Detailed Description
Based on the above disclosure, other modifications, substitutions and alterations can be made without departing from the basic technical concept of the present disclosure as it is known and customary in the art.
I. Definition of
Intestinal microbiota
"intestinal microbiota" (formerly known as intestinal flora or microbial community) means a population of microorganisms in the intestine of any organism (human, animal, insect, etc.) belonging to the kingdom animalia. Although each individual has a unique microbiota composition (of a total of 400 and 500 different bacterial species/individual, 60 to 80 bacterial species are shared by more than 50% of the sampled population), it always fulfills a similar major physiological function and has a direct impact on the health of the individual: digestion of certain foods (mainly indigestible fibers) that contribute to the inability of the stomach and small intestine to digest; contribute to the production of some vitamins (B and K); protecting against other microorganisms, and maintaining the integrity of the small intestinal mucosa; plays an important role in the development of the appropriate immune system; a healthy, diverse and balanced intestinal microbiota is critical to ensure proper functioning of the intestine.
Given the main role played by the gut microbiota in the normal function of the body and the different functions it carries out, it is nowadays considered as an "organ". However, because the infant is sterile in nature, it is an "acquired" organ; that is, intestinal colonization starts immediately after birth and evolves later.
The development of the gut microbiota starts at birth. The sterile, neonatal gut inside the uterus is rapidly colonized by microorganisms from the mother (vagina, skin, breast, etc.), the environment in which delivery occurs, the air, etc. Starting from day 3, the composition of the gut microbiota is directly dependent on the infant if fed: the intestinal microbiota of infants fed with breast milk is dominated, for example, by bifidobacteria, compared to infants fed with infant formula.
The composition of the gut microbiota evolves throughout life, from birth to the elderly, and is the result of different environmental influences. The balance of gut microbiota can be affected during aging, and thus, elderly people have a significantly different microbiota than young adults.
While the general composition of the dominant gut microbiota is similar in most healthy people (4 major phyla, the firmicutes, bacteroidetes, actinomycetes and proteobacteria), the composition at the seed level is highly personalized and is largely determined by the genetics, environment and diet of the individual. The composition of the gut microbiota may become accustomed to the dietary composition (temporarily or permanently). For example, Japanese people can digest marine algae (part of their daily diet) due to special enzymes that their microflora have acquired from marine bacteria.
Ecological disorder
While it can accommodate changes and has a high elastic capacity, a loss of balance in gut microbiota composition can occur in certain specific situations. This is known as "dysbiosis", an imbalance between potentially "harmful" bacteria and known "beneficial" bacteria in the gut or any deviation in the microbiota considered to be "healthy" microbiota in terms of composition and diversity of the main microbiota. Dysbiosis may be associated with health problems such as functional bowel disease, inflammatory bowel disease, allergies, obesity, and diabetes. It may also be the result of therapy, such as cytotoxic therapy or antibiotic therapy.
In colorectal cancer (CRC) patients, intestinal microbial dysbiosis involves the enrichment of bacterial species from the genus Bacteroides and the reduction of species belonging to Faecalibacterium and Roseburia (Roseburia) (Sobhani et al, 2011; Wu et al, 2013). In particular, the genus clostridium (Fusobacterium) and Campylobacter (Campylobacter) were found to increase consistently in both stool and mucosa in CRC patients.
In the case of cancer, the "beneficial" or "advantageous" bacteria are essentially of the genus lactobacillus and bifidobacterium, while the "harmful" or "disadvantageous" bacteria are essentially of the following species: parabacteroides gibsonii and the following genera: germinium, Alisipes.
As used herein, "microbiota" and "flora" refer to a population of microorganisms, including eukaryotes, archaea, bacteria, and viruses, including bacterial viruses (i.e., bacteriophage), that live in or on a subject's body, both persistently and transiently.
As used herein, "colony forming units (cfu)" refers to an estimate of the number of viable microbial cells in a given sample.
As used herein, "viable" means having the ability to proliferate.
As used herein, "isolated" or "purified" refers to a bacterium or other entity or substance that has been (1) isolated from at least some of the components with which it was associated at the time of initial production (whether naturally or in a laboratory setting), and/or (2) produced, prepared, purified, and/or manufactured by the hands of a human. An isolated or purified bacterium can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which it is initially associated.
