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WO2024235910A1 - Nanocorps pour traiter l'inflammation, l'auto-inflammation ou le cancer - Google Patents

Nanocorps pour traiter l'inflammation, l'auto-inflammation ou le cancer Download PDF

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Publication number
WO2024235910A1
WO2024235910A1 PCT/EP2024/063091 EP2024063091W WO2024235910A1 WO 2024235910 A1 WO2024235910 A1 WO 2024235910A1 EP 2024063091 W EP2024063091 W EP 2024063091W WO 2024235910 A1 WO2024235910 A1 WO 2024235910A1
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Prior art keywords
seq
sequence
nanobody
catenin
identity
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English (en)
Inventor
Ranja SALVAMOSER
Chris A. BALDWIN
James E. VINCE
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Mermaid Bio GmbH
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Mermaid Bio GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to a heavy chain variable domain (VHH) of an anti- beta-catenin nanobody, anti- beta-catenin nanobody comprising said VHH, as well as said VHH for use and said nanobody for use in treating inflammation, autoinflammation or cancer.
  • VHH heavy chain variable domain
  • a nanobody is an antibody comprising a single variable domain that is located on a heavy chain.
  • nanobody comprise a heavy chain variable domain (VHH)
  • VHH heavy chain variable domain
  • Nanobodies are small, stable, have high target affinity and specificity and can be designed against a wide range of intracellular targets. They represent a next-frontier technology for intracellular protein interaction.
  • scFvs single-chain variable fragments
  • camelid and cartilaginous fish naturally produce heavy chain-only antibodies presents a promising alternative. These antibodies are small ( ⁇ 75 kDa versus the conventional Ab size of ⁇ 150 kDa) with a highly stable antigen-binding V-domain-designated VHH or nanobody ( ⁇ 15 kDa) (Diagram 1). conventional antibody camelid heavy-chain antibody fluorobody chromobody
  • Diagram 1 Schematic representation of antibody formation shown to be functional within living cells, source: (2, 4).
  • Nanobodies or VHH from such camelid antibodies are reduced down to just the fragment of the variable heavy-chain domain.
  • One of the major drawbacks of conventional antibodies is their requirement for multiple disulfide bonds for structural integrity and stability (5).
  • Many VHH do not rely on disulfide bonds for folding and antigen-specificity, thus they can be expressed in producer cell lines or other expression systems (6).
  • VHH are highly soluble, stable (VHH can be stored at 4°C for months without their binding capacity being affected) and have shown excellent tissue penetration in vivo (6). Taking all these aspects together, nanobodies hold immense potential in applications such as oncology, infection and disease.
  • nanobody single domain antibody (sdAb)
  • VHH single domain antibody
  • VHH can refer to “nanobody” or “single domain antibody (sdAb)” according to the context, which would be understood by a person skilled in the art
  • VHH domain or “a heavy chain variable domain (VHH)” can refer to a heavy chain variable domain contained in the antibody according to the context, which would be understood by a person skilled in the art.
  • VHH can refer to “nanobody” or “single domain antibody (sdAb)” according to the context, which would be understood by a person skilled in the art
  • VHH domain or “a heavy chain variable domain (VHH)” can refer to a heavy chain variable domain contained in the antibody according to the context, which would be understood by a person skilled in the art.
  • a skilled person with a mind willing to understand would be able to differentiate the meaning of above terms according to different context wherein said terms are involved.
  • beta-catenin In cancer cells the activity of beta-catenin is hyperactive wherein cancer cells stop beta-catenin from being phosphorylated then ubiquitinated and consequently targeted for proteasomal degradation. Mutations in cancer cells can occur in the complex required for its phosphorylation and degradation (e.g., APC/AXIN 1 mutations), other proteins involved in the Wnt-signalling pathway, or in beta-catenin itself.
  • an antibody which can efficiently target beta-catenin for therapeutic purpose is desirable.
  • the present invention provides a VHH domain and a nanobody comprising said VHH domain which can bind to beta-catenin.
  • a new platform is established which can efficiently target the nanobody to beta-catenin for therapeutic purpose, wherein for example the nanobody fused with pertinent kinase can target beta-catenin for phosphorylation or/and degradation.
  • the targeting efficacy is further improved.
  • the term “comprise”, “comprises” or “comprising”, should be understood as the inclusion of a stated member, integer, or step but not the exclusion of any other non-stated member, integer or step.
  • the term “consisting of” is a particular embodiment of the term “comprise”, indicating that any other non-stated member, integer, or step is excluded.
  • the term “comprise” encompasses the term “consist of”.
  • the term “comprising” encompasses “including” as well as “consisting”, for example, a composition “comprising” X may consist exclusively of X or may include something additional, for example, X + Y.
  • the present invention provides a heavy chain variable domain (VHH) of an anti-beta-catenin nanobody, wherein the amino acid sequence of said VHH comprises a sequence exhibiting at least 70% identity to one of SEQ ID NOs 1 -15 (Table 1), and preferably wherein said sequence exhibiting at least 70% identity to one of SEQ ID NOs 1-15 mediates the binding of said nanobody with beta-catenin.
  • VHH heavy chain variable domain
  • the amino acid sequence of said VHH comprises a sequence exhibiting at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibiting 100% identity to one of SEQ ID NOs 1 -15.
  • the amino acid sequence of said VHH comprises two or more sequences, wherein each of said two or more sequences exhibits at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibiting 100% identity to one of SEQ ID NOs 1 -15.
  • the amino acid sequence of said VHH comprises a sequence of SEQ ID NOs 1 -15 (Table 1).
