Combined vaccine for preventing or treating tetanus

Abstract

Disclosed in the present invention is a pharmaceutical combination comprising an adsorbed tetanus vaccine and an antibody or an antigen-binding fragment thereof against tetanus toxin. The pharmaceutical combination is used for preventing and / or treating tetanus.

Description

A combined vaccine for preventing or treating tetanus

[0001] Cross-reference to Related Applications

[0002] This patent application claims the priority benefit of Chinese Patent Application No. CN202411344714.4, filed on September 25, 2024, the entire contents of which are incorporated herein for all purposes. TECHNICAL FIELD

[0003] The present application relates to biological vaccine, in particular, the present application relates to a kind of drug combination for preventing and treating tetanus and application thereof. BACKGROUND

[0004] Tetanus is caused by tetanus toxin produced by Clostridium tetani, which causes tetanic muscle spasm and leads to an acute infectious disease. Clostridium tetani exists in large quantities in the intestines of humans and animals, and often invades the human body through skin or mucous membrane wounds after being contaminated by feces in the soil, and produces tetanus toxin by growing and breeding in anoxic environment. Tetanus toxin is a neurotoxic protein that blocks the transmission of information between nerves and muscles by hindering the release of inhibitory neurotransmitters, causing muscle spasm, cranial nerve paralysis and central nervous system dysfunction.

[0005] Depending on the level of local health care, the prevalence and mortality of tetanus patients vary greatly around the world, but the mortality rate of those who have not received treatment, especially the elderly and infants, is almost 100%. Tetanus toxin is highly toxic and acts rapidly, and when a patient is diagnosed with Clostridium tetani infection, there is usually a large amount of bacteria and toxin in the body, and antibiotics cannot save the patient's life. Therefore, since 1978, China has implemented childhood immunization, and the combined vaccine for pertussis, diphtheria and tetanus has been included in the routine childhood immunization program. In addition, after the disposal of wounds caused by trauma, tetanus immunization preparations are often used to prevent tetanus infection.

[0006] Tetanus immunization agents are divided into active immunization agents and passive immunization agents. Tetanus active immunization agents are tetanus toxoid-containing vaccines (TTCV). TTCV includes tetanus vaccine, adsorbed (TT), diphtheria and tetanus combined vaccine, adsorbed (DT), diphtheria, tetanus and acellular pertussis combined vaccine, adsorbed (DTaP), etc. Tetanus passive immunization agents include tetanus antitoxin (TAT), equine anti-tetanus F(ab')2 (F(ab')2) and human tetanus immunoglobulin (HTIG), etc.

[0007] However, it is found in clinical application that the protection time of tetanus active immunization agents is relatively long, but the induction of the body to produce antibodies against tetanus toxoid in the body is slow. Generally, the antibodies induced after injection for about 2 weeks reach a protective level. Moreover, patients who have never received tetanus immunization need to be injected continuously for 3 doses to reach sufficient antibody titer. Furthermore, there are often large differences in potency between batches of agents, and it is difficult to control quality.

[0008] Therefore, there is still a need in the art for a new vaccine for preventing or treating tetanus without adverse effects. SUMMARY

[0009] In order to solve the above technical problems, the purpose of the present application is to provide a combination of tetanus active immunization agents and passive immunization agents, and the application of the combination in the prevention and / or treatment of tetanus.

[0010] The present application particularly relates to the following terms.

[0011] As used herein, the term "tetanus" refers to a specific infection caused by Clostridium tetani invading the human body and growing and breeding in anoxic environment to produce toxins (tetanus toxin) and cause muscle spasm. According to the 2020 edition of "Non-neonatal tetanus diagnosis and treatment standard" issued by the National Health Commission, the diagnosis of non-neonatal tetanus mainly depends on the typical clinical manifestations, and at least one of the following manifestations is required: (1) trismus or risus sardonicus; (2) painful muscle spasm. For patients with questionable diagnosis, a tongue depressor test can be used, which involves using a tongue depressor to gently touch the posterior pharynx of the patient, and a positive result is a bite muscle reflexive spasm rather than a normal reflexive nausea. Tetanus can cause temporary functional changes in the central nervous system, manifested as generalized skeletal muscle spastic contraction and paroxysmal spasm. In particular, the symptoms of tetanus are facial expression muscles, neck, back, abdominal, limb muscles, diaphragm spasm.

[0012] As used herein, the term "tetanus toxoid-containing vaccine" refers to a vaccine containing tetanus toxoid as an effective component, including but not limited to monovalent vaccines (such as adsorbed tetanus vaccine), bivalent vaccines (such as adsorbed tetanus and diphtheria combined vaccine), multivalent vaccines (such as adsorbed acellular pertussis, diphtheria, tetanus combined vaccine), etc. The above vaccines can be prepared by the following process: culturing Clostridium tetani and other strains in a suitable culture medium to produce toxins, which are refined, detoxified (for example, formaldehyde detoxification), and adsorbed (for example, aluminum hydroxide adsorption) to prepare tetanus and other toxoids, which are used as antigens for preparation.

[0013] A specific example of the "tetanus toxoid-containing vaccine" presented in the present application is an adsorbed tetanus vaccine (monovalent vaccine), for example, as described above, aluminum hydroxide adjuvant is added during the preparation of the vaccine to prepare an aluminum adsorbed vaccine. This vaccine is also known as "adsorbed tetanus vaccine" or "tetanus adsorbed vaccine".

[0014] Another specific example is an adsorbed tetanus and diphtheria combined vaccine (bivalent vaccine), for example, as described above, Clostridium tetani and Corynebacterium diphtheriae are cultured in a suitable culture medium to produce tetanus toxin and diphtheria toxin, which are refined, detoxified, and adsorbed (for example, aluminum phosphate adjuvant) to prepare an adsorbed tetanus and diphtheria combined vaccine, also known as "tetanus and diphtheria adsorbed vaccine".

[0015] In another specific example, the "tetanus toxoid-containing vaccine" can also be a multivalent vaccine, such as diphtheria, tetanus, and pertussis adsorbed trivalent vaccine; diphtheria, tetanus, pertussis, hepatitis B (recombinant), and polio pentavalent vaccine; diphtheria, tetanus, pertussis, inactivated polio, Haemophilus influenzae type b conjugate vaccine, and hepatitis B hexavalent vaccine, etc.

[0016] As used herein, the term "combination therapy" refers to the simultaneous or sequential use of two or more agents to achieve a particular purpose or effect.

[0017] 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 reference is made herein to "antigen-binding fragment", the antigen refers to "tetanus toxin", which is a secreted protein produced by Clostridium tetani under anaerobic conditions.

[0018] As used herein, the term "mammal" refers to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, mice, pigs, monkeys, apes, and the like. The mammal preferably refers to a rodent or a primate, more preferably to a human. In case the subject is a human, an adult of 18 years of age or older is preferred.

[0019] The technical solutions of the present application are as follows.

[0020] In a first aspect, the present application provides a pharmaceutical combination comprising: an antibody or antigen-binding fragment thereof against tetanus toxin; and, a tetanus toxoid-containing vaccine. The pharmaceutical combination is used for preventing and / or treating tetanus.

[0021] As to the antibody or antigen-binding fragment thereof comprised in the pharmaceutical combination, the antibody or antigen-binding fragment thereof against tetanus toxin comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising three heavy chain CDRs, i.e. HCDR1, HCDR2, HCDR3, the light chain variable region comprising three light chain CDRs, i.e. LCDR1, LCDR2, LCDR3, wherein:

[0022] the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 2, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 6, the L-CDR2 comprises the amino acid sequence set forth in SEQ ID NO. 7, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 8.

