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WO2024084203A1 - Anticorps à domaine unique se liant à l'albumine - Google Patents

Anticorps à domaine unique se liant à l'albumine Download PDF

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Publication number
WO2024084203A1
WO2024084203A1 PCT/GB2023/052694 GB2023052694W WO2024084203A1 WO 2024084203 A1 WO2024084203 A1 WO 2024084203A1 GB 2023052694 W GB2023052694 W GB 2023052694W WO 2024084203 A1 WO2024084203 A1 WO 2024084203A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
modifications
variant
Prior art date
Application number
PCT/GB2023/052694
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English (en)
Inventor
Edward MCGOWAN
Simon Cooper
Sophie WATCHAM
Lurdes DUARTE
Marion CUBITT
Ines BARBOSA
Anna BOCZKAJ
Clare Simpson
Original Assignee
Isogenica Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2215365.4A external-priority patent/GB202215365D0/en
Priority claimed from GBGB2312183.3A external-priority patent/GB202312183D0/en
Application filed by Isogenica Limited filed Critical Isogenica Limited
Publication of WO2024084203A1 publication Critical patent/WO2024084203A1/fr

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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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to serum albumin-binding single domain antibodies, in particular serum albumin-binding VHHs, and molecules comprising said single domain antibodies.
  • the invention also relates to methods utilising the single domain antibodies to simultaneously improve the half-life of and purification methods for therapeutic and diagnostic agents.
  • Fc domains have many other functions such as immune cell regulation and inclusion of an Fc domain in a bio-therapeutic molecule has, in some instances, been shown to induce toxicity in the liver or reduce the potency of IgG-based bi-specifics in solid tumours, e.g. as was observed for the catumaxomab (Middleburg et al., Cancers. 2021, 13, 287).
  • possible adverse side effects such as cytokine release, can result from Fc interaction with Fc-gamma receptors (FcyRs) which are ubiquitously expressed on immune cells.
  • FcyRs Fc-gamma receptors
  • HSA human serum albumin
  • HSA is the most abundant protein in plasma, with a circulatory half-life of approximately 3 weeks.
  • Serum albumin is actively recycled through the endocytic compartment in peripheral tissue by binding to FcRn.
  • FcRn acidic pH-dependent binding protects the FcRn: albumin complex from degradation within the endosomal pathway.
  • Albumin is then released back into the bloodstream upon exocytosis and exposure to neutral blood pH. Conjugation or tight binding to albumin allows a drug to ‘piggyback’ this serum albumin endocytic recycling pathway, thereby extending half-life. Accordingly, there remains a need for safe and effective products and methods which increase the half-life of biotherapeutics without compromising the drug’s pharmacokinetics, thereby prolonging efficacy and reducing pronounced dose fluctuation.
  • T0023500 also known as T023500029; corresponds to SEQ ID NO: 19 of WO 2018/134235.
  • Alb23 and T0023500 were considered best-in-class anti-HSA single domain antibodies.
  • these antibodies suffer from disadvantages.
  • T0023500 demonstrates little or no binding to protein A, which is widely used as an affinity ligand for the purification of antibodies, and thus must be purified by less desirable tag-based methods.
  • Alb23 demonstrates limited cross-reactivity across albumin proteins of different species, limiting their capacity for use in animal-based trials and in agricultural or veterinary settings.
  • albumin-binding single domain antibodies which display a full suite of high-affinity binding to the target albumin of interest (usually HSA but other species of serum albumin may be preferred depending on intended use), high affinity for protein A for ease of purification, and broad cross-reactivity.
  • the present invention relates to serum albumin-binding single domain antibodies suitable for use in extending the half-life of human and other animal diagnostic and therapeutic agents, and/or use as process reagent in protein A purification.
  • the anti-albumin single domain antibodies of the invention are multi-functional: they provide the dual benefit of strong binding to protein A, thereby allowing manufacturing options and flexibility, as well as cross-reactivity to serum albumin from a broad range of animal species. To the best of the present inventors’ knowledge, these benefits were previously considered mutually exclusive. For example, as discussed above, existing state- of-the-art anti-albumin VHH molecules may demonstrate either the ability to be purified effectively via protein A or broad-range albumin species cross-reactivity but not both.
  • the single domain antibodies described herein advantageously bind to serum albumin with high specificity and binding affinity across a wide pH range, including acidic endosomal pHs, without disrupting the serum albumi FcRn binding interaction. These properties advantageously allow single domain antibodies of the invention to ‘piggyback’ through the endocytic recycling compartment, preventing renal clearance and lysosomal degradation and significantly increasing half-life of such antibodies.
  • the serum albumin-binding single domain antibodies of the invention are readily amenable to chemical and genetic conjugation to one or more agents without deleteriously impacting the properties of said agent or said single domain antibodies. It has also been demonstrated that the single domain antibodies described herein are tolerant to the addition of linker sequences at their N- and C- termini. The conjugated agent can thereby be escorted through the endocytic recycling pathway and, as demonstrated herein, its half-life significantly prolonged. Single domain antibodies of the invention bind HSA with improved binding affinity than other anti-albumin single domain antibodies, for example Alb23.
  • the single domain antibodies described herein bind well to protein A.
  • the single domain antibodies described herein exhibit superior binding to protein A relative to known anti-albumin single domain antibodies (for example T0023500), such that they can be purified effectively from complex biological mixtures.
  • Protein A purification techniques have become a standard method for purification of antibody-based drugs, since full-length antibodies bind almost universally to protein A. However, most VHHs exhibit no protein A binding (De Genst et al. Developmental & Comparative Immunology. 2006, 30, 1-2, 187-198).
  • Purification of single domain antibodies using protein A affinity chromatography is desirable since it avoids the need to generate and optimise a construct in which a tag (for example a His-tag or cleavable purification tag) is fused to the single domain antibody sequence. This can be a time-consuming and costly process.