  • the beta-catenin is of mammalian origin, e.g. of human, rat, mouse, rabbit, or goat.
  • the present invention also provides an anti-beta-catenin nanobody, wherein said nanobody comprises the VHH disclosed above.
  • nanobody in the present application can also refer to single domain antibody.
  • the numbering scheme for defining the CDR is IMGT.
  • the CDR3 domain of said nanobody is one of SEQ ID NOs 1 -15.
  • the CDR1 domain of said nanobody is any one of SEQ ID NOs 18-32.
  • the CDR2 domain of said nanobody is any one of SEQ ID NOs 33-47.
  • the nanobody or the heavy chain variable domain comprises an amino acid sequence of any one of SEQ ID NOs 48-62 which contains a CDR1 domain, a CDR2 domain and a CDR3 domain, wherein the sequence of said CDR1 domain, the sequence of said CDR2 domain and the sequence of said CDR3 domain can be found in above SEQ ID NOs 1 -15, SEQ ID NOs 18-32 and SEQ ID NOs 18-32 accordingly.
  • the nanobody or the heavy chain variable domain comprises an amino acid sequence exhibiting at least 70% identity to any one of SEQ ID NOs 48-62, more preferably wherein the sequences of CDR1, CDR2 and CDR3 domains contained in said amino acid sequence are not altered, when compared with those in corresponding sequence of any one of SEQ ID NOs 48-62.
  • the nanobody or the heavy chain variable domain comprises an amino acid sequence exhibiting at least 80% identity to any one of SEQ ID NOs 48-62, more preferably wherein the sequences of CDR1, CDR2 and CDR3 domains contained in said amino acid sequence are not altered, when compared with those in corresponding sequence of any one of SEQ ID NOs 48-62.
  • the nanobody or the heavy chain variable domain comprises an amino acid sequence exhibiting at least 90% identity to any one of SEQ ID NOs 48-62, more preferably wherein the sequences of CDR1, CDR2 and CDR3 domains contained in said amino acid sequence are not altered, when compared with those in corresponding sequence of any one of SEQ ID NOs 48-62.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 1, a CDR1 having a sequence of SEQ ID NO 18 and a CDR2 having a sequence of SEQ ID NO 33.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 1, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 18 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 33.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 1, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 18 and a CDR2 having a sequence that exhibits at least 80% identity with SEQ ID NO 33.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 1, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 18 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 33.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 2, a CDR1 having a sequence of SEQ ID NO 19 and a CDR2 having a sequence of SEQ ID NO 34.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 2, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 19 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 34.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 2, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 19 and a CDR2 having a sequence that exhibits at least 80% identity with SEQ ID NO 34.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 2, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 19 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 34.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 3, a CDR1 having a sequence of SEQ ID NO 20 and a CDR2 having a sequence of SEQ ID NO 35.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 3, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 20 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 35.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 3, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 20 and a CDR2 having a sequence that exhibits at least 80% identity with SEQ ID NO 35.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 3, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 20 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 35.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 4, a CDR1 having a sequence of SEQ ID NO 21 and a CDR2 having a sequence of SEQ ID NO 36.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 4, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 21 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 36.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 4, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 21 and a CDR2 having a sequence that exhibits at least 80% identity with SEQ ID NO 36.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 4, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 21 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 36.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 5, a CDR1 having a sequence of SEQ ID NO 22 and a CDR2 having a sequence of SEQ ID NO 37.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 5, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 22 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 37.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 5, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 22 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 37.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 5, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 22 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 37.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 6, a CDR1 having a sequence of SEQ ID NO 23 and a CDR2 having a sequence of SEQ ID NO 38.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 6, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 23 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 38.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 6, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 23 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 38.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 6, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 23 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 38.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 7, a CDR1 having a sequence of SEQ ID NO 24 and a CDR2 having a sequence of SEQ ID NO 39.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 7, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 24 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 39.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 7, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 24 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 39.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 7, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 24 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 39.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 8, a CDR1 having a sequence of SEQ ID NO 25 and a CDR2 having a sequence of SEQ ID NO 40.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 8, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 25 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 40.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 8, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 25 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 40.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 8, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 25 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 40.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 9, a CDR1 having a sequence of SEQ ID NO 26 and a CDR2 having a sequence of SEQ ID NO 41.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 9, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 26 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 41.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 9, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 26 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 41.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 9, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 26 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 41.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 10, a CDR1 having a sequence of SEQ ID NO 27 and a CDR2 having a sequence of SEQ ID NO 42.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 10, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 27 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 42.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 10, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 27 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 42.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 10, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 27 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 42.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 11, a CDR1 having a sequence of SEQ ID NO 28 and a CDR2 having a sequence of SEQ ID NO 43.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 11, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 28 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 43.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 11, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 28 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 43.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 11, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 28 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 43.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 12, a CDR1 having a sequence of SEQ ID NO 29 and a CDR2 having a sequence of SEQ ID NO 44.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 12, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 29 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 44.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 12, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 29 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 44.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 12, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 29 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 44.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 13, a CDR1 having a sequence of SEQ ID NO 30 and a CDR2 having a sequence of SEQ ID NO 45.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 13, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 30 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 45.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 13, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 30 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 45.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 13, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 30 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 45.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 14, a CDR1 having a sequence of SEQ ID NO 31 and a CDR2 having a sequence of SEQ ID NO 46.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 14, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 31 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 46.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 14, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 31 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 46.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 14, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 31 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 46.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence of SEQ ID NO 15, a CDR1 having a sequence of SEQ ID NO 32 and a CDR2 having a sequence of SEQ ID NO 47.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 70% identity with SEQ ID NO 15, a CDR1 having a sequence that exhibits at least 70% identity with SEQ ID NO 32 and a CDR2 having a sequence that exhibits at least 70% identity with SEQ ID NO 47.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 80% identity with SEQ ID NO 15, a CDR1 having a sequence that exhibits at least 80% identity with SEQ ID NO 32 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 47.
