a portion of a blood cell preparation (e.g., leukocytes or lymphocytes) and plasma. Blood cell preparations include purified natural cells and derivatives thereof. For example, a mixture of plasma and a blood cell purification material, such as cell pellet, white blood cell pellet, red blood cell pellet isolated from natural blood according to the prior art. Also for example, a mixture of plasma and blood cell derivatives, such as blood cells induced, activated, expanded in vitro according to the prior art (e.g., DC cells, LAK cells, TIL cells, CIK cells, DC-CIK, CTL cells, TCR-T cells, CAR-T cells, NK cells, γ δ stem cells, etc.). Also for example, a mixture of plasma and blood cell purities derived from other enriched tissues. Wherein said hemocyte purification, such as lymphocytes obtained after a treatment of removing erythrocytes from the spleen of an animal according to the prior art, is prepared, for example: taking spleen after the animal is killed, crushing the spleen gently, adding serum-free DMEM culture solution and erythrocyte lysate (0.0075% ammonium chloride/0.0026% Tris is added into sterile aqueous solution for dissolving and diluting to 500mL), stirring gently, standing for 5 minutes, performing centrifugal washing for 3 times (the centrifugal condition is 1000 rpm/min, and the sediment resuspension is serum-free DMEM), and finally obtaining lymphocyte sediment.

1. Semi-fluid containing blood and preparation of vaccine

The semifluids of the present invention, and vaccines comprising the same, may be prepared by the semifluid process disclosed herein. The table below lists some of the preparations of this example (preparation numbers), the blood from which it was prepared, the main preparation steps, and the effect achieved by the preparation and its nodular nature.

TABLE 3

Specific volume: hematocrit is measured according to conventional blood measurement methods (e.g., using a fully automated hematology analyzer BC 5000).

For example, the cell concentration in blood of a 43% hematocrit mouse is 11X 10⁹One per ml.

Hematocrit: provided by veterinarians of relevant animal testing companies;

the function is as follows: a indicates the presence of semifluidization (+ presence), and B indicates the presence of severe damage (+ presence).

And (3) nodule: indicates whether a semifluid nodule (+ energy) can be formed at the injection site, as follows: 100 μ l of the above table

The preparation is injected into the left axilla of BALB/c mouse to form a nodule, and the nodule is pressed between the index finger and thumb

Irreversible deformation occurs. Skin of semifluid nodules except for semifluid dope (X10) for 0.1-0.5 days

The second half of the disappearance period is 1 to 30 days. When the hematocrit of the cell suspension is 77% or more, it turns into a semi-fluid thick matter.

Mice: the mice in the table are BALB/c mice.

Several examples of the preparation tests of the semifluids according to the invention are listed below.

Example 1 a: one new zealand immune was sacrificed and 15ml of the immune blood was taken and allowed to stand at room temperature for 30 minutes to obtain self-coagulated blood (X1 in the above table).

Example 1 b: one new zealand immune was sacrificed and 15ml of the immune blood was placed in a cup pre-filled with anticoagulant sodium citrate, slowly stirred well, then a gas permeable lid was added to the cup, and then placed in steam (about 100 ℃) to steam for 20-50 minutes to obtain heat coagulated blood (X2 in the above table).

The preparation of preparations X3 and X4 in the above table can be carried out separately using the same method as that for X2.

Example 1 c: about 6ml of blood was obtained from each of the orbits of a plurality of mice (with or without anticoagulant), mixed in a cup, treated in an ultrasonic instrument (temperature 5-25 ℃ C., operating frequency 10-30kHZ,;) for 1-30 minutes, then covered with a gas-permeable lid, and steamed in steam (about 100 ℃ C.) for 20 minutes to obtain preparation X5 in the above table.

Example 1 d: separately collecting blood (with or without anticoagulant) from orbit of multiple mice, adding preset coagulant (such as thrombin with final concentration of 10-100U/1ml and calcium chloride with concentration of 5-25 mmol/L) into the cup, mixing well, standing for 30 min, and stirring and crushing (rotation speed 3000 and 10000 rpm, total time 1-3 min) to obtain preparation X6 in the above table.

Example 1 e: preparation X7 in the above table was obtained by separately bleeding the orbit of a plurality of mice, centrifuging to remove 40% serum, adding to the cup a gas permeable lid, and steaming in steam (about 100 ℃ C.) for 20 minutes. Preparation X8 in the above table was obtained by the same preparation using human blood.

Example 1 f: approximately 10ml of blood was drawn from the horse ear using a blood drawing needle, and then the syringe containing the blood was heated in a microwave oven for 1 minute with the small needle tip set upright to obtain preparation X9 in the above table.

Example 1 g: preparation X10 in the above table was obtained by separately bleeding the orbit of a plurality of mice and centrifuging to remove 45% of the serum.

Example 1 h: 2g of human plasma and 3g of human leukocyte precipitate were stirred in a cup, and then covered with a gas-permeable lid, and steamed in steam (about 100 ℃ C.) for 5-15 minutes to obtain preparation X11.

The preparations are injectable semifluids and can be used as vaccines after being subpackaged into injectors.

2. Composition comprising a semi-fluid of blood and an active ingredient and preparation of a vaccine

The blood-containing semifluid of the invention as an antigen can be used in the preparation of a medicament as a synergistic component of the antigen and the active ingredient. Wherein the active ingredient comprises a biologic and/or a tissue disrupting agent.

Chemotherapeutic drugs and most biologicals are commercially available (e.g., table 1). Cell preparations were prepared in the laboratory. For example, normal blood is extracted and obtained according to the prior art, and white blood cells, red blood cells, and platelets are obtained by centrifugation. As another example, lymphocyte fluid is obtained from animal spleen following a treatment of removal by erythrocyte lysis according to the prior art: taking spleen after the animal is killed, crushing the spleen gently, adding serum-free DMEM culture solution and erythrocyte lysate (0.0075% ammonium chloride/0.0026% Tris sterile aqueous solution for hydrolysis and diluting to 500mL), stirring gently, standing for 5 minutes, performing centrifugal washing for 3 times (the centrifugal condition is 1000 rpm/min, and the sediment resuspension is serum-free DMEM), and finally obtaining lymphocyte sediment.

The tumor antigens used in this example were prepared as follows: tumor cell fluid (10)⁸-10⁹Individual cells/ml) were subjected to conventional freeze-thaw inactivation treatment (by placing in liquid nitrogen at a temperature of less than-80 ℃ for 30 minutes and then thawing at 37 ℃ for 3 times), and then verified as non-tumorigenic by conventional tumor cell transplantation tumor experiments, i.e., as tumor antigens, the tumor antigens obtained from the respective tumor cell fluids included the following tumor antigens obtained from the respective tumor cell fluids: sarcoma antigen (S180 cell), hepatocarcinoma antigen (H22 cell), colon cancer antigen (CT26 cell), breast cancer antigen (4T1 cell), and melaninTumor antigen (B16 cells), lung cancer antigen (LLC cells).

The composition comprising the blood semifluid and the active ingredient of the invention is prepared by the preparation method disclosed by the invention by adding the active ingredient before, during and after the blood semifluid or/and severe injury treatment and mixing the active ingredient appropriately. The following table lists a portion of the composition semifluid (preparation number) prepared in this example, the preferred blood and preferred active ingredients from which it was prepared, the major preparation steps, and the effects achieved by the preparation and its nodular nature.

TABLE 4

Irreversible deformation occurs. The subcutaneous half-disappearance period of the above semifluid nodules is 1-30 days.

Mice: the mice in the table are BALB/c mice.

Mixing and stirring: in the present example, the mixing is mechanical unless otherwise stated (rotational speed 10-1000 rpm ^ er)

Minute and total time is 1-3 minutes).

Several examples of the preparation test of the semi-fluid containing blood and active ingredient of the present invention are listed below.

Example 1 i: separately, blood (with or without anticoagulant) was drawn from each of the orbit of a plurality of mice and mixed, 4.9g of the blood was mixed with 0.1g of 5-Fu in a cup, and then a gas-permeable lid was attached to the cup and steamed in steam (about 100 ℃) for 5-50 minutes to obtain preparation Y1 in the above table.