Immune signaling pathway modulators
Immune cell signaling pathways may be modulated by one or more of the following exemplary receptor/ligand pairs on a cell: PD1/PDL1, PD1/PDL2, CD28/B7-1(CD80), CD28/B7-2(CD86), CTLA4/B7-1(CD80), CILA4/B7-2(CD86), 4-1BB (CD137)/4-1BBL (CD137L), ICOS/B7RP1, CD40/CD40L, herpes virus entry regulatory factor (Herpesvirus entry mediator, HVEM)/B-and T-lymphocyte attenuation factor (B-and T-lymphotetainer, BTLA); OX40/OX40L, CD27/CD70, GITR/GITRL, KIR/MHC, lymphocyte activation gene 3(LAG3 or CD223)/MHC, Hepatitis A virus cell receptor 2 (HAVCR 2; also known as T cell immunoglobulin and mucin domain containing-3 (TIM3))/TIM3 ligand, T cell immunoreceptor with Ig and ITIM domains (TIGIT)/CD96, and TIGIT/CD 226. The immune cell signaling pathway may also be modulated by one or more of the following exemplary cytokines/chemokines and their cognate cell surface receptors: interleukin 2(IL-2)/CD122, adenosine/adenosine A2A receptor (adenosine A2A receptor, A2AR), interleukin 6(IL-6)/IL6R (CD126), interleukin 10(IL-10)/IL-10R, interleukin 15(IL-15)/IL-15R, transforming growth factor beta (TGF beta)/TGF beta R, and macrophage colony stimulating factor 1 (CSF-1)/CSF-1R. Other immune molecules include, but are not limited to, KIR2DL, VISTA, HLLA2, TLIA, DNAM-1, CEACAM1, CD155, and indoleamine 2, 3-dioxygenase (IDO), such as IDO 1. Any of the above immune molecules may be targeted by an anti-cancer therapy as described in the present disclosure.
As used herein, an "immune signaling pathway modulator" refers to an agent that alters the activity of an immune molecule (e.g., any of those described herein) relative to a control vehicle. The term "modulator" is used in the broadest sense in this disclosure and includes any molecule that partially or completely alters an information pathway modulated by one or more immune molecules, including an information transduction pathway modulated by a molecule as described in this disclosure.
In some cases, an immune signaling pathway modulator is an inhibitor of an immune checkpoint molecule that can reduce, slow, stop, and/or prevent activity modulated by the checkpoint molecule. The term "inhibitor" is used in the broadest sense in this disclosure and includes any molecule that partially or completely blocks, inhibits, or neutralizes an informative pathway modulated by one or more immune checkpoint molecules, including a regulatory pathway modulated by a molecule as described in this disclosure. Suitable inhibitory molecules include in particular antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides and the like.
In other cases, an immune signaling pathway modulator is an activator of an immune molecule that enhances and improves the activity modulated by the immune molecule. The term "activator" is used in the broadest sense in this disclosure and includes any molecule that enhances an information pathway modulated by one or more immune molecules, including an information transduction pathway modulated by a molecule as described in this disclosure. Suitable activating agents include agonistic antibodies or antibody fragments, small organic molecules, recombinant proteins or peptides, and the like. In some cases, the activator can be an agonist antibody to an immune molecule, such as MEDI0562 (humanized OX40 agonist antibody), MEDI6469 (mouse OX4 agonist); and MEDI6383(OX40 agonist).
Methods of identifying such modulators are well known in the art. For example, a candidate modulator can be contacted with a suitable target of an immune molecule, and the intensity of information transduction modulated by the immune molecule can be measured by conventional assays. A detectable change in information transduction in the presence of the candidate modulator relative to the blank indicates that the candidate modulator has modulating activity of an immune molecule.
Medicament or pharmaceutical composition
The bacterial solution of healthy human intestinal flora according to the present disclosure, together with one or more adjuvants such as adjuvants, carriers or diluents, may be placed in the form of pharmaceutical compositions, unit doses or dosage forms. The pharmaceutical compositions may be employed in solid dosage forms (such as powders, granules, pellets, coated or uncoated tablets or filled capsules) or in liquid dosage forms (such as solutions, suspensions, emulsions or capsules filled therewith) or in semisolid dosage forms (such as gels, creams and ointments). The dissolution and release characteristics of one or more active ingredients of a pharmaceutical dosage form may vary from seconds to months.
The "medicaments" or "pharmaceutical compositions" are designed for use in animals and humans and can be administered via all routes of administration. Preferred routes of administration are oral, pulmonary, nasal, rectal, parenteral. Such pharmaceutical compositions and unit dosage forms thereof may contain conventional or novel ingredients in conventional or special proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient to be employed commensurate with the intended daily dosage range.