[0023] Heavy chain CDR1 (HCDR1): GGPFTGSY; SEQ ID NO. 1;

[0024] Heavy chain CDR2 (HCDR2): VSQSGST; SEQ ID NO. 2;

[0025] Heavy chain CDR3 (HCDR3): ARLTKHYINSAY; SEQ ID NO. 3;

[0026] Light chain CDR1 (LCDR1): QIIGSW; SEQ ID NO. 6;

[0027] Light chain CDR2 (LCDR2): KAS; SEQ ID NO. 7;

[0028] Light chain CDR3 (LCDR3): QQYNSYPYT; SEQ ID NO. 8.

[0029] Further preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 4 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 9.

[0030] Heavy chain variable region (VH):

[0031] Light chain variable region (VL):

[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 can be of the IgG, IgA, IgM, IgD or IgE type, preferably of the IgG type, and the light chain constant region can be of the kappa or lambda type.

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

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

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

[0036] “TRN0011” is an immunoglobulin G1 (IgG1) monoclonal antibody, having two heavy chains comprising the amino acid sequence of SEQ ID NO. 5 and two light chains comprising the amino acid sequence of SEQ ID NO. 10. TRN0011 is capable of binding to specific surface of tetanus toxin protein with high affinity, preventing the enzymatic activity of tetanus toxin by neutralizing the toxin, thereby protecting the body.

[0037] As to the tetanus toxoid-containing vaccine comprised in the pharmaceutical combination, the vaccine can be a monovalent vaccine, such as 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.

[0038] The vaccine can also be a bivalent vaccine, such as adsorbed tetanus and diphtheria combined vaccine. Preferably, the tetanus toxoid contained in a single human dose of the adsorbed tetanus and diphtheria combined vaccine is 5 Lf.

[0039] The vaccine can also be a multivalent vaccine, such as diphtheria, tetanus and pertussis adsorbed trivalent vaccine; diphtheria, tetanus, pertussis, hepatitis B (recombinant) and poliomyelitis pentavalent vaccine; diphtheria, tetanus, pertussis, inactivated poliomyelitis, Haemophilus influenzae type b conjugate vaccine and hepatitis B hexavalent vaccine, etc.

[0040] According to the present application, the tetanus is prevented or treated by administering (e.g. injecting) the pharmaceutical combination to the subject. Herein, the pharmaceutical combination can be used for a subject who has been infected or has not been infected with Clostridium tetani. In theory, the subject can be infected with tetanus and needs to be prevented as long as the wound or break is caused. Common causes include: (1) history of trauma or breakage of skin, mucosa (such as animal injury, injection of drugs, childbirth or abortion); (2) history of bacterial infection of skin, mucosa, soft tissue (such as chronic otitis media, chronic sinusitis, periodontal infection, etc.); (3) history of digestive tract breakage (such as history of digestive tract surgery).

[0041] The subject is a mammal, preferably a rodent or a primate, more preferably a human. In the case where the subject is a human, the subject is preferably an adult over 18 years old.

[0042] Preferably, the antibody or antigen-binding fragment thereof against tetanus toxin in the pharmaceutical combination is administered to the subject by injection, preferably by single injection. Preferably, the antibody or antigen-binding fragment thereof against tetanus toxin is administered to the subject by intramuscular injection.

[0043] Further, in the pharmaceutical combination provided by the present application, the administration dosage of the antibody or antigen-binding fragment thereof against tetanus toxin is 55-275 pg / kg body weight, such as 55 pg / kg, 60 pg / kg, 83 pg / kg, 100 pg / kg, 167 pg / kg or 250 pg / kg, based on the body weight of the subject; preferably, the administration dosage is 83-250 pg / kg; more preferably, the administration dosage is 167 pg / kg. Alternatively, the administration dosage of the antibody or antigen-binding fragment thereof against tetanus toxin is 0.6-15 mg, such as 3.3 mg, 4 mg, 5 mg, 6 mg, 10 mg or 15 mg; preferably, the administration dosage is 5-15 mg; more preferably, the administration dosage is 10 mg.

[0044] 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 by intramuscular injection.

[0045] Further, in the pharmaceutical combination provided by the present application, the tetanus toxoid-containing vaccine is adsorbed tetanus vaccine, which is administered by intramuscular injection, and the administration dosage can be 0.5 ml / person / time.

[0046] According to the present application, the antibody or antigen-binding fragment thereof against tetanus toxin and the tetanus toxoid-containing vaccine in the pharmaceutical combination are administered simultaneously; or administered at intervals in time. For example, the antibody or antigen-binding fragment thereof against tetanus toxin is administered simultaneously with the first injection of adsorbed tetanus vaccine.

[0047] In a second aspect, the present application provides the use of an antibody or antigen-binding fragment thereof against tetanus toxin and a tetanus toxoid-containing vaccine in the preparation of a medicament for preventing or treating tetanus.

[0048] In this aspect, the antibody or antigen-binding fragment thereof against tetanus toxin and the tetanus toxoid-containing vaccine are as described above in the first aspect.

[0049] According to the present application, the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are used for preparing a medicine capable of preventing or treating the tetanus, which can comprise the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine in any form, so that the tetanus can be prevented or treated by administering (e.g. injecting) the medicine to a subject. The medicine is used for a subject who has been infected or not yet infected with Clostridium tetani. In theory, a subject can be infected with tetanus and needs to be prevented as long as a wound or opening is caused. Common causes include: (1) a history of trauma or damage to the skin, mucosa (such as animal injury, injection of drugs, childbirth or abortion); (2) a history of bacterial infection of the skin, mucosa, 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).

[0050] The subject is a mammal, preferably a rodent or a primate, more preferably a human. In the case where the subject is a human, an adult of 18 years of age or older is preferred.

[0051] Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by injection, preferably by single injection. Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by intramuscular injection.

[0052] Further, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered at a dose of 55-275 μg / kg of 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 administration dose is 83-250 μg / kg; more preferably, the administration dose is 167 μg / kg. Alternatively, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered at a dose of 3.3-16.5 mg, such as 3.3 mg, 4 mg, 5 mg, 6 mg, 10 mg or 15 mg; preferably, the administration dose is 5-15 mg; more preferably, the administration dose is 10 mg.

[0053] The above administration mode and dose of the anti-tetanus toxin antibody or antigen-binding fragment thereof can be achieved via the prepared medicine.

[0054] 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 by intramuscular injection.

[0055] Further, the tetanus toxoid-containing vaccine is an adsorbed tetanus vaccine, which is administered via intramuscular injection, and the administration dose can be 0.5 ml per person per time.

[0056] The above administration mode and dose of the tetanus toxoid-containing vaccine can be achieved by the prepared medicine.

[0057] According to the present application, the antibody against tetanus toxin or the antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are administered simultaneously or at intervals in time. For example, the antibody against tetanus toxin or the antigen-binding fragment thereof is administered simultaneously with the injection of the adsorbed tetanus vaccine.

[0058] In a third aspect, the present application provides a method for preventing or treating tetanus, which comprises administering a pharmaceutical combination to a subject in need, wherein the pharmaceutical combination comprises: an antibody against tetanus toxin or an antigen-binding fragment thereof; and a tetanus toxoid-containing vaccine.