  • said nanobody or said heavy chain variable domain comprise a CDR3 having a sequence that exhibits at least 90% identity with SEQ ID NO 15, a CDR1 having a sequence that exhibits at least 90% identity with SEQ ID NO 32 and a CDR2 having a sequence that exhibits at least 90% identity with SEQ ID NO 47.
  • said nanobody is derived from mammals e.g. camelid, mouse, rat, pig, donkey, rabbit, sheep or goat, or from a library of VHH or VHH-like structures assembled from same.
  • said nanobody is an intrabody.
  • intrabody refers to a nanobody which takes a function in the intracellular region and binds with an intracellular protein.
  • said anti-beta-catenin nanobody disclosed above is conjugated with another polypeptide.
  • said another polypeptide is a degrader, a biodegrader, a kinase, a kinase domain, a trafficking motif which is directed to a specific region or part of a cell, or a nuclear localization sequence (N LS), wherein more preferably said kinase or kinase domain is responsible for beta- catenin phosphorylation and/or degradation.
  • N LS nuclear localization sequence
  • the present invention also provides a multivalent nanobody, comprising two or more anti- beta-catenin nanobodies as disclosed above.
  • said multivalent nanobody is an intrabody.
  • said two or more anti-beta-catenin nanobodies are nanobodies wherein each nanobody comprises a sequence exhibiting at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibiting 100% identity to the same sequence selected from SEQ ID NOs 1 -15, or each nanobody comprises a combination of sequences wherein each of them exhibits at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibits 100% identity to one of SEQ ID NOs 1 -15, and each nanobody comprises the same combination of sequences corresponding to SEQ ID NOs 1 -15. All of combinations raised from SEQ ID NOs 1 -15 which can be envisaged by a skilled person are disclosed herewith.
  • two or more anti-beta-catenin nanobodies are nanobodies wherein each nanobody comprises a distinct sequence exhibiting at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibiting 100% identity one of SEQ ID NOs 1 -15, or each nanobody comprises a combination of sequences wherein each of them exhibits at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibits 100% identity to one of SEQ ID NOs 1 -15, and each nanobody comprises a distinct combination of sequences corresponding to SEQ ID NOs 1 -15. All of combinations raised from SEQ ID NOs 1-15 which can be envisaged by a skilled person are disclosed herewith.
  • each of two or more anti-beta-catenin nanobodies comprises a combination of sequences wherein each of them exhibits at least 70%, 75%, 80%, 85%, 90%, 95% identity or exhibits 100% identity to one of SEQ ID NOs 1 -15
  • said two or more anti-beta-catenin nanobodies overlap with each other in respect of the combination of sequences corresponding to SEQ ID NOs 1 -15. All of combinations raised from SEQ ID NOs 1 -15 which can be envisaged by a skilled person are disclosed herewith.
  • the multivalent nanobody is a bivalent nanobody.
  • the two or more anti-beta-catenin nanobodies, or fusions of an anti-beta-catenin nanobody with another nanobody targeting other desirable proteins are conjugated through one or more linkers, and wherein more preferably at least one linker is cleavable, furthermore preferably said cleavable linker comprising a sequence which can be cleaved by caspase-8.
  • a cleavable linker can be introduced for separating nanobodies.
  • a cleavable linker is created so that nanobodies can be dosed in known ratios, and so that inactive multivalent nanobody, e.g. bivalent o trivalent nanobody fusions, can be separated in relevant cells to cause their activation (l.e. targeting of the intended protein).
  • inactive multivalent nanobody e.g. bivalent o trivalent nanobody fusions
  • a bivalent beta-catenin nanobody that has a cleavable linker to a monovalent RAS nanobody.
  • one site to introduce into a linker region is the portion of R.IPK1 or a region of Bid, that is processed by caspase-8 and/or cathepsin proteases either at steady state (human R.IPK1, up to and including residues 309-340, which is laid out as SEQ ID NO: 16) or upon cellular stress (human Bid, up to and including residues 41 -82, i.e. Glycine 41 to isoleucine 82 of human BID, which is laid out as SEQ ID NO: 17).
  • the sequences of SEQ ID Nos 16-17 are laid out in Table 2. Most cells express the protease caspase-8 in the cytosol that is active.
  • R.IPK1 cleaves R.IPK1 to prevent autoinflammation or cell death, wherein human mutants exist where the caspase-8 cleavage site of Rl PK1 is lost, and they have severe autoinflammation, so we know it is conserved in people.
  • cathepsins and granzymes can be released from the endosomal-lysosomal compartments and cleave Bid. Fusing nanobodies that contain a RIPK1 sequence cleaved by caspase-8 at steady state (residues 309- 340) will allow their separation and targeting of distinct cellular proteins (e.g.
  • the length and composition of one or more linkers are adjusted to allow for proper structure or to maintain the biological activity of the conjugated anti-beta-catenin nanobodies. More preferably, in order to maintain said biological activity, the linker is composed of 10-80 amino acids, 15-95 amino acids, 20-90 amino acids, 25-85 amino acids, 30-80 amino acids, 35-75 amino acids, 40-70 amino acids, 45-65 amino acids, 50-60 amino acids, or 15-30 amino acids.
