Combined vaccine for preventing or treating tetanus

Abstract

The present invention provides a pharmaceutical combination comprising a tetanus-adsorbing vaccine and an antibody against tetanus toxin or an antigen-binding fragment thereof. The pharmaceutical composition is used for preventing and / or treating tetanus. According to the present invention, the combination of the recombinant natural completely humanized monoclonal antibody and the tetanus adsorption vaccine proves that the detection time of the antibody induced by the tetanus adsorption vaccine is advanced, and the high titer level can last for a longer time;

Description

Technical Field

[0001] The present invention relates to a biological preparation vaccine, and in particular to a drug combination and application thereof for preventing and treating tetanus. Background Art

[0002] Tetanus is an acute infectious disease caused by paroxysmal muscle spasms caused by tetanus toxin produced by Clostridium tetani. Clostridium tetani exists in large quantities in the intestines of humans and animals. It often invades the human body through skin or mucosal wounds after feces contaminates the soil, and grows and reproduces in an oxygen-deficient environment to produce tetanus toxin. Tetanus toxin is a neuroprotein toxin that blocks the release of inhibitory neurotransmitters, blocks the transmission of information between nerves and muscles, and causes muscle spasms, cranial nerve paralysis, and central nervous system dysfunction.

[0003] Depending on the level of local medical care, the morbidity and mortality of tetanus patients around the world vary greatly, but the mortality rate of those who do not receive treatment, especially the elderly and infants, is almost 100%. Tetanus toxin is highly toxic and acts quickly. When a patient is diagnosed with Clostridium tetani infection, there are generally a large number of bacteria and toxins in the body, and the use of antibiotics is too late to save the patient's life. Therefore, since 1978, my country has implemented the child immunization program, and the mixed vaccine of pertussis, diphtheria and tetanus has been included in the routine immunization program for children; in addition, tetanus immunization preparations are often used after wound treatment to prevent tetanus infection.

[0004] Tetanus immunization preparations are divided into active immunization preparations and passive immunization preparations. Active tetanus immunization preparations are tetanus toxoid-containing vaccines (TTCV). TTCV includes adsorbed tetanus vaccine (TT), adsorbed diphtheria and tetanus combined vaccine (DT), and adsorbed acellular pertussis, diphtheria, tetanus and acellular pertussis combined vaccine (DTaP). Passive tetanus immunization preparations include tetanus antitoxin (TAT), equine anti-tetanus F(ab')2 (F(ab')2), and human tetanus immunoglobulin (HTIG).

[0005] However, in clinical applications, it has been found that although the protection time of tetanus active immunization preparations is relatively long, the onset of inducing the body to produce antibodies against tetanus toxoid is slow; generally, the induced antibodies reach a protective level about 2 weeks after injection; and patients who have never received tetanus immunization require three consecutive injections to achieve sufficient antibody titers; furthermore, there are often problems such as large potency differences between batches of preparations and difficulty in quality control.

[0006] Therefore, there is still a need in the art for new vaccines for preventing or treating tetanus without adverse effects. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a combination preparation of a tetanus active immunization preparation and a passive immunization preparation, and the use of the combination preparation in preventing and / or treating tetanus.

[0008] The present invention particularly relates to the following terms.

[0009] As used herein, the term "tetanus" refers to a specific infection in which Clostridium tetani invades the human body and grows and reproduces in an oxygen-deficient environment to produce toxins (tetanus toxin) and cause muscle spasms. According to the 2020 version of the "Non-neonatal Tetanus Diagnosis and Treatment Guidelines" issued by the National Health Commission, the diagnosis of non-neonatal tetanus is mainly based on typical clinical manifestations, and at least one of the following manifestations is required: (1) clenched teeth or a wry smile; (2) painful muscle spasms. For patients with doubtful diagnosis, a tongue depressor test can be used. The method is to use a tongue depressor to gently touch the back of the patient's pharynx. The occurrence of masseter reflex spasm instead of normal reflex nausea is positive. Tetanus can cause temporary functional changes in the central nervous system, manifested as tonic contraction and paroxysmal spasm of skeletal muscles throughout the body. In particular, the symptoms of tetanus are spasms of facial expression muscles, neck, back, abdomen, limb muscles, and diaphragm muscles.

[0010] As used herein, the term "tetanus toxoid-containing vaccine" refers to a vaccine containing tetanus toxoid as an active ingredient, including but not limited to monovalent vaccines (such as adsorbed tetanus vaccine), bivalent vaccines (such as adsorbed tetanus-diphtheria combined vaccine), multivalent vaccines (such as adsorbed acellular pertussis, diphtheria, tetanus mixed vaccine), etc. The above-mentioned vaccine can be prepared by the following process: the toxin produced by culturing Clostridium tetani and other strains in a suitable culture medium is purified, detoxified (such as formaldehyde detoxification), and adsorbed (such as aluminum hydroxide adsorption) to make tetanus toxoids, etc., and the tetanus toxoids, etc. are used as antigens for preparation.

[0011] A specific example of the "tetanus toxoid-containing vaccine" proposed in the present invention is an adsorbed tetanus vaccine (monovalent vaccine). For example, as described above, an aluminum hydroxide adjuvant is added during the preparation of the vaccine to prepare an aluminum adsorbed vaccine. The vaccine is also called "adsorbed tetanus vaccine" or "tetanus adsorbed vaccine".

[0012] Another specific example is an adsorbed tetanus-diphtheria combined vaccine (bivalent vaccine). For example, as described above, when preparing the vaccine, Clostridium tetani and Corynebacterium diphtheriae are cultured in a suitable culture medium, and the produced tetanus toxin and diphtheria toxin are refined, detoxified, and adsorbed (for example, with aluminum phosphate adjuvant) to prepare an adsorbed tetanus-diphtheria combined vaccine, also known as "tetanus-diphtheria adsorbed vaccine."

[0013] In another specific example, "tetanus toxoid-containing vaccine" can also be a multivalent vaccine, such as a trivalent diphtheria, tetanus and pertussis adsorbed vaccine; a pentavalent diphtheria, tetanus, pertussis, hepatitis B (recombinant) and polio vaccine; a hexavalent diphtheria, tetanus, pertussis, inactivated poliomyelitis, Haemophilus type B conjugate vaccine and hepatitis B vaccine, etc.

[0014] As used herein, the term "combined use" refers to the simultaneous or sequential use of two or more agents to achieve a specific purpose or effect.

[0015] As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability of the full-length antibody to specifically bind to an antigen. When referring to "antigen-binding fragment" in the present invention, the antigen refers to "tetanus toxin", which is a secretory protein produced by Clostridium tetani under anaerobic conditions.

[0016] As used herein, the term "mammal" refers to any animal classified as a mammal, including but not limited to humans, domestic and agricultural animals, and zoo, sports or pet animals, such as sheep, dogs, horses, cats, cows, rats, mice, pigs, such as macaques and apes, etc. The mammal preferably refers to a rodent or a primate, more preferably a human. In the case where the subject is a human, an adult over 18 years old is preferred.

[0017] The technical solution of the present invention is as follows.

[0018] In a first aspect, the present invention provides a drug combination, comprising: an antibody or an antigen-binding fragment thereof against tetanus toxin; and a vaccine containing tetanus toxoid. The drug combination is used for preventing and / or treating tetanus.

[0019] As for the antibody or antigen-binding fragment thereof contained in the drug combination, the anti-tetanus toxin antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain CDRs, namely HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain CDRs, namely LCDR1, LCDR2, and LCDR3, wherein:

[0020] The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, the L-CDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.8.

