JPWO2019129214A5

JPWO2019129214A5 -

Info

Publication number: JPWO2019129214A5
Application number: JP2020555284A
Authority: JP
Publication date: 2023-05-30
Anticipated expiration: 2038-12-28

Description

結果を図１３～１６に示す。ＬＤ_５０破傷風毒素を２０倍攻撃した場合、陰性対照群のマウスは、１．８５μｇ／ｍｌのＴＲＮ００１０抗体を有する実験群のマウスを除いて、７日後に死亡し、他のすべての実験群のマウス（０．６２μｇ／ｍｌ、５．５６μｇ／ｍｌ、１６．６７μｇ／ｍｌ、５０μｇ／ｍｌ）及び陽性対照群の全マウスが生存した。これは、０．６２μｇ／ｍｌという低用量モノクロナル抗体が１０ＵＩ／ｍｌの標準抗毒素に相当することを示している。モノクロナル抗体は、破傷風毒素の致死量攻撃から動物を保護することができ、標準抗毒素とほぼ同じ防御活性を有する。本発明のモノクロナル抗体の実際の用量は、標準抗毒素の用量よりもはるかに低く、その効果が抗毒素のそれよりも優れていることを示した。結果は、本発明のモノクロナル抗体が、マウスの体内の毒素を中和し、マウスを保護し、インビボ活性を有することを示す。
The results are shown in Figures 13-16. When challenged 20-fold with LD ₅₀ tetanus toxin, mice in the negative control group died after 7 days, except mice in the experimental group with 1.85 μg /ml TRN0010 antibody; of mice (0.62 μg /ml, 5.56 μg /ml, 16.67 μg /ml, 50 μg /ml) and all mice in the positive control group survived. This indicates that a low dose of 0.62 μg /ml monoclonal antibody is equivalent to 10 UI/ml standard antitoxin. Monoclonal antibodies can protect animals from lethal challenge with tetanus toxin and have approximately the same protective activity as standard antitoxin. The actual dose of the monoclonal antibody of the invention was much lower than that of the standard antitoxin, indicating that its efficacy was superior to that of the antitoxin. The results show that the monoclonal antibodies of the invention neutralize toxins in mice, protect mice, and have in vivo activity.

図１７に示すように、より高用量の破傷風毒素攻撃（ＬＤ_５０の６０倍）下で、陰性対照群のマウスが２４時間以内に死亡した場合、低用量実験群のマウス（０．６２μｇ／ｍｌ、１．８５μｇ／ｍｌ）は７日以内に死亡し、中及び高用量実験群のマウス（５．５６μｇ／ｍｌ、１６．６７μｇ／ｍｌ、及び５０μｇ／ｍｌ）は全て７日以内に生存し、本発明のモノクロナル抗体が高用量の毒素攻撃を防御することができ、モノクロナル抗体がインビボ中和活性が極めて強いことを示す。
As shown in Figure 17, under higher dose tetanus toxin challenge (60 times the _LD50 ), mice in the negative control group died within 24 hours, whereas mice in the low dose experimental group (0.62 µg /ml, 1.85 μg /ml) died within 7 days, and mice in the mid- and high-dose experimental groups (5.56 μg /ml, 16.67 μg /ml, and 50 μg /ml) died within 7 days. ) all survived within 7 days, demonstrating that the monoclonal antibodies of the invention can protect against high-dose toxin challenge and that the monoclonal antibodies have very strong in vivo neutralizing activity.

Claims

A fully native human neutralizing monoclonal antibody or antigen-binding fragment thereof against tetanus toxoid, wherein the neutralizing monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) having three CDRs , and a light chain variable domain (VL) with three CDRs:
wherein CDR1, CDR2 and CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; comprising the amino acid sequences set forth in SEQ ID NO: 24 and SEQ ID NO: 25;
wherein CDR1, CDR2 and CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; comprising the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6;
wherein CDR1, CDR2 and CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; 14, and the amino acid sequence set forth in SEQ ID NO: 15; or
wherein CDR1, CDR2 and CDR3 of VH comprise the amino acid sequences set forth in SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, respectively; No. 31, and the neutralizing monoclonal antibody or antigen-binding fragment thereof, comprising the amino acid sequences set forth in SEQ ID NO: 32.

wherein VH comprises the amino acid sequence set forth in SEQ ID NO: 22 and VL comprises the amino acid sequence set forth in SEQ ID NO: 26;
wherein VH comprises the amino acid sequence set forth in SEQ ID NO:7 and VL comprises the amino acid sequence set forth in SEQ ID NO:8;
wherein VH comprises the amino acid sequence set forth in SEQ ID NO: 17 and VL comprises the amino acid sequence set forth in SEQ ID NO: 18 ; or
2. The neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein VH comprises the amino acid sequence set forth in SEQ ID NO:33 and VL comprises the amino acid sequence set forth in SEQ ID NO:34.

A neutralizing monoclonal antibody or antigen-binding fragment thereof immunospecifically binds to tetanus toxin, wherein the neutralizing monoclonal antibody or antigen-binding fragment thereof binds to tetanus toxin or B. tetanus with an equilibrium dissociation constant greater than ^10-6 M. 3. The neutralizing monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which does not dissociate from Clostridium tetani.

A nucleotide molecule encoding the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3.

A recombinant expression vector comprising the nucleotide molecule of claim 4 .

A recombinant cell obtained by transforming or transfecting a host cell with the recombinant expression vector according to claim 5 .

4. A composition comprising the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3 covalently attached to a chemical or biological label .

wherein said chemical label comprises an isotope label, an immunotoxin label, or a chemical drug label, and said biological label comprises a biotin label, an avidin label, an enzyme label, a fluorescent label, or an electron transfer agent label; Item 8. The composition according to item 7.

A conjugate comprising the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3, and/or the composition of claim 7, coupled to a solid or semi-solid medium.

A combination composition comprising a neutralizing monoclonal antibody or antigen-binding fragment thereof according to claim 1, 2 or 3, and/or a composition according to claim 7, and/or a conjugate according to claim 9 . .

A neutralizing monoclonal antibody or antigen-binding fragment thereof according to claim 1, 2 or 3 and/or a composition according to claim 7 and/or a conjugate according to claim 9 and/or claims A test product comprising the combination composition of paragraph 10 .

A pharmaceutical composition comprising the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3,
(1) to determine the presence or level of tetanus toxin or bacillus tetani in a sample;
(2) for detecting whether a subject is infected with tetanus toxin or bacillus tetani; or (3) for use as a medicament to neutralize the toxicity of tetanus toxin.

(1) the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3, and/or the composition of claim 7, and/or the conjugate of claim 9 , and/ or the use of a combination composition according to claim 10 to prepare a test product for tetanus toxin or tetanus infection;
(2) the neutralizing monoclonal antibody or antigen-binding fragment thereof of claim 1, 2 or 3, and/or the composition of claim 7, and/or the conjugate of claim 9 , and/ or (3) the nucleotide molecule of claim 4 , Use of the recombinant expression vector according to claim 5 or the recombinant cell according to claim 6 for producing a prophylactic or therapeutic agent for diseases induced by tetanus toxin or B. tetanus infection.