The preparations of preparations Y2, Y3 and Y4 in the above table can be carried out, respectively, using the same method as the preparation of Y1.

Example 1 j: separately, blood (with or without anticoagulant) was drawn from each of the orbit of a plurality of mice and mixed, 5g of the blood was mixed with 500 ten thousand units of interferon alpha in a cup, and then a gas-permeable lid was attached to the cup and the cup was steamed in steam (about 100 ℃) for 20 to 15 minutes to obtain 500 ten thousand units of interferon alpha/5 g of a mouse blood semifluid composition (preparation Y5).

Using the same procedure, a cytokine/blood semi-fluid composition (e.g., Y6) can be prepared separately when blood from other animals is used, or when other cytokines are used.

Example 1 k: blood (with or without anticoagulant) was collected from each of the orbit of a plurality of mice and mixed, 4.95g of the blood was mixed with 0.05g of anti-PD-1 antibody in a cup, and then a gas-permeable lid was attached to the cup and steamed in steam (about 100 ℃) for 20-15 minutes to obtain a 1% anti-PD-1 antibody/99% mouse blood semifluid composition (preparation Y7).

The same procedure is used when using blood from other animals, or when using other immunomodulatory antibodies, to prepare immunomodulatory antibody/blood semi-fluid compositions, respectively.

Example 1 l: 0.6g of mouse lymphocyte precipitate and 0.4ml of mouse blood were added to the cup and gently mixed, and the cup was covered with a gas-permeable cap and steamed in steam (about 100 ℃ C.) for 20 minutes to obtain preparation Y8 in the above table.

Example 1 m: preparation Y9 in the above table was obtained by mixing 6g of human blood with 4g of human leukocyte pellet taken from a leukocyte-removing filter, and steaming the cup with a gas-permeable lid in steam (about 100 ℃ C.) for 10 minutes.

Example 1 n: respectively collecting blood (with or without anticoagulant) from orbit of multiple mice, mixing, and mixing 1.5ml of the blood with 1ml of liver cancer cell freeze-thawing inactivation solution (10)⁹Individual cells/ml) was stirred well in the cup, and then a gas-permeable lid was added to the cup, which was put into steam (about 100 ℃) to steam for 5-50 minutes, to obtain preparation Y10 in the above table.

Example 1 o: separately, blood (with or without anticoagulant) was collected from each orbit of a plurality of mice and mixed, and 1.5ml of the blood was mixed with 1ml of lung cancer cell fluid (10)⁹Individual cells/ml) in a cup, and then placed in a plastic bagSealed, frozen in liquid nitrogen (below-80 ℃) for 30 minutes and thawed at 37 ℃, and this freeze-thaw may be performed one or more times to obtain preparation Y11 in the above table.

Example 2: condition study of the use of blood-containing semifluids as antigens

The immune system is a multi-level network system. The interaction and the relation of an intracellular biochemical network, an intercellular transmission network and an organ intracellular transmission network are complex. Under what conditions the exogenous material is sufficient to activate at least one level and induce a response in the whole immune system, such that the tumor-specific immunologically active component proliferates and migrates through systemic circulation to the tumor site in sufficient quantity to exert its effector function, which may be a key issue for its use as a vaccine antigen. For this reason, the following experiments investigated the requirements in the technical solution of the present invention.

In one experiment, the experimental animals were BALB/c mice, the modeled cells were sarcoma S180 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 342 mm)³) The test groups were randomly divided into 12 test groups (as shown in the table below). The test groups were 2 series, the tail vein injection series had 1 negative control group (group 01) and 2 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 9 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the C mice were C57BL/6 mice, and the B mice were BALB/C mice. Blood from 4 mice was prepared as follows: the natural blood (hematocrit 41%) was fresh blood taken from mice with an anticoagulant (sodium citrate). 6ml of the natural blood is separated out and centrifuged, 3ml of serum is taken out, and the rest part in the centrifuge tube is mixed uniformly to obtain the concentrated blood of the mouse (the hematocrit is 68%). 2ml of serum was mixed with 2ml of natural blood to give diluted blood (1) for mice (hematocrit 22%). Mixing the remaining 1ml of serum with 2ml of natural mouse blood to obtain diluted mouse blood (2)) (hematocrit 33%). The hematocrit of each blood was measured according to a conventional method. Each of the mouse blood semifluids was a semifluid obtained by heat coagulation of the corresponding mouse blood (prepared by the methods of preparation X4 and X7 in example 1, respectively). Semi-solid of mouse blood is prepared by adding porcine thrombin (final concentration of 100U/1ml) and calcium chloride (final concentration of 20mmol/L) to mouse blood to form gel (semi-solid), and cutting into volume of about 400mm³The small blocks are used.

Each experimental group was administered 1 time in the following administration mode (subcutaneous injection is administered in the left axilla) and each time 400. mu.l/patient was administered. Study group 10 mice were subcutaneously implanted with semisolid by surgery. The drugs of the other experimental groups were injected by syringe. In this experiment, administration to study groups 1, 3, 5 and 7-10 can be considered an allogeneic administration model (which may represent more than 99% of allogeneic administrations), and administration to other study groups can be considered a fully compatible administration model and an autologous administration model. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 5.

TABLE 5

Note: + is the formation of nodules, -is the absence of nodules; x is the tumor weight average (g, the same table below).

In the above table, the inhibition rates of study groups 1, 2, 3 and 4 were low, and the difference in tumor weights between them and the respective negative control groups was not statistically significant (P: 0.9332>0.05, P: 0.5340>0.05, P: 0.3788>0.05 and P: 0.5458> 0.05). This result indicates that liquid blood does not show a significant tumor burden reduction effect, whether it is syngeneic or allogeneic, intravenous or subcutaneous.

In fact, it is generally believed that allogeneic blood transfusion to a patient with a tumor will result in increased secretion of interleukin-2, interleukin-10 and interleukin-24, inhibition of interleukin-2 secretion by helper T lymphocytes, a reduction in the B lymphocyte stimulatory response and antibody production, and a negative modulation of cellular immunity. Allogeneic blood is thus considered as a potential inhibitor of immune effector cells and also as a stimulator of immunosuppressive cells, which down-regulates the beneficial tumor-suppressing immune function. However, in the above table, study groups 5 and 6 both showed higher tumor inhibition rates. The difference in tumor weights between each of them and the negative control group was statistically significant (P0.0004 <0.05, P0.0002 < 0.05). Furthermore, tumor weights between study groups 5 and 3 (P ═ 0.0005), 5 and 1 (P ═ 0.0004), 6 and 4 (P ═ 0.0001), 6 and 2 (P ═ 0.0002) were statistically significant (P < 0.05). While the tumor weight difference between study groups 5 and 6 was not statistically significant (P ═ 0.1375> 0.05). The above results indicate that the correlation between the ability of blood as a solid tumor vaccine antigen to significantly reduce the tumor mass and its status (whether liquid or semi-fluid) is significantly higher than its genetic identity.

In the C mouse blood semifluid study group, the order of the tumor inhibition rates from large to small is: study group 5, study group 9, study group 8, and study group 7. Among them, the difference in tumor weight between study groups 7 and 3 (P ═ 0.2139) was statistically insignificant (P >0.05), while the differences in tumor weight between study groups 8, 9, 5 and 7, respectively, were statistically significant (P ═ 0.0107<0.05, P ═ 0.0227<0.05, P ═ 0.0008<0.05, respectively). This result indicates whether the blood semifluid can be correlated with the specific volume (> 22%, preferably ≧ 33%) of the cells it contains as a solid tumor vaccine antigen that significantly reduces the tumor volume.

In the above table, study group 10 had a lower tumor inhibition rate than study group 9, while the difference in tumor weight between study groups 10 and 9 was statistically significant (P ═ 0.0147< 0.05). This result demonstrates that the preferred state of blood as a solid tumor vaccine antigen for significant reduction of tumor mass is semi-fluid, not semi-solid. In addition, semi-fluid phases are more semi-solid with greater ease of handling and patient compliance for clinical use.

In addition to the natural blood, natural blood components studied in the above experiments, the following experiments investigated the requirements of engineered blood containing blood cells and plasma as solid tumor antigens.