  • the nanobody fusions are only activated after cleavage.
  • the present invention also provides the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above for use as a medicament.
  • the present invention also provides the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above for use in treatment of inflammation, autoinflammation, or cancer.
  • the VHH disclosed above, the anti-beta- catenin nanobody disclosed above, and the multivalent nanobody disclosed above can be used in combination.
  • the VHH can be combined with the anti-beta-catenin nanobody, or with the multivalent nanobody.
  • the anti-beta-catenin nanobody can be combined with the multivalent nanobody.
  • the VHH can be combined with the anti-beta-catenin nanobody and the multivalent nanobody.
  • one or more of the VHH disclosed above, the anti-beta-catenin nanobody disclosed above, and the multivalent nanobody disclosed above can also be combined with currently known agents for cancer therapy, for example with Sorafenib/ PD-L1 inhibition.
  • examples of cancer included in the present invention comprise ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, craniopharyrgioma, bile duct carcinoma, choriocarcinoma, Wil
  • examples of cancer included in the present invention also comprises head and neck squamous cell cancer, pancreatic cancer; colorectal cancer; breast cancer; cervical squamous cell cancer; esophageal squamous cell cancer; lung squamous cell cancer, colorectal cancer, solid tumor, colorectal adenocarcinoma; gastric adenocarcinoma; pancreatic adenocarcinoma; bile duct carcinoma; hepatocellular carcinoma, esophageal carcinoma, gastrointestinal cancer, synovial sarcoma, gastroenteric tumor,; gastric cancer; bladder cancer; lung cancer; melanoma, ovarian cancer; osteosarcoma, colon carcinoma, colonic carcinoma, colon cancer, relapsed or refractory AML and MDS, AML; CML, ALL; CML; MM BL, cHL, T-ALL; APL, CLL; MCL.
  • examples of cancer included in the present invention also comprisesBC, cervix cancer, , , , endometrial cancer HER2- BC, sarcoma, basal cell cancer, PC, , metastatic pancreatic cancer, esophageal gastric cancer, MM, NSCLC, biliary tract cancer, CLL, small lymphocytic lymphoma, BC, MCL, HL, AML, pancreatic cancer, and desmoid tumor.
  • Other non-cancer diseases b-catenin dysregulation plays a role in (7) are also included.
  • Atherosclerosis arrhythmic cardiomyopathy, Ml, cardiac fibrosis, congenital heart disease, hair disorders, in the lungs COPD, IPF, BPD, in the bones osteogenesis dysregulation, osteoporosis an in the nervous system diseases include Parkinson's and Alzheimer's, Amyotrophic lateral sclerosis.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above may be used to delay development of a disease or disorder or to slow the progression of a disease or disorder.
  • the term "treating”, “treatment” or “alleviation” refers to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated.
  • treating cancer refers to improving the patient's condition, alleviating, delaying or slowing progression or onset, decreasing the severity of one or more symptoms of cancer.
  • treating cancer includes any one or more of: decreasing tumor size, decreasing rate of tumor size increase, halting increase in size, decreasing the number of metastases, decreasing pain, increasing survival, and increasing progression free survival.
  • the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above are administered in a therapeutically effective amount to the subject ( ⁇ 8).
  • a “therapeutically effective amount” in the present invention is variable according to factors such as age, sex, the disease state, weight of the subject, and the ability of the substance/molecule, to elicit a desired response in the subject.
  • a therapeutically effective amount may encompass an amount in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • the therapeutically effective amount for administration is 0.1 -1.0 mg of mRNA per kilogram (Kg) dosed monthly, 0.2-0.9 mg of mRNA per Kg dosed monthly, 0.3-0.9 mg of mRNA per Kg dosed monthly, 0.3-0.8 mg of mRNA per Kg dosed monthly, 0.3-0.7 mg of mRNA per Kg dosed monthly, 0.3-0.6 mg of mRNA per Kg dosed monthly, 0.3-0.5 mg of mRNA per Kg dosed monthly, 0.3-0.4 mg of mRNA per Kg dosed monthly, or 0.3 mg of mRNA per Kg dosed weekly. More preferably, the therapeutically effective amount for administration is 0.1 -0.3 mg of mRNA per Kg dosed monthly. More preferably, the administration is performed every three weeks, every two weeks, weekly, or twice a week.
  • above administration to one subject is performed in a total number of 2-20 times, 3-19 times, 4-18 times, 5-17 times, 6-16 times, 7-15 times, 8-14 times, 9-13 times, 10-12 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times.
  • the interval of above two administration is 1 -10 days, 2-9 days, 3-8 days, 4-7 days, 5-6 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 -5 weeks, 2-4 weeks, 2-3 weeks, 1 -2 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks.
  • the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above is administered into the subject together with pharmaceutically acceptable carriers and/or excipients including but not being limited to: stabilizing agents, surface-active agents, salts, buffers, colouring agents etc.
  • the present application also provides a composition, preferably a pharmaceutical composition, comprising the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above.
  • said composition comprises pharmaceutically acceptable carriers and/or excipients including but not being limited to: stabilizing agents, surface-active agents, salts, buffers, colouring agents etc.
  • the present invention also provides a polynucleotide, wherein said polynucleotide encodes the VHH disclosed above, the anti-beta-catenin nanobody disclosed above, or the multivalent nanobody disclosed above.
  • said polynucleotide is a messenger RNA (mRNA), a ssDNA, a dsDNA, or a complementary DNA (cDNA). More preferably, said polynucleotide is a mRNA.