The term "carrier" as applied to the pharmaceutical compositions of the present disclosure relates to a diluent, adjuvant or vehicle with which the active compound is administered.
The medicaments or pharmaceutical compositions of the present disclosure may be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is often desirable to use the oral route. The active agent may be administered orally in The form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).
For oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be combined with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweetening agents, natural and synthetic gums (e.g., acacia, tragacanth or alginate), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, and the like. For oral administration in liquid form, the pharmaceutical components may be combined with non-toxic, pharmaceutically acceptable inert carriers (e.g., ethanol, glycerol, water), anti-settling agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous carriers (e.g., almond oil, oleyl esters, ethanol, or fractionated vegetable oils), preserving agents (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), and the like. Stabilizers such as antioxidants (BHA, BHT, propyl gallate, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.
Tablets containing the active compound may be coated by methods well known in the art. The compositions of the present disclosure comprising as active compound a compound of formula I may also incorporate beads, microspheres or microcapsules, for example constructed from polyglycolic acid/lactic acid (PGLA). Liquid preparations for oral administration may take the form of, for example, solutions, syrups, emulsions or suspensions or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Formulations for oral administration may suitably be formulated so as to provide controlled or delayed release of the active compound.
The medicaments or pharmaceutical compositions of the present disclosure may be delivered parenterally, i.e., by intravenous (i.v.), intracerebroventricular (i.c.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.d.), or intradermal (i.d.) administration, by direct injection, via, for example, bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers with added preservative. The compositions may take the form of an excipient (excipient), a suspension, solution or emulsion in an oil or aqueous carrier, and may contain formulatory agents such as anti-settling agents, stabilising agents and/or dispersing agents. Alternatively, the active ingredient may be reconstituted with a suitable carrier (e.g., sterile pyrogen-free water) in powder form prior to use.
The medicaments or pharmaceutical compositions of the present disclosure may also be formulated for rectal administration, for example, in the form of suppositories or retention enemas (e.g., containing conventional suppository bases such as cocoa butter or other glycerides).
Combination therapy method
The present disclosure provides a combination cancer therapy using immune signaling pathway modulators as described in the present disclosure and bacterial solutions of healthy human intestinal flora. The term "combination therapy" as used in this disclosure includes the administration of these agents (e.g., immune signaling pathway modulators and bacterial solutions of healthy human intestinal flora) in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time, and the administration of these therapeutic agents, or at least two agents, is performed substantially simultaneously. The sequential, or substantially simultaneous, administration of each agent may be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, subcutaneous routes, and direct absorption through mucosal tissue. The agents may be administered by the same route or by different routes. For example, a first agent (e.g., a bacterial suspension of healthy human intestinal flora) can be administered orally, while a second agent (e.g., an anti-checkpoint antibody such as an anti-PD 1 antibody) is administered intravenously. Furthermore, selected combinations may be administered by intravenous injection, while other agents of the combination may be administered orally. Alternatively, two or more agents may be administered by intravenous or subcutaneous injection, for example.
Combination therapy may also include administration of the agents as described in the present disclosure (e.g., immune signaling pathway modulators and fermented compositions) further in combination with other bioactive ingredients (e.g., different antineoplastic agents) as well as non-drug treatments (e.g., surgery or radiation therapy). Where the combination therapy further comprises radiation therapy, the radiation therapy can be administered at any suitable time, so long as a beneficial effect is obtained from the combined action of the therapeutic agent and the radiation therapy. For example, where appropriate, this beneficial effect may be achieved when radiation therapy is temporarily removed from administration of the therapeutic agent, perhaps for days or even weeks.
It is to be understood that any combination of immune signaling pathway modulators and bacterial solutions as described in the present disclosure may be used in any order to treat cancer. The combination of the present disclosure may be selected based on a number of factors including, but not limited to, effectiveness in inhibiting or preventing progression of cancer, effectiveness in alleviating a side effect of another drug of the combination, or effectiveness in alleviating a symptom associated with cancer. For example, combination therapy as described in the present disclosure may reduce any side effects associated with each individual member of the combination. Some examples are provided in the following tables. For example, the bacterial fluid of healthy human intestinal flora may be used daily during a treatment involving an immune signaling pathway modulator (e.g., anti-PD 1 antibody).