  • recombinant tags may negatively affect pharmacokinetic, immunogenicity and binding properties of the single domain antibody product, or require additional purification steps such as tag cleavage.
  • protein A affinity purification is cheaper and more efficient than tag-based purification.
  • single domain antibodies described herein may be purified more easily, more cheaply, and in a form better suited to therapeutic use.
  • a further advantage of the single domain antibodies described herein lies in their cross reactivity to serum albumin from different species, such as human (HSA), cynomolgus monkey (CSA) and murine (MSA) serum albumin, as well as feline serum albumin, bovine serum albumin, canine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • Cross-reactivity of single domain antibodies described herein is thus broader than that seen for other anti-albumin single domain antibodies, for example Alb23 or T0023500.
  • the dual benefits of easy manufacturing and broad cross-reactivity have to date been considered mutually exclusive. Modular approaches result in bulky molecules which cannot readily be purified and manufactured. Further, multivalent binding of FcRn-associated HSA can lead to FcRn crosslinking and trafficking of the drug to the lysosome for degradation (Weflen et al Mol. Biol. Cell 2013 1; 24(15): 2398-2405). This would lead to targeting of the conjugated agent for destruction, instead of prolonging its survival in the bloodstream.
  • the single domain antibodies of the invention advantageously address this drawback.
  • the single domain antibodies of the invention possess intrinsic cross-species reactivity and can also be purified using protein A chromatography, thereby allowing cost- effective manufacture, including through the use of “gold standard” mammalian expression systems.
  • the present invention thus provides a single domain antibody that specifically binds albumin, has multi-species cross-reactivity and binds to protein A.
  • the single domain antibody comprises a complementarity determining region (CDR) 1 comprising the amino sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications, a CDR2 having the amino acid sequence of SEQ ID NO: 189 or a variant thereof comprising up to three modifications, and a CDR3 having an amino acid sequence SEQ ID NO: 33 or a variant thereof comprising up to three modifications.
  • CDR complementarity determining region
  • the single domain antibody comprises a CDR1 comprising the amino sequence of SEQ ID NO: 1 or a variant thereof comprising up to three modifications, a CDR2 comprising the amino acid sequence of SEQ ID NO: 190 or a variant thereof comprising up to three modifications, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191 or a variant thereof comprising up to three modifications.
  • a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 13, 15, 16, 17, 18, 19, 164, and variants thereof comprising up to three modifications;
  • a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 166, and variants thereof comprising up to three modifications;
  • a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 35, 36, 37, 38, 168, and variants thereof comprising up to three modifications.
  • the single domain antibody comprises:
  • a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 13, and variants thereof comprising up to three modifications;
  • a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 23, 24, and variants thereof comprising up to three modifications;
  • a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 33, and variants thereof comprising up to three modifications.
  • the single domain antibody comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising up to three modifications, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications.
  • the single domain antibody comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof comprising up to three modifications, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications.
  • the single domain antibody comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to three modifications, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to three modifications, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to three modifications.
  • the single domain antibody comprises the amino acid sequence of:
  • SEQ ID NO: 131 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 132 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto; or
  • SEQ ID NO: 124 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto.
  • the present invention also provides a single domain antibody that specifically binds serum albumin, wherein the single domain antibody comprises:
  • a CDR1 comprising the amino sequence of SEQ ID NO: 1 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino sequence of SEQ ID NO: 2 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino sequence of SEQ ID NO: 3 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 164 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 166 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 168 or a variant thereof comprising up to three modifications.
  • the single domain antibody comprises the amino acid sequence of:
  • SEQ ID NO: 153 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 154 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 155 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 156 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 157 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 158 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 159 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 160 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 161 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 126 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 127 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 128 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 130 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 131 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 132 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 133 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 174 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 142 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 136 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 140 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto;
  • SEQ ID NO: 141 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto; or
  • SEQ ID NO: 175 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity thereto.
  • the present invention also provides a binding molecule comprising a single domain antibody of the invention and one or more agents, optionally wherein the one or more agents comprises a therapeutic agent or a diagnostic agent.
  • the present invention further provides one or more polynucleotides encoding a single domain antibody of the invention and/or a binding molecule of the invention.
  • the present invention also provides a vector comprising the one or more polynucleotides of the invention.
  • the present invention further provides a cell comprising the one or more polynucleotides of the invention and/or the vector of the invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a single domain antibody of the invention, a binding molecule of the invention, the one or more polynucleotides of the invention, the vector of the invention, and/or the cell of the invention, optionally wherein the composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the present invention further provides a method of extending the half-life of an agent in vivo, comprising administering a single domain antibody of the invention, a binding molecule of the invention, the one or more polynucleotides of the invention, and/or the vector of the invention to a patient simultaneously or sequentially with the agent.
  • Also provided by the present invention is the use of a single domain antibody of the invention, a binding molecule of the invention, the one or more polynucleotides of the invention, and/or the vector of the invention in a method of extending the half-life of an agent in vivo.
  • a method of manufacturing a single domain antibody of the invention or a binding molecule of the invention comprising expressing in a host cell the one or more polynucleotides of the invention, optionally wherein the method further comprises recovering the single domain antibody or binding molecule.
  • FIG. 1 Evaluation of binding of VHHs to recombinant serum albumin. Binding curves, performed by ELISA, showing binding of 12 selected and purified VHHs from master plates ISA-1 and ISA -2 to (A) immobilised HSA (HSA-H5220) and (B) immobilised MSA (MSA-M52H8).
  • the VHHs evaluated are ISA1-D3, ISA1-C8, ISA1-B7, ISA2-F8, ISA2-A8, ISA1-F11, ISA1-E6, ISA1-F6, ISA1-E7, ISA1-B5, ISA1-B9, ISA2-E8, as indicated.
