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JP-7857080-B2 - Single-domain antibody that binds to tetanus neurotoxin

JP7857080B2JP 7857080 B2JP7857080 B2JP 7857080B2JP-7857080-B2

Inventors

  • デ スミット， アブラハム ヨハネス
  • ハームセン， マイケル マリー

Assignees

  • スミヴェット ビー．ブイ．

Dates

Publication Date
20260512
Application Date
20190313
Priority Date
20180313

Claims (18)

  1. A single-domain antibody (SDA) having the ability to bind to tetanus neurotoxin (TeNT) with a KD value of less than 1 nM, having at least 90% overall amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 17 , 15 , 20 , and 25 , wherein the amino acid sequence identity of CDR1, CDR2, and CDR3 is 100%.
  2. The SDA according to claim 1, having at least 95% overall amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs : 17, 15 , 20 , and 25 .
  3. The SDA according to claim 1 or 2, having 100% overall amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 17 , 15 , 20 , and 25.
  4. A polypeptide construct comprising at least one SDA having the ability to bind to TeNT as described in any one of claims 1 to 3, and at least one SDA having the ability to bind to serum proteins.
  5. The polypeptide construct according to claim 4, wherein the serum protein is serum albumin or immunoglobulin.
  6. The polypeptide construct according to claim 5, wherein the SDA having the ability to bind to serum albumin has 100% overall amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 40, 37, 38, 39, 41, and 42, provided that the amino acid sequence identity of CDR1, CDR2, and CDR3 is 100%.
  7. The polypeptide construct according to claim 5, wherein the immunoglobulin is immunoglobulin G (IgG).
  8. The polypeptide construct according to claim 5 or 7, wherein the SDA having the ability to bind to the immunoglobulin has 100% overall amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 30, 27, 28, 29, 31, 32, 33, and 34, provided that the amino acid sequence identity of CDR1, CDR2, and CDR3 is 100%.
  9. A polypeptide construct according to any one of claims 4 to 8, comprising at least two SDAs capable of binding to TeNT, wherein each of the at least two SDAs capable of binding to TeNT has at least 95% overall amino acid sequence identity with a sequence selected from option A: SEQ ID NO: 24, or option B: SEQ ID NO: 25, or option C: SEQ ID NO: 20, or option D: SEQ ID NO: 17 or 19, or option E: SEQ ID NO: 22, 15, 23, or 14, provided that the at least two SDAs do not contain sequences derived from the same option, and the amino acid sequence identity of CDR1, CDR2, and CDR3 is 100%.
  10. A pharmaceutical composition comprising at least one SDA according to any one of claims 1 to 3, and/or at least one polypeptide construct according to any one of claims 4 to 9, and a pharmaceutically acceptable carrier , wherein at least one SDA having the ability to bind to TeNT according to any one of claims 1 to 3 has at least 90% overall amino acid sequence identity with SEQ ID NO: 17 or 20, provided that the amino acid sequence identity of CDR1, CDR2, and CDR3 is 100% .
  11. The pharmaceutical composition according to claim 10, comprising at least two SDAs having the ability to bind to TeNT as described in any one of claims 1 to 3, and/or at least one polypeptide construct as described in claim 9.
  12. A composition for use as a pharmaceutical, comprising the SDA according to any one of claims 1 to 3, and/or the polypeptide construct according to any one of claims 4 to 9.
  13. A composition comprising the SDA described in any one of claims 1 to 3 and/or the polypeptide construct described in any one of claims 4 to 9, or the pharmaceutical composition described in claim 10 or 11, for use in the prevention or treatment of tetanus (Clostridium tetanus) disease/symptoms.
  14. A DNA fragment encoding an SDA according to any one of claims 1 to 3, or a polypeptide construct according to any one of claims 4 to 9.
  15. A nucleic acid comprising the DNA fragment described in claim 14, wherein the DNA fragment described in claim 14 is operably linked to a promoter and, optionally, other regulatory elements.
  16. A host cell containing the nucleic acid described in claim 15.
  17. A method for producing an SDA according to any one of claims 1 to 3, or a polypeptide construct according to any one of claims 4 to 9, comprising: a) culturing the host cells according to claim 16 under conditions that enable the expression of the SDA or polypeptide construct; and optionally b) recovering the SDA or polypeptide construct from at least one of the host cells and the culture medium.
  18. A diagnostic kit comprising at least one SDA having the ability to bind to the TeNT described in any one of claims 1 to 3.