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 206 mm)³) The test groups were randomly divided into 9 test groups (as shown in the table below). The test groups were divided into 1 negative control group (group 0) and 8 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the human leukocyte precipitate and human plasma are obtained by extracting and obtaining normal blood according to the prior art and then centrifuging. The human leukocyte solution is diluted solution with hematocrit of 35% obtained by adding human leukocyte precipitate (hematocrit of 70.5%) into physiological saline of the same volume. 50% human plasma is a mixture of human plasma and an equal volume of physiological saline. Human leukocyte/plasma human leukocyte pellets were added to a mixture of equal volumes of plasma (hematocrit 35%). The hematocrit is determined by conventional methods. The human leukocyte/human plasma semifluid was a semifluid obtained by heat-setting human leukocyte/human plasma (prepared by the method of preparation X11 in example 1). The human leukocyte/human plasma semisolid is a gel (semisolid) prepared by adding porcine thrombin (final concentration of 100U/1ml) and calcium chloride (final concentration of 20mmol/L) into human leukocyte/human plasma, and cutting into volume of about 400mm³The small blocks are used.

Each experimental group was administered subcutaneously to the left axilla once at 200. mu.l/patient. Study group 5 semisolid was surgically implanted subcutaneously into the left flank of mice. The drugs of the other experimental groups were injected by syringe. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 6.

TABLE 6

Group number	Research medicine	Form of the composition	Node (B)	Tumor weight (x + -s)	Tumor inhibition rate
						0	Physiological saline	Liquid, method for producing the same and use thereof	-	0.92±0.18g	-
1	50% human plasma	Liquid, method for producing the same and use thereof	-	0.89±0.11g	3％
						2	Human leukocyte blood/human plasma	Liquid, method for producing the same and use thereof	-	0.84±0.19g	8％
3	Human leukocyte liquid	Liquid, method for producing the same and use thereof	-	0.87±0.10g	5％
						4	Human leukocyte/plasma semifluid	Semifluid	+	0.61±0.10g	43％
5	Human leukocyte/plasma semisolid	Semi-solid	+	0.75±0.26g	19％

*: + is for the formation of nodules, -is for the absence of nodules

In the above table, the tumor inhibition rates of the study groups 1, 2, and 3 were all low. While the q-score (q ═ 1.13) for study group 2 showed additive effects between study groups 1, 2, and 3, the tumor weights between study groups 2 and 1 and 2 and 3 had no statistical significance (P ═ 0.0931>0.05 and P ═ 0.3762>0.05, respectively), and the additive effects shown for group 2 were not significant. The results indicate that neither leukocyte (even very high concentrations of xenogenous leukocytes) fluids, human plasma fluids, nor leukocyte/human plasma composition fluids show a significant tumor burden reduction effect.

However, the tumor inhibition rates of study groups 2, 4, and 5 were very different. The drug effect of study group 4 not only exceeded that of study group 2 and study group 5, but even showed drug effects exceeding the additive effect of the two groups (q ═ 1.69>1.15), and the tumor weight differences between study groups 4 and 2, and 4 and 5 were statistically significant (P ═ 0.0271<0.05, P ═ 0.0472<0.05, respectively). This result demonstrates that even with the same composition, concentration and administration, the blood may exhibit significantly different tumor burden reduction effects if it is morphologically different (liquid, semi-fluid, semi-solid). Semi-fluids exhibit a theoretical expected efficacy that significantly exceeds the sum of the liquid, semi-solid, and even liquid and semi-solid effects of the same composition. This shows that semifluids may have a completely different mechanism of immune action than liquids and semisolids.

According to the above studies and more similar studies, the essential requirements of the pathogen disease vaccine antigens of the present invention are: the composition and morphology of the blood-containing semifluid is such that it forms a semifluid nodule at the site of administration that activates an effective immune response. This requirement appears to be the local formation in vivo of a specific immune response that is physiologically similar in composition and morphology to the pathogen invading tissue (diseased tissue), yet is effectively recognized by the body and elicits against it and against pathogens like immunogens. Thus, the basic technical scheme of the composition of the invention is as follows:

the blood in the semifluid is preferably in a state that is readily recognized by the immune system as severely damaging tissue, apart from its natural state. Furthermore, the blood contained in the semifluid according to the invention is preferably blood comprising severely damaged blood cells (e.g. blood cells which have lost proliferative activity). Severely damaged cells can often expose cellular contents (e.g., cellular contents released by cell membrane disruption);

the above-mentioned blood-containing semifluid is preferably one or more selected from the group consisting of: a semi-fluid dope comprising blood, a semi-fluid coagulum comprising blood, a morselized coagulum comprising blood, more preferably one or more selected from the group consisting of: a semi-fluid coagulation comprising blood, a disrupted coagulation comprising blood coagulation. These blood-containing semifluids exhibit a state of high deviation from the natural state or high damage, as compared with natural blood;

the semifluid is contained in the lesion application and/or the topical application outside the lesion of the vaccine;

the semi-fluid is preferably one or more selected from the group consisting of: a semi-fluid dope comprising blood, a semi-fluid coagulum comprising blood, a morselized coagulum comprising blood, more preferably one or more selected from the group consisting of: a semi-fluid coagulation comprising blood, a disrupted coagulation comprising blood coagulation.

The semifluid comprises blood having a hematocrit of>22% (or cell concentration of>5.6×10⁹Individual cells/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml), 45% -86% (or cell concentration 11.5X 10)⁹-22×10⁹Individual cells/ml), or 55% -86% (or cell concentration 14.0X 10)⁹-22×10⁹Individual cells/ml);

under these conditions, the following examples further optimize the technical solutions.

Example 3: specific immunogen study and tissue optimization of the present semifluid antigens

In one experiment, the experimental animals were CB6F1 mice, and the modeled cells were hepatoma H22 cells at 2X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Of the successfully modeled test animals, one part was used to prepare the tumor particle semifluid, and the other part (tumor mean volume 213 mm)³) The groups were randomized into 6 test groups (as shown in the table below, 1 negative control group (group 0) and 5 study groups were included). The negative control was physiological saline and study drugs are shown in the table below. The study drug was prepared as follows:

the preparation of CB6F1 mouse concentrated blood and CB6F1 mouse concentrated blood semifluid was the same as in example 2. CB6F1 mouse muscle mass is about 400mm of a volume³The block-shaped mouse meat. The preparation of a semi-fluid of tumor tissue particles of CB6F1 mice was as follows: stripping tumor bodies of the liver cancer-bearing CB6F1 mice, and then placing the tumor bodies in a stirrer for crushing (the rotating speed is 1000-10000 r/min, the total time is 1-3 minutes) to obtain macroscopic tissue particles with the average cross section size of less than 1mm multiplied by 1 mm. Horse blood semifluid was preparation X9 in example 1.

Each experimental group was administered subcutaneously 1 time to the left axilla, 400. mu.l/mouse. Study group 3 mice muscle mass was surgically implanted subcutaneously in the left rib of mice. The drugs of the other experimental groups were injected subcutaneously by syringe into the left flank of CB6F1 mice. In this experiment, administration to study groups 1-4 and 8 can be considered a full-phase transplantation model or an autologous administration model, administration to study group 5 can be considered an allogeneic semi-phase administration model, administration to study group 6 can be considered an allogeneic administration model, and administration to study group 7 can be considered a xenogeneic administration model. Animals were measured for graft versus host disease score on day 14 post-dose, then euthanized, tumor weight determined after dissection, and tumor inhibition rate calculated from the negative control group, with results shown in table 7.

TABLE 7

It is generally believed that antigens in molecular form (e.g., whole tumor cell antigens, heterologous glycoprotein antigens, allogeneic cell antigens, subunit antigens) have distinct structural components (e.g., Pathogen-associated molecular patterns, PAMPs) that are distinct from those of normal organisms, whereas Pattern-recognition receptors (PRRs) that recognize these structural components (e.g., PAMPs) are present on the cells of the innate immune system in the body, which PRRs are stimulated and initiate an adaptive immune response to attack the same or similar structural components (e.g., cross-antigen components in tumor cells). Thus, a higher graft-versus-host response mediated by a heterologous or allogeneic graft through its antigenic molecules (e.g., heterologous glycoproteins, allogeneic cells, etc.) can result in higher graft-versus-tumor reactivity.