  • FIG. 1 Cross-species binding of VHHs to recombinant serum albumin and binding to human Domain II.
  • Dose response curves performed by ELISA, for 10 VHHs against immobilised recombinant: (A) human serum albumin (HSA; HSA-H5220), (B) cynomolgus serum albumin (CSA; CSA-C52H4), (C) murine serum albumin (MSA; MSA-M52H8) and (D) Domain II (Albumin Bioscience 1002). Curves analysed to determine ECso with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.2.0).
  • VHHs evaluated are: ISA1-D3, ISA1-C8, ISA1-B7, ISA2-F8, ISA2-A8, ISA1-F11, ISA1- E6, ISA1-F6, ISA1-E7, ISA1-B5, as indicated.
  • FIG. 3 Evaluation of binding of VHHs to albumin in serum in a competition ELISA to recombinant serum albumin.
  • Dose response curves performed by competitive ELISA, to measure competing binding to native serum albumin of 10 VHHs against immobilised recombinant (A) HSA (HSA-H5220), (B) CSA (CSA-C52H4), (C) MSA (MSA-M52H8). Curves analysed with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.2.0).
  • FIG. 4 Evaluation of binding of VHHs with N- or C-terminal link to recombinant serum albumin.
  • Dose response curves performed by ELISA, for 10 N-terminally or C- terminally linked VHHs against recombinant HSA (HSA-H5220) or MSA (MSA-M52H8).
  • HSA-H5220 HSA-H5220
  • MSA-M52H8 MSA-M52H8
  • FIG. 5 Evaluation of binding of parental (non-humanised) or humanised VHHs to recombinant human and murine serum albumin.
  • A parental VHH against HSA;
  • B aggressively humanised VHH against HSA;
  • C lightly humanised VHH against HSA;
  • D parental VHH against MSA;
  • E aggressively humanised VHH against MSA; and
  • FIG. 6 Evaluation of binding of parental (non-humanised) or humanised VHHs to albumin in serum in a competition ELISA to recombinant serum albumin.
  • C lightly humanised VHH against HSA;
  • D parental VHH against MSA;
  • E aggressively humanised VHH against MSA; and
  • FIG. 7 Evaluation of cross-species binding of VHHs to recombinant serum albumin and human Domain II at pH 7.4 and pH 6.0.
  • Dose response curves performed by ELISA, for 4 lead VHHs - C8Light, F8Light, Fl lLight and A8Light- against: (A) recombinant HSA (HSA-H5220) at pH 7.4; (B) recombinant CSA (CSA-C52H4) at pH 7.4; (C) recombinant MSA (MSA-M52H8) at pH 7.4; (D) recombinant Domain II (Albumin Bioscience 1002) at pH 7.4; (E) recombinant HSA at pH 6.0; (F) recombinant CSA at pH 6.0; (G) recombinant MSA at pH 6.0; and (H) recombinant Domain II at pH 6.0. Curves analysed with log( agonist) vs. response - Variable slope
  • Figure 8 Evaluation of cross-species binding of C8Light to recombinant serum albumin and human Domains I, II and III.
  • Dose response curves performed by ELISA, for C8Light against recombinant HSA, CSA, MSA and Domain I, Domain II and Domain III as indicated. Curves analysed with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.2.0).
  • Figure 9 Evaluation of specificity of lead clones to recombinant serum albumin.
  • Dose response curves performed by ELISA, for selected lead clones Fl 1 Light, F8Light, C8Light, C8 R100>Q, C8 non-humanised, and A8 non-humanised as indicated against (A) recombinant HSA and off-target (B) cardiolipin, (C) keyhole limpet hemocyanin (KLH), (D) lipopolysaccharide (LPS), (E) ssDNA, (F) dsDNA, (G) insulin and (H) streptavidin. Curves analysed with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.2.0).
  • FIG. 11 Evaluation of binding of bi-specific C8Light:scFv to recombinant serum albumin via Domain II.
  • Dose response curves performed by ELISA, of C8Light:scFv bi- specific against recombinant HSA, CSA, MSA and Domain I, Domain II and Domain III as indicated. Curves analysed with log( agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.2.0).
  • FIG. 12 Evaluation of binding of VHH:VHH bi-specifics to recombinant serum albumin via Domain II.
  • Dose response curves performed by ELISA, of (A) IRR:F 11 Light bi-specific and (B) IRR:C8R100>Q bi-specific, wherein ‘IRR’ is an irrelevant (non-human target binding) VHH having the amino acid sequence of SEQ ID NO: 150, against recombinant HSA, CSA, MSA and Domain I, Domain II and Domain III as indicated.
  • FIG. 13 Evaluation of cross-species bi-specific binding at pH 5.0.
  • Figure 16 Evaluation of cross-reactivity. Side-by-side comparison of dose response curves against recombinant HSA (A and D), CSA (B and E), MSA (C and F), and native purified canine (G), feline (H) and bovine (I) serum albumin for a panel of anti-albumin clones and comparator molecules, as indicated. (Curves analysed with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.4.2).
  • Figure 21 Evaluation of binding to recombinant albumin at pH 6.0. Side-by-side comparison of dose response curves for VHHs A8-Light, B7-Light, B9-Light, B9-Parental, C8-Light, E6-Light, F8-Light and Fl l-Light against recombinant HSA (A), MSA (B) and CSA (C) for panel of anti-albumin clones and comparator molecules at pH 6.0. Comparator molecules Alb23 and T0023500 were used as positive controls. (Curves analysed with log(agonist) vs. response - Variable slope (four parameters) in GraphPad (v. 8.4.2).
  • Figure 22 Protein A purification of anti-albumin clones. Percentage recovery measured by protein A pull-down and elution for B7-Light, C8-Light and F8-Light. Comparator molecules Alb23 and T0023500 were used as controls. Full data is provided in Table 9 below.