[0059] In this aspect, the antibody against tetanus toxin or the antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are as described above in the first aspect.

[0060] The method for preventing or treating tetanus provided by the present application can comprise administering (e.g., injecting) the pharmaceutical combination to the subject to prevent or treat tetanus. The subject is a subject who has been infected or has not been infected with Clostridium tetani. In theory, as long as the subject has a wound or a break, the subject can be infected with tetanus and needs to be prevented. Common causes include: (1) a history of trauma or breakage of the skin or mucous membrane (such as animal injury, injection of drugs, childbirth or abortion, etc.); (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).

[0061] The subject is a mammal, preferably a rodent or a primate, more preferably a human. In the case where the subject is a human, the subject is preferably an adult over 18 years old.

[0062] Preferably, the antibody against tetanus toxin or the antigen-binding fragment thereof in the pharmaceutical combination is administered to the subject in the form of injection, preferably single injection. Preferably, the antibody against tetanus toxin or the antigen-binding fragment thereof is administered to the subject via intramuscular injection.

[0063] Further, the antibody or antigen-binding fragment thereof against tetanus toxin is administered at a dose of 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 at a dose of 83-250 μg / kg, more preferably at a dose of 167 μg / kg, based on the body weight of the subject. Alternatively, the antibody or antigen-binding fragment thereof against tetanus toxin is administered at a dose of 3.3-16.5 mg, such as 3.3 mg, 4 mg, 5 mg, 6 mg, 10 mg or 15 mg, preferably at a dose of 5-15 mg, more preferably at a dose of 10 mg.

[0064] The above administration mode and dose of the antibody or antigen-binding fragment thereof against tetanus toxin can be achieved via the pharmaceutical combination.

[0065] Preferably, the tetanus toxoid-containing vaccine in the pharmaceutical combination is also administered to the subject in an injection mode. Preferably, the tetanus toxoid-containing vaccine is administered to the subject via intramuscular injection.

[0066] Further, the tetanus toxoid-containing vaccine is an adsorbed tetanus vaccine, which is administered via intramuscular injection at a dose of 0.5 ml per person per time.

[0067] The above administration mode and dose of the tetanus toxoid-containing vaccine can be achieved via the pharmaceutical combination.

[0068] According to the present application, the antibody or antigen-binding fragment thereof against tetanus toxin and the tetanus toxoid-containing vaccine in the pharmaceutical combination are administered simultaneously or at intervals in time. For example, the antibody or antigen-binding fragment thereof against tetanus toxin is administered simultaneously with the injection of the adsorbed tetanus vaccine.

[0069] Compared with the prior art, the present application proposes a combined use of a tetanus toxoid-containing vaccine and an antibody or antigen-binding fragment thereof against tetanus toxin, such as a pharmaceutical combination of the two, for the prevention and treatment of tetanus infection.

[0070] Tetanus toxoid-containing vaccines are usually prepared from tetanus toxin produced by Clostridium tetani, detoxified, refined, and added with adjuvant. After vaccination with such vaccines, the immune system of the body will produce an immune response to the tetanus toxoid contained therein, and produce neutralizing antibodies against it. When the body contacts tetanus toxin, these antibodies bind to the toxin, preventing it 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 and tetanus toxoid-containing vaccines may offset the immune effect 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 eliciting an effective immune response in the body, thereby reducing the concentration of antibodies produced by the vaccine and affecting the immune effect. Moreover, exogenous antibodies can also cause immune suppression in the body, such as inhibition of immune cell activation and proliferation through various mechanisms, further reducing the immune effect of the vaccine.

[0071] The present application found that the combination of a recombinant natural full human monoclonal tetanus antibody and a tetanus toxoid-containing vaccine does not substantially weaken the immunogenicity of the adsorbed tetanus vaccine, while the detection time of the antibodies induced by the tetanus toxoid-containing vaccine is advanced, and the higher titer level can be sustained for a longer period of time, and there is no obvious immunotoxicity when the two are used in combination. Without being bound by any theory, it is speculated that the combination of the antibody and the tetanus toxoid-containing vaccine can induce the production of anti-tetanus toxin neutralizing antibodies by the tetanus toxoid-containing vaccine 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 full human monoclonal tetanus antibody and a tetanus toxoid-containing vaccine, such as a combination preparation, can achieve the dual prevention and treatment effects of active and passive tetanus immunization, and can enhance the protective effect of the tetanus toxoid-containing vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0072] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0073] Figure 1 is a graph showing the change in the geometric mean of anti-tetanus antibody titers over time after administration of a vaccine combined with TRN0011 and HTIG to subjects.

[0074] Figure 2 shows the interaction of TRN0011 with DT-NFDC, with data expressed as the mean ± SD of duplicate wells.

[0075] Figure 3 shows the saturation concentration of TD binding to TRN0011.

[0076] Figure 4 shows the in vitro binding of TRN0011 to adsorbed tetanus vaccine and diphtheria-tetanus vaccine, with data expressed as the mean ± SD of duplicate wells.

[0077] Best mode for carrying out the invention

[0078] The present application is illustrated below with reference to specific examples. Those skilled in the art will appreciate that the examples are for illustration only and do not limit the scope of the present application in any way.

[0079] In the following examples, the experimental methods are conventional unless otherwise specified. In the following examples, the raw materials, reagents, etc. used are commercially available products unless otherwise specified.

[0080] In the following examples, a fully humanized monoclonal antibody against tetanus toxin, referred to as TRN0011 (hereinafter referred to as "the antibody" or "the previously proposed antibody" in the context of the present application), is used. The amino acid sequence and other characteristics of the antibody are as defined above.

[0081] In the following examples, the test drug is an injection solution containing TRN0011; or a placebo containing the same excipients as the injection solution but no antibody.

[0082] Regardless of the specific amount of administration, the group in which TRN0011 is administered alone is collectively referred to as the "TRN0011 group".

[0083] The titer of the anti-tetanus toxin antibody is detected in serum using enzyme-linked immunosorbent assay (ELISA).

[0084] In the following Examples 1 and 2, the tetanus toxoid-containing vaccine used is an adsorbed tetanus vaccine, which is purchased from Chengdu Oulin Biotechnology Co., Ltd.

[0085] In the following Example 3, the HTIG used is immunoglobulin from Huazhen Biotechnology Co., Ltd.

[0086] Example 1 animal experiment

[0087] The effect of the combination of TRN0011 and a vaccine is studied by administering TRN0011 alone and administering TRN0011 in combination with an adsorbed tetanus vaccine to the muscles of mice.

[0088] (I) Grouping and administration of mice experiments and detection items

[0089] 1. Group administration

[0090] 460 healthy ICR mice were randomly divided into the following 6 groups according to body weight:

[0091] (1) Solvent control group;

[0092] (2) TRN0011 low-dose group (500 μg / kg of TRN0011);

[0093] (3) TRN0011 high dose group (2500 pg / kg of TRN0011);

[0094] (4) Adsorbed tetanus vaccine administration group (1 pL per mouse of vaccine);

[0095] (5) Combination low dose group (500 pg / kg of TRN0011 + 1 pL per mouse of vaccine);

[0096] (6) Combination high dose group (2500 pg / kg of TRN0011 + 1 pL per mouse of vaccine).