In the above table, the comparison between study groups 2 and 1 shows that the score for the resistance to host disease is not statistically significant (P ═ 0.3016>0.05), indicating that the immunogenicity of transplant rejection (against normal host cells and tumor cells) is comparable, whereas the difference in tumor weight (against tumor bodies in the host) is statistically significant (P ═ 0.0023< 0.05). Comparison between study groups 2 and 3 showed that the anti-host disease score was statistically significant (P0.0002 <0.05), indicating that the immunogenicity of graft rejection (against host cells) was significantly different, whereas the tumor weight difference (against host tumor bodies) was not statistically significant (P0.5417 > 0.05). Furthermore, although the difference in graft versus host disease scores between study groups 5 (including horse blood) and 2 (including mouse blood) was indeed statistically significant (P0.0005 <0.05), the difference in tumor weight between them was not statistically significant (P0.7295 > 0.05). According to these results, the expression of blood-containing semifluids as solid tumor vaccine antigens was not clearly directly correlated with their anti-host response. Thus, the antigenicity of the blood-containing semifluid of the present invention, where necessary as disclosed herein, appears to be significantly different from that of the grafts of the prior art (usually semi-solid or liquid), the latter often having an anti-tumor immunogenicity that is positively correlated with its graft rejection immunogenicity.

Furthermore, the difference in tumor weights between study 4 (containing tumor cells) and study 2 (containing blood) was not statistically significant (P ═ 0.7295> 0.05).

Similar results were obtained with the other preparations of example 1.

According to the above and further similar studies, the technical solution of the semi-fluid comprising blood according to the present invention is further preferred as follows: the semifluid of the present invention is preferably a semifluid having dominant antigenicity against pathogen-invading tissue (diseased tissue, e.g., tumor tissue) rather than against host antigenicity (e.g., having a tumor suppression rate of. gtoreq. against host rate, preferably. gtoreq.150% against host rate). Thus:

the essential composition of the semifluid of the invention does not comprise blood with a strong graft-versus-host response (e.g. xenogeneic blood), preferably selected from blood with a weak graft-versus-host response, such as one or more of the following groups: allogeneic blood, allogeneic blood and autologous blood with same ABO blood type or similar HLA.

Under the above preferred conditions, the blood contained in the semifluid of the present invention is preferably selected from cell-enriched components of natural blood (e.g., blood cell-enriched blood having a plasma content of 30 to 80% of the plasma content of natural blood, and blood cell-enriched blood having a blood cell content of 130% or more of natural blood), and/or engineered blood (e.g., leukocyte and/or a mixture of lymphocyte and plasma) containing a portion of the blood cell preparation and plasma. Wherein the blood cells are selected from one or more of: red blood cells, white blood cells, platelets, wherein the white blood cells are selected from one or more of the following: granulocytes, monocytes, lymphocytes, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells.

Example 4: targeted studies and indications for the semifluid antigens of the invention

The results of the above examples demonstrate that the semifluid antigens of the invention target the tumor body of tumor-bearing mice. The following experiments were conducted on tumor-bearing rabbits.

In one experiment, the test animal is a New Zealand white rabbit (with the weight of 2.0-2.5 kg and unlimited male and female), and the modeling tissue is rabbit VX 2 tumor body tissue inoculated by 2 passages. The tumor body tissue fine block (about 1 mm) is implanted into the right lung of the animal by operation³Block, total amount about 500mm³Only). Successfully modeled lung cancer-bearing rabbits (subjected to CT examination 14 days after inoculation and with tumor diameter of 6-13 mm) were randomly divided into 2 test groups (1 negative control group and 1 study group), and each group had 6 rabbits. The negative controls were all saline and the study drug was an allogeneic concentrated blood semifluid prepared from blood taken from rabbits in california by the method of preparation X7 in example 1. Each experimental group was administered subcutaneously 3 times to the left axilla, 1 time every 7 days, 2 ml/dose. Animals were euthanized on day 7 after dosing, tumor weights were determined after dissection, and tumor inhibition rates of the study groups were calculated from the negative control group. .

In the literature, almost all solid tumor vaccine studies are performed in the mean tumor volume<100mm³The animal model of (1) was studied. In fact, tumor vaccine antigens in the prior art almost target tumor cells and structural components therein (e.g., cross-antigens of grafts), rather than tumor bodies, especially larger tumor bodies. Taking an allogeneic hematopoietic stem cell graft (allo-HSCT, usually liquid) as an example, it targets antigen-mediated immune responses unspecifically against both host healthy cells (graft versus host disease (GVHD)) and also against tumor cells (e.g., anti-leukemia response (CVL)). Thus, they may show the best therapeutic effect on non-solid tumors (e.g., leukemia), as well as on smaller solid tumorsHas better curative effect and is often ineffective for solid tumors with larger tumor bodies.

In the above experiment, the tumor inhibition rate was 27%. In addition, 5 rabbits of the study group developed subcutaneous palpable hard nodules after treatment. It is well known that such nodules are often a complication of local acute inflammation in the body. The results further show that the semifluid antigens of the invention appear to target the tumor mass as a semifluid nodule antigen by inducing acute inflammation of the tumor mass, and have an average volume much greater than 100mm³The tumor body of (a) also shows a drug effect of reducing tumor body load.

According to the results of the above studies and more similar studies, the semifluid antigens of the invention differ from other antigens of the prior art (in particular antigens in molecular form that are freely distributed in the blood) and in their targeting and thus their indications. Tumor antigens target tumor cells, and thus their indications of application are associated with specific tumor species. Conventional transplants (usually semi-solid) or allo-HSCT (usually liquid) target cells (normal and tumor cells), and thus their indications for use are related to the ease of exposure of tumor cells (liquid cancer is easy, solid tumors are difficult, the larger the tumor volume is, the more difficult).

These results further enhance the analysis of specific immunogens described above with respect to the semifluids of the present invention. One possible explanation is: the immunization strategy of the invention, the necessary conditions of which simulate the three-dimensional characteristics of the components, shape, charge, size and the like of the pathogen invading tissue (tumor body) but not the pathogen (tumor cells), and the four-dimensional dynamic reconstruction phenomenon (viscoelastic soft matter). Thus, the semifluid antigen of the invention is essentially a pathogen-like invading tissue (tumor) invading tissue, but is more readily recognized by the immune system than diseased tissue (tumor) to elicit a novel antigen that targets an effective immune response to the diseased tissue (tumor) it also mimics. Of course, effective destruction of diseased tissue (tumor mass) may in turn secondarily release the in situ antigen and result in an autologous vaccine. Thus, the indications for the semifluid antigens of the invention are related to the invasion of the pathogen into the tissue (tumor volume), in particular preferably larger rather than smaller volumes, and are not strongly dependent on what pathogen (tumor cell) is in particular inside the diseased tissue (tumor volume), whether it is in molecular form or not, and the action of the immune system (exposition).

According to these results, the semifluid of the invention is useful as a vaccine antigen for the treatment of pathogenic diseases, preferably for the total volume of the pathogen-invading tissue (e.g., tumor mass)>85mm³Preferably ≥ 200mm³More preferably ≥ 400mm³A pathogenic disease (e.g., a solid tumor). Depending on the indication, the semifluid antigen of the invention is administered in a single animal in an amount of 0.2ml or more, preferably 0.2-25ml or 0.4-30 ml.

The semifluid antigens of the invention are thus of broad application range, for example, with a large spectrum of neoplastic cell types. This was confirmed in the following experiments, in addition to the liver cancer, breast cancer, and sarcoma in the above experiments.

In the following experimental series, which included 3 experiments, the experimental animals and the modeled cells were each as follows. Modeling cells conventional transplantation tumor modeling was performed subcutaneously in the right axilla of animals, respectively. The successfully modeled test animals in each series of experiments were randomly divided into 4 experimental groups, 1 negative control group and 3 study groups (A, B, C groups). The negative control was physiological saline and the A, B, C group study drugs were preparations X7, X8, and X10 in example 1, respectively. Each experimental group was administered to the left axilla by subcutaneous injection 1 time, 250. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from each series of negative control groups, and the experimental results are shown below.