[0021] Heavy chain CDR1 (HCDR1): GGPFTGSY; SEQ ID NO. 1;

[0022] Heavy chain CDR2 (HCDR2): VSQSGST; SEQ ID NO. 2;

[0023] Heavy chain CDR3 (HCDR3): ARLTKHYINSAY; SEQ ID NO. 3;

[0024] Light chain CDR1 (LCDR1): QIIGSW; SEQ ID NO. 6;

[0025] Light chain CDR2 (LCDR2): KAS; SEQ ID NO.7;

[0026] Light chain CDR3 (LCDR3): QQYNSYPYT; SEQ ID NO.8.

[0027] Further preferably, the anti-tetanus toxin antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.9.

[0028] Heavy chain variable region (VH):

[0029] SEQ ID NO.4

[0030] Light chain variable region (VL):

[0031] SEQ ID NO.9.

[0032] More preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be of IgG, IgA, IgM, IgD or IgE type, preferably IgG type, and the light chain constant region may be of κ or λ type.

[0033] Preferably, in the pharmaceutical combination of the present invention, the anti-tetanus toxin antibody is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Alternatively, the anti-tetanus toxin antibody is a monoclonal antibody or a single-chain antibody. According to a specific embodiment of the present invention, the anti-tetanus toxin antibody comprises a heavy chain (HC) and a light chain (LC), the heavy chain comprises the amino acid sequence shown in SEQ ID NO.5, and the light chain comprises the amino acid sequence shown in SEQ ID NO.10.

[0034] According to a specific embodiment of the present invention, the anti-tetanus toxin antibody is a monoclonal antibody.

[0035] According to a specific embodiment of the present invention, in the pharmaceutical combination of the present invention, the anti-tetanus toxin antibody is a recombinant natural fully human monoclonal antibody, which is referred to as "TRN0011" in the present invention. The amino acid sequence of TRN0011 is as follows.

[0036]

[0037]

[0038] "TRN0011" is an immunoglobulin G1 (IgG1) monoclonal antibody with two heavy chains containing the amino acid sequence shown in SEQ ID NO.5 and two light chains containing the amino acid sequence shown in SEQ ID NO.10. TRN0011 can bind to the specific surface of tetanus toxin protein with high affinity, and prevent the enzymatic cleavage activity of tetanus toxin by neutralizing the toxin, thereby protecting the body.

[0039] As for the tetanus toxoid-containing vaccine contained in the pharmaceutical combination, the vaccine may be a monovalent vaccine, such as an adsorbed tetanus vaccine. Preferably, the potency of tetanus toxoid contained in a single human dose of the adsorbed tetanus vaccine is not less than 40 IU.

[0040] The vaccine may also be a bivalent vaccine, such as an adsorbed tetanus-diphtheria combined vaccine. Preferably, the tetanus toxoid contained in a single human dose of the adsorbed tetanus-diphtheria combined vaccine is 5Lf.

[0041] The vaccine can also be a multivalent vaccine, such as a trivalent vaccine for diphtheria, tetanus and pertussis adsorbed; a pentavalent vaccine for diphtheria, tetanus, pertussis, hepatitis B (recombinant) and poliomyelitis; a hexavalent vaccine for diphtheria, tetanus, pertussis, inactivated poliomyelitis, Haemophilus type B conjugate vaccine and hepatitis B, etc.

[0042] According to the present invention, the tetanus is prevented or treated by administering (e.g., injecting) the drug combination to the subject. The drug combination can be used for subjects who have been infected or not yet infected with Clostridium tetani. Theoretically, as long as a wound or break is caused, the subject may be infected with tetanus and needs to be prevented. Common causes include: (1) a history of trauma or damage to the skin or mucous membrane (such as animal injuries, injection of drugs such as drugs, childbirth or miscarriage); (2) a history of bacterial infection of the skin, mucous membrane, or soft tissue (such as chronic otitis media, chronic sinusitis, periodontal infection, etc.); (3) a history of digestive tract damage (such as a history of digestive tract surgery).

[0043] The subject is a mammal, preferably a rodent or a primate, more preferably a human. When the subject is a human, an adult over 18 years old is preferred.

[0044] Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof in the pharmaceutical combination is administered to the subject by injection, preferably a single injection. Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject via intramuscular injection.

[0045] Further, in the drug combination provided by the present invention, the dosage of the anti-tetanus toxin antibody or its antigen-binding fragment is 55-275 μg / kg body weight, such as 55 μg / kg, 60 μg / kg, 83 μg / kg, 100 μg / kg, 167 μg / kg or 250 μg / kg, based on the body weight of the subject; preferably, the dosage is 83-250 μg / kg; more preferably, the dosage is 167 μg / kg. Alternatively, the dosage of the anti-tetanus toxin antibody or its antigen-binding fragment is 0.6-15 mg, such as 3.3 mg, 4 mg, 5 mg, 6 mg, 10 mg or 15 mg; preferably, the dosage is 5-15 mg; more preferably, the dosage is 10 mg.

[0046] Preferably, the tetanus toxoid-containing vaccine in the pharmaceutical combination is also administered to the subject by injection. Preferably, the tetanus toxoid-containing vaccine is administered to the subject via intramuscular injection.

[0047] Furthermore, in the pharmaceutical combination provided by the present invention, the tetanus toxoid-containing vaccine is an adsorbed tetanus vaccine, which is administered via intramuscular injection at a dosage of 0.5 ml / person / time.

[0048] According to the present invention, the anti-tetanus toxin antibody or antigen-binding fragment thereof in the pharmaceutical combination is administered simultaneously with the tetanus toxoid-containing vaccine, or is administered at intervals in time. For example, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered simultaneously with the first injection of the adsorbed tetanus vaccine.

[0049] In a second aspect, the present invention provides the use of an anti-tetanus toxin antibody or an antigen-binding fragment thereof and a tetanus toxoid-containing vaccine in the preparation of a medicament for preventing or treating tetanus.

[0050] In this aspect, the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are as described above in the first aspect.

[0051] According to the present invention, the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are used to prepare a drug capable of preventing or treating tetanus. The drug may contain the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine in any form, thereby preventing or treating tetanus by administering (e.g., injecting) the drug to a subject. The drug is used for subjects who have been infected or have not yet been infected with Clostridium tetani. Theoretically, as long as a wound or a break is caused, the subject may be infected with tetanus and needs to be prevented. Common causes include: (1) a history of trauma or damage to the skin or mucous membrane (such as animal injuries, injection of drugs such as drugs, childbirth or abortion); (2) a history of bacterial infection of the skin, mucous membrane, or soft tissue (such as chronic otitis media, chronic sinusitis, periodontal infection, etc.); (3) a history of digestive tract damage (such as a history of digestive tract surgery).

[0052] The subject is a mammal, preferably a rodent or a primate, more preferably a human. When the subject is a human, an adult over 18 years old is preferred.

[0053] Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by injection, preferably a single injection. Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject via intramuscular injection.

[0054] Further, based on the subject's body weight, the dosage of the anti-tetanus toxin antibody or its antigen-binding fragment is 55-275 μg / kg body weight, such as 55 μg / kg, 60 μg / kg, 83 μg / kg, 100 μg / kg, 167 μg / kg or 250 μg / kg; preferably, the dosage is 83-250 μg / kg; more preferably, the dosage is 167 μg / kg. Alternatively, the dosage of the anti-tetanus toxin antibody or its antigen-binding fragment is 3.3-16.5 mg, such as 3.3 mg, 4 mg, 5 mg, 6 mg, 10 mg or 15 mg; preferably, the dosage is 5-15 mg; more preferably, the dosage is 10 mg.