[0097] The number of animals in each group was 70, 78, 78, 78, 78, and 78, respectively, with half male and half female. The animals in each group were further divided into: the main test group, a total of 240, 40 in each of (1) to (6); the toxicokinetic (TK) (including ADA) satellite group, a total of 112, 12 in the (1) solvent control group and 20 in each of the remaining (2) to (6); the immunotoxicity satellite group, a total of 108, 18 in each of (1) to (6).

[0098] The first day of administration was defined as the first day of the test (D1). The mice in each group were administered 3 mL / kg of solvent (TRN0011 placebo) or the corresponding dose of TRN0011 by bilateral intramuscular injection in the left hind limb, single administration (D1); 20 pL per mouse of sodium chloride injection or 20 pL per mouse of 0.05 pL (vaccine stock solution) / pL concentration of adsorbed tetanus vaccine by intramuscular injection in the right hind limb, administered twice with an interval of 4 weeks (D1, D29).

[0099] Determination of the administration dose of adsorbed tetanus vaccine and antibody in the above mouse experiment:

[0100] The dose of adsorbed tetanus vaccine - The clinical dose of adsorbed tetanus vaccine is 1 dose (0.5 mL), which is about 8.33 pL / kg (based on an adult body weight of 60 kg), and the dose for mice is about 0.167 pL per mouse (based on a mouse body weight of 20 g). However, the results of the pre-experiment conducted before the present combination experiment showed that the positive rates of polyclonal antibodies induced by intramuscular injection of 0.2, 2, and 20 pL per mouse of adsorbed tetanus vaccine in ICR mice on the 15th day of the test were 2 / 10, 10 / 10, and 10 / 10, respectively, and the minimum dose for generating effective antibodies was 0.2-2 pL per mouse. In order to ensure that the vaccine can stimulate the mice to produce antibodies sufficiently, the dose of the vaccine was selected to be 1 pL per mouse.

[0101] TRN0011 dose - The inventors of the present application preliminarily confirmed in other studies on the passive immunoprotective effect of TRN0011 against tetanus infection that the clinically expected dose of TRN0011 is 100 μg / kg. However, as mentioned above, the tested dose of adsorbed tetanus vaccine in mice is about 5 times the human dose. Therefore, the low-dose group of TRN0011 alone or in combination used 5 times, i.e. 500 μg / kg, and the high-dose group used 25 times, i.e. 2500 μg / kg, accordingly.

[0102] 2. Test items:

[0103] The general state and death of the animals in the main test group were observed every day, and the body weight and food intake of the animals were measured once a week. Ophthalmic examination was performed before the end of administration (D30) and the end of the recovery period (D56), and blood samples were collected from the abdominal aorta of 10 mice / group / sex under anesthesia for hematological (including blood coagulation) and / or blood biochemical tests, respectively. Euthanasia was performed and gross autopsy observation, organ weighing, and histopathological examination were carried out.

[0104] The TK (with ADA) satellite group animals were collected blood samples before the first administration, and at 6, 24, 72, 168 hours after administration, and on D15, D22, D29 (before administration), D36, D43, D57 for detection of TRN0011 content and adsorbed tetanus vaccine-induced polyclonal antibody titers in serum; anti-TRN0011 antibody (ADA) was detected before the first administration, and on D15, D29 (before administration), D57. The immunotoxicity satellite group animals were detected for cytokines (IL-2, IL-4, IL-5, IL-6, TNF-ɑ, IFN-γ) before the first administration, and at 6, 24 hours after the first and last administration.

[0105] (ii) Results

[0106] 1. Dead / moribund animals

[0107] During the test period, a total of 4 dead animals were found, all of which were satellite group animals, 2 in the high-dose group of TRN0011, 1 in the low-dose group of TRN0011, and 1 in the high-dose group of combined use, which died on D1, D30, D16, D15, respectively, after blood sampling. It was judged that the animals died of excessive blood loss after blood sampling operation, which was not related to the test substance.

[0108] 2. General condition

[0109] Except for the dead animals in the satellite group, no obvious abnormal changes related to the test substance were found in the administration site, appearance, general behavior, mental state, gland secretion, respiratory status, fecal and urinary color, respiratory status, and genital organs of mice in each group during the test period.

[0110] 3. Body weight, food intake

[0111] During the test period, the body weight and food consumption of each group of mice did not show obvious abnormal changes related to the test substance.

[0112] 4. Ophthalmic examination

[0113] At the end of administration (D29) and the end of the recovery period (D57), the eyelids, conjunctiva, cornea, pupil, retina, fundus vascular morphology, and optic disc of mice in each administration group showed no obvious abnormal changes related to the test substance.

[0114] 5. Hematology (including blood clotting)

[0115] At the end of administration (D30) and the end of the recovery period (D57), the red blood cells, white blood cells and each classification cell count or percentage, PLT, PT, APTT, and other hematology (including blood clotting) indicators of each group of mice showed no obvious abnormal changes related to the test substance.

[0116] 6. Blood biochemistry

[0117] At the end of administration (D30) and the end of the recovery period (D57), the AST, ALT, Urea, Crea, GLU, TC, TG, Na + , K + , Cl - , and other blood biochemistry indicators of each group of mice showed no obvious abnormal changes related to the test substance.

[0118] 7. Cytokines

[0119] At 6 and 24 hours after the first and last administration, the cytokines IL-2, IL-4, IL-5, IL-6, TNF-ɑ, and IFN-γ of each group of mice showed no obvious abnormal changes related to the test substance.

[0120] 8. Organ weight and coefficient

[0121] At the end of administration (D30) and the end of the recovery period (D57), the organ weights and coefficients of the main organs such as liver, kidney, thymus, uterus, ovary, testis, and epididymis of female and male mice in each administration group showed no obvious abnormal changes related to the test substance.

[0122] 9. Gross anatomy

[0123] At the end of administration and the end of the recovery period, the administration sites of mice in each group showed no obvious abnormalities under gross macroscopic observation. The size, shape, color, or texture of the main organs and tissues such as brain, heart, liver, lung, kidney, adrenal gland, gastrointestinal tract, and reproductive organs showed no obvious abnormalities.

[0124] 10. Histopathology

[0125] No obvious abnormality related to TRN0011 was observed in each group of mice at the end of administration (D30) and the end of recovery period (D57). Single nuclear cell / neutrophil infiltration, muscle fiber degeneration / necrosis, lymphocyte infiltration, fibrosis and other pathological changes were observed in the right gastrocnemius muscle (absorbed tetanus vaccine or sodium chloride injection was administered) of each group, which was considered to be caused by local injection or immune response of absorbed tetanus vaccine. Compared with the solvent control group at the same period, no histopathological changes related to the test substance were observed in each tissue and organ of the high-dose TRN0011 group and the high-dose combination group.

[0126] 11. Toxicokinetics

[0127] Based on no obvious gender difference observed in each group, the data of male and female animals were comprehensively analyzed:

[0128] At low dose, the average T 1 / 2 was 292h and 450h, respectively, the average T max was 48h and 39h, respectively, the average C max was 4.8μg / mL and 4.2μg / mL, respectively, and the average AUC last was 1936h*μg / mL and 1736h*μg / mL, respectively.

[0129] At high dose, the average T 1 / 2 was 289h and 414h, respectively, the average T max was 24h and 15h, respectively, the average C max was 21.6μg / mL and 21.3μg / mL, respectively, and the average AUC last was 8806h*μg / mL and 7943h*μg / mL, respectively.