Description

This invention particularly relates to single-domain antibodies (SDAs) having the ability to bind to tetanus neurotoxin, polypeptide constructs comprising such SDAs, compositions comprising such SDAs and/or polypeptide constructs, and DNA fragments encoding such SDAs and/or polypeptide constructs. Furthermore, this invention relates to host cells comprising such DNA fragments, methods for producing such SDAs and/or polypeptide constructs, and the use of such SDAs and/or polypeptide constructs and/or compositions in treating or preventing disease after Clostridium tetanus (Tetani) infection. Tetanus is a disease caused by the bacterium *Clostridium tetanus*, and was first described in Egypt approximately 3,000 years ago. Tetanus toxemia is caused by a specific neurotoxin; tetanus neurotoxin (TeNT) produced by the bacterium *Clostridium tetani*. Almost all mammals, including humans, are susceptible. Humans, horses, and lambs are among the most susceptible species. Dogs and cats are relatively resistant. Tetanus is found worldwide, however, soil tetanus incidence, as well as tetanus morbidity in humans, horses, and lambs, is higher in the temperate parts of various continents. In most cases, *Clostridium tetani* bacteria are introduced into tissue through wounds. Tetanus commonly results from deep, penetrating wounds that promote the growth of anaerobic bacteria. However, in lambs, and sometimes in other species, tetanus can also result from tail docking or castration. Tetanus neurotoxin is a zinc-binding protease that cleaves synaptobrevin, a vesicle-associated membrane protein. Cleavage of this protein inhibits neurotransmitter release. The toxin is absorbed by motor neurons within the infected area and travels retrogradely from the nerve cords to the spinal cord, where it causes ascending tetanus. TeNT is synthesized as a single polypeptide chain of 150 kDa. This polypeptide is cleaved into a 100 kDa heavy chain (H) and a 50 kDa light chain (L) that are held together by disulfide bonds to form an active toxin. Digestion of the holotoxin with papain yields fragment B, which consists of the TeNT light chain and the amino-terminal half ( H₁N₁ ) of the TeNT heavy chain, and fragment C ( H₂C or TTC), which contains the carboxyl-terminal half of the heavy chain. In vitro experiments suggested that H₂C is involved in binding to neurons via gangliosides, while the H₁N₁ fragment plays a role in internalization and membrane translocation. The incubation period (the time from infection to the first symptom) can range from as short as 24 hours to as long as several months after tetanus inoculation. This interval may reflect the distance the toxin must travel within the nervous system and may also be related to the amount of toxin released. The symptomatic period is the time between the first symptom and the onset of spastic paralysis. The incubation period is usually 10 to 14 days on average. Localized rigidity is often the first symptom observed, including in the masticatory and neck muscles, hind limbs, and the area of the infected wound; generalized rigidity becomes prominent after about one day, and then tonic spasms and hyperesthesia become apparent. Due to their relatively high resistance to tetanus toxin, dogs and cats often have long incubation periods and frequently develop localized tetanus; however, systemic tetanus certainly develops in these species as well. A comprehensive description of tetanus neurotoxin (and related botulinum neurotoxin) can be found in *BOTULINUM AND TETANUS NEUROTOXINS*, ISBN 978-1-4757-9544-8, (C) 1993 Springer Science & Business Media New York. It was originally published in Plenum Press, New York in 1993. Reviews related to tetanus, its causes and effects, and treatment methods can be found, for example, in *J. Neurol Neurosurg Psychiatry* 69:292-301 (2000) by Farrar, J. J. et al. Passive immunization using polyclonal human or, for example, equine tetanus antitoxin can shorten the progression of tetanus and reduce its severity. Equine antiserum (Fab) is prepared from pooled serum collected from immunized horses and has a half-life of 12–20 hours in humans (Flanagan RJ, Jones AL. Drug Saf. 2004;27(14):1115–33). Equine (or bovine) forms are used throughout the developing world and, although they can incidentally cause anaphylactic reactions, their production is far cheaper and easier than that of human donor serum. Treatment for tetanus disease consists of administration of antibiotics or metronidazole, treatment of the infection site (e.g., flushing, draining, and dissection), administration of antitoxin, and supportive care (e.g., skeletal muscle relaxants, sedatives, and hydration). Passive immunization using preparations containing immunoglobulins (e.g., purified and fragmented) obtained from actively immunized sheep or horses provides effective protection in unimmunized animals and humans. Tetanus antitoxin (e.g., in the form of SDA or immunoserum) can be used in at least three