  • said polynucleotide is suitable for administration into a subject to express the VHH disclosed above, and/or the anti-beta-catenin nanobody disclosed above, and/or the multivalent nanobody disclosed above in cells of the subject.
  • the commonly used method for administration of above polynucleotide is known to a person skilled in the art, e.g. inhalation or intertumoral or intravenous injection, topical, intrathecal, intranasal, transdermal, intracranial .
  • the present invention also provides the polynucleotide disclosed above for use as a medicament, preferably for use in treatment of inflammation, autoinflammation or cancer.
  • the examples of inflammation, autoinflammation or cancer are disclosed above.
  • said polynucleotide is delivered into cells of the subject by lipid nanoparticles (LNPs).
  • LNPs contains four components: 1) Charged lipid or ionisable lipid, 2) Neutral or helper lipid, 3) Cholesterol and 4) Pegylated lipid (peg-lipid).
  • the charged or ionisable lipid forms a complex with polynucleotide via electrostatic interactions and aggregates to form the core of the LNPs.
  • the neutral lipid and the cholesterol self-assemble onto the core to form outer layer and provide structural stability and rigidity for the LNP.
  • the Peg-lipid primarily reside in the outermost layer due to the hydrophilic nature of the peg and prevents aggregation via steric hindrance.
  • a modified peg-lipid can be used proportionately to the total peg-lipid, which can be used to attach an active targeting agent.
  • An active targeting agent can be a protein, small molecule, or polynucleotide, which increases the uptake of LNP by a target cell or tissue.
  • the LNPs are produced using microfluidic technology wherein the lipid component in ethanol and polynucleotide in sodium acetate buffer at pH 4-5 are mixed rapidly.
  • the following parameter or features can be optimised depending upon the length of the polynucleotide: 1) the N to P ratio (the ratio between the positively charged lipid or ionisable lipid (Nitrogen atom) to the negatively charged nucleic acid (Phosphate molecule) can be determined dependent on the length of the polynucleotide. 2) the more ratio of the charged- or ionisable- lipid, the helper lipid, cholesterol, and peg-lipid. 3) the flow rate and the volume ratio at which the lipid solution and polynucleotide can be mixed.
  • formed LNPs can deliver the polynucleotide to a desired organ, tissue, or a particular type of cell.
  • the LNP carrying polynucleotide can preferentially accumulate in an organ or tissue of interest, depending on the lipid composition.
  • the LNPs can accumulate in disease sites characterized by abnormal blood vessels. For example, LNPs accumulate in tumor tissue due to enhanced permeation and retention as the tumor blood vessels are leaky with poorly lined endothelial cells.
  • natural polymers such as polysialic acid, chitosan, dextran, hyaluronic acid, cyclodextrin, alginate, and gelatin pullulan have been explored and can be used for preparation LNPs. Even synthetic polymers can be used in the present invention.
  • Synthetic polymers can be chitosan derivatives, , poly(oxazolines) (POXs), poly(carboxybetaine acrylamide) (PCBAA), poly( hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(carboxybetaine) (PCB), poly(sulfobetaine) (PSB), dendrimers, poly(Bamino ester) (PAE), polyanhydride, polyethylene glycol) (PEG) ,and so on which can be envisaged by a person skilled in the art.
  • POXs poly(oxazolines)
  • PCBAA poly(carboxybetaine acrylamide)
  • PPMA poly( hydroxypropyl methacrylate)
  • PHEMA poly(2-hydroxyethyl methacrylate)
  • PSB poly(sulfobetaine)
  • dendrimers poly(Bamino ester) (PAE), polyanhydride,
  • two or more different type of polynucleotides for encoding different target polypeptides are administered into the subject at the same time. More preferably, the administration is performed in the same way as in above preferred embodiments.
  • Table 1 Sequences of SEQ ID NOs 1 -15. The detailed description with regard to nanobody plasmid ID and sequences of CDR3 can be found in Examples with regard to Table 3.
  • Figure 1 Pre and Post immunisation sera reactivity ELISA. Microtiter wells were coated with 125 nM non-biotinylated target antigen. Binding of varying concentrations of alpaca serum (x-axis) was detected with HRP-conjugated anti-alpaca antibodies. Absorbance was measured at 405 nm with technical duplicates.
  • FIG. 2 ELISA screen of phage supernatants against antigen to determine positive hits. 3 rounds of immobilised panning was performed. Microtiter wells were coated with 250 nM nonbiotinylated target antigen. Binding of the nanobodies was detected with an anti-phage M13 antibody. The dotted line represents the cut off OD 405 for a positive hit. H1 is a media control and H12 is a negative control with an irrelevant Nb.
  • FIG. 3 Summary of ELISA screening and nanobody sequence analyses. 94 individual clones were randomly selected for the ELISA screen. ELISA-positive clones were sent for sequencing and high quality Sanger-sequences (Phred score > 20) were translated and annotated using the International ImMunoGeneTics database (IMGT). The translated VHH and CDR3 sequences were aligned and clustered using PipeBio.
  • IMGT International ImMunoGeneTics database
  • FIG. 4 Nanobody Purification. Nanobodies were purified using a single-step Ni-NTA affinity. Reducing SDS PAGE gel of purified Nb. Yield: Beta_R3_A9 Nb of ⁇ mg/mL in ⁇ uL PBS Buffer was obtained from 250 mL Terrific Broth culture.