[0130] Compared with TRN0011 alone, the average C max ratio (low-dose combination group / TRN0011 low-dose group, high-dose combination group / TRN0011 high-dose group) was 0.87 and 0.99, respectively, and the average AUC last ratio (low-dose combination group / TRN0011 low-dose group, high-dose combination group / TRN0011 high-dose group) was 0.90 and 0.90, respectively, and no obvious difference was observed.

[0131] 12. Anti-tetanus vaccine induced polyclonal antibodies

[0132] The antibody titer of the adsorbed tetanus vaccine group D15 began to be higher than the titer threshold, the median titer was 6619, and the titer showed a trend of first increasing and then decreasing after the first administration. The titer level sharply increased at D36 (7 days after the second administration), and the antibody titer reached the maximum at D43 (14 days after the second administration), with a median titer of 216699. The titer decreased significantly at D57.

[0133] The antibody titer of the combination low and high dose groups began to be higher than the titer threshold at D7 after the first administration, with median titers of 275 and 145, respectively. The titer showed a trend of first increasing and then decreasing after the first administration, and reached the maximum at D43 and D36, respectively, with median titers of 394455 and 262850, respectively. The antibody titer decreased at D57, but the decrease was smaller than that of the adsorbed tetanus vaccine group.

[0134] The results suggest that the antibody appeared in the animals at D15 after the first administration of the adsorbed tetanus vaccine alone. The antibody titer level sharply increased at D36 (7 days after the second administration), and the titer level reached the maximum at D43 (14 days after the second administration). The antibody titer decreased significantly at D57. When combined with TRN0011, the adsorbed tetanus vaccine induced the appearance of antibodies earlier, and the high antibody titer lasted for a longer time.

[0135] 13. Anti-TRN0011 antibody

[0136] In the solvent control group, the results of anti-drug antibody (ADA) detection in the serum of all animals before administration were negative.

[0137] After the mice were injected with TRN0011, 0 / 10, 1 / 10, and 1 / 10 of the low dose group and 1 / 10, 3 / 10, and 3 / 10 of the high dose group developed ADA at D15, D29, and D57, respectively. After the mice were injected with TRN0011 combined with the adsorbed tetanus vaccine, 7 / 10, 7 / 10, and 6 / 10 of the low dose combination group and 4 / 10, 6 / 10, and 6 / 10 of the high dose combination group developed ADA at D15, D29, and D57, respectively.

[0138] The experimental results show that the ICR mice were injected with 500, 2500 μg / kg of TRN0011 by single intramuscular injection within 4 weeks, and the antibody induced by the adsorbed tetanus vaccine was detected for the first time on D8, and the antibody titer was higher than that of the adsorbed tetanus vaccine alone, indicating that TRN0011 can induce animals to produce antibodies in advance, and at the same time, it can improve the antibody titer level induced by the adsorbed tetanus vaccine. Moreover, the data show that the combination of TRN0011 and the adsorbed tetanus vaccine has no negative effect on the induction of antibodies by the adsorbed tetanus vaccine.

[0139] After the first administration, the exposure of TRN0011 in the dose range of 500-2500 μg / kg in male and female mice max and AUC last increased with the increase of the dose, and no significant gender difference was found. After the combination of 1 μL / animal of the adsorbed tetanus vaccine, there was no significant effect on the exposure of TRN0011 at different doses.

[0140] Among the above data, it is particularly noteworthy that the adsorbed tetanus vaccine alone induces antibodies for the first time on D15, and the antibody titer shows a trend of first increasing and then decreasing after the first administration, and sharply increases on D36 (7 days after the second administration), and shows a downward trend on D57. After the combination of 500, 2500 μg / kg of TRN0011, the adsorbed tetanus vaccine induces antibodies for the first time on D8, and the antibody titer is higher than that of the adsorbed tetanus vaccine alone, indicating that TRN0011 can induce animals to produce antibodies in advance, and at the same time, it can improve the antibody titer level induced by the adsorbed tetanus vaccine. Moreover, the data show that the combination of TRN0011 and the adsorbed tetanus vaccine has no negative effect on the induction of antibodies by the adsorbed tetanus vaccine.

[0141] After the combination with the vaccine, the antibody titer level and positive rate of anti-TRN0011 antibody increased, but had no effect on the exposure of the drug, and no immunotoxicity and other related findings were found.

[0142] (Three) Calculation of human dose

[0143] The following calculation formula is used to calculate the administration dose of TRN0011 combined with the adsorbed tetanus vaccine:

[0144] Human dose (mg / kg) = W x mouse dose (mg / kg), where W (conversion factor) = 0.11

[0145] The TRN0011 low and high doses in the mouse combination experiment were brought into the formula, and the human dose range of TRN0011 was calculated to be 55 μg / kg-275 μg / kg, i.e. 3.3 mg-16.5 mg (calculated according to an adult body weight of 60 kg) when combined with adsorbed tetanus vaccine. Further, according to the clinical dose and actual application, the human dose is preferably 167 μg / kg, i.e. 10 mg.

[0146] In summary, the first time of detecting the induced antibody in the animals of the above-mentioned combination (low and high doses) groups was Day 7, and the median of the peak titer was 394455 and 262850, respectively. The first time of detecting the induced antibody in the animals of the adsorbed tetanus vaccine group was Day 15, and the median of the peak titer was 216699. It can be seen from the results that, compared with the vaccine alone, the combination of TRN0011 and the vaccine can enhance the body's response level to the vaccine, advance the time of the vaccine-induced antibody, and increase the titer level of the induced antibody.

[0147] Moreover, whether the vaccine is used alone or in combination, the antibody titer induced by the vaccine undergoes a process of first rising and then falling. At the last time point (Day 57) of the experimental detection, the median of the antibody titer of the combination low and high dose groups was 274540 and 227087, respectively, and the median of the antibody titer of the adsorbed tetanus vaccine group was 85046. It can be seen from the results that the combination of TRN0011 and the vaccine can prolong the duration of the body's response to the vaccine, and prolong the time of maintaining the antibody induced by the vaccine at a high titer level.

[0148] In addition, the average T 1 / 2 of the low-dose TRN0011 alone group and the combination group was 292 h and 450 h, respectively, and the average T 1 / 2 of the high-dose TRN0011 alone group and the combination group was 289 h and 414 h, respectively. It can be seen from the results that the combination of TRN0011 and the vaccine can prolong the half-life of TRN0011, which is beneficial to maintaining TRN0011 at a high level in the body.

[0149] Overall, the combination of TRN0011 and adsorbed tetanus vaccine does not weaken the immunogenicity of adsorbed tetanus vaccine, and does not increase adverse reactions, and has good safety.

[0150] Example 2 DDI experiment research

[0151] A random, open, parallel design pharmacokinetic and pharmacodynamic interaction study of TRN0011 and adsorbed tetanus vaccine alone and in combination was carried out in Chinese healthy subjects.

[0152] Study Objectives:

[0153] Primary Objective -

[0154] • To evaluate the effect of TRN0011 on the PD characteristics of adsorbed tetanus vaccine after administration of TRN0011 in combination with adsorbed tetanus vaccine in healthy adult subjects.

[0155] Secondary Objective -

[0156] • To evaluate the safety and tolerability of TRN0011 alone or in combination with adsorbed tetanus vaccine in healthy adult subjects.