  • SEQ ID NO: 1 is the consensus amino acid sequence of CDR1 of VHHs F8, F6, C8, and B7.
  • SEQ ID NO: 2 is the consensus amino acid sequence of CDR1 of VHHs E6, A8, Fl 1, and B9.
  • SEQ ID NO: 3 is the consensus amino acid sequence of CDR1 of VHHs D3, E7, and B5.
  • SEQ ID NO: 4 is the consensus amino acid sequence of CDR2 of VHHs F8, F6, C8, and B7.
  • SEQ ID NO: 5 is the consensus amino acid sequence of CDR2 of VHHs E6, A8, Fl 1, and B9.
  • SEQ ID NO: 6 is the consensus amino acid sequence of CDR2 of VHHs D3, E7, and B5.
  • SEQ ID NO: 7 is the consensus amino acid sequence of CDR3 of VHHs F8, F6, C8, and B7.
  • SEQ ID NO: 8 is the consensus amino acid sequence of CDR3 of VHHs E6, A8, Fl 1, and B9.
  • SEQ ID NO: 9 is the consensus amino acid sequence of CDR3 of VHHs D3, E7, and B5.
  • SEQ ID NOs: 10 is the amino acid sequence of the CDR1 of F8 and E8
  • SEQ IDs NO: 11 is the amino acid sequence of the CDR1 of D3
  • SEQ ID NO: 13 is the amino acid sequence of the CDR1 of B7, C8 and F6
  • SEQ ID NOs 15-19 and 164 are the amino acid sequences of the CDR1 of A8, Fl 1, E7, E6,
  • SEQ ID NOs: 20 to 29, 166 and 167 are the amino acid sequences of the CDR2 of D3, F6, C8, B7, A8, Fl 1, E7, E6, B5, B9 and E8, respectively
  • SEQ ID Nos: 30 to 32 are the amino acid sequences of the CDR3 of F8, D3 and F6, respectively
  • SEQ ID NO: 33 is the amino acid sequence of the CDR3 of B7 and C8
  • SEQ ID NOs: 35 and 36 are the amino acid sequences of the CDR3 of A8 and Fl l, respectively.
  • SEQ ID NO: 37 is the amino acid sequence of the CDR3 of B5 and E7.
  • SEQ ID NOs: 38, 168 and 169 are the amino acid sequences of the CDR3 of E6, B9 and E8, respectively
  • SEQ ID Nos: 40 to 50 and 170 are the amino acid sequences of the FR1 of parental F8, B9,
  • SEQ ID NO: 51 is the amino acid sequence of the FR1 of light and agg F8
  • SEQ ID NO: 52 is the amino acid sequence of the FR1 of light A8
  • SEQ ID NO: 53 is the amino acid sequence of the FR1 of light and agg Fl l
  • SEQ ID NO: 54 is the amino acid sequence of the FR1 of light and agg F6
  • SEQ ID NO: 55 is the amino acid sequence of the FR1 of light and agg E7
  • SEQ ID NO: 56 is the amino acid sequence of the FR1 of light E6
  • SEQ ID NO: 57 is the amino acid sequence of the FR1 of light and agg D3
  • SEQ ID NO: 58 is the amino acid sequence of the FR1 of light and agg C8
  • SEQ ID NO: 59 is the amino acid sequence of the FR1 of light and agg B7
  • SEQ ID NO: 60 is the amino acid sequence of the FR1 of light and agg B5
  • SEQ ID NO: 61 is the amino acid sequence of the FR2 of parental and light F8
  • SEQ ID NO: 62 is the amino acid sequence of the FR2 of parental and light B9
  • SEQ ID NO: 63 is the amino acid sequence of the FR2 of parental and light A8
  • SEQ ID NO: 64 is the amino acid sequence of the FR2 of parental and light Fl l
  • SEQ ID NO: 65 is the amino acid sequence of the FR2 of parental and light F6
  • SEQ ID NO: 66 is the amino acid sequence of the FR2 of parental and light E7 SEQ ID NO: 67 is the amino acid sequence of the FR2 of parental and light E6 SEQ ID NO: 68 is the amino acid sequence of the FR2 of parental and light D3 SEQ ID NO: 69 is the amino acid sequence of the FR2 of parental and light C8 SEQ ID NO: 70 is the amino acid sequence of the FR2 of parental and light B7 SEQ ID NO: 71 is the amino acid sequence of the FR2 of parental and light B5 SEQ ID NO: 72 is the amino acid sequence of the FR2 of agg F8 SEQ ID NO: 73 is the amino acid sequence of the FR2 of agg Fl 1 SEQ ID NO: 74 is the amino acid sequence of the FR2 of agg F6 SEQ ID NO: 75 is the amino acid sequence of the FR2 of aggE7 SEQ ID NO: 76 is the amino acid sequence of the FR2 of agg D3 SEQ ID NO
  • SEQ ID NO: 97 is the amino acid sequence of the FR3 of light and agg D3 SEQ ID NO: 98 is the amino acid sequence of the FR3 of light and agg C8 SEQ ID NO: 99 is the amino acid sequence of the FR3 of light and agg B7 SEQ ID NO: 100 is the amino acid sequence of the FR3 of light and agg B5 SEQ ID NO: 101 is the amino acid sequence of the FR3 of agg F8
  • SEQ ID NO: 113 is the amino acid sequence of the FR4 of light and agg F8
  • SEQ ID NO: 114 is the amino acid sequence of the FR4 of light A8
  • SEQ ID NO: 115 is the amino acid sequence of the FR4 of light and agg Fl 1
  • SEQ ID NO: 116 is the amino acid sequence of the FR4 of light and agg F6
  • SEQ ID NO: 117 is the amino acid sequence of the FR4 of light and agg E7