In some embodiments, the combination of an immune signaling pathway modulator described in the present disclosure and a bacterial fluid as described in the present disclosure is provided in the form of a kit. The kit comprises an immune signal pathway regulator and the bacterial liquid disclosed by the disclosure. In some cases, the kits of the present disclosure may also contain other necessary reagents. It is to be understood that the various components of the kits of the present disclosure may be used in any order for treating cancer.
Any combination of an immune signaling pathway modulator as described in the present disclosure and bacterial fluid of healthy human intestinal flora can be used to treat cancer. The term "cancer" as used in this disclosure refers to medical conditions mediated by tumors or malignant cell populations, proliferation or metastasis, including solid and non-solid cancers. Examples of cancer include, but are not limited to, lung cancer, kidney cancer, stomach cancer, breast cancer, brain cancer, prostate cancer, hepatocellular cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, thyroid cancer, melanoma, head and neck cancer, colon cancer, leukemia, lymphoma, skin cancer, stomach cancer, esophageal cancer, myeloma, rectal cancer, bone cancer, uterine cancer, prostate cancer, and hematological malignancies.
The terms "individual," "individual," and "patient" are used interchangeably in this disclosure and refer to a mammal that is evaluated for treatment and/or is being treated. The subject may be a human, but also includes other mammals, particularly those useful as laboratory models of human disease, e.g., mice, rats, rabbits, dogs, and the like.
As used in this disclosure, "effective amount" refers to the amount of each active agent (e.g., an immune signaling pathway modulator, e.g., an anti-PD 1 antibody or a bacterial solution of healthy human intestinal flora) required to confer a therapeutic effect on an individual, whether administered alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is inhibition of cancer cell growth and/or reduction of tumor burden. In some embodiments, the amount of bacterial fluid of healthy human intestinal flora is effective to enhance the anti-cancer effect of the immune signaling pathway modulator. In other embodiments, the amount of bacterial fluid of healthy human intestinal flora is effective to enhance the immunity of the individual to cancer cells. In some embodiments, the therapeutic effect is prevention or inhibition of tumor growth. In some embodiments, the therapeutic effect is a reduction in side effects associated with one or more drugs/medicaments. For example, side effects that may result from inhibition of the PD-1 pathway (e.g., fatigue, peripheral edema, chills, fever, diarrhea, nausea, abdominal pain, cough, dyspnea, rash, itch, leukoderma, arthralgia, myalgia, back pain, headache, dizziness, and/or increased aspartate Aminotransferase (AST)) may be alleviated by co-treatment with another agent (e.g., an immune signaling pathway modulator with a bacterial solution of a healthy human intestinal flora as described in this disclosure).
As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to an individual who has the disease or condition of interest, a symptom of the disease/condition, or a predisposition toward the disease/condition, and whose purpose is to cure, treat, alleviate, alter, remedy, ameliorate, augment, or otherwise affect the disease, disease symptom, or predisposition toward the disease or condition.
Ameliorating the target disease/disorder includes delaying the development or progression of the disease, or reducing the severity of the disease. Relief from the disease does not necessarily require a therapeutic outcome. As used herein, "delaying" the progression of a target disease or disorder means delaying, arresting, slowing, impeding, stabilizing, and/or delaying disease progression. Such delays may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. A method of "delaying" or reducing the progression of, or delaying the onset of, a disease by reducing the likelihood of developing one or more symptoms of the disease within a given time frame, and/or by reducing the extent of symptoms within a given time frame, as compared to a person not using the method. Such comparisons are typically based on clinical studies, using a number of individuals sufficient to give statistically significant results.
"progression" or "progression" of a disease means the initial manifestation and/or subsequent progression of the disease. Development of the disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may not be detectable. For the purposes of the present invention, development or progression refers to the biological process of the condition. "development" includes occurrence, recurrence and onset. As used in this disclosure, "onset" or "occurrence" of a disease or disorder of interest includes initial onset and/or recurrence.
In some embodiments, the combination of an immune signaling pathway modulator as described in the present disclosure and a bacterial solution of healthy human intestinal flora is administered to an individual in need of treatment in an amount sufficient to inhibit the activity of one or more target information transduction pathways in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, the combination is administered in an amount effective to reduce the degree of activity of one or more antigens of interest by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more).
In some embodiments, a bacterial solution of healthy human intestinal flora as described in the present disclosure is administered to an individual (e.g., a human cancer patient) who has received or is receiving an anti-cancer therapy involving the use of an immune signaling pathway modulator (e.g., inhibitor), as described in the present disclosure.