[0157] (I) Study Design

[0158] A total of 60 healthy subjects were enrolled in this study, divided into 3 parallel groups, 20 subjects in each group. The eligible subjects were allocated to TRN0011 alone (referred to as TRN0011 group), vaccine alone (referred to as vaccine group), or TRN0011 in combination with vaccine (referred to as combination group) in a random ratio of 1:1:1. The specific dosing information of each group is shown in Table 1.

[0159] Table 1: Specific dosing information of each group

[0160] The study included a selection period (Day -28 to Day -2), an inpatient period (Day -1 to Day 4), and a follow-up period (Day 5 to Day 106). The eligible subjects were admitted to the study center on Day -1 and completed the relevant safety checks and randomization. On Day 1, serum samples were collected from the subjects before administration for PK, PD, and anti-drug antibody (ADA) analysis, and the corresponding test drug was administered according to the randomization. The subjects were hospitalized in the study center until Day 4, when they left after completing the relevant safety checks and PK, PD sampling. From Day 5 to Day 106, all subjects returned to the study center at the specified time (i.e., Day 6, 8, 11, 15, 18, 22, 29, 57, 85, and Day 106) for safety assessment and / or PK, PD, and ADA blood sample collection. The above-mentioned PK sample collection was limited to the TRN0011 group and the combination group, and the PD samples included TRN0011 PD and vaccine PD for all groups.

[0161] (II) Selection of Enrolled Subjects

[0162] This study is a study on the interaction of TRN0011 in combination with adsorbed tetanus vaccine. The indication of TRN0011 is intended for tetanus prevention in the presence of unclean or contaminated wounds after trauma. In addition to trauma, the population of the indication of TRN0011 is mostly in a healthy state. At the same time, the population of the indication of adsorbed tetanus vaccine is mainly healthy people. Therefore, adult healthy subjects were recruited for this study.

[0163] The study participants were selected according to the following criteria.

[0164] 1. Healthy male or female, aged 18 to 55 years (inclusive) at the time of enrollment;

[0165] 2. Body mass index (BMI) in the range of 19.0 to 26.0 kg / m2 (inclusive) and body weight ≥ 50 kg. 2

[0166] (III) Evaluation of the study indexes - the effect of TRN0011 on the PD of the vaccine after combination

[0167] 1) Evaluation of the PD of the vaccine

[0168] Primary endpoint

[0169] The percentage of subjects with serum anti-tetanus antibody level ≥ 0.1 IU / mL at 28 days after immunization with tetanus adsorbed vaccine alone and tetanus adsorbed vaccine combined with TRN0011

[0170] Secondary endpoints

[0171] The percentage of subjects with serum anti-tetanus antibody level ≥ 0.1 IU / mL at other visit points after immunization with tetanus adsorbed vaccine alone and tetanus adsorbed vaccine combined with TRN0011, in addition to 28 days

[0172] Serum anti-tetanus antibody level, increase from baseline and fold increase from baseline at each visit point after immunization with tetanus adsorbed vaccine alone and tetanus adsorbed vaccine combined with TRN0011

[0173] 2) Evaluation of safety

[0174] Adverse events (AEs), safety laboratory tests (blood routine, blood biochemistry and urine routine), 12-lead electrocardiogram, physical examination and vital signs

[0175] (IV) Statistical methods

[0176] Full analysis set (FAS): all subjects who were randomized and received trial drug administration.

[0177] Safety analysis data set (SS): all subjects who were randomized, received trial drug administration and had post-administration safety evaluation data.

[0178] PD analysis data set (PDS): all subjects who were randomized, received trial drug administration and had at least one evaluable PD data, and no occurrence of events affecting the PD results and detection.

[0179] (V) Analysis of the effect of combination on the PD of the vaccine​

[0180] Primary endpoint analysis:

[0181] The number, percentage, and 90% CI (based on Clopper-Pearson method) of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL at 28 days post-dose were calculated by dosing group, and the difference in the percentage of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL and the 90% CI for the difference (based on stratified Miettinen-Nurminen method, stratified by anti-tetanus antibody titer test results at randomization) between the two groups were provided.

[0182] Secondary endpoint analysis:

[0183] The same statistical method as the primary endpoint was used to analyze the percentage of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL and ≥ 1.0 IU / mL at other visit time points post-dose, respectively.

[0184] The anti-tetanus antibody level, increase from baseline, and fold increase from baseline at other visit time points post-dose were listed and descriptively summarized by actual dosing group, respectively.

[0185] (VI) Results (Effect of TRN0011 on the vaccine after combination)

[0186] 1) Primary endpoint analysis

[0187] As shown in Table 2, at 28 days post-dose, the proportion of subjects with serum anti-tetanus antibody level ≥ 0.1 IU / mL was 90% (18 / 20) in both the combination group and the vaccine group, and the difference between the two groups (combination group - vaccine group) was 0.0% (90% CI: -16.2, 21.3), and no significant difference was observed between the two groups.

[0188] Table 2: Proportion of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL at 28 days post-dose in the combination group and the vaccine group (PDS) Note: PDS: pharmacodynamic analysis dataset; CI: confidence interval.

[0189] The proportion confidence interval is based on the Clopper-Pearson method; the difference between the groups represents the proportion of the combination group minus the proportion of the vaccine group, and the confidence interval for the difference is based on the stratified Miettinen-Nurminen method.

[0190] 2) Secondary endpoint analysis

[0191] As shown in Table 3, no subject in the vaccine group had anti-tetanus antibody level ≥ 0.1 IU / mL within 5 days after administration, and the proportion of such subjects gradually increased starting from 7 days after administration, and rose to 80% (16 / 20) on day 17 after administration. The proportion of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL gradually increased in the combination group after 24 hours of administration, and rose to 70% (14 / 20) on day 5 after administration, and reached the highest level (94.4%-95.0%) during the period of 14-21 days.

[0192] At the time point of 56 days after administration and later, the proportion of the combination group was slightly lower than that of the vaccine group, but there was a large degree of overlap in the 90% CI of the two groups, indicating that there was no significant difference between the two administration groups.

[0193] As can be seen from the change of anti-tetanus antibody geometric mean titer over time in the two groups, whether the vaccine was used alone or in combination with TRN0011, both showed that the anti-tetanus antibody began to rapidly increase around 7 days after administration, and both could reach a high level. Before the time point of 14 days after administration, the anti-tetanus antibody titer geometric mean of the combination group was higher than that of the vaccine group, and there was no significant difference between the two administration groups.

[0194] Table 3: Proportion of subjects with anti-tetanus antibody level ≥ 0.1 IU / mL at the visit point other than 28 days (PDS)

[0195] Therefore, the above results can show that when 10 mg of TRN0011 is combined with tetanus vaccine, it does not have a clinically significant adverse effect on the active immunization effect of the vaccine. When the two are combined, the in vivo tetanus antibody level can rapidly increase after administration, and protection can be provided in most people 5 days after administration, which is earlier than when the vaccine is used alone, and the combination of the two can make the subjects achieve protection earlier.

[0196] Example 3 Effect of TRN0011, HTIG (human immunoglobulin) and tetanus vaccine combination on the vaccine

[0197] A multicenter, randomized, double-blind, parallel-group, positive control trial to compare the effectiveness and safety of TRN0011 injection and tetanus human immunoglobulin passive immunization in preventing tetanus infection (I) Study design

[0198] This is a multicenter, randomized, double-blind, positive control, parallel-group trial conducted in subjects who have unclean or contaminated wounds due to various trauma and need to receive passive immunization against tetanus, aiming to compare the effectiveness and safety of TRN0011 10 mg and HTIG 250 IU in preventing tetanus infection in the study population.