[0055] The above-mentioned administration method and dosage of the anti-tetanus toxin antibody or antigen-binding fragment thereof can be achieved through the prepared medicine.

[0056] Preferably, the tetanus toxoid-containing vaccine is also administered to the subject by injection. Preferably, the tetanus toxoid-containing vaccine is administered to the subject via intramuscular injection.

[0057] Furthermore, the tetanus toxoid-containing vaccine is an adsorbed tetanus vaccine, which is administered via intramuscular injection, and the dosage may be 0.5 ml / person / time.

[0058] The above-mentioned administration method and dosage of the tetanus toxoid-containing vaccine can be achieved through the prepared medicine.

[0059] According to the present invention, the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are administered simultaneously or at intervals in time. For example, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered simultaneously with the adsorbed tetanus vaccine injection.

[0060] Compared with the prior art, the present invention proposes a scheme for the combined use of a tetanus toxoid vaccine and an anti-tetanus toxin antibody or an antigen-binding fragment thereof, for example, the two are implemented as a drug combination for the prevention and treatment of tetanus infection.

[0061] Tetanus toxoid vaccines are usually made from tetanus toxin produced by Clostridium tetani, which is detoxified, refined, and adjuvanted. After vaccination with this type of vaccine, the body's immune system will produce an immune response to the toxins therein, producing neutralizing antibodies against them. These antibodies bind to the toxins when the body comes into contact with the tetanus toxin, preventing them from binding to neurons and exerting toxic effects, thereby achieving the effect of preventing tetanus. Therefore, it is generally believed that the combination of antibodies against tetanus toxin with vaccines containing tetanus toxin may offset the immune effects of such vaccines. For example, exogenous antibodies against tetanus toxin may bind to antigens in the vaccine to form antigen-antibody complexes, thereby preventing the antigens in the vaccine from triggering an effective immune response in the body, thereby causing a decrease in the antibody concentration produced by the vaccine and affecting the immune effect. In addition, exogenous antibodies may also cause an immunosuppressive response in the body, such as ultimately leading to the inhibition of immune cell activation and proliferation through a variety of mechanisms, further reducing the immune effect of the vaccine.

[0062] The present application found that when a recombinant natural fully human monoclonal tetanus antibody is used in combination with a tetanus toxoid-containing vaccine, the immunogenicity of the adsorbed tetanus vaccine is basically not weakened. At the same time, the detection time of the antibody induced by the tetanus toxoid-containing vaccine is advanced, and the higher titer level can last longer. In addition, there is no obvious immunotoxicity when the two are used in combination. Without being limited to any theory, it is speculated that the combined use of the antibody and the tetanus toxoid-containing vaccine can enable the tetanus toxoid-containing vaccine to induce the production of anti-tetanus toxin neutralizing antibodies earlier, and can prolong the duration of the higher titer of anti-tetanus toxin neutralizing antibodies induced by the tetanus toxoid-containing vaccine. Therefore, the combination of a recombinant natural fully human monoclonal tetanus antibody and a tetanus toxoid-containing vaccine, for example, prepared into a combined preparation, can achieve the dual prevention and treatment effects of active and passive immunity to tetanus, and can enhance the protective effect of the tetanus toxoid-containing vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0064] Figure 1 This is the curve of the geometric mean of anti-tetanus antibody titer changing over time after administration of TRN0011 and HTIG combined vaccines to subjects.

[0065] Figure 2 The interaction of TRN0011 with DT-NFDC is shown, and the data are presented as the mean ± SD of duplicate wells.

[0066] Figure 3 Saturating concentrations of TD binding to TRN0011 are shown.

[0067] Figure 4 The in vitro binding of TRN0011 to tetanus adsorbed vaccine and tetanus-diphtheria vaccine is shown, and the data are presented as the mean ± SD of duplicate wells. DETAILED DESCRIPTION

[0068] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0069] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0070] The following examples use a fully humanized monoclonal antibody against tetanus toxin, referred to as TRN0011 (referred to as "antibody" or "previously proposed antibody" in the context of the present invention), and the amino acid sequence and other characteristics of the antibody are as defined above.

[0071] The test drug in the following examples is an injection containing TRN0011; or a placebo, which contains the same excipients as the injection but does not contain antibodies.

[0072] Regardless of the specific dosage, the groups that received TRN0011 alone are collectively referred to as "TRN0011 groups".

[0073] Anti-tetanus toxin antibody titers were measured in serum using enzyme-linked immunosorbent assay (ELISA).

[0074] In the following Examples 1 and 2, the tetanus toxoid-containing vaccine used was the adsorbed tetanus vaccine purchased from Chengdu Olin Biotechnology Co., Ltd.

[0075] The HTIG used in the following Example 3 is immunoglobulin from Hualan Biotechnology Co., Ltd.

[0076] Example 1 Animal experiments

[0077] The effects of TRN0011 combined with vaccine were studied by intramuscular administration of TRN0011 alone and in combination with adsorbed tetanus vaccine in mice.

[0078] (I) Grouping, medication and test items of mouse experiments

[0079] 1. Grouped medication

[0080] 460 healthy ICR mice were randomly divided into the following 6 groups according to body weight:

[0081] (1) solvent control group;

[0082] (2) TRN0011 low-dose group (500 μg / kg of TRN0011);

[0083] (3) TRN0011 high-dose group (2500 μg / kg of TRN0011);

[0084] (4) adsorbed tetanus vaccine administration group (1 μL / vaccine);

[0085] (5) low-dose combination group (500 μg / kg of TRN0011 + 1 μL / vaccine);

[0086] (6) High-dose combination group (2500 μg / kg of TRN0011 + 1 μL / vaccine).

[0087] The number of animals in each group was 70, 78, 78, 78, 78, and 78, respectively, with half male and half female. Each group was further divided into: main test group, a total of 240 animals, 40 animals in each of groups (1) to (6); toxicokinetics (TK) (including ADA) satellite group, a total of 112 animals, 12 animals in the (1) solvent control group, and 20 animals in the remaining (2) to (6) groups; immunotoxicity satellite group, a total of 108 animals, 18 animals in each of groups (1) to (6).

[0088] The first day of administration was defined as the first day of the experiment (D1). Each group of mice was given bilateral administration, with a single injection of 3 mL / kg of solvent (TRN0011 placebo) or corresponding dose of TRN0011 through the left hind limb muscle (D1); 20 μL / mouse sodium chloride injection or 20 μL / mouse adsorbed tetanus vaccine at a concentration of 0.05 μL (vaccine stock solution) / μL was injected through the right hind limb muscle, and the administration was given twice, with an interval of 4 weeks (D1, D29).

[0089] Determination of the dosage of adsorbed tetanus vaccine and antibodies in the above mouse experiment:

[0090] Adsorbed tetanus vaccine dosage - The clinical dosage of adsorbed tetanus vaccine is 1 dose (0.5mL), which is about 8.33μL / kg (based on an adult weight of 60kg), and the dosage for mice converted by body weight is about 0.167μL / mouse (based on a mouse weight of 20g). However, the results of the preliminary experiment conducted before this combined experiment showed that the induced polyantibody positive rates of ICR mice injected intramuscularly with 0.2, 2, and 20μL / mouse of adsorbed tetanus vaccine were 2 / 10, 10 / 10, and 10 / 10 on the 15th day of the experiment, respectively. Therefore, the minimum dose for producing effective antibodies is 0.2-2μL / mouse. In order to ensure that the vaccine can stimulate mice to produce antibodies, the selected vaccine dosage is 1μL / mouse.