  • SEQ ID NO: 118 is the amino acid sequence of the FR4 of light E6
  • SEQ ID NO: 119 is the amino acid sequence of the FR4 of light and agg D3
  • SEQ ID NO: 120 is the amino acid sequence of the FR4 of light and agg C8
  • SEQ ID NO: 121 is the amino acid sequence of the FR4 of light and agg B7
  • SEQ ID NO: 122 is the amino acid sequence of the FR4 of light and agg B5
  • SEQ ID NO: 123 is the amino acid sequence of a (648)2 linker
  • SEQ ID NO: 124 is the amino acid sequence of F8 light (‘ISA2-F8 Light’)
  • SEQ ID NO: 125 is the amino acid sequence of A8 light (‘ISA2-A8 Light’)
  • SEQ ID NO: 126 is the amino acid sequence of Fl 1 light (‘ISA1-F11 Light’)
  • SEQ ID NO: 127 is the amino acid sequence of F6 light (‘ISA1-F6 Light’)
  • SEQ ID NO: 128 is the amino acid sequence of E7 light (‘ISA1-E7 Light’)
  • SEQ ID NO: 129 is the amino acid sequence of E6 light (‘ISA1-E6 Light’)
  • SEQ ID NO: 130 is the amino acid sequence of D3 light (‘ISA1-D3 Light’)
  • SEQ ID NO: 131 is the amino acid sequence of C8 light (‘ISA1-C8 Light’)
  • SEQ ID NO: 132 is the amino acid sequence of B7 light (‘ISA1-B7 Light’)
  • SEQ ID NO: 134 is the amino acid sequence of F8 Agg (‘ISA2-A8 Agg)
  • SEQ ID NO: 135 is the amino acid sequence of Fl 1 Agg (‘ISA1-F11 Agg’)
  • SEQ ID NO: 136 is the amino acid sequence of F6 Agg (‘ISA1-F6 Agg’)
  • SEQ ID NO: 137 is the amino acid sequence of E7 Agg (‘ISA1-E7 Agg’)
  • SEQ ID NO: 138 is the amino acid sequence of D3 Agg (‘ISA1-D3 Agg’)
  • SEQ ID NO: 139 is the amino acid sequence of C8 Agg (‘ISA1-C8 Agg’)
  • SEQ ID NO: 140 is the amino acid sequence of B7 Agg (‘ISA1-B7 Agg’)
  • SEQ ID NO: 141 is the amino acid sequence of B5 Agg (‘ISA1-B5 Agg’)
  • SEQ ID NO: 142 is the amino acid sequence of C8 light R100>Q
  • SEQ ID NO: 149 is the amino acid sequence of anti-CD3 scFv
  • SEQ ID NO: 150 is the amino acid sequence of ‘IRR’ (‘VHHirr’, anti-lysozyme VHH)
  • SEQ ID NO: 151 is the amino acid sequence of bi-specific C8Light:scFv
  • SEQ ID NO: 152 is the amino acid sequence of HEK_IRR_F1 Iph
  • SEQ ID NOs: 153-163 and 176 are the amino acid sequences of parents VHHs E7, Fl 1, E6,
  • SEQ ID NO: 171 is the amino acid sequence of the FR2 of light E8
  • SEQ ID NO: 172 is the amino acid sequence of the FR3 of parental E8
  • SEQ ID NO: 173 is the amino acid sequence of the FR4 of parental E8
  • SEQ ID NO: 174 is the amino acid sequence of B9 light (‘ISA1-B9 Light’)
  • SEQ ID NO: 175 is the amino acid sequence of B9 Agg (‘ISA1-B9 Agg’)
  • SEQ ID NO: 177 is the amino acid sequence of the FR1 of light B9
  • SEQ ID NO: 178 is the amino acid sequence of the FR1 of agg B9
  • SEQ ID NO: 179 is the amino acid sequence of the FR2 of agg B9
  • SEQ ID NO: 180 is the amino acid sequence of the FR3 of light and agg B9
  • SEQ ID NO: 181 is the amino acid sequence of the FR4 of light and agg B9
  • SEQ ID NO: 182 is the amino acid sequence of IRR C8 R100>Q
  • SEQ ID NO: 183 is the amino acid sequence of F8Light:scFv
  • SEQ ID NO: 184 is the amino acid sequence of Fl lLight:scFv
  • SEQ ID NO: 185 is the amino acid sequence of A8Light:scFv
  • SEQ ID NO: 186 is the amino acid sequence of the G4S linker
  • SEQ ID NO: 187 is the amino acid sequence of Alb-23
  • SEQ ID NO: 188 is the amino acid sequence of T0023500
  • SEQ ID NO: 189 is the consensus amino acid sequence of CDR2 ofVHHs C8 and B7
  • SEQ ID NO: 190 is the consensus amino acid sequence of CDR2 ofVHHs C8, B7 and F8
  • SEQ ID NO: 191 is the consensus amino acid sequence of CDR3 ofVHHs C8, B7 and F8
  • SEQ ID NO: 192 is the amino acid sequence of VHH C8 R100Q
  • a single-domain antibody also known as ‘ nanobody’ or ‘sdAb’
  • sdAb is a monomeric antigenbinding polypeptide comprising a variable antibody domain and lacking the constant domains present in conventional immunoglobulins (‘antibodies’), but achieving binding affinities on a par with conventional immunoglobulins. Binding properties are primarily conferred by three linear peptide portions known as the complementarity determining regions (CDRs) 1, 2 and 3 (from N-terminus to C-terminus) interspersed between four structural framework regions (FRs) 1, 2, 3 and 4.
  • CDRs complementarity determining regions
  • FRs structural framework regions
  • the variable antibody domain is usually a heavy chain variable region (VH) but may also be a light chain variable region (VL).