[0199] In this study, 71 subjects received both the investigational product TRN0011 and the adsorbed tetanus vaccine, with 47 and 24 subjects receiving the vaccine concurrently in the TRN0011 and HTIG groups, respectively. The second dose of vaccine was administered 4-8 weeks after the first dose, in accordance with the adult adsorbed tetanus vaccine package insert. A total of 60 subjects completed the second dose of vaccine, with 41 (9.3%) and 19 (8.6%) in the TRN0011 and HTIG groups, respectively. Most second doses of vaccine were completed at the visit on Day 28 after dosing.

[0200] (B) Selection of subjects

[0201] TRN0011 is a passive immunization preparation for tetanus, intended for use in the prevention of tetanus in the presence of unclean or contaminated wounds following trauma. Therefore, subjects enrolled in this study were patients who had unclean or contaminated wounds due to trauma such as a cut, burn, traffic injury, animal scratch or bite, and needed to receive passive immunization to prevent tetanus infection.

[0202] The subjects enrolled in the study were selected primarily according to the following criteria.

[0203] 1. Age ≥ 18 years, male or female;

[0204] 2. Subjects who had unclean or contaminated wounds due to trauma such as a cut, burn, traffic injury, animal scratch or bite, and needed to receive passive immunization to prevent tetanus infection;

[0205] 3. The time from the trauma to the administration of the investigational product was expected to be no more than 24 hours.

[0206] (C) Investigational product

[0207] Table 4

[0208] (D) Analysis data set

[0209] (E) Analysis of effectiveness endpoints

[0210] A subgroup analysis was performed on the 71 subjects in this study who received the vaccine. Based on the results of the HTIG PD bioanalysis method, the anti-tetanus antibody titers at each time point after dosing were compared between the subjects who received the vaccine in the TRN0011 and HTIG groups, including the anti-tetanus antibody GMT and the proportion of subjects with an anti-tetanus antibody titer of ≥ 0.1 IU / mL.

[0211] (F) Analysis of results

[0212] At each time point post-dose (12 hours and days 3, 7, 28 and 90), the anti-tetanus antibody GMTs were higher in subjects who received TRN0011 co-administered vaccine (TRN0011 co-administration group) than in subjects who received HTIG co-administered vaccine (HTIG co-administration group), and the proportion of subjects with anti-tetanus antibody titres > 0.1 IU / mL was also generally higher in the TRN0011 co-administration group than in the HTIG co-administration group.

[0213] At the key time point for assessing the immune effect of tetanus vaccine, i.e. at day 28 post-dose, the anti-tetanus antibody GMTs were 2.09 IU / mL (95% CI: 1.35, 3.25) in the TRN0011 co-administration group and 0.504 IU / mL (95% CI: 0.230, 1.10) in the HTIG co-administration group. The proportion of subjects with anti-tetanus antibody titres > 0.1 IU / mL at day 28 post-dose was 100% (95% CI: 92.5%, 100.0%) in the TRN0011 co-administration group and 87.5% (95% CI: 67.6%, 97.3%) in the HTIG co-administration group.

[0214] At day 90 post-dose (after the second dose of vaccine), the anti-tetanus antibody GMTs were 2.29 IU / mL (95% CI: 1.60, 3.27) in the TRN0011 co-administration group and 1.24 IU / mL (95% CI: 0.555, 2.77) in the HTIG co-administration group, and the proportion of subjects with anti-tetanus antibody titres > 0.1 IU / mL was 100% (95% CI: 92.5%, 100.0%) and 91.7% (95% CI: 73.0%, 99.0%) respectively. This indicates that the anti-tetanus antibody levels were higher in subjects who received TRN0011 co-administered vaccine than in subjects who received HTIG co-administered vaccine. The geometric mean time profiles of anti-tetanus antibody levels in the two groups are shown in Figure 1 (in Figure 1, a is a linear plot and b is a log plot).

[0215] Thus, the results described above show that the anti-tetanus antibody levels were higher after 10 mg of TRN0011 co-administered vaccine than after 250 IU of HTIG co-administered vaccine, indicating that the effect of TRN0011 on the suppression of active immunisation with vaccine was less than the effect of HTIG on the suppression of active immunisation with vaccine.

[0216] Note: CI = confidence interval; GMT = geometric mean titre.

[0217] Example 4 Comparison of the in vitro interaction of TRN0011 with tetanus vaccine, tetanus-diphtheria adsorbed vaccine

[0218] From the above experiments, it can be known that TRN0011 has an unexpected effect when combined with tetanus vaccine (single-valent vaccine). To further verify the effect of TRN0011 combined with bivalent vaccine (such as diphtheria and tetanus adsorbed vaccine) and the like, the following exploratory experiments are carried out in vitro to understand whether there is a difference between TRN0011 combined with diphtheria and tetanus adsorbed vaccine (bivalent vaccine) and TRN0011 combined with tetanus adsorbed vaccine (single-valent vaccine).

[0219] (I) List of abbreviations

[0220] (II) Key reagents

[0221] (III) Experimental methods and results

[0222] I. Assessing whether diphtheria toxoid (DT-NIFDC) and TRN0011 form a complex in direct ELISA

[0223] 1. Procedure:

[0224] Step 1: Coat 11 serial gradient concentrations (dilution factor: 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400) of DT-NIFDC in weakly acidic (pH = 6) coating solution at 100 μL / well in a 96-well ELISA plate, incubate overnight at 2-8°C, and wash 3 times with 0.05% PBST 300 μL / well.

[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 to 500 ng / mL with 3% BSA, add 100 μL / well to different column positions 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 color developing solution, incubate at room temperature for 10 minutes in the dark.

[0228] Step 5: Add 50 μL / well of stop solution to terminate color development, and immediately collect data by plate reading.

[0229] 2. Results

[0230] The experiment was repeated three times, and the representative results are shown in Figure 2. The red curve is the reaction curve of anti-DT-PAb-HRP binding with 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 indicates that DT-NIFDC is successfully coated on the 96-well plate within the dilution concentration range. However, when TRN0011-HRP is used instead of anti-DT-PAb-HRP under the same coating conditions, the binding curve (green) remains at an OD value of about 0.01 without a dose response, indicating that TRM0011 does not bind to DT-NIFDC and there is no interaction between them.

[0231] II. Determining the saturation concentration of TRN0011 binding to Td in indirect ELISA

[0232] 1. Steps:

[0233] Step 1: Dilute Td with weak acid (pH = 6) solution by three different multiples (133 times, 266 times and 1333 times), add 100 μL / well to 96-well ELISA coating, ～ coat overnight at 8°C, then wash three times with 0.05% PBST at 300 μL / well.

[0234] Step 2: Add 250 μL of 3% BSA solution per well for blocking, incubate at 25°C for 2 hours, then wash three times as in Step 1.

[0235] Step 3: Prepare TRN0011 into 12 different concentrations (0, 9.77, 19.53, 39.06, 78.125, 156.25, 625, 1250, 2500, 5000, 10000, 20000 ng / mL) and add to the coated microplate, incubate at 25°C for 2 hours, then wash three times as in Step 1.

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

[0237] Step 5: Add TMB color developing solution 100 μL / well, 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 immediately collect data.