Conventional methods known to those of ordinary skill in the medical arts can be used to administer a pharmaceutical composition to an individual depending on the type of disease to be treated or the site of the disease. The composition may also be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenteral" as used in this disclosure includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intracerebral, and intracranial injection or infusion techniques. Furthermore, they may be administered to a subject by an injectable depot route, for example using 1, 3 or 6 month storage tanks or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.
The active ingredients of formula I of the present disclosure may be administered to a subject in need thereof, such as a living animal (including a human) body, in an effective amount, whether by oral, rectal or parenteral (including intravenous and subcutaneous) or, in some cases, even topical routes, either with one or more pharmaceutically-acceptable adjuvants, carriers or diluents, particularly and preferably in the form of their pharmaceutical compositions, for the treatment, alleviation or amelioration, palliation or elimination of the indications or conditions to which they are sensitive or which are set forth elsewhere in the application.
Detailed description of the preferred embodiments
The disclosure is further illustrated with reference to the following examples. The description of the specific exemplary embodiments of the present disclosure has been presented for purposes of illustration and description. It is not intended to limit the disclosure to the precise form disclosed, and obviously many modifications and variations are possible in light of the teaching of this specification. The exemplary embodiments were chosen and described in order to explain certain principles of the disclosure and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the disclosure and various alternatives and modifications thereof.
One object of the present disclosure is to provide a method for preparing a healthy human intestinal flora holobacteria composition, which is characterized by comprising the following steps:
adding sterile deoxidized normal saline into excrement of a healthy person according to the mass volume ratio of 1:3-5, and stirring a sample;
homogenizing the sample;
filtering the homogenized liquid step by step;
centrifuging the filtrate for 2 times, and re-suspending to obtain suspension, i.e. the whole bacteria composition of human intestinal flora;
the process is carried out under anaerobic conditions.
In a preferred embodiment, the first centrifugation conditions are 0-25 ℃, 50-200g, centrifugation for 1-15 minutes; centrifuging for 1-15 min at 4000-5500 deg.C for the second time;
the first and second centrifugation temperatures are preferably 0-15 deg.C, or 1-10 deg.C, or 2-9 deg.C, or 3-8 deg.C, or 4-7 deg.C, or 5-6 deg.C, and most preferably 4 deg.C;
the first centrifugation rotating speed is preferably 100-200g, or 150-200g, and most preferably 200 g;
the second centrifugation rotating speed is preferably 4500-;
the first and second centrifugation times are preferably 3 to 10 minutes, more preferably 5 to 7 minutes, and most preferably 7 minutes.
In a preferred embodiment, the particle size of the step-wise filtration is selected from the group consisting of 1mm to 2mm, 400 μm and 20 to 80 μm.
In a preferred embodiment, the preparation method further comprises the steps of:
collecting a fresh excrement sample by using an aseptic excrement collecting box, covering a cover after sampling, conveying the sample to a laboratory for weighing, and recording the quality;
placing the sampling box into a transfer window of an anaerobic operation table, opening a box cover to ensure gas exchange, covering the transfer window cover, and starting a deoxidation program; after 5 minutes, transferring the sampling box to the interior of an operation table;
sterile deoxidized normal saline is added according to the mass volume ratio of 1:3-5, and a sample is stirred to ensure that excrement is not excessively adhered to a sampling box;
homogenizing the sample feces;
the homogenized liquid is filtered step by step, preferably, the step by step filtration is 1mm-2mm, 400-600 μm and 20-80 μm;
transferring the filtrate into a centrifugal tube, centrifuging for 2 times, wherein the first centrifugation condition is 1-10 ℃ and 50-200g, centrifuging for 5-7 minutes, the second centrifugation condition is 1-10 ℃ and 4000-5500 g, centrifuging for 5-7 minutes, and collecting thallus precipitate;
and (3) resuspending the mixture by using 50mL of sterile deoxidized physiological saline, and obtaining suspension, namely the human intestinal flora whole bacteria composition.
In a preferred embodiment, the method further comprises the steps of:
quality control of the excrement source; and/or
Quality control of the human intestinal flora whole bacteria composition.
In a preferred embodiment, the quality control step of the fecal source comprises: healthy persons 18-40 years old are selected, passed health screening and clinical examination.