  • Figure 5 Reactivity of purified nanobody to antigen. Microtiter wells were coated with 250 nM of biotinylated antigen. Binding of varying concentrations of purified nanobody (x-axis) was detected with mouse-anti-his followed by HRP-conjugated anti-mouse antibodies. Absorbance was measured at 405 nm with technical duplicates.
  • FIG. 6 B -catenin VHH expression blocks Wnt signalling in Hek293T cells.
  • FIG. 7 B-catenin sdAb expression blocks Wnt signalling in Hek293T cells. Wnt/B-catenin signalling was measured in Hek293T cells transfected with vectors expressing various B-catenin binding sdAb constructs (i.e. sdAb-RFP, bivalent sdAb-RFP, sdAb, sdAb-RFP mutant (with limited binding to B-catenin), bivalent sdAb-RFP mutant (with limited binding to B-catenin)).
  • sdAb-RFP bivalent sdAb-RFP, sdAb, sdAb-RFP mutant (with limited binding to B-catenin)
  • FIG. 8 Bivalent B-catenin VHH intra body blocks Wnt signalling in HepG2 cells. Wnt/B-catenin signalling was measured in HepG2 cells incubated with LNPs loaded with B-catenin VHH-VHH mRNA, B-catenin VHH-VHH mutant mRNA or empty LNPs at 100ng/mL, 500ng/mL or 2500ng/mL.
  • the mutant control (VHH-VHH mutant mRNA) contains three Ala mutations (AAA) ablating intrabody binding to B-catenin. Data is pooled from 3 independent experiments, triplicate wells.
  • Bivalent B-catenin sdAb blocks Wnt signalling in HepG2 cells. Wnt/B-catenin signalling was measured in HepG2 cells incubated with LNPs loaded with bivalent B-catenin sdAb-RFP mRNA, bivalent B-catenin sdAb-RFP mutant mRNA or empty LNPs at 100ng/mL, 500ng/mL or 2500ng/mL. Bivalent B-catenin sdAb-RFP mutant has reduced binding to B-catenin. Data is pooled from 3 independent experiments, triplicate wells. For normalisation, the ratio of Firefly luminescence from TCF/LEF reporter to Renilla luminescence from Renilla control was calculated.
  • FIG. 10 Bivalent VHH prevents translocation of B-catenin in HepG2 cells.
  • B-catenin presence (8, E) was measured within HepG2 cells incubated following Wnt-signalling activation and after activation with LNPs loaded with B-catenin VHH-VHH mRNA at 4000ng/mL. Nuclei were identified with DAPI (A, D).
  • Merged images (C, F) illustrate that B-catenin is preferentially localised within nuclei rather than the cytosol where not VHH treatment was performed, while B-catenin was distributed evenly across the cytosol and nuclei when VHH was present.
  • FIG 11 [3-catenin binding intrabody (bivalent sdAb-RFP) expression reduces nuclear translocation of B-catenin in HepG2 cells.
  • HepG2 cells were treated with LNPs loaded with bivalent sdAb-RFP mRNA and B-catenin localisation was analysed (B,E). Nuclei identified by DAPI staining (A, D). Nuclear translocation of B-catenin upon Wnt activation (C) is prevented upon intrabody treatment. mCherry not shown. Brightness and contrast of all images were set identically.
  • Figure 12 Quantitation of translocation prevention of B-catenin in HepG2 cells by Bivalent VHH.
  • B-catenin presence was measured within HepG2 cells incubated following Wnt-signalling activation with no treatment, LNPs loaded with B-catenin VHH-VHH mRNA mutated to impair binding at 4000ng/mL, MSAB (a known Wnt-signal inhibitor) at 0.3 uM, LNPs loaded B-catenin VHH-VHH mRNA at 1000ng/mL and LNPs loaded B-catenin VHH-VHH mRNA at 4000ng/mL.
  • MSAB a known Wnt-signal inhibitor
  • Ratios of B-catenin presence in the nuclei vs the cytosol was compared demonstrating highly significant reductions in nuclear B-catenin when treated with bivalent VHH and either concentration. Error bars represent SEM. ns P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001 in a two-way Anova.
  • Figure 13 Quantification of reduced B-catenin nuclear translocation in HepG2 cells upon bivalent B-catenin sdAb expression. Using fluorescence, B-catenin localisation in HepG2 cells was quantified following treatment with LNPs loaded with bivalent B-catenin sdAb-RFP mRNA. Treatments: Untreated control, sdAb-sdAb-RFP mutant 4000, MSAB (Wnt inhibitor), sdAb- sdAb-RFP 1000, sdAb-sdAb-RFP 4000. Fluorescent intensities of nuclear and cytosolic B- catenin were measured by confocal microscopy and ratios calculated. Error bars represent SEM. ns P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001 in a two-way Anova.
  • FIG 14 Quantitation of translocation prevention of B-catenin in multiple cell types by Bivalent VHH.
  • B-catenin presence was measured within HepG2, HUH7 and SK-HEP1 cells incubated following Wnt-signalling activation and after activation with no treatment and with LNPs loaded with B-catenin VHH-VHH mRNA at 4000ng/mL.
  • a Student t-test demonstrated significance (P ⁇ 0.05).
  • Figure 15 Bivalent B-catenin sdAb expression reduces B-catenin nuclear localisation in liver cancer cell lines.
  • FIG. 16 B-catenin sdAbs expression blocks Wnt signalling in Hek293T Wnt reporter cells. Expression of B-catenin sdAbs in a Wnt reporter cell line significantly reduce Wnt signalling. Activatedcontrol (lipofectamine only) and control sdAb. Treatments normalised to cell number and relative to activated lipofectamine control.