[0239] 2. Results

[0240] The experiment was repeated three times, and the representative results are shown in Figure 3. When TRN0011 increased from 0 to 20 pg / 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 pg / mL, the OD value tended to be flat, indicating that TRN0011 at a concentration higher than 1 pg / mL was excessive under this Td coating condition.

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

[0242] 1. Procedure

[0243] Step 1: Same as Experiment 2, use three different dilutions (133 times, 266 times and 1333 times) of Td to coat 96-well ELISA plates in an acidic coating solution at a volume of 100 pL / well, overnight at 2-8°C, and then wash three times with 0.05% PBST at a volume of 300 pL / well.

[0244] Step 2: Add 250 pL of 3% BSA solution per well, incubate at 25°C for 2 hours for blocking, and then wash three times as in Step 1.

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

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

[0247] Step 5: Add 50 pL / well stop solution to stop the color development reaction and immediately read the plate on a 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-fold, 266-fold and 1333-fold). When Td was diluted 133-fold, the OD value of all wells peaked at about 1.0, regardless of the presence of excess TRN0011. When Td was diluted 266-fold, the OD value of all wells peaked at about 0.5, regardless of the presence of excess TRN0011. When Td was diluted 1333-fold, the OD value of all wells peaked at about 0.1, regardless of the presence of excess TRN0011. These results show that the detection of DT-Td by anti-DT-PAb-HRP was not affected by excess TRN0011, demonstrating that TRN0011 does not bind to DT-Td in vitro, i.e. there is no interaction between the two.

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

[0251] Four, Effect of TRN0011 on the use of tetanus vaccine and diphtheria-tetanus vaccine in combination

[0252] 1. Procedure

[0253] Step 1 : Two vaccines (Td and TT Olymvax) were separately coated in microplates overnight at low temperature (2-8°C) after gradient dilution (dilution factor 200 to 12800-fold) with weak alkaline (pH=8) solution, then washed three times with 300 μL / well of 0.05% PBST solution.

[0254] Step 2: 250 μL / well of 3% BSA was added for blocking, incubated at 25°C for 2 hours, then washed three times according to the method of Step 1.

[0255] Step 3: TRN0011-HRP (66.67 ng / mL) was added, incubated at 25°C for 1 hour, then washed three times according to the method of Step 1.

[0256] Step 4: 100 μL / well of TMB color developing solution was added, incubated at room temperature for 10 minutes in the dark.

[0257] Step 5: 50 μL / well of stop solution was added to stop the color developing reaction, and the plate was immediately read to collect data.

[0258] 2. Results

[0259] The experiment was repeated three times, and representative results are shown in Figure 4. The red response curve is from the binding of TRN0011-HRP to coated TT-Olympvax (dilution range: 200-12800). As the dilution factor increased from 200 to 12800, the OD value decreased from 2.6 to 0.01, forming a good linear fit. The green response curve is from the binding of TRN0011-HRP to coated TT-Td (dilution factors: 200, 400, 800, 1600, 3200, 6400, 12,800), and as the dilution factor increased from 200 to 12800, the OD value on the curve also decreased from 2.6 to 0.01.

[0260] Overall, the two curves show the same binding trend, indicating that TRN0011 binds to tetanus toxoid in both vaccines similarly.

[0261] Thus, from the above four experimental results, we can conclude that (1) TRN0011 does not bind to diphtheria toxoid in the bivalent vaccine. (2) TRN0011 shows similar binding to tetanus toxoid in the bivalent vaccine compared to the monovalent vaccine, i.e., the drug binding of TRN0011 in combination with the monovalent vaccine and the bivalent vaccine in vivo is similar.

[0262] The above description of specific embodiments of the present application is not intended to limit the present application, and those skilled in the art can make various changes or modifications to the present application without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of the appended claims of the present application.

Claims

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

8. the anti-tetanus toxin antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 4, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO.

9.

2. The pharmaceutical combination according to claim 1, characterized in that, the anti-tetanus toxin antibody or the antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; 3. The pharmaceutical combination according to claim 1 or 2, characterized in that, preferably, the heavy chain constant region is of IgG, IgA, IgM, IgD or IgE type, and further preferably of IgG type; preferably, the light chain constant region is of κ or λ type. the anti-tetanus toxin antibody is murine, chimeric, humanized or fully human; or, the anti-tetanus toxin antibody is monoclonal or single-chain antibody; 4. The pharmaceutical combination according to any one of claims 1 to 3, characterized in that, preferably, the anti-tetanus toxin antibody comprises a heavy chain (HC) and a light chain (LC), the heavy chain comprises the amino acid sequence set forth in SEQ ID NO. 5, and the light chain comprises the amino acid sequence set forth in SEQ ID NO.

10. the tetanus toxoid-containing vaccine is adsorbed tetanus monovalent or multivalent vaccine.

5. The pharmaceutical combination according to any one of claims 1 to 4, characterized in that, the tetanus toxoid-containing vaccine is monovalent vaccine, such as adsorbed tetanus vaccine, which contains tetanus toxoid with a potency of not less than 40 IU per single human dose; 6. The pharmaceutical combination according to any one of claims 1 to 4, characterized in that, alternatively, the tetanus toxoid-containing vaccine is bivalent vaccine, such as adsorbed tetanus and diphtheria combined vaccine; alternatively, the tetanus toxoid-containing vaccine is multivalent vaccine, such as adsorbed diphtheria, tetanus and pertussis trivalent vaccine; diphtheria, tetanus, pertussis, hepatitis B (recombinant) and poliomyelitis pentavalent vaccine; diphtheria, tetanus, pertussis, inactivated poliomyelitis, Haemophilus influenzae type b conjugate vaccine and hepatitis B hexavalent vaccine. 7.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; the anti-tetanus toxin antibody or the antigen-binding fragment thereof and the tetanus toxoid-containing vaccine are each as defined in any one of claims 1 to 6. wherein ​ 8. Use according to claim 7, characterized in that, The medicament comprises the anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine.

9. 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 as a single injection; preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by 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 by intramuscular injection.

10. 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 a time interval.

11. A method for preventing or treating tetanus, the method comprising administering to a subject in need thereof a pharmaceutical combination comprising: an anti-tetanus toxin antibody or antigen-binding fragment thereof; and, a tetanus toxoid-containing vaccine; wherein 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.

12. The method of claim 11, wherein, The subject is a subject who has been infected or has not been infected with Clostridium tetani; and / or The subject is a mammal, preferably a rodent or a primate, more preferably a human; further preferably, the subject is an adult human of 18 years of age or older.

13. The method according to claim 11 or 12, characterized in that, The anti-tetanus toxin antibody or antigen-binding fragment thereof in the pharmaceutical combination is administered to the subject by injection, preferably as a single injection; preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject by intramuscular injection. Preferably, the anti-tetanus toxin antibody or antigen-binding fragment thereof is administered to the subject at a dose of 55-275 pg / kg body weight.

14. The method according to any one of claims 11 to 13, characterized in that, The tetanus toxoid-containing vaccine in the pharmaceutical combination is administered to the subject by injection; further preferably, the tetanus toxoid-containing vaccine is administered to the subject by intramuscular injection. Preferably, the tetanus toxoid-containing vaccine is 0.5 ml per person per time.

15. The method according to any one of claims 11 to 14, characterized in that, The anti-tetanus toxin antibody or antigen-binding fragment thereof and the tetanus toxoid-containing vaccine in the pharmaceutical combination are administered simultaneously; or are administered at a time interval.

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