In a more preferred embodiment, the health screening and clinical examination comprises: the blood examination has no infection of HIV, hepatitis B virus, EB virus, etc.; diseases of the body without digestive tract and other diseases which can affect the intestinal flora, such as metabolic syndrome and the like; stool examination for infection by rotavirus, norovirus, salmonella, shigella, carbapenem-resistant bacteria of the family Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Enterobacteriaceae producing extended spectrum beta-lactamase, Candida albicans, Cryptosporidium, and Cyclosporidium; three meals and regular work and rest.
In a preferred embodiment, the quality control step of the human intestinal flora holothurian composition comprises: the viable bacteria rate is counted in the bacterial liquid obtained by resuspending sterile deoxidized normal saline>5x1010Individual/ml was considered acceptable.
The invention also aims to provide the human intestinal flora whole bacteria composition prepared by any one of the preparation methods.
The invention also aims to provide a pharmaceutical composition containing the human intestinal flora whole bacteria composition prepared by any one of the preparation methods and a pharmaceutically acceptable carrier.
The invention also aims to provide a kit for treating cancer, which comprises the human intestinal flora holobacteria composition or the pharmaceutical composition and the PD-1 antibody.
The present disclosure also provides the use of the above-mentioned human intestinal flora holobacteria composition in the preparation of a medicament or a kit for treating cancer.
In a preferred embodiment, the cancer is a tumor of the digestive tract.
In a more preferred embodiment, the cancer is colorectal cancer.
In a preferred embodiment, the above use for the manufacture of a medicament or kit for the treatment of cancer, characterized in that said medicament or kit further comprises one or more immune signaling pathway modulators.
In a more preferred embodiment, the immune signaling pathway modulator comprises an optional immune signaling pathway modulator known in the art, including, but not limited to, PD1/PDL1, PD1/PDL2, CD28/B7-1(CD80), CD28/B7-2(CD86), CTLA4/B7-1(CD80), CILA4/B7-2(CD86), 4-1BB (CD137)/4-1BBL (CD137L), ICOS/B7RP1, CD40/CD40 40, herpes virus entry regulatory factor (Herpesvirus entry mediator, HVEM)/B-and T-lymphocyte attenuation factor (B-and T-lymphotactuenter, LA), 40/CD40, GITR/TRL, KIR gene, MHC activated cell gene (MHC) and MHC activating domain of TIM/TAX 40, MHC activating protein/TAX 40, CD40/CD40, CD-and MHC binding protein of the TAX 40/CD 843, (ii) a T cell immunoreceptor with Ig and ITIM domain (TIGIT)/CD96, and one or more of TIGIT/CD 226; preferably, the immune signaling pathway modulator is a PD-1 inhibitor, most preferably a PD-1 antibody.
Preferably, the use as described above for the preparation of a medicament or kit for the treatment of cancer, said medicament being capable of increasing the therapeutic response of a patient to a PD-1 inhibitor.
Preferably, the use as described above for the preparation of a medicament or kit for the treatment of cancer, said medicament being capable of enhancing the response of a PD-1 inhibitor.
Example 1: preparation of healthy human intestinal fecal flora whole bacteria composition
A 32 year old healthy person was selected, and by health screening and clinical examination: blood examination for infection with HIV, hepatitis B, EB virus (EBV), etc.; diseases of the body without digestive tract and other diseases which can affect the intestinal flora, such as metabolic syndrome and the like; stool examination for infection by rotavirus, norovirus, salmonella, shigella, carbapenem-resistant bacteria of the family Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Enterobacteriaceae producing extended spectrum beta-lactamase, Candida albicans, Cryptosporidium, and Cyclosporidium; three meals and regular work and rest.
Collecting a fresh excrement sample by using a specific sterile excrement collecting box, covering a cover after sampling, conveying the sample to a laboratory for weighing, and recording the quality; and (3) placing the sampling box into a transfer window of an anaerobic operating platform, and opening a box cover to ensure gas exchange. Covering a transfer window cover, and starting a deoxidation program; after 5 minutes, the cartridge was transferred to the inside of the console and the experimental procedure was started.
According to the weighed sample mass, 5 times volume of sterile deoxygenated physiological saline is poured. For example, if the mass of the excrement is 100g, 500mL is poured; stirring the sample to ensure that the excrement is not excessively adhered to the sampling box; pouring the whole sample into a sealed bag, and homogenizing the sample excrement; then, filtering step by step, wherein the particle size of the step by step filtration is selected from 1mm-2mm, 400-600 μm and 20-80 μm; then using an electric pipettor and a pipette to transfer the filtrate of the last time into 50mL centrifuge tubes, and filling each tube with 50 mL; performing two-step centrifugation, the first step: centrifuging at 4 ℃ for 7 minutes at 200 g; the second step is that: centrifuging at 4 ℃ for 7 minutes at 4500g, removing supernatant, and collecting thalli sediment; the bacteria are resuspended in 50mL sterile deoxygenated normal saline, and the suspension is a microbial solution containing the whole bacteria, namely FMT bacteria solution, and can be used for subsequent treatment.