[0091] TRN0011 dosage - The inventors of this application have preliminarily confirmed in other studies on the passive immune protection effect of TRN0011 against tetanus infection that the expected clinical dose of TRN0011 is 100μg / kg. However, as mentioned above, the test dose of adsorbed tetanus vaccine in mice is about 5 times the human dose. Therefore, correspondingly, the low-dose group of TRN0011 used alone or in combination used 5 times the dose, i.e. 500μg / kg, and the high-dose group used 25 times the dose, i.e. 2500μg / kg.

[0092] 2. Inspection items:

[0093] The general condition and death of the animals in the main test group were observed every day; the body weight and food intake of the animals were measured once a week; an ophthalmological examination was performed before the end of administration (D30) and the end of the recovery period (D56), and blood was collected from the abdominal aorta of 10 mice / group / sex after anesthesia for hematological (including blood coagulation) and / or blood biochemical tests, and euthanasia was performed for gross anatomical observation, organ weighing and histopathological examination.

[0094] Blood samples were collected from the animals in the TK (containing ADA) satellite group before the first administration, and 6, 24, 72, 168 hours after administration, and on D15, D22, D29 (before administration), D36, D43, and D57 to detect the TRN0011 content in serum and the polyclonal antibody titer induced by adsorbed tetanus vaccine; anti-TRN0011 antibodies (ADA) were detected before the first administration, and on D15, D29 (before administration), and D57. Cytokines (IL-2, IL-4, IL-5, IL-6, TNF-ɑ, IFN-γ) were detected in the animals in the immunotoxic satellite group before the first administration and 6 and 24 hours after the first and last administration.

[0095] (II) Results

[0096] 1. Dead / dying animals

[0097] During the trial, a total of 4 dead animals were found, all of which were satellite group animals, including 2 in the TRN0011 high-dose group, 1 in the TRN0011 low-dose group, and 1 in the combined high-dose group. They died on D1, D30, D16, and D15 after TK / ADA blood collection. It was determined that the death was caused by excessive blood loss after the blood collection operation, which was not related to the test substance.

[0098] 2. General Condition

[0099] Except for the animals that died in the satellite group, no obvious abnormal changes related to the test substance were observed in the administration site, appearance signs, general behavioral activities, mental state, glandular secretions, respiratory status, feces and urine characteristics and color, respiratory status, and genitals of mice in each group during the experiment.

[0100] 3. Body weight and food intake

[0101] At each testing point during the experiment, no obvious abnormal changes related to the test substance were found in the body weight and food intake of mice in each group.

[0102] 4. Eye examination

[0103] At the end of drug administration (D29) and the end of the recovery period (D57), examinations of the eyelids, conjunctiva, cornea, pupil, retina, fundus vascular morphology, and optic disc of mice in each drug administration group showed no obvious abnormal changes related to the test substance.

[0104] 5. Hematology (including blood coagulation)

[0105] At the end of drug administration (D30) and the end of the recovery period (D57), no obvious abnormal changes related to the test substance were found in the counts or percentages of red blood cells, white blood cells and various classified cells, PLT, PT, APTT and other hematological (including blood coagulation) indicators of mice in each group.

[0106] 6. Blood biochemistry

[0107] At the end of drug administration (D30) and recovery period (D57), the levels of AST, ALT, Urea, Crea, GLU, TC, TG, and Na in each group of mice + , K + , Cl - No obvious abnormal changes related to the test substance were found in blood biochemical indices.

[0108] 7. Cytokines

[0109] 6 and 24 hours after the first and last administration, no obvious abnormal changes related to the test substance were found in the levels of IL-2, IL-4, IL-5, IL-6, TNF-ɑ, and IFN-γ cytokines in each group of mice.

[0110] 8. Organ weight and coefficient

[0111] At the end of dosing (D30) and the end of the recovery period (D57), no obvious abnormal changes related to the test substance were found in the organ weights and coefficients of the liver, kidney, thymus, uterus, ovary, testis, epididymis and other major organs of female and male mice in each dosing group.

[0112] 9. Gross anatomy

[0113] At the end of the administration and the recovery period, gross visual observation of the mice dissected in each group showed no obvious abnormalities in the administration site, and no obvious abnormalities in the size, shape, color or texture of the major organs and tissues such as the brain, heart, liver, lungs, kidneys, adrenal glands, gastrointestinal tract, and reproductive organs.

[0114] 10. Histopathology

[0115] At the end of administration (D30) and the end of the recovery period (D57), pathological examination of mice in each administration group showed no obvious abnormalities related to the test substance TRN0011. Pathological changes such as mononuclear cell / neutrophil infiltration, muscle fiber degeneration / necrosis, lymphocyte infiltration, and fibrosis were observed in the right gastrocnemius muscle of each group (administered with adsorbed tetanus vaccine or sodium chloride injection), which were considered to be immune reactions caused by local injection or adsorption of tetanus vaccine. Compared with the solvent control group at the same time, no histopathological changes related to the test substance were found in the tissues and organs of the high-dose TRN0011 group and the high-dose combination group.

[0116] 11. Toxicokinetics

[0117] Since there was no obvious gender difference between the groups, the data of male and female animals were comprehensively analyzed:

[0118] At low dose, the average T 1 / 2 292h and 450h respectively, with an average T max 48h and 39h respectively, with an average C max The average AUC was 4.8 μg / mL and 4.2 μg / mL respectively. last They are 1936h*μg / mL and 1736h*μg / mL respectively.

[0119] At high dose, the average T 1 / 2 289h and 414h respectively, with an average T max 24h and 15h respectively, with an average C max The average AUC was 21.6 μg / mL and 21.3 μg / mL respectively. last They were 8806h*μg / mL and 7943h*μg / mL respectively.

[0120] Compared with TRN0011 alone, the average C max The ratios (low-dose combination group / TRN0011 low-dose group, high-dose combination group / TRN0011 high-dose group) were 0.87 and 0.99, respectively, and the average AUC last The ratios (low-dose combination medication group / low-dose TRN0011 group, high-dose combination medication group / high-dose TRN0011 group) were 0.90 and 0.90, respectively, with no significant difference.

[0121] 12. Adsorption of tetanus vaccine to induce polyclonal antibodies

[0122] Starting from D15, the antibody titer of the adsorbed tetanus vaccine group was higher than the titer threshold, with a median titer of 6619. After the first dose, it showed a trend of first increasing and then decreasing. The titer level increased sharply on D36 (7 days after the second dose), and reached the maximum on D43 (14 days after the second dose), with a median titer of 216699. The titer decreased significantly on D57.

[0123] Starting from D7, the first dose of the combination drug, the antibody titers of the low- and high-dose groups were higher than the titer threshold, and the median titers were 275 and 145, respectively. After the first dose, the titers first increased and then decreased, and reached the maximum on D43 and D36, respectively, with the median antibody titers of 394455 and 262850, respectively. The antibody titer decreased on D57, but the decrease was smaller than that of the adsorbed tetanus vaccine group.

[0124] The results showed that antibodies began to appear in the animals on D15 after the first administration of adsorbed tetanus vaccine alone; the antibody titer level increased sharply on D36 (7 days after the second administration), reached the maximum titer level on D43 (14 days after the second administration), and the antibody titer dropped significantly on D57. When used in combination with TRN0011, the adsorbed tetanus vaccine induced polyantibodies to appear earlier and the polyantibody titer could last longer.