  • VHH VHH or VHH
  • camelids camels, llamas, alpacas etc
  • VNAR or ‘VNAR’
  • cartilaginous fish e.g. sharks
  • conventional immunoglobulins Single domain antibodies occur in nature as VHH (VHH or VHH) in camelids (camels, llamas, alpacas etc) and VNAR (or ‘VNAR’) in cartilaginous fish (e.g. sharks) or can be engineered from, for example, conventional immunoglobulins.
  • Single domain antibodies such as VHHs
  • VHHs are ideal building blocks for next-generation multi-specific biotherapeutics. They have binding capabilities similar to those of conventional monoclonal antibodies but are typically less immunogenic and can target antigens and epitopes that are considered difficult or intractable for typical antibodies.
  • Single domain antibodies canonically do not pair with a respective light (or heavy) chain, avoiding improper chain pairing. Their adaptability in formatting and engineering into multiple therapeutic formats, as well as their high thermal stability, make them an essential asset in drug development.
  • Specific binding when referring to an antibody or single domain antibody, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biologies.
  • an antibody or single domain antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as albumin) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject.
  • a single domain antibody can be said to specifically bind a target molecule or target class of molecules, such as serum albumin, with a binding affinity that is greater than for a non-target molecule.
  • a single domain antibody that is specific for a particular target molecule may bind to that target molecule with a binding affinity that is at least 2-fold, 10-fold, 50-fold or 100-fold greater than its binding affinity for binding to another non-target molecule. Specific binding can be determined by methods known in the art.
  • Serum albumin is the most abundant protein in mammalian blood plasma. It is a water-soluble, globular protein in the albumin family comprising three helical domains (Domains I, II, and III) and several hydrophobic binding pockets which allow the transport of hydrophobic molecules such as steroid hormones and fatty acids in the blood. Homologs are found in all vertebrates and are named accordingly: human serum albumin (HSA) in humans, murine serum albumin (MSA) in mice, bovine serum albumin (BSA) in cows, etc.
  • HSA human serum albumin
  • MSA murine serum albumin
  • BSA bovine serum albumin
  • Serum albumin binds the neonatal Fc Receptor (‘FcRn’), a transmembrane endosomal receptor, at low endosomal pH and is diverted away from the lysosomal degradation pathway for release at the cell membrane (‘endocytic recycling’). HSA:FcRn binding occurs predominantly via Domain III (Dill) with contributions from DI residues.
  • Serum albumin as used herein refers exclusively to the protein entity and not to its bound (immobilised or otherwise) state. Thus, specific binding of the single domain antibodies of the invention to serum albumin should be taken to mean binding to the serum albumin polypeptide or a fragment and/or domain thereof, and not to mean binding to albumin only when solubilised within serum plasma.
  • the single domain antibodies described herein bind specifically to serum albumin.
  • specific binding means that a single domain antibody preferentially binds to a serum albumin with a binding affinity that is greater than for a non-target molecule.
  • Single domain antibodies described herein may bind serum albumin homologs from different species. Thus, in this context, specific binding does not mean that the single domain antibody binds to a particular serum albumin homolog from one species in preference to other albumin homologs. Rather, a single domain antibody described herein may specifically bind to multiple different serum albumin homologs, e.g. those from different species.
  • single domain antibodies of the invention specifically bind to serum albumin, optionally wherein the serum albumin is human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and/or primate serum albumin.
  • the serum albumin is human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and/or primate serum albumin.
  • Cross-reactive single domain antibodies are those that specifically bind more than one serum albumin homolog, for example single domain antibodies that bind both HSA and MSA.
  • the single domain antibody is cross-reactive for (i.e. binds to) human and murine serum albumin and/or cross-reactive for human and cynomolgus monkey serum albumin.
  • single domain antibodies described herein are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and/or primate serum albumin.
  • single domain antibodies described herein are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and/or mini pig serum albumin.
  • the single domain antibodies are cross-reactive for three or more of: human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and primate serum albumin, optionally comprising: human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for four or more, or five or more of: human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for six or more of: human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin and two or more of: murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin and three or more of: murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for four or more, five or more, six or more, or seven of said serum albumins.
  • the single domain antibodies are cross-reactive for human serum albumin and five or more of: murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin, murine serum albumin and cynomolgus monkey serum albumin, as well as at least one of: bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and primate serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin, murine serum albumin and cynomolgus monkey serum albumin, as well as at least one of: canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin, murine serum albumin and cynomolgus monkey serum albumin, as well as two or more of: bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and primate serum albumin.
  • the single domain antibodies are cross-reactive for human serum albumin, murine serum albumin and cynomolgus monkey serum albumin, as well as two or more of: canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin. In some embodiments, the single domain antibodies are cross-reactive for human serum albumin, murine serum albumin and cynomolgus monkey serum albumin, as well as three or more of: canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • the single domain antibodies have broader cross-reactivity then Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 1 as CDR1, SEQ ID NO: 190 as CDR2 and SEQ ID NO: 191 as CDR3 are cross- reactive for human serum albumin and five or more species selected from the group comprising: murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 189 as CDR2 and SEQ ID NO: 33 as CDR3 are cross- reactive for human serum albumin and five or more species selected from the group comprising: murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 13, 24 and 34 as CDR1, CDR2 and CDR3, respectively, are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rat serum albumin and mini pig serum albumin.
  • single domain antibodies comprising the amino acid sequences of SEQ ID NO: 132 or variants thereof are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin, rat serum albumin and mini pig serum albumin.
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 13, 23 and 33 as CDR1, CDR2 and CDR3, respectively, are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin and rat serum albumin.
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 131 or variants thereof are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, bovine serum albumin and rat serum albumin.
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3, respectively, are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 124 or variants thereof are cross-reactive for human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, canine serum albumin, feline serum albumin, rabbit serum albumin, rat serum albumin and mini pig serum albumin.