  • FIG 17 Location of protease cleavage sites in Bid.
  • Bid is shown with a ribbon plot.
  • the N- terminal helices, which are removed to convert to the pro-apoptotic form, are colored magenta; the "bait loop," where most of the protease cleavage sites reside, is yellow; and the C-terminal part of Bid, which is implicated in progression of apoptosis, is green. All of the cleavage sites are marked together with the cleaving proteases.
  • the coordinates were taken from the Protein Data Base (2BID).
  • the figure was prepared using WebLabViewer Lite Version 4.0 (Molecular Simulations Inc.).
  • Figures 18a and 18b Predicted binding sites and their relevance for B-catenin. Structural predictions of the binding sites associated with three sdAbs (A-C). The physical structure of B- catenin is well-known from x-ray crystallography and domains relevant to its many interactions have been collectively documented as well ( ). Our analysis confirms that that sdAbs bind to B-catenin using their respective CDR3 loops. Furthermore, their binding can be seen to epitopes within B-catenin that are critical for Wnt-signalling providing results consistent with other functional assays.
  • Intrabodies against the beta-catenin are identified through three different routes:
  • Multivalent intrabodies Multiple intrabodies were conjugated with a linker to generate multivalent intrabodies, which can significantly enhance inhibition of the target. Preliminary data shows that bivalent ASC nanobodies can inhibit inflammasome activity far more efficiently than the published monovalent ASC nanobody.
  • alpaca lymphocyte mRNA was extracted and amplified by RT-PCR with specific primers to generate a cDNA library size of 105 nanobodies with 80% correct sized nanobody insert.
  • the library was cloned into a pMES4 phagemid vector amplified in E. coli TG1 strain and subsequently infected with M 13K07 helper phage for recombinant phage expression.
  • Pre and post-immunization serum was tested for reactivity to human beta-catenin using ELISA and a dilution series of alpaca serum.
  • biopanning for beta-catenin nanobodies using phage display was performed as previously described in Pardon et al. (2014). Phages displaying beta-catenin nanobodies were enriched after three rounds of biopanning on 10 ug/ml (or 1 ug/well) of immobilized beta- catenin protein. After the third round of panning, individual clones were selected for further analyses by ELISA for the presence of beta-catenin nanobodies.
  • microtiter wells were coated with 250 nM of beta-catenin. Binding of the nanobodies was detected with an anti-phage M13-HRP antibody. The dotted line represents the cut off absorbance measurement (OD 405) for a positive hit.
  • H1 is a media control and H12 is a negative control with an irrelevant nanobody.
  • Positive clones were sequenced and annotated using the International ImMunoGeneTics database (IMGT) and aligned in Geneious Prime. After three rounds of bio-panning, distinct nanobody clonal groups were identified based on differences in the amino acid sequence of the complementary determining region 3 (CDR3).
  • IMGT International ImMunoGeneTics database
  • nanobodies were expressed in Escherichia coli WK6 cells. Bacteria were grown in Terrific Broth at 37 °C to an OD600 of 0.7, induced with 1 mM IPTG and grown overnight at 28 °C for 16 h. Cell pellets were harvested and resuspended in 20% sucrose, 20 mM imidazole, 150 mM NaCI DPBS and incubated for 15 min on ice. 5 mM EDTA then added and incubated on ice for 20 minutes.
  • HRP horseradish peroxidase
  • B -catenin VHH-RFP bivalent B-catenin VHH-RFP, B -catenin VHH- FLAG, B -catenin VHH-RFP mutant with reduced binding to B -catenin , bivalent B -catenin VHH-RFP mutant with reduced binding to B -catenin
  • Cells were then stimulated with Wnt3a (100 ng/mL) or R-Spondin (200 ng/mL) overnight to activate the Wnt signalling pathway.
  • the amount of Wnt signalling was quantified by cellular luciferase activity according to the manufacturers protocol (PierceTM Firefly Luc One-Step Glow Assay Kit).
  • Wnt3a 100 ng/mL
  • R-Spondin 200 ng/mL
  • the amount of Wnt signalling was quantified by cellular luciferase activity according to the manufacturers protocol (PierceTM Firefly Luc One-Step Glow Assay Kit).
  • HepG2 cells were seeded (triplicates wells per treatment) on flat-bottom 96-well plates and incubated overnight (37°C, 5%CO2).
  • cells were transfected as per manufacturer's instructions (BPS biosciences, Transfection CollectionTM: TCF/LEF Transient Pack Wnt I
  • 7 h post transfection the media was replaced with fresh media or indicated mRNA/LNPs treatments diluted in cell media. Treatments were incubated at 37°C 5%CO2. 48h post-transfection Luciferase activity was measured following manufacturer's instructions, plate was read on TecanTM Spark plate reader. Both VHH mRNA constructs contain a RFP sequence.
  • HepG2 cells were seeded (triplicates wells per treatment) and incubated overnight (37°C, 5% CO2). On day 2, cells were transfected as per manufacturer's instructions (BPS biosciences, Transfection CollectionTM: TCF/LEF Transient Pack Wnt/
  • HepG2 cells were seeded on 1.8 cm square slide coverslips and incubated overnight (37°C, 5%CO2). On day 2, cells were treated with B-catenin VHH-VHH mRNA at 4000ng/mL. On day 3 cells were stimulated with Wnt3a (100ng/mL) or R-Spondin (200ng/mL) to activate the Wnt signalling pathway. The cells were fixed to the slide covers on day 4 and stained using a standard antibody staining protocol. Nuclei were stained with SlowFade Gold antifade DAPI (INVITROGEN).