[0125] 13. Anti-TRN0011 antibody

[0126] In the solvent control group, the anti-drug antibody (ADA) test results in the sera of all animals before administration were negative.

[0127] After intramuscular injection of TRN0011 in mice, 0 / 10, 1 / 10 and 1 / 10 ADA appeared in the low-dose group at D15, D29 and D57, respectively, and 1 / 10, 3 / 10 and 3 / 10 ADA appeared in the high-dose group at D15, D29 and D57, respectively; after intramuscular injection of TRN0011 combined with adsorbed tetanus vaccine in mice, 7 / 10, 7 / 10 and 6 / 10 ADA appeared in the low-dose group at D15, D29 and D57, respectively, and 4 / 10, 6 / 10 and 6 / 10 ADA appeared in the high-dose group at D15, D29 and D57, respectively.

[0128] The experimental results showed that after a single intramuscular injection of 500 and 2500 μg / kg of TRN0011 within 4 weeks, or a single intramuscular injection of 500 and 2500 μg / kg of TRN0011 combined with two intramuscular injections of 1 μL / mouse adsorbed tetanus vaccine, no animal deaths related to the test substance were observed after 4 weeks of recovery. No obvious abnormalities related to the test substance were observed in clinical observations, body weight, food intake, hematology, coagulation, blood biochemistry, cytokines, gross anatomy, organ weights and coefficients, and pathological examinations. The no observed toxic effect dose (NOAEL) was 2500 μg / kg.

[0129] After the first administration, TRN0011 exposure C in male and female mice was within the range of 500-2500 μg / kg. max and AUC last Both increased with the increase of dose, and no significant gender difference was observed. After co-administration of 1 μL / head of adsorbed tetanus vaccine, there was no significant effect on the exposure of TRN0011 at different doses.

[0130] Of particular note in the above data is that the first detection time of antibodies induced by adsorbed tetanus vaccine alone was D15, and after the first administration, it showed a trend of first increasing and then decreasing, and it increased sharply on D36 (7 days after the second administration), and a downward trend appeared on D57. After the combined administration of 500 and 2500 μg / kg doses of TRN0011, the first detection time of antibodies induced by adsorbed tetanus vaccine was D8, and the antibody titer was higher than that of the group using adsorbed tetanus vaccine alone, indicating that TRN0011 can induce animals to produce antibodies in advance with adsorbed tetanus vaccine, and at the same time increase the antibody titer level induced by adsorbed tetanus vaccine. In addition, the data suggest that the combination of TRN0011 and adsorbed tetanus vaccine has no negative impact on the production of antibodies induced by adsorbed tetanus vaccine.

[0131] After combined use with the vaccine, the titer level and positive rate of anti-TRN0011 antibodies increased, but had no effect on drug exposure and no related findings such as immunotoxicity.

[0132] (III) Calculation of human dose

[0133] The dosage of TRN0011 combined with adsorbed tetanus vaccine was calculated using the following formula:

[0134] Human dose (mg / kg) = W × mouse dose (mg / kg), where W (conversion factor) = 0.11

[0135] Substituting the low and high doses of TRN0011 in the mouse combination experiment into the formula, it is calculated that when used in combination with adsorbed tetanus vaccine, the human dose range of TRN0011 is 55μg / kg-275μg / kg, i.e. 3.3mg-16.5mg (based on an adult weight of 60kg). Furthermore, based on clinical doses and actual applications, the preferred human dose is 167μg / kg, i.e. 10mg.

[0136] In summary, the time of first detection of induced antibodies in animals in the above-mentioned combination drug (low and high dose) groups was Day 7, and the median peak titers were 394455 and 262850 respectively; while the time of first detection of induced antibodies in animals after administration of adsorbed tetanus vaccine alone was Day 15, and the median peak titer was 216699. From this result, it can be seen that compared with the use of vaccines alone, the combination of TRN0011 and vaccines can enhance the body's response to vaccines, advance the time of vaccine-induced antibody production, and at the same time increase the titer of induced antibodies.

[0137] Moreover, whether the vaccine is used alone or in combination, the antibody titer induced by the vaccine has experienced a process of first rising and then falling. At the last time point of the experimental test (Day 57), the median antibody titers of the low-dose and high-dose combination groups were 274540 and 227087, respectively, while the median antibody titer of the adsorbed tetanus vaccine group was 85046. From this result, it can be seen that the combination of TRN0011 and the vaccine can prolong the duration of the body's response to the vaccine and prolong the time that the vaccine-induced antibodies are maintained at a higher titer level.

[0138] In addition, the average T 1 / 2 The average T 1 / 2 The results show that the combination of TRN0011 and vaccine can prolong the half-life of TRN0011, which is beneficial to maintain TRN0011 at a higher level in the body.

[0139] In general, when TRN0011 is used in combination with adsorbed tetanus vaccine, TRN0011 will not weaken the immunogenicity of adsorbed tetanus vaccine, will not increase adverse reactions, and has good safety.

[0140] Example 2 DDI Experimental Study

[0141] A randomized, open-label, parallel-design pharmacokinetic and pharmacodynamic interaction study of TRN0011 and adsorbed tetanus vaccine alone or in combination was conducted in healthy Chinese subjects.

[0142] Purpose of the study:

[0143] Main purpose:

[0144] To evaluate the effect of TRN0011 combined with adsorbed tetanus vaccine on the PD characteristics of adsorbed tetanus vaccine in healthy adult subjects;

[0145] Secondary purpose:

[0146] To evaluate the safety and tolerability of TRN0011 alone or in combination with adsorbed tetanus vaccine in healthy adult subjects.

[0147] 1. Research design

[0148] A total of 60 healthy subjects were enrolled in this study and divided into three parallel groups, with 20 subjects in each group. The qualified subjects were randomly assigned to TRN0011 alone (referred to as TRN0011 group), vaccine alone (referred to as vaccine group), or TRN0011 and vaccine combination (referred to as combination group) in a 1:1:1 ratio. The specific dosing information of each group is shown in Table 1.

[0149] Table 1: Specific dosing information for each group

[0150]

[0151] The study includes the selection period (day -28 to day -2), the hospitalization period (day -1 to day 4), and the follow-up period (day 5 to day 106). Qualified subjects were admitted to the research center on day -1 and completed relevant safety checks and random grouping. On day 1, serum samples of subjects were collected before administration for PK, PD and anti-drug antibody (ADA) analysis, and the administration of the corresponding trial drugs was completed according to random grouping. The subjects were hospitalized in the research center until they left after completing relevant safety checks and PK and PD sampling on day 4. From day 5 to day 106, all subjects returned to the research center at the specified time (i.e., day 6, 8, 11, 15, 18, 22, 29, 57, 85 and 106) for safety assessment and / or PK, PD and ADA blood sample collection. The above PK sample collection is limited to the TRN0011 group and the combination group, and the PD samples include TRN0011 PD and vaccine PD of all groups.

[0152] (II) Selection of participants

[0153] This study is a study on the interaction between TRN0011 and adsorbed tetanus vaccine when used in combination. The indication of TRN0011 is intended to prevent tetanus when there is an unclean or contaminated wound after trauma. The population of patients for whom TRN0011 is indicated is mostly healthy except for trauma. At the same time, the population for which adsorbed tetanus vaccine is indicated is also mainly healthy people. Therefore, this study recruited healthy adult subjects to participate in this study.