1) Application in treatment of colon cancer

In one experiment, the experimental animals were BALB/c mice and the modeled cells were colon cancer CT26 cells (1X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 203 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 36%, 31%, and 29%, respectively.

2) Application of the compound in treating melanoma

At one isIn the experiment, the test animal is a C57BL/6 mouse, and the modeled cell is melanoma B16 cell (1 × 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 403 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The average tumor inhibition rates of the B, C groups were 41%, 35% and 33%, respectively.

3) Application of the same in lung cancer treatment

In one experiment, the test animals were C57BL/6 mice, and the modeled cells were Lewis Lung Carcinoma (LLC) (1X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 532 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 32%, 31%, and 28%, respectively.

Similar results as above were obtained using the other preparations of example 1.

Based on the above results and preliminary analysis of the immunization strategies leading to these results, under the basic technical solution (requirements) disclosed in the present invention, the semifluid (morphological) antigens in the preferred technical solution of the present invention can be distinguished from the molecular morphological antigens in the prior art (such as pathogen antigens, conventional graft antigens, and cellular antigens in non-semifluid), showing specific immunogens against diseased tissues similar to those of highly damaged pathogen invading tissues (diseased tissues such as tumor tissues). It is well known that the main feature of chemotherapeutic drugs is their composition, whereas the main feature of vaccine antigens is their specific immunogen. The immunogen is likely because it forms a pathogen-like invasive tissue nodule, but is semi-fluid and highly invasive (blood in a semi-fluid is largely different from liquid) but is more readily recognized by the immune system than the pathogen invasive tissue, thereby eliciting an effective immune response against the pathogen invasive tissue it mimics. The following experiments will further investigate this.

According to the above studies and more similar studies, the composition of the present invention can be applied to a wide spectrum of pathogen diseases, such as tumors, microbial infections. Wherein the tumor is preferably a solid tumor, wherein the solid tumor is one or more selected from the group consisting of: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma. Wherein the microbial infection is selected from one or more of the following groups: HIV, hepatitis B, hepatitis C, leprosy, chronic mucocutaneous candidiasis.

Example 5: synergistic study of semi-fluid compositions comprising blood

The semifluid according to the invention may also be a semifluid composition comprising blood and an active ingredient against pathogenic diseases. The blood-containing semifluid of the present invention further comprises, as an antigen, an immunopotentiating antigen which is an active ingredient against pathogenic diseases, wherein the immunopotentiation means that the combination of the semifluid and the active ingredient produces an immunopotentiating effect higher than that of either one of the compositions taken alone. The effectiveness of the immune response can be observed by a single immune response or by a combined immune response of immuno-chemotherapy (especially considering in situ antigen release). The immune synergistic effect is researched by taking a tumor-bearing mouse as a test animal model, and the chemotherapy synergistic effect is researched by taking a tumor-bearing nude mouse as a test animal model.

1. Synergistic regimen for semifluid compositions comprising blood and biological products

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 236 mm)³) The test groups were randomly divided into 16 test groups (as shown in the table below). The test groups were 2 series, the tail vein injection series had 1 negative control group (group 01) and 3 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 11 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the breast cancer antigen is a freeze-thaw inactivation solution (10) of breast cancer cells prepared by the method of example 1 from the tumor body of a breast cancer-bearing mouse obtained by the same modeling method⁹Individual cells/ml). Blood from 4 mice was prepared as follows: the natural blood (hematocrit 41%) was fresh blood taken from mice with an anticoagulant (sodium citrate). 6ml of the natural blood is separated out and centrifuged, 3ml of serum is taken out, and the rest part in the centrifuge tube is mixed uniformly to obtain the concentrated blood of the mouse (the hematocrit is 68%). 2ml of serum was mixed with 2ml of natural blood to give diluted blood (1) for mice (hematocrit 22%). The remaining 1ml of serum was then mixed with 2ml of native mouse blood to give diluted mouse blood (2) (hematocrit 33%). The hematocrit of each blood was measured according to a conventional method. Each breast cancer antigen/mouse blood is 40% breast cancer cell freeze-thaw inactivation solution (10)⁹Individual cells/ml) and 60% of each mouse blood. Each breast cancer antigen/mouse plasma semifluid was a semifluid formed by heat coagulation of each breast cancer antigen/mouse blood (prepared according to the preparation method of preparation Y10 in example 1). The mouse native blood semifluid is a semifluid formed by heating and coagulating mouse blood (prepared by the method of preparation X4 in example 1).

In this experiment, the administration to the study group can be considered an allogeneic administration model (which may represent more than 99% of allogeneic administrations). Each experimental group was administered once in the administration mode shown in the following table (subcutaneous injections were all administered subcutaneously in the left axilla), and each administration was 400. mu.l/tube. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 8.

TABLE 8

And (3) nodule: + is for the formation of nodules, -is for the absence of nodules

*: mouse blood semifluid tumor inhibition rate: according to the related experiments, the tumor inhibition effect of the semifluid of diluted mouse blood is less than that of the semifluid of 100 percent of mouse blood

In the above table, the q of the composition group (q 0.83<0.85) showed antagonism among the study groups 3, 2 and 1 subjected to intravenous injection, whereas the tumor weights of the study groups 3 and 1 and 3 and 2 had no statistical significance (P0.7662 >0.05 and P0.9317 >0.05, respectively), and thus the composition group showed no significant antagonism. The q of the composition group (q 0.79<0.85) showed antagonism among study groups 6, 5 and 4 injected subcutaneously, whereas the tumor weights of study groups 6 and 4 and 6 and 5 had no statistical significance (P0.3274 >0.05 and P0.7771 >0.05, respectively), and thus the composition group showed no significant antagonism. The results demonstrate that the liquid compositions show no synergistic or even no significant additive effect, whether injected intravenously or subcutaneously.

However, between study groups 8, 7, 5 injected subcutaneously, the q-judged (q >1.77>1.15) of the composition group showed synergy, and the tumor weight differences between study groups 8 and 5, and 8 and 7 were all statistically significant (P ═ 0.0006<0.05, P ═ 0.0327<0.05, respectively), so the composition group showed significant synergy. This result demonstrates that there are completely different interactions between blood-containing liquids and semi-fluids and active components.

In the above table, the tumor inhibition rates in the study group of compositions injected subcutaneously are in the order of magnitude: study group 14, study group 8, study group 12, study group 10. Between study groups 10, 9, 5, the q-judged (q ═ 0.95) of the composition group showed additive effects, but the difference in tumor weight between study groups 10 and 5 had statistical significance (P ═ 0.0288<0.05), and the difference in tumor weight between 10 and 9 had no statistical significance (P ═ 0.2850>0.05), so the composition group showed no significant additive effects, more no synergistic effects. As the hematocrit increased, the q-judged (q ═ 1.29>1.15) of the composition group showed synergy among the study groups 12, 11, 5, and the tumor weight differences between the study groups 12 and 5, 12 and 11 were all statistically significant (P ═ 0.0012<0.05, P ═ 0.0274<0.05, respectively), so the composition group showed significant synergy. Further, between study groups 14, 13, and 5, the q of the composition group (q ═ 1.25>1.15) showed synergy, and the tumor weights of study groups 14 and 5 and 14 and 13 were statistically significant (P ═ 0.0001<0.05 and P ═ 0.0283<0.05, respectively), so that the composition group showed significant synergy. The results demonstrate that the synergistic effect of the blood-containing semifluid on the biological product is dependent on the hematocrit. The hematocrit may be involved in determining many properties (e.g., softness) of the nodules formed in vivo by the semifluid, and thus in determining its synergistic effects.

Similar results were obtained using other preparations from example 1 (e.g., Y8, Y9, etc.).

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 215 mm)³) The groups were randomized into 8 experimental groups, including 1 negative control group and 7 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the PD- (L)1 antibody used is commercially available. The PD- (L)1 antibody drugs were all aqueous solutions of indicated concentrations, the mouse blood was taken from fresh blood of BALB/c mice to which an anticoagulant (sodium citrate) was added, the mouse blood semifluid was preparation X4 in example 1, and PD- (L)1 antibody/mouse blood semifluid at different weight ratios were prepared according to the preparation method of preparation Y7 in example 1.