Taking 1ul of the bacterial liquid sample for quality inspection, diluting the bacterial liquid sample in 1ml of sterile physiological saline, and adding thermo Fisher LIVE/DEAD into the bacterial liquid sampleTM BacLightTMThe SYTO 9 stain and PI stain were each 1. mu.l in the Bacterial Viability Kit. Shaking and mixing evenly, and standing for 15 minutes in the dark. After 15 minutes, 2. mu.l of the solution was diluted in 1ml of sterile physiological saline and mixed by shaking. The staining dilutions were examined using flow cytometry, with the FITC channel gain adjusted to 150, the PC5.5 channel gain adjusted to 200, the primary threshold set to FSC, manual, 400, the secondary threshold set to SSC, manual, 4500, all other parameters set as default, and the collection volume fixed. And setting a flow cytometer acquisition diagram, taking the log value of FITC-A as an abscissa and the log value of PC5.5-A as an ordinate, and quantitatively acquiring data. And (4) in the acquired scatter diagram, the scatter points gathered at the lower right part in the diagram are circled, and the scatter points are the viable bacteria. And counting the number of the bacteria, and calculating the viable bacteria rate in the original bacteria liquid according to the dilution multiple of the experimental process.
The quality inspection standard is that the viable bacteria rate>5x1010Per ml, e.g. to achieve thisThe standard is considered to be qualified.
The viable bacteria rate of the product prepared by the bacteria liquid is 1.21x10 detected by a flow cytometer11and/mL. And (5) checking to be qualified.
Example 2: healthy human intestinal flora combined with PD-1 immunosuppressant for treating digestive tract tumor
Test agent Rat IgG2a
Supplier: zhongmeike biological technology Limited
The goods number is: CVP039
Batch number: 0119L220
Packaging: 2.8mg/ml,96mg
test agent aPD-1 antibody (RMP1-14)
Supplier: zhongmeike biological technology Limited
The goods number is: CVP033
Batch number: 0119L225
Packaging: 4.1mg/ml,96mg
antibiotic
Day-8 to Day-4: the formula comprises 1mg/ml of Ampicillin (Ampicillin), 1mg/ml of Neomycin (Neomycin), 1mg/ml of Metronidazole (Metronidazole) and 0.5mg/ml of Vancomycin (Vancomycin). Antibiotics are added into sterile water in proportion for mice to drink, the treatment time of the antibiotics is 4 days, and the water bottle containing the antibiotics is replaced every three days.
Day-3 to Day 7: the formulation comprises 1mg/ml Ampicillin (Ampicillin), 10mg/ml Neomycin (Neomycin), 10mg/ml Metronidazole (Metronidazole), 5mg/ml Vancomycin (Vancomycin) and 0.1mg/ml amphotericin B (Amphotericin B). Ampicillin was added to sterile water for mice to drink, and the mice were gavaged with a mixture of other antibiotics twice a day, 200 μ l each time.
24h before receiving microbial treatment (Day 7), antibiotic treatment was discontinued and the mice were transferred to new cages to prevent the mice from eating antibiotic-containing faeces.
Day8 mice were again transferred to new cages before the first microbial treatment to prevent rats from eating feces that also had residual antibiotic.
Cell culture
CT26 cells (SIBS: Shanghai Life sciences research institute, CAT #: TCM37) were cultured in RPMI-1640 medium containing 10% fetal bovine serum. CT26 cells were collected in the exponential growth phase and PBS was resuspended to appropriate concentration for subcutaneous tumor inoculation in mice.
Animal modeling and random grouping
The administration method of the mouse colon cancer CT26 model is shown in the figure. Specifically, SPF grade 6-8 week old female Balb/c mice were purchased. And (5) adaptively feeding for one week. Mice belonging to the same group, housed in different cages, were randomly exchanged during the experiment.
Mice were divided into 4 groups: control group (sterile saline + isotype control), FMT treatment alone group (FMT + isotype control), PD-1 treatment alone group (sterile saline + PD-1) and FMT combination treatment group (FMT + PD-1), with 10 mice per group.