  • Single domain antibodies comprising amino acid sequences of: SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3, respectively; SEQ ID NOs: 13, 23 and 33 as CDR1, CDR2 and CDR3, respectively; or SEQ ID NOs: 13, 24 and 34 as CDR1, CDR2 and CDR3 respectively, have broader cross-reactivity for albumin binding than Alb23 (SEQ ID NO: 187).
  • Single domain antibodies comprising amino acid sequences SEQ ID NOs: 124, 131 or 132, or variants thereof, have broader cross-reactivity for albumin binding than Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 13, 23 and 33 as CDR1, CDR2 and CDR3, respectively, or SEQ ID NOs: 13, 24 and 34 as CDR1, CDR2 and CDR3, respectively, show significant improvement relative to T0023500 (SEQ ID NO: 188) in binding to feline and bovine serum albumin.
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 13, 23 and 33 as CDR1, CDR2 and CDR3, respectively, or SEQ ID NOs: 13, 24 and 34 as CDR1, CDR2 and CDR3, respectively, have a reduced ECso value relative to T0023500 (SEQ ID NO: 188) when binding feline and bovine serum albumin.
  • single domain antibodies comprising amino acid sequences SEQ ID NOs: 131 or 132, or variants thereof, show significant improvement relative to T0023500 (SEQ ID NO: 188) in binding to feline and bovine serum albumin.
  • single domain antibodies comprising amino acid sequences SEQ ID NOs: 131 or 132, or variants thereof have a reduced ECso value relative to T0023500 (SEQ ID NO: 188) when binding to feline and bovine serum albumin.
  • the single domain antibodies are suitable for purification by protein A pull-down.
  • single domain antibodies described herein have improved protein A binding relative to other anti-albumin single domain antibodies, such as Alb-23 and/or T0023500.
  • improved binding it is meant that the single domain antibodies described herein are able to be recovered in greater proportions through binding to protein A beads compared to a control anti-albumin single domain antibody.
  • Efficient protein A binding is advantageous in that it allows for purification by protein A, which is superior to, and cheaper than, tag purification (for example His-tag purification).
  • tag purification for example His-tag purification
  • the protein A binding characteristics of the single domain antibodies described herein allow for their reliable but low-cost purification and production.
  • the protein A-binding properties of a single domain antibody can be assessed, for example, using a pull-down assay on protein A coated beads (see Example 24 below). Other suitable methods known in the art may also be used.
  • the single domain antibodies described herein demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 124, 125, 126, 129, 131 or 132, or variants thereof, demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 1 as CDR1, SEQ ID NO: 190 as CDR2 and SEQ ID NO: 191 as CDR3 demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 189 as CDR2 and SEQ ID NO: 33 as CDR3 demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188).
  • single domain antibodies comprising: the amino acid sequences of SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3, respectively; SEQ ID NOs: 13, 23 and 33 as CDR1, CDR2 and CDR3, respectively; or SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3, respectively, demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188).
  • the single domain antibodies described herein demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188) and Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 132, or variants thereof demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188) and Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 131, or variants thereof demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188) and Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 124, or variants thereof demonstrate improved binding to protein A relative to T0023500 (SEQ ID NO: 188) and Alb23 (SEQ ID NO: 187).
  • the single domain antibodies described herein demonstrate improved binding to protein A relative to Alb23 (SEQ ID NO: 187).
  • single domain antibodies the comprising amino acid sequence of SEQ ID NO: 132, or variants thereof demonstrate improved binding to protein A relative to Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 131, or variants thereof demonstrate improved binding to protein A relative to Alb23 (SEQ ID NO: 187).
  • single domain antibodies comprising the amino acid sequence of SEQ ID NO: 124, or variants thereof demonstrate improved binding to protein A relative to Alb23 (SEQ ID NO: 187).
  • improved binding to protein A means recovery of at least 5% more of the single domain antibody input in the protein A pull-down fraction is observed relative to the comparator molecule of interest. As an illustrative example, if 20% recovery is observed with a comparator molecule, and 25% recovery is observed with a test molecule, then 5% more recovery of the test molecule is observed relative to the comparator molecule. Percentage recovery means pull down fraction as a percentage of input fraction.
  • At least 10% more of the single domain antibody input in the protein A pull-down fraction, at least 1 % more of the single domain antibody input in the protein A pull-down fraction, at least 20% more of the single domain antibody input in the protein A pull-down fraction, at least 25% more of the single domain antibody input in the protein A pull-down fraction, at least 30% more of the single domain antibody input in the protein A pull-down fraction or at least 35% more of the single domain antibody input in the protein A pull-down fraction is observed relative to the comparator molecule of interest.
  • At least 10% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 15% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 15% more of a single domain antibody comprising the amino acid sequence of SEQ ID NO: 1 as CDR1, SEQ ID NO: 190 as CDR2 and SEQ ID NO: 191 as CDR3 is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 20% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188).
  • At least 25% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 25% more of a single domain antibody comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 189 as CDR2 and SEQ ID NO: 33 as CDR3 is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 30% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 35% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188).
  • At least 40% more of the single domain antibody is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188). In some embodiments, at least 30%, at least 35%, or at least 40% more of a single domain antibody comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 23 as CDR2 and SEQ ID NO: 33 as CDR3 is recovered by protein A pull-down than T0023500 (SEQ ID NO: 188).
  • the single domain antibody demonstrates at least a 5% improvement in percentage recovery on protein A beads relative to the comparator anti-albumin single domain antibody. In some embodiments the single domain antibody demonstrates at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30% or at least a 35% improvement in percentage recovery on protein A beads relative to the comparator antialbumin single domain antibody.
  • the single domain antibody demonstrates at least a 5% improvement in percentage recovery on a protein A substrate relative to the comparator anti-albumin single domain antibody, i.e. protein A recovery is improved by at least 5% relative to the comparator single domain antibody.
  • protein A recovery is improved by at least 5% relative to the comparator single domain antibody.