In this experiment, the administration to the study group can be considered as an isogenic administration model (which can represent allogenic and autologous administration). The left axilla of each experimental group was administered by subcutaneous injection once in the dosage form shown in the table (subcutaneous injection means subcutaneous injection in the left axilla) and 250. mu.l/patient was administered each time. Animals were euthanized at 14 days post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 9.

TABLE 9

In the above table, in the PD- (L)1 antibody/blood semifluid composition study group, the tumor inhibition rates are ranked from large to small: study group 7, study group 6, study group 5. Between study groups 5, 2, 1, the q-judged (q ═ 1.11) of the composition group showed additive effects, but the difference in tumor weight between study groups 5 and 2 had statistical significance (P ═ 0.0019<0.05) while the difference in tumor weight between study groups 5 and 1 had no statistical significance (P ═ 0.2980>0.05), so the composition group showed no significant additive effects. However, between study groups 6, 3, 1, the q of the composition group was judged (q ═ 1.30>1.15) to show synergy, and the tumor weight differences between study groups 6 and 3, and between 6 and 1 were all statistically significant (P ═ 0.0003<0.05, P ═ 0.0060<0.05, respectively), so the composition group showed significant synergy. Further, between study groups 7, 4, and 1, the q of the composition group was judged (q ═ 1.54>1.15) to show synergy, and the tumor weight differences between study groups 7 and 4, and 7 and 1 were statistically significant (P ═ 0.0003<0.05, and P ═ 0.0038<0.05, respectively), so the composition group showed significant synergy. According to this and further results, the synergistic quantitative ratio (w: w) of PD- (L)1 antibodies to the semi-fluid composition (more generally, to their cognate immunomodulatory antibodies to the semi-fluid composition) is >0.05/100, preferably ≧ 0.1/100.

The following further experiments confirm the above ranges of the synergistic amount ratio.

In one experiment, the experimental animals were BALB/c mice, the modeled cells were sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animal (Holland sarcoma mouse, average tumor volume 251mm³) The animals were randomized into 8 experimental groups (1 negative control group and 7 study groups) and intratumorally administered to the animals. The negative control was physiological saline and study drugs are shown in the table below. The study drug was prepared as follows:

0.1 percent of BCG vaccine and 50 ten thousand IU/ml of human recombinant interferon are respectively liquid prepared by water for injection. The mouse blood semifluid was X4 of example 1, 0.1% BCG/99.9 mouse blood semifluid, and the human recombinant interferon/mouse blood (50 ten thousand IU/ml) semifluid were thermal coagulates of the indicated amounts of the mixture of the biological product and the mouse blood, respectively (prepared by the method of Y5 of example 1).

Each experimental group was administered intratumorally once, 250. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 10.

Watch 10

Group number	Research medicine	Ratio of measurement	Tumor weight (x + -s)	Tumor inhibition rate
					0	Physiological saline	-	1.73±0.26g	-
1	Mouse blood semifluid	-	1.28±0.33g	26％
					2	0.1% BCG vaccine	-	1.45±0.31g	16％
3	0.1% BCG/99.9% mouse blood semifluid	0.1/100	0.81±0.22g	53％
					4	Human recombinant interferon 50 ten thousand IU/ml	-	1.51±0.35g	13％
5	Human recombinant interferon/mouse blood (50 ten thousand IU/ml) semifluid	1/100	0.90±0.20g	48％

In the above table, the q-judged (q ═ 1.20>1.15) of the composition group showed a synergistic effect among the study groups 3, 2, and 1, and the tumor weight differences among the study groups 3 and 2 and 3 and 1 were all statistically significant (P ═ 0.0022<0.05, and P ═ 0.0166<0.05, respectively), so that the composition group showed a significant synergistic effect. Between study groups 5, 4 and 1, the q-judged (q ═ 1.33>1.15) of the composition group showed synergy, and the tumor weights between study groups 5 and 4 and between 5 and 1 were statistically significant (P ═ 0.004<0.05 and P ═ 0.0379<0.05, respectively), so that the composition group showed significant synergy.

The above results and more similar studies indicate that a semi-fluid composition comprising blood and an anti-pathogenic disease biological product of the invention provides a synergistic disruption of the invasion of tissue by pathogenic variants of the pathogen. This synergy is likely to be due to the blood-containing semifluid of the invention acting synergistically as an immunological component (antigen) or/and as a slow-release carrier. The increased destruction of pathogenic variants of the pathogen into tissues facilitates the production of endogenous vaccines.

2. Study of the synergy regimen of semi-fluid compositions comprising blood and chemotherapeutic drugs

The tissue-destructive or/and sustained-release properties of the blood-containing semifluid are the basis for its use as a tissue-destructive or/and sustained-release synergistic component. This was investigated in the following experiments.

In one experiment, the test animal was a nude mouse, and the modeled cell was sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (Holland sarcoma mice, average tumor volume 218mm³) The test groups were randomly divided into 6 test groups (as shown in the table below). The test groups were 2 series, the intratumoral injection series had 1 negative control group (group 01) and 3 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 1 study group. The negative controls were all saline and the study drugs were as shown in the table below, respectively: BALB/c mouse fresh blood, absolute ethanol (positive control for ablator), mouse blood semifluid (X4 of example 1), each semifluid containing blood cells. Each experimental group was administered once, 200. mu.l/patient, and the administration mode (subcutaneous injection is subcutaneous injection in the left axilla) is shown in the following table.

The nude mouse is a congenital athymic nude mouse, wherein a recessive mutant gene 'nu' positioned on the 11 th chromosome pair is introduced into a BALB/c mouse. The thymus of the nude mouse only has remnant or abnormal epithelium, which can not lead the T cell to be normally differentiated, lacks the auxiliary, inhibiting and killing functions of mature T cell and has low cell immunity. The animals were euthanized on day 7 after drug administration of the sarcoma nude mice, tumor weights were determined after dissection, and the tumor inhibition rates were calculated from the negative control group, and the results are shown in table 11.

TABLE 11

Group number	Research medicine	Mode of administration	Tumor weight (x + -s)	Tumor inhibition rate
					01	Physiological saline	Intratumoral injection	1.91±0.34g	-
1	Mouse blood semifluid	Intratumoral injection	1.30±0.15g	32％
					2	Blood of mouse	Intratumoral injection	1.70±0.22g	11％
3	Anhydrous ethanol	Intratumoral injection	1.13±0.27g	41％
					02	Physiological saline	Subcutaneous injection	1.99±0.21g	-
4	Mouse blood semifluid	Subcutaneous injection	1.87±0.15g	5％

In the above table, the difference in tumor weight between study groups 4 and 02 was not statistically significant (P ═ 0.437> 0.05). This result and more similar studies indicate that the blood-containing semifluid of the composition of the present invention shows immunosuppressive effects mainly as a thymus-dependent antigen in previous subcutaneous injections of tumor-bearing mice.

However, in the above table, the tumor weights were statistically significant between study groups 1 and 2 and between study groups 1 and 01 (P ═ 0.0043<0.05 and P ═ 0.0014<0.05, respectively). While the tumor weight difference between study groups 1 and 3 was not statistically significant (P-0.2096 > 0.05). This result and more similar studies suggest that the semifluid of the present invention can be used in immune synergy as a thymus-dependent antigen, and in chemotherapy synergy as a thymus-independent tissue-disrupting component. It is well known that destruction of tumor body tissue may give secondary release of tumor antigens (in situ) and produce a vaccine effect.

In one experiment, the test animal was a nude mouse, and the modeled cell was sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Test animals successfully modeled (Holland sarcoma mice, mean tumor volume 209mm³) The test groups were randomly divided into 10 test groups (1 negative control group and 9 study groups) and intratumorally administered to animals. The negative control was physiological saline and study drugs are shown in the table below. The study drug was prepared as follows: blood semifluid is blood taken from other nude mice prepared according to the method of preparation X16 in example 1, 0.5% 5-Fu, 0.5% methylene blue, 10% arginine/10% glycine respectively as aqueous solutions of tissue-damaging agents, and other drugs are tissue-damaging agent/autologous blood semifluid compositions prepared according to the methods of preparation Y6, Y8, Y9 in example 1, respectively, from blood taken from other nude mice.