[0154] The study participants were selected according to the following criteria.

[0155] 1. Healthy men or women aged between 18 and 55 years (inclusive) at the time of enrollment;

[0156] 2. Body mass index (BMI) between 19.0 and 26.0 kg / m 2 Within the range (including the limit), body weight ≥50kg.

[0157] (III) Evaluation of research indicators: the impact of TRN0011 combined with vaccines on vaccines

[0158] 1) Vaccine PD evaluation

[0159] Primary End Point

[0160] Percentage of subjects with serum anti-tetanus antibody levels ≥ 0.1 IU / mL 28 days after immunization with tetanus vaccine adsorbed alone or TRN0011 combined with tetanus vaccine adsorbed

[0161] Secondary End Points

[0162] Percentage of subjects with serum anti-tetanus antibody levels ≥ 0.1 IU / mL at all visit points except day 28 after immunization with tetanus vaccine adsorbed alone or TRN0011 combined with tetanus vaccine adsorbed

[0163] Anti-tetanus antibody levels, baseline elevations, and multiple increases in serum at each visit point after immunization with adsorbed tetanus vaccine alone and TRN0011 combined with adsorbed tetanus vaccine

[0164] 2) Safety evaluation

[0165] Adverse events (AEs), safety laboratory tests (blood routine, blood biochemistry and urine routine), 12-lead electrocardiogram, physical examination and vital signs

[0166] 4. Statistical methods

[0167] Full Analysis Set (FAS): All randomized subjects who received the investigational drug.

[0168] Safety analysis data set (SS): all subjects who were randomized, received the investigational drug, and had post-drug safety evaluation data.

[0169] PD analysis data set (PDS): All subjects who were randomized, received investigational drugs, had at least one evaluable PD data, and did not have any events that affected the PD results and detection.

[0170] (V) Analysis of vaccine PD after combined use

[0171] Primary endpoint analysis:

[0172] According to the dosing group, the number, percentage and 90% CI (based on the Clopper-Pearson method) of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL 28 days after administration were calculated, and the difference in the percentage of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL between the two groups and the 90% CI of the difference were provided (based on the stratified Miettinen-Nurminen method, stratified according to the anti-tetanus antibody titer test results at the time of random stratification).

[0173] Secondary endpoint analysis:

[0174] The same statistical method as the primary endpoint was used to analyze the percentage of subjects with anti-tetanus antibody levels ≥0.1 IU / mL and ≥1.0 IU / mL at other visit time points after drug administration.

[0175] According to the actual medication groups, the anti-tetanus antibody levels at other visit time points after medication, the increased values ​​compared with the baseline, and the multiples of increase compared with the baseline were listed and summarized descriptively.

[0176] (VI) Results (Effects of TRN0011 combined with vaccines on vaccines)

[0177] 1) Primary endpoint analysis

[0178] As shown in Table 2, on the 28th day after administration, the proportion of subjects with serum anti-tetanus antibody levels ≥ 0.1 IU / mL in both the combination group and the vaccine group was 90% (18 / 20), and the inter-group difference (combination group-vaccine group) was 0.0% (90% CI: -16.2, 21.3). No significant difference was observed between the two groups.

[0179] Table 2: Proportion of subjects with anti-tetanus antibody levels ≥ 0.1 IU / mL in the combination group and vaccine group 28 days after administration (PDS)

[0180]

[0181] Note: PDS: pharmacodynamic analysis data set; CI: confidence interval.

[0182] Confidence intervals for proportions are based on the Clopper–Pearson method; between-group differences, which represent the proportion in the combination group minus the proportion in the vaccine group, are based on the stratified Miettinen–Nurminen method.

[0183] 2) Secondary endpoint analysis

[0184] As shown in Table 3, within 5 days after administration, no subject in the vaccine group had an anti-tetanus antibody level ≥ 0.1 IU / mL. Starting from 7 days after administration, the proportion of such subjects gradually increased, reaching 80% (16 / 20) on 17 days after administration. In the combination group, the proportion of anti-tetanus antibody levels ≥ 0.1 IU / mL gradually increased 24 hours after administration, reaching 70% (14 / 20) on 5 days after administration, and reached the highest level (94.4%-95.0%) during the 14-21 days.

[0185] At 56 days after administration and later time points, the proportion in the combination group was slightly lower than that in the vaccine group, but the 90% CI of the two groups overlapped to a large extent, indicating that there was no significant difference between the two administration groups.

[0186] From the changes in the geometric mean titers of anti-tetanus antibodies in the two groups over time, it can be seen that both the single vaccine and the TRN0011 combined vaccine showed that anti-tetanus antibodies began to increase rapidly around 7 days after administration, and both reached a high level. Before 14 days after administration, the geometric mean titer of anti-tetanus antibodies in the combination group was higher than that in the vaccine group, and there was no significant difference between the two administration groups.

[0187] Table 3: Proportion of subjects with anti-tetanus antibody levels ≥ 0.1 IU / mL at visit points beyond day 28 (PDS)

[0188]

[0189]

[0190] Therefore, the above results show that when 10mg TRN0011 is used in combination with tetanus vaccine, it will not have a clinically significant adverse effect on the active immune effect of the vaccine. When the two are used in combination, the level of tetanus antibodies in the body can increase rapidly after administration, and can provide protection in most people 5 days after administration. Compared with using the vaccine alone, the combination of the two can enable the subjects to achieve protection earlier.

[0191] Example 3 Effect of TRN0011, HTIG (human immunoglobulin) and tetanus vaccine on vaccines

[0192] A multicenter, randomized, double-blind, parallel, positive-controlled trial to compare the efficacy and safety of passive immunization with TRN0011 injection and tetanus human immunoglobulin in preventing tetanus infection (I) Study design

[0193] This is a multicenter, randomized, double-blind, active-controlled, parallel-group trial conducted in subjects with unclean or contaminated wounds due to various traumas who require passive immunization against tetanus, aiming to compare the efficacy and safety of TRN0011 10 mg and HTIG 250 IU in preventing tetanus infection in this study population.

[0194] In this study, a total of 71 subjects received adsorbed tetanus vaccine while receiving the investigational drug TRN0011 and HTIG, of which 47 and 24 subjects received the vaccine simultaneously. According to the instructions for adult adsorbed tetanus vaccine, the second dose of vaccination was given 4-8 weeks after the first dose. A total of 60 subjects completed the second dose of vaccination, 41 (9.3%) in the TRN0011 group and 19 (8.6%) in the HTIG group. Most of the second dose of vaccine was completed at the 28th day visit after administration.

[0195] (II) Selection of participants

[0196] TRN0011 is a passive tetanus immunization agent, intended for the prevention of tetanus when there is an unclean or contaminated wound after trauma. Therefore, the subjects recruited in this study are patients who have unclean or contaminated wounds caused by various traumas such as cuts, burns, traffic injuries, animal bites, etc. and need to receive passive immunization to prevent tetanus infection.

[0197] The study participants were selected mainly according to the following criteria.

[0198] 1. Aged ≥ 18 years old, male or female;

[0199] 2. Subjects who have unclean or contaminated wounds caused by various traumas such as cuts, burns, traffic injuries, animal scratches and bites and need to receive passive immunization to prevent tetanus infection;

[0200] 3. The time from the above-mentioned injury to the administration of the investigational drug is expected to be no more than 24 hours.