  • B-catenin was stained using a primary antibody of Anti B-catenin -Mouse (Thermo CAT-5H10) at 1.5 ug/ml and a secondary antibody of Anti-mouse in Donkey attached to AlexaFluor 488 (Invitrogen A21202) at 2 ug/ml. Images were obtained on a Nikon TiE spinning disk confocal microscope equipped with 100 mW Andro LC 600 series lasers at 405, 488, and 561 nm and a 140 mW 638 nm Andro LC 600 series laser. Image analysis was performed in ImageJ.
  • HepG2 cells were seeded on 1.8 cm square slide coverslips and incubated overnight (37°C, 5% CO2).
  • cells were treated with bivalent B-catenin sdAb-RFP mRNA at 4000ng/mL.
  • cells were stimulated with Wnt3a (100ng/mL) and R-Spondin (200ng/mL) to activate Wnt signalling.
  • the cells were fixed to the slide covers on day 4 and stained using a standard antibody staining protocol. Nuclei were stained with SlowFade Gold antifade DAPI (INVITROGEN).
  • B-catenin was stained using a primary antibody of Anti B-catenin -Mouse (Thermo CAT-5H10) at 1.5 ug/ml and a secondary antibody of Anti-mouse in Donkey attached to AlexaFluor 488 (Invitrogen A21202) at 2 ug/ml. Images were obtained on a Nikon TiE spinning disk confocal microscope equipped with 100 mW Andro LC 600 series lasers at 405, 488, and 561 nm and a 140 mW 638 nm Andro LC 600 series laser. Image analysis was performed in ImageJ.
  • HepG2 cells were seeded on 1.8 cm square slide coverslips and incubated overnight (37°C, 5%CC>2).
  • cells were treated as indicated with 0.3 uM MSAB, LNPs loaded with bivalent B-catenin sdAb-RFP mRNA at 1000ng/mL or 4000ng/mL, respectively.
  • cells were stimulated with Wnt3a (100ng/mL) and R-Spondin (200ng/mL) to activate Wnt signalling.
  • Wnt3a 100ng/mL
  • R-Spondin 200ng/mL
  • Dual Wnt reporter cells (GFP and luciferase reporter) were seeded and incubated overnight (37°C 5%CC>2).
  • Lipofectamine 2000 was used to transfect cells with a vector expressing a sdAb construct of interest (i.e. sdAb mutant with reduced binding to B-catenin, or constructs expressing a B-catenin- specific sdAb labelled A7, A8, A9, B5, B10, B11, C8, D2, D6, F11, G3, G8 or H9, respectively) for 24 hours.
  • Cells were then stimulated with Wnt3a (100 ng/mL) and R-Spondin (200 ng/mL) overnight to activate the Wnt signalling pathway.
  • nanobodies can be delivered into the cell via mRNA LNP technology.
  • Various nanobody complexes such as a bivalent nanobody could also be connected to a binder and/ or a degrader fused to it, to attain the desired therapeutic effects.
  • the present application provides a plausible technical concept, and the desired effects connected with said nanobodies conjugated with binder and/or a degrader and/or a localization signal can be envisaged by a person skilled in the art.
  • Generating sequences by cloning is not difficult, however the CDR3 contains a minimum of 13 amino acids representing 3 base pairs per amino acids is 39 base pairs at 4 potential bases 2
  • a 78 potential sequence arrangements possibilities for a good binder Generating all potential binders can only be achieved either using phage display libraries or by immunization of relevant animals such as Alpacas. Both of these approaches require a very skilled person to obtain only the potential binders.
  • evaluating potential binders for functionality of inhibiting Wnt signaling requires various established Wnt readouts.
  • a successful binder to a Wnt protein does not necessarily make this binder therapeutically relevant as the binder additionally has to be stable in the cytosol, non-toxic to cells and interact directly with b-catenin in very specific ways.
  • Establishing this therapeutically relevant and specific inhibitory activity of a binder requires various assays, expertise in cell signalling pathwasy as well as therapeutics.
  • b-catenin overexpression is the driving factor of overproliferation and tumorigenesis via Wnt-signaling.
  • Targeting b-catenin during Wnt-signalling is an effective treatment during cancer development 8).
  • Elevated Wnt signaling has been linked to a higher rate of proliferation and thus disease progression (9, 70) .Therefore, the level of Wnt inhibition is an important indication for therapeutic efficacy.
  • sdAb A9 attains increased inhibition of Wnt-signalling and has furthermore shown significantly increased degradation of b-catenin (compared to control sdAb). Since b-catenin suppression has been shown to be therapeutically effective, this increased inhibitory effect of the A9 sdAb is a direct readout for a superior therapeutic effect.

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Abstract

La présente demande concerne un domaine variable de chaîne lourde (VHH) d'un nanocorps anti-beta-caténine, la séquence d'acides aminés dudit VHH comprenant une séquence présentant au moins 70% d'identité avec l'une des SEQ ID NO 1-15, et ladite séquence présentant au moins 70% d'identité avec l'une des SEQ ID NO 1-15 médiant la liaison dudit nanocorps avec la beta-caténine, et concerne également un nanocorps anti-beta-caténine comprenant le VHH ci-dessus, et ledit nanocorps étant de préférence un intracorporel.
PCT/EP2024/063091 2023-05-12 2024-05-13 Nanocorps pour traiter l'inflammation, l'auto-inflammation ou le cancer Pending WO2024235910A1 (fr)

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