After the adaptive feeding is finished, the mice are subjected to antibiotic treatment in an intragastric manner, wherein the treatment time is 16 days (from Day-8 to Day7), and the intragastric administration is carried out for 1 time every Day.
During the antibiotic treatment, cultured CT26 cells were injected into mice at Day0 for molding. 256 female mice were inoculated subcutaneously on the right side with 5X105CT26 cells. The Day of vaccination was defined as Day0 (Day 0).
The tumors were randomly grouped 8 days after inoculation according to their volume, with the average tumor volume in each group being 87mm3。
After completion of the gavage by Day7, the antibiotic treatment was discontinued and the mice were transferred to new cages to prevent the mice from eating antibiotic-containing feces.
Day9 once again transferred the mice to new cages before the first microbial treatment.
FMT cell suspension prepared in example 1, resuspended at 2.5X10 in sterile saline10One per ml.
Mice in the alone-FMT treatment group and the combination-FMT treatment group were administered Day8, Day11, Day14 and Day17, respectively, for gavage, and each mouse was gavage 1 time per Day at a gavage volume of 200 μ l, i.e., at a dose of 5X109One/only/time; the control group and the PD-1 treatment-alone group were infused with an equal volume of physiological saline.
Antibody injections were performed at 200 ug/dose in Day9, Day12, Day15 and Day18, respectively. Wherein the PD-1 single treatment group and the combined treatment group are injected with PD-1 antibody, and the control group and the single treatment group are injected with isotype control.
Day24 mice were sacrificed, tumors were collected, tumor tissue was ground after weighing and measuring tumor area, and spleens were collected for flow cytometry analysis.
During the experiment, the growth of the mouse tumors after tumor cell inoculation was monitored periodically: directly measuring the tumor volume by using a vernier caliper, wherein the tumor volume is calculated according to the formula:
tumor volume (mm)3)=1/2×(a×b2) (wherein a represents a long diameter and b represents a short diameter).
The monitoring time points were: days 5, 8, 11, 14, 17, 21 and 24; monitoring of mouse body weight after tumor inoculation.
Relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treated and control groups at a certain time point. The calculation formula is as follows:
T/C%=TRTV/CRTV×100%(TRTV: treatment group mean RTV; cRTV: vehicle control mean RTV; RTV ═ Vt/V0,V0Is the tumor volume of the animal in the group, VtIs the tumor volume of the animal after treatment); or the like, or, alternatively,
T/C%=TTW/CTW×100%(TTW: mean tumor weights at the end of treatment group experiments; cTW: mean tumor weight at the end of the vehicle control experiment).
Relative tumor inhibition, TGI (%), calculated as follows:
TGI% (1-T/C) × 100% (T and C are the Relative Tumor Volume (RTV) or Tumor Weight (TW) at a particular time point in the treated and control groups, respectively).
Conclusion
All groups of mice of the CT26 model were observed at Day 13 after the administration (Day 21 after cell inoculation, Day21) from the growth of each group of mice (table 1):
mean tumor volume of 1916.88mm in mice from sterile saline plus isotype control group3;
The mean tumor volume of the FMT + isotype control group was 1790.40mm3Compared with a control group, the tumor suppressor has no statistically significant difference, the p value is 0.679, and the relative tumor inhibition rate TGI (%) is 6%;
mean tumor volume of 1402.64mm in the sterile saline + aPD-1 group3Compared with the control group, the tumor suppressor has no statistically significant difference, the p value is 0.159, and the relative tumor inhibition rate TGI (%) is 26%;
mean tumor volume of 1206.56mm in FMT + aPD-1 group3Compared with the control group, the difference was statistically significant, the p value was 0.003, and the relative tumor inhibition rate TGI (%) was 37% (table 1).
TABLE 1 table of drug effect analysis of each group in mouse colon cancer CT26 model
All groups of mice of the CT26 model were subjected to survival analysis (Table 2) at Day15 after administration (Day 24 after cell inoculation, Day24), and survival curves of the treated groups were analyzed by Kaplan-Meier method (FIG. 2), and the FMT + aPD-1 treated group was able to significantly promote survival of mice, which was statistically significantly different from the control group (p value was 0.002). And FMT + aPD-1 and treatment groups further promoted survival of mice compared to the sterile saline + aPD-1 treatment group (p value 0.039).
TABLE 2 survival analysis in murine Colon cancer CT26 model
*MST: median lifetime (Median survivval Time)
**ILS: survival extension rate (Increase of Life Span)