[0201] (III) Investigational Drugs

[0202] Table 4

[0203] Trial Drugs Comparator drugs Product Name TRN0011 (injection) Tetanus immune globulin (HTIG) Specification 10mg / 0.5mL / bottle 250IU / bottle Dosage Buttocks intramuscular injection Intramuscular injection into the deltoid muscle of the upper arm

[0204] 4. Analyzing the Dataset

[0205]

[0206] 5. Effectiveness endpoint analysis

[0207] Subgroup analysis was performed on the 71 subjects who received the combination vaccine in this study. Based on the results of the HTIG PD bioassay, the anti-tetanus antibody titers at various time points after administration were compared between the TRN0011 group and the HTIG group, including the anti-tetanus antibody GMT and the proportion of subjects with an anti-tetanus antibody titer ≥ 0.1 IU / mL.

[0208] (VI) Results Analysis

[0209] At each time point after administration (12th hour and 3rd, 7th, 28th and 90th days), the anti-tetanus antibody GMT of subjects receiving the TRN0011 combination vaccine (TRN0011 combination group) was higher than that of subjects receiving the HTIG combination vaccine (HTIG combination group), and the proportion of subjects with anti-tetanus antibody ≥ 0.1 IU / mL was also generally higher than that of the HTIG combination group.

[0210] Among them, at the recognized key time point for evaluating the immune effect of tetanus vaccine, that is, on the 28th day after administration, the anti-tetanus antibody GMT of the TRN0011 combination group reached 2.09IU / mL (95% CI: 1.35, 3.25), while that of the HTIG combination group was 0.504IU / mL (95% CI: 0.230, 1.10). In terms of the proportion of subjects with anti-tetanus antibody titers ≥ 0.1IU / mL on the 28th day after administration, the TRN0011 combination group was 100% (95% CI: 92.5%, 100.0%), while that of the HTIG combination group was 87.5% (95% CI: 67.6%, 97.3%).

[0211] On the 90th day after administration (after the second dose of vaccination), the GMT of anti-tetanus antibodies in the TRN0011 combination group and the HTIG combination group were 2.29 IU / mL (95% CI: 1.60, 3.27) and 1.24 IU / mL (95% CI: 0.555, 2.77), respectively. The proportion of subjects with anti-tetanus antibody titers ≥ 0.1 IU / mL was 100% (95% CI: 92.5%, 100.0%) and 91.7% (95% CI: 73.0%, 99.0%), respectively. This suggests that the anti-tetanus antibody levels of subjects receiving the TRN0011 combination vaccine were higher than those of subjects receiving the HTIG combination vaccine. The geometric mean time curve of the anti-tetanus antibody levels of the two groups under the combination condition is shown in Figure 1 ( Figure 1 In the figure, a is a linear coordinate graph and b is a logarithmic coordinate graph).

[0212] Therefore, the above results show that after 10mg TRN0011 combined with vaccine, the level of anti-tetanus antibodies is higher than that of 250IUHITG combined with vaccine, indicating that compared with the combination of HTIG and vaccine, the combination of TRN0011 and vaccine has less inhibitory effect on the active immunity of the vaccine.

[0213] Note: CI = confidence interval; GMT = geometric mean titer.

[0214] Example 4 Comparison of in vitro interactions between TRN0011 and tetanus vaccine and tetanus-diphtheria adsorbed vaccine

[0215] The above experiments show that the combination of TRN0011 and tetanus vaccine (monovalent vaccine) has an unexpected effect. In order to further verify the effect of TRN0011 combined with bivalent vaccines (such as tetanus diphtheria adsorbed vaccine), the following exploratory experiments were carried out in vitro to understand whether there are differences between the combination of TRN0011 and tetanus diphtheria adsorbed vaccine (bivalent vaccine) and the combination of TRN0011 and tetanus adsorbed vaccine (monovalent vaccine).

[0216] (I) List of abbreviations

[0217]

[0218]

[0219] (II) Key Reagents

[0220]

[0221] (III) Experimental methods and results

[0222] 1. Evaluation of the binding interaction between diphtheria toxoid (DT-NIFDC) and TRN0011 in direct ELISA

[0223] 1. Steps:

[0224] Step 1: DT-NIFDC of 11 series gradient concentrations (dilution factor: 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400) was coated on a 96-well ELISA plate at 100 μL / well in weakly acidic (pH = 6) coating solution, incubated overnight at 2-8°C, and washed three times with 300 μL / well of 0.05% PBST.

[0225] Step 2: Add 250 μL / well of 3% BSA to block the microwells, incubate at 25°C for 2 hours, and then wash 3 times according to step 1.

[0226] Step 3: Dilute anti-DT-PAb-HRP and TRN0011-HRP with 3% BSA to 500 ng / mL, add 100 μL / well to different columns of the ELISA plate, incubate at 25°C for 1 hour, and then wash 3 times according to step 1.

[0227] Step 4: Add 100 μL / well of TMB colorimetric solution and incubate at room temperature in the dark for 10 minutes.

[0228] Step 5: Add 50 μL / well of stop solution to stop color development and immediately read the plate to collect data.

[0229] 2. Results

[0230] The experiment was repeated three times, and representative results are shown in Figure 2As shown. The red curve is the reaction curve of anti-DT-PAb-HRP binding to DT-NIFDC (11 gradient dilutions: 100-102400). As the dilution factor of DT-NIFDC increased from 100 to 102400, the OD value (red curve) decreased from 3.46 to 0.01, forming a good linear fit. This shows that DT-NIFDC was successfully coated on the 96-well plate within the dilution concentration range. However, under the same coating conditions, when TRN0011-HRP was used instead of anti-DT-PAb-HRP, the binding curve (green) remained at an OD value of around 0.01, with no dose response, indicating that TRM0011 did not bind to DT-NIFDC and there was no interaction between them.

[0231] 2. Determination of the saturation concentration of TRN0011 binding to Td in an indirect ELISA

[0232] 1. Steps:

[0233] Step 1: Dilute Td to three different multiples (133 times, 266 times and 1333 times) with weakly acidic solution (pH = 6), add 100 μL / well to 96-well ELISA coating, and ～ After overnight coating at 8°C, the plates were washed three times with 0.05% PBST at 300 μL / well.

[0234] Step 2: Add 250 μL of 3% BSA solution to each well for blocking, incubate at 25°C for 2 hours, and then wash three times according to the method in step 1.

[0235] Step 3: TRN0011 was prepared into 12 different concentrations (0, 9.77, 19.53, 39.06, 78.125, 156.25, 625, 1250, 2500, 5000, 10000, 20000 ng / mL) and added to the coated microplate, incubated at 25°C for 2 hours, and then washed three times according to the method in step 1.

[0236] Step 4: Dilute TRN0011-PAb-HRP to 500 ng / mL, prepare with 3% BSA solution, add to the treated microwells, incubate at 25°C for 1 hour, and then wash three times according to the method in step 1.

[0237] Step 5: Add 100 μL / well of TMB colorimetric solution and incubate at room temperature for 10 minutes in the dark.

[0238] Step 6: Add 50 μL / well of stop solution to stop color development and collect data immediately.

[0239] 2. Results

[0240] The experiment was repeated three times and representative results are shown in Figure 3 As shown. When TRN0011 increased from 0 to 20 μg / mL, the three 4-parameter logistic curves (4-PL curves) corresponding to the three different dilutions of Td coated on the plate all showed a good gradient response. When Td was diluted 133 times, the OD value increased from 0.016 to 2.059 (red curve). When Td was diluted 266 times, meaning less antigen, the OD value rose from 0.017 to 1.287. When Td was diluted 1333 times (even less antigen), the OD value rose from 0.013 to a lower 0.678. In addition, on all three curves, when the concentration of TRN0011 was higher than 1 μg / mL, the OD value tended to stabilize, indicating that under this Td coating condition, TRN0011 at a concentration higher than 1 μg / mL was excessive.