Each experimental group was administered intratumorally once, 200. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 12.

TABLE 12

Group number	Research medicine	Tumor weight (x + -s)	Tumor inhibition rate
				0	Physiological saline	1.87±0.17g	-
1	Blood semifluid of nude mice	1.35±0.21g	28％
				2	0.5％5-Fu	1.27±0.18g	32％
3	0.5% 5-Fu/99.5% nude mouse blood semifluid	0.71±0.25g	62％
				4	1% 5-Fu/99% nude mouse blood semifluid	0.49±0.08g	74％
5	0.5% methylene blue	1.63±0.28g	13％
				6	0.5% methylene blue/99.5% nude mouse blood semifluid	0.92±0.21g	51％
7	1% methylene blue/99% nude mouse blood semifluid	0.65±0.07g	65％
				8	10% arginine/10% glycine	1.40±0.29g	25％
9	10% arginine/10% glycine/80% nude mouse blood semifluid	0.92±0.12g	51％

Mouse blood semifluid tumor inhibition rate: according to the related experiments, the tumor inhibition effect of the semifluid of diluted mouse blood is less than that of the semifluid of 100 percent of mouse blood

In the above table, the tumor inhibition rate of study group 4 was higher than that of study group 3, and that of study group 7 was higher than that of study group 6. Between study groups 3, 2 and 1, the q of the composition group (q ═ 1.22>1.15) showed synergy, and the tumor weight differences between study groups 3 and 2 and 3 and 1 were all statistically significant (P ═ 0.0012<0.05 and P ═ 0.0007<0.05, respectively), so the composition group showed significant synergy. Between study groups 6, 5 and 1, the q-judged (q ═ 1.38>1.15) of the composition group showed a synergistic effect, and the tumor weight differences between study groups 6 and 5 and between 6 and 1 were all statistically significant (P ═ 0.0006<0.05 and P ═ 0.0054<0.05, respectively), so that the composition group showed a significant synergistic effect. Between study groups 9, 8, 1, the q-judged (q >1.17>1.15) of the composition group showed synergy, and the tumor weight differences between study groups 9 and 8, 9 and 1 were all statistically significant (P ═ 0.0041<0.05, P ═ 0.0015<0.05, respectively), so the composition group showed significant synergy.

The above results and more similar studies indicate that a semi-fluid composition comprising blood of the invention and an anti-pathogenic disease chemotherapeutic agent provides a synergistic destructive effect on the invasion of tissues by pathogenic variants of the pathogen. This synergy is likely to be due to the blood-containing semifluid of the invention acting synergistically as an immunological component (antigen), synergistically as a tissue destruction agent, or/and synergistically as a slow release carrier. The increased destruction of pathogenic variants of the pathogen into tissues facilitates the production of endogenous vaccines.

According to the above and more similar studies, the blood-containing semifluid can be used as an antigen to form a composition with other immune drugs (such as immune biological products) against solid tumors to improve immune effect, and can also be used as a tissue destruction component or/and a carrier to form a composition with chemotherapeutic drugs to improve immune-chemotherapeutic composite effect while being used as an antigen.

The requirements of the composition of the invention are: its composition and morphology is such that it elicits a more effective immune response and/or a more effective chemotherapeutic effect than the semifluid antigen alone. Thus, the basic technical scheme of the composition of the invention is as follows:

the use of a semifluid comprising blood as a synergistic component of an antigen and an active ingredient against pathogenic diseases;

the above-mentioned semi-fluid comprising blood and active ingredient is preferably one or more selected from the group consisting of: a semi-fluid dope comprising the active ingredient and blood, a coagulum comprising the active ingredient and blood, a morselized coagulum comprising the active ingredient and blood, more preferably one or more selected from the group consisting of: a coagulum containing the active ingredient and blood, a morselized product of a coagulum containing the active ingredient and blood;

the above-mentioned semifluid comprising blood and active ingredient is contained in the topical administration of said vaccine;

the semi-fluid comprising blood and active ingredient is a semi-fluid implant, preferably a semi-fluid injection. The semi-fluid injection is an injection which can be directly administered by a conventional injection system in a semi-fluid manner, and the semi-solid implant is usually implanted by surgery or forms a semi-solid (e.g. gelated) nodule at the administration site after being administered by a conventional injection system in a fluid (liquid) manner.

Under more preferred conditions, the semi-fluid composition of the invention comprising blood and an anti-solid tumor active ingredient exhibits an immune-synergistic effect, a chemotherapeutic-synergistic effect and/orThe carrier acts synergistically. The synergistic conditions for the semi-fluid composition are: the ratio of the amount of the active ingredient to the amount of the composition (w/w or v/v) is (0.1-30)/100, and the ratio of the amount of the cell to the amount of the composition (v/v) is>22% (or cell concentration of>5.6×10⁹Individual cells/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml) or 45% -86% (or cell concentration 11.5X 10)⁹-22×10⁹Individual cells/ml). In which the semi-fluid dope composition, the ratio of the amount of the cells to the amount of the composition (v/v) is 70% or more (or the cell concentration is 17.9X 10 or more)⁹Individual cell/ml), preferably 70% to 86% (or a cell concentration of 17.9X 10)⁹-22×10⁹Individual cells/ml).

More preferably, the concentration of the active ingredient is greater than or equal to the concentration at which it acts when administered topically alone, wherein:

the concentration of the biological product is greater than or equal to the concentration at which it acts when administered topically alone, for example: the content of the tumor antigen is more than 10⁵Is preferably 10/ml⁵～10⁹Tumor antigens contained in each ml of tumor cells, the concentration of the microbial antigens being>0.1 percent, the content of the immunoregulation antibody medicine is more than or equal to 0.1 percent, preferably 0.25 to 5 percent, and the like; or/and

the concentration of the chemotherapeutic agent is greater than or equal to the concentration at which it acts when administered topically alone, for example: the concentration of the cytotoxic drug is more than 50% of the saturation concentration of the cytotoxic drug, and preferably 50% -500% of the saturation concentration of the cytotoxic drug; the concentration of the conventional ineffective compound is > 0.25%, preferably 0.35-30% (for example, the local administration concentration of the amino acid nutrient is more than 5%, preferably 5-30%, the local administration concentration of the ineffective aromatic compound is more than 0.25%, preferably 0.35-10%, and the local administration concentration of the plant or fungus active ingredient is more than 0.25%, preferably 0.75-15%).

The present disclosure includes the following items:

item 1, use of a semifluid comprising or formed from blood as an antigen for the preparation of a vaccine for the treatment or inhibition of a pathogenic disease.

Item 2, a vaccine for treating or inhibiting a pathogenic disease, comprising as an antigen blood-containing or blood-forming semifluid.

Item 3, a method of treating a disease or inhibiting a pathogenic disease comprising topically administering, preferably by local injection, to a subject individual in need thereof a vaccine comprising as an antigen blood or a semi-fluid formed from blood.

Item 4, the use, vaccine or method according to one of items 1 to 3, wherein the vaccine is in a form suitable for topical administration, and wherein the composition and morphology of the semifluid is such that it forms a semifluid nodule at the site of administration.

Item 5, the use, vaccine or method according to one of items 1 to 4, wherein the blood is one or more selected from the group consisting of: natural blood, blood cell-enriched components of natural blood (e.g., concentrated blood of blood cells having a plasma content of 30-80% of the plasma content of natural blood), and engineered blood comprising a blood cell preparation and plasma (e.g., a mixture of leukocytes or/and lymphocytes and plasma).

Item 6, the use, vaccine or method according to one of items 1 to 5, wherein the blood is distant from its native state, preferably in a state that is easily recognized by the immune system as severely damaged tissue.

Item 7, the use, vaccine or method according to one of items 1 to 6, wherein the semifluid is one or more selected from the group consisting of: a semi-fluid dope comprising said blood, a semi-fluid coagulum comprising said blood, a disruption of a coagulum comprising said blood, preferably one or more selected from the group consisting of: a semi-fluid coagulum containing the blood, a disrupted coagulum containing the blood.

Item 8, the use, vaccine or method according to one of items 1 to 7, wherein the hematocrit of the blood is>22% (or cell concentration of>5.6×10⁹Individual cells/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml) or 45% -86% (or cell)The concentration is 11.5X 10⁹-22×10⁹Individual cells/ml).