[0241] 3. Study whether TRN0011 interacts with DT-Td in another indirect ELISA

[0242] 1. Steps

[0243] Step 1: Same as Experiment 2, three different dilution multiples (133-fold, 266-fold and 1333-fold) of Td were used to coat a 96-well ELISA plate at 100 μL / well in acidic coating solution, incubated at 2°C to 8°C overnight, and then washed three times with 0.05% PBST at 300 μL / well.

[0244] Step 2: Add 250 μL of 3% BSA solution to each well, incubate at 25°C for 2 hours for blocking, and then wash three times according to the method in step 1.

[0245] Step 3: Prepare TRN0011 into 6 solutions with different concentrations (0, 12.5, 25, 50, 100, 200 μg / mL) and add them to the same well at the same time with anti-DT-PAb-HRP (1000 ng / mL), incubate at 25°C for 1 hour, and then wash three times according to the method in step 1.

[0246] Step 4: Add 100 μL / well TMB colorimetric solution and incubate at room temperature for 10 minutes in the dark.

[0247] Step 5: Add 50 μL / well stop solution to terminate the color reaction, and immediately read the plate on the microplate reader to collect data.

[0248] 2. Results

[0249] The experiment was repeated three times, and representative results are shown in Table 7. The three OD value levels (1.0, 0.5, and 0.1) correspond to three different dilutions of Td coating solution (133 times, 266 times, and 1333 times). When Td was diluted 133 times, the OD value peaks of all wells were about 1.0, regardless of whether there was an excess of TRN0011. When Td was diluted 266 times, the OD value peaks of all wells were about 0.5, regardless of whether there was an excess of TRN0011. When Td was diluted 1333 times, the OD value peaks of all wells were about 0.1, regardless of whether there was an excess of TRN0011. These results indicate that the detection of DT-Td by anti-DT-PAb-HRP is not affected by excess TRN0011, thus proving that TRN0011 does not bind to DT-Td in vitro, that is, the two do not interact.

[0250] Table 7 TRN0011 does not bind to DT-Td in vitro

[0251]

[0252]

[0253]

[0254] 4. Effect of TRN0011 combined with tetanus vaccine and diphtheria-tetanus vaccine

[0255] 1. Steps

[0256] Step 1: Two vaccines (Td and TT Olymvax) were diluted in a weak alkaline (pH = 8) solution in a gradient manner (dilution factor 200 to 12800 times) in a microplate, coated overnight at low temperature (2-8°C), and then washed three times with 0.05% PBST at 300 μL / well.

[0257] Step 2: Add 250 μL / well of 3% BSA for blocking, incubate at 25°C for 2 hours, and then wash 3 times according to the method in step 1.

[0258] Step 3: Add TRN0011-HRP (66.67 ng / mL), incubate at 25°C for 1 hour, and then wash 3 times according to the method in step 1.

[0259] Step 4: Add 100 μL / well of TMB colorimetric solution and incubate at room temperature in the dark for 10 minutes.

[0260] Step 5: Add 50 μL / well of stop solution to terminate the color reaction and read the plate immediately to collect data.

[0261] 2. Results

[0262] The experiment was repeated three times, and representative results are shown in Figure 4 As shown. The red response curve comes from the combination of TRN0011-HRP and coated TT-Olymvax (dilution range: 200-12800). As the dilution factor increases from 200 to 12800, the OD value decreases from 2.6 to 0.01, forming a good linear fit. The green response curve comes from the combination of TRN0011-HRP and coated TT-Td (dilution factors: 200, 400, 800, 1600, 3200, 6400, 12,800). As the dilution factor increases from 200 to 12800, the OD value on the curve also decreases from 2.6 to 0.01.

[0263] Overall, both curves showed the same binding trend, indicating that TRN0011 bound similarly to tetanus toxoid in both vaccines.

[0264] Therefore, from the above four experimental results, we can conclude that (1) TRN0011 does not bind to the diphtheria toxoid in the bivalent vaccine. (2) Compared with the monovalent vaccine, TRN0011 exhibits similar binding to the tetanus toxoid in the bivalent vaccine, that is, the drug binding effects of TRN0011 in vivo with the monovalent vaccine and the bivalent vaccine are similar.

[0265] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. A pharmaceutical combination for preventing or treating tetanus, comprising: an antibody or an antigen-binding fragment thereof against tetanus toxin; and a vaccine containing tetanus toxoid; in, The anti-tetanus toxin antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain CDRs, namely HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain CDRs, namely LCDR1, LCDR2, and LCDR3, wherein: The HCDR1 comprises the amino acid sequence shown in SEQ ID NO.1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO.2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.6, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.

8.

2. The drug combination according to claim 1, characterized in that The anti-tetanus toxin antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.4, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

9.

3. The drug combination according to claim 1 or 2, characterized in that: The anti-tetanus toxin antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is of IgG, IgA, IgM, IgD or IgE type, more preferably of IgG type; Preferably, the light chain constant region is of the kappa or lambda type.

4. The pharmaceutical combination according to any one of claims 1 to 3, characterized in that The anti-tetanus toxin antibody is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody; or, the anti-tetanus toxin antibody is a monoclonal antibody or a single-chain antibody; Preferably, the anti-tetanus toxin antibody comprises a heavy chain (HC) and a light chain (LC), the heavy chain comprises the amino acid sequence shown in SEQ ID NO.5, and the light chain comprises the amino acid sequence shown in SEQ ID NO.

10.

5. The pharmaceutical combination according to any one of claims 1 to 4, characterized in that The tetanus toxoid-containing vaccine is an adsorbed tetanus monovalent or multivalent vaccine.

6. The pharmaceutical combination according to any one of claims 1 to 4, characterized in that The tetanus toxoid-containing vaccine is a monovalent vaccine, such as an adsorbed tetanus vaccine, and the potency of the tetanus toxoid contained in a single human dose is not less than 40 IU; Alternatively, the tetanus toxoid-containing vaccine is a bivalent vaccine, such as an adsorbed tetanus-diphtheria combined vaccine; Alternatively, the tetanus toxoid-containing vaccine is a multivalent vaccine, such as a diphtheria, tetanus and pertussis adsorbed trivalent vaccine; a diphtheria, tetanus, pertussis, hepatitis B (recombinant) and polio pentavalent vaccine; a diphtheria, tetanus, pertussis, inactivated poliomyelitis, Haemophilus type B conjugate vaccine and hepatitis B hexavalent vaccine.

7. Use of an antibody or antigen-binding fragment thereof against tetanus toxin and a vaccine containing tetanus toxoid in the preparation of a medicament for preventing or treating tetanus; in, The anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are as defined in any one of claims 1 to 6, respectively.

8. The use according to claim 7, characterized in that The medicine comprises the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine.

9. The use according to claim 7 or 8, characterized in that The anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by injection, preferably a single injection; preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject via intramuscular injection; Preferably, the tetanus toxoid-containing vaccine is administered to the subject by injection; preferably, the tetanus toxoid-containing vaccine is administered to the subject via intramuscular injection.

10. The use according to any one of claims 7 to 9, characterized in that The anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are administered simultaneously; or are administered at intervals in time.

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