Item 9, the use, the vaccine or the method according to one of items 1 to 8, wherein the hematocrit of the blood is not less than 55% (or the cell concentration is not less than 14.0 x10)⁹Individual cells/ml), preferably 55% to 86% (or a cell concentration of 14.0X 10)⁹-22×10⁹Individual cells/ml).

Item 10, the use, vaccine or method according to one of items 5 to 9, wherein the blood cells are selected from one or more of the following: red blood cells, white blood cells, platelets and derivatives thereof, wherein the white blood cells are selected from one or more of the following: granulocytes, monocytes, lymphocytes and derivatives thereof, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells, and derivatives thereof.

Item 11, the use, vaccine or method according to one of items 5 to 10, wherein the blood cells comprise leukocytes, wherein the leukocytes are selected from one or more of: granulocytes, monocytes, lymphocytes, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells.

Item 12, the use, vaccine or method according to one of items 1 to 11, wherein the blood is one or more selected from the group consisting of: allogeneic blood, allogeneic blood and autologous blood with same ABO blood type or similar HLA.

Item 13, the use, vaccine or method according to one of items 1 to 11, wherein the blood comprises autologous blood.

14. The use, vaccine or method according to one of items 1 to 11, wherein the blood comprises autologous blood and allogeneic blood cells.

15. The use, vaccine or method according to one of items 1 to 14, wherein the vaccine further comprises an active ingredient for combating pathogenic disease.

16. The use, vaccine or method according to clause 15, wherein the amount ratio (w/w or v/v) of the blood-containing or blood-forming semifluid in the vaccine is (30-99.9)/100; the amount ratio (w/w or v/v) of the active ingredients in the vaccine is (0.1-85)/100, and wherein the active ingredients are selected from one or more of chemotherapeutic drugs or/and biologicals against the pathogen diseases.

Item 17, the use, vaccine or method according to item 16, wherein the biological product is selected from one or more of the following group: a cell preparation, an immunomodulatory antibody, a cytokine, a pathogen, or a pathogen subunit, and the amount ratio (w/w) of the biological preparation to the semifluid is (0.1-30)/100.

Item 18, the use, vaccine or method according to item 16, wherein the cell preparation is a cell preparation derived from allogeneic and autologous cells.

Item 19, the use, vaccine or method according to item 16, wherein the cell preparation is a preparation of natural or engineered cells selected from the group consisting of one or more of: tissue-rich cells such as muscle cells; immune cells such as peripheral blood mononuclear cells, T cells, B cells, NK cells, lymphocytes; stem cells such as mesenchymal stem cells, hematopoietic stem cells.

Item 20, the use, vaccine or method according to item 16, wherein the immunomodulatory antibody is one or more selected from the group consisting of: antibody blocking agents against inhibitory receptors, such as blocking antibodies against CTLA-4 molecules and PD-1 molecules; antibody blockers against ligands for inhibitory receptors, activating antibodies against immune response cell surface stimulatory molecules, such as anti-OX 40 antibodies, anti-CD 137 antibodies, anti-4-1 BB antibodies; neutralizing antibodies against immunosuppressive molecules in the solid tumor microenvironment, such as anti-TGF-p 1 antibodies.

Item 21, the use, vaccine or method according to item 16, wherein the cytokine is one or more selected from the group consisting of: tumor necrosis factor, interferon, interleukin.

Item 22, the use, vaccine or method according to item 16, wherein the pathogen in the pathogen or subunit of pathogens is selected from one or more of the following group: tumor cells, bacteria, viruses.

Item 23, the use, vaccine or method according to item 15, wherein the chemotherapeutic drug is selected from a cytotoxic drug and/or one or more of a conventional ineffective but topically effective compound and the amount ratio (w/w) of the chemotherapeutic drug to the semi-fluid is (0.1-30)/100.

Item 24, the use, vaccine or method according to item 23, wherein the cytotoxic drug comprises one or more selected from the group consisting of: 5-fluorouracil, gemcitabine, epirubicin, antibiotics against pathogen diseases, teniposide, metal platinum complex, paclitaxel.

Item 25, the use, medicament or method according to item 23, wherein the conventionally ineffective but topically effective compound comprises one or more of the following groups: amino acid nutrient, ineffective aromatic compound, and bioactive component.

Item 26, the use, vaccine or method according to item 25, wherein the conventionally ineffective but topically effective compound comprises one or more selected from the group consisting of: amino acid nutrients such as arginine, lysine, glycine, cysteine, glutamic acid, or salts thereof, or oligopeptides comprising the same, ineffective aromatic compounds such as methylene blue, acetylsalicylic acid, quinine monohydrochloride, quinine dihydrochloride, bioactive ingredients such as algal polysaccharides, medicinal plant polysaccharides, fungal polysaccharides, artemisinin.

Item 27, the use, vaccine or method according to one of items 1 to 26, wherein the pathogenic disease is selected from one or more of the following group: tumors, microbial infections.

Item 28, the use, vaccine or method according to item 27, wherein the tumor is preferably selected from the group consisting of tumor volume>85mm³Preferably ≥ 200mm³More preferably not less than 300mm³The solid tumor of (3).

Item 29, a use, vaccine or method according to item 27 or 28, wherein the tumor is selected from one or more of the group consisting of: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma.

Item 30, the use, vaccine or method according to item 27, wherein the microbial infection is selected from one or more of the following group: HIV, hepatitis B, hepatitis C, leprosy, chronic mucocutaneous candidiasis.

Item 31, the method of item 3, wherein the method further comprises administering to the subject one or more of: interventional therapy, chemotherapy, other immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention.

Item 32, a method for preparing a vaccine for treating or inhibiting a pathogenic disease, said vaccine comprising as an antigen blood-containing or blood-forming semifluid, the method comprising the steps of:

a. providing blood;

Item 33, the method of item 32, wherein the semi-fluidizing process comprises one or more of: semi-fluid visco-thickening, thermal solidification, solidification with a solidifying agent, and wherein the thermal solidification is selected from one or more of: direct heat treatment, steam heat treatment, microwave heat treatment, radio frequency heat treatment, laser heat treatment, preferably microwave heat treatment.

Item 34 the method of item 32, wherein the semi-fluidizing treatment is further followed or preceded by adding an active ingredient for combating pathogenic disease.

Item 35, a vaccine prepared according to the method of one of items 32-34.

Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.

Claims

1. Use of a semifluid comprising or formed from blood as an antigen in the manufacture of a vaccine for the treatment or inhibition of a pathogenic disease.

2. A vaccine for treating or inhibiting a pathogenic disease comprising blood or a semifluid formed from blood as an antigen.

3. The use, vaccine or method according to claim 1 or 2, wherein the vaccine is in a form suitable for topical administration, and wherein the composition and morphology of the semifluid is such that it forms a semifluid nodule at the site of administration.

4. Use, vaccine or method according to one of claims 1 to 3, wherein the blood is kept away from its natural state, preferably in a state that is easily recognized by the immune system as severely damaged tissue.

5. Use, vaccine or method according to one of claims 1 to 4, wherein the tumour is preferably selected from the group consisting of tumour volume>85mm³Preferably ≥ 200mm³More preferably not less than 300mm³The solid tumor of (3).

6. The use, vaccine or method according to claim 5, wherein the tumour is selected from one or more of the group consisting of: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma.

7. A method of preparing a vaccine for treating or inhibiting a pathogenic disease, said vaccine comprising as an antigen blood-containing or blood-forming semifluid, the method comprising the steps of:

a. providing blood;

8. The method of claim 7, wherein the semi-fluidized treatment comprises one or more of: semi-fluid visco-thickening, thermal solidification, solidification with a solidifying agent, and wherein the thermal solidification is selected from one or more of: direct heat treatment, steam heat treatment, microwave heat treatment, radio frequency heat treatment, laser heat treatment, preferably microwave heat treatment.

9. The method according to claim 7, wherein the semi-fluidized treatment is further mixed with the addition of an active ingredient for combating pathogenic diseases before or after the semi-fluidized treatment.

10. A vaccine prepared according to the method of any one of claims 7 to 9.