  • the single domain antibody demonstrates at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30% or at least a 35% improvement in percentage recovery on a protein A substrate relative to the comparator antialbumin single domain antibody.
  • the single domain antibody demonstrates at least a 20%, at least a 25% or at least a 30% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, the single domain antibody demonstrates at least a 35% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, a single domain antibody comprising the amino acid sequence of SEQ ID NO: 1 as CDR1, SEQ ID NO: 190 as CDR2 and SEQ ID NO: 191 as CDR3 demonstrates at least a 35% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188).
  • the single domain antibody demonstrates at least a 40% improvement, 45% improvement or at least a 50% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, the single domain antibody demonstrates at least a 55% improvement or at least 60% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, a single domain antibody comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 189 as CDR2 and SEQ ID NO: 33 as CDR3 demonstrates at least a 60% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188).
  • the single domain antibody demonstrates at least a 65%, at least a 70%, at least a 75%, at least a 80%, or at least a 85% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, the single domain antibody demonstrates at least a 90% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188). In some embodiments, the single domain antibody demonstrates at least a 95% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188).
  • the single domain antibody demonstrates at least a 96%, 97% or 98% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188).
  • a single domain antibody comprising the amino acid sequence of SEQ ID NO: 13 as CDR1, SEQ ID NO: 23 as CDR2 and SEQ ID NO: 33 as CDR3 demonstrates at least a 96%, 97% or 98% improvement in percentage recovery on a protein A substrate relative to T0023500 (SEQ ID NO: 188).
  • Relative percentage of the single domain antibody input in the protein A pull-down fraction may be measured by any means known to the skilled person, for example by quantitative western blot analysis of a protein A bead pull-down fraction.
  • Single domain antibodies of the invention may be further engineered, for example via modification of component amino acid residues or via conjugation to biological or chemical moieties.
  • Conjugation refers to the covalent linkage of molecules. Covalent conjugation may be achieved via genetic engineering of the encoding polynucleotides into a recombinant open reading frame, optionally separated via a polynucleotide sequence encoding a linker, or via post-translational chemical reaction, for example mediated by chemical tethering, ‘plug and play’ reagents, SpyTag/SpyCatcher reagents, and so on.
  • Moieties (or 'agents’) suitable for conjugation to the single domain antibodies of the invention may include, but are not limited to, antibodies or antigen-binding fragments thereof, single domain antibodies including single domain antibodies of the invention, VHHs including VHHs of the invention, VNARS, single chain variable fragments (scFvs), receptors and/or canonical binding partners, small molecule drugs, radioactive labels, nanoparticles, viral particles, cytokines, fluorescent labels, cells, receptors, nucleic acids, liposomes, peptides, and/or toxins.
  • antigen-binding fragment refers to a portion of a single domain antibody that retains the capacity to specifically bind the same antigen that is recognised by said single domain antibody.
  • Coupled or conjugated moieties may be therapeutic agents and/or diagnostic agents in that they possess therapeutic and/or diagnostic capability, thereby enabling use of the single domain antibody: agent construct in a therapeutic and/or diagnostic context.
  • Therapeutic and/or diagnostic agents may also be administered separately from - i.e. not coupled to - single domain antibodies of the invention.
  • a linker is a chemical or biochemical structure, typically a polymer, which spatially separates two or more functionally relevant domains.
  • Linkers are usually unstructured allowing the two or more domains to re-orientate relative to each other to accommodate relative configurations of binding partners.
  • Many linkers are known in the art and suitable linkers will be immediately apparent to the skilled person, for example but not limited to polyethylene glycol, alkyls, alkynyls, heteroaromatic or heterocyclic chains, poly- and oligopeptides, such as but not limited to poly-glycine linkers and poly-glycine/serine linkers.
  • amino acid sequences of the single domain antibodies herein may be modified or optimised by modification according to standard techniques known in the art, provided that serum albumin binding activity is retained. By “retained” it is to be understood that the single domain antibody of the modified sequence does not exhibit significantly reduced binding affinity to serum albumin compared against the unmodified sequence. Any comparison of binding affinity between sequences is to be conducted using the same assay.
  • a modification may be post-translational modification or, preferably, amino acid mutation. Examples of mutations are substitution, deletion or insertion. Substitution may be non-conservative or, preferably, conservative in that the original residue’s chemical character is maintained. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume.
  • amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.
  • a conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid.
  • Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table Al below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in Table A2.
  • Table A2 - hydropathy scale Variants of amino acid sequences provided herein are sequences which are identical to said amino acid sequences except for the presence of one or more modifications which do not compromise serum albumin binding activity. Generally, therefore, more mutations may be tolerated in framework regions than in CDRs.
  • CDRs of the VHHs described herein may comprise one modification, optionally one mutation, optionally one substitution, preferably wherein the mutation or substitution is conservative.
  • CDRs of the VHHs described herein may comprise two or three modifications, optionally two or three mutations, optionally two or three substitutions, preferably wherein the mutations or substitutions are conservative.
  • Framework regions of the VHHs described herein may comprise one, two, three, four, five or six modifications, optionally one, two, three, four, five or six mutations, optionally one, two, three, four, five or six substitutions.
  • polynucleotide sequences of the invention encoding single domain antibodies of the invention may be modified or optimised by modification according to standard techniques known in the art, as long as serum albumin binding activity of the encoded single domain antibody is retained.
  • modification may be addition of chemical and/or epigenetic markers or by substitution, deletion or insertion of nucleotides
  • the single domain antibodies of the invention are humanised.
  • Humanisation is the modification of amino acid residues within non-human derived sequences to reduce immunogenicity when administered to a human subject.
  • Humanisation may be by grafting of non-human functional sequences (e.g. CDRs) onto human structural or scaffolding sequences (e.g. FRs), but is now more commonly achieved by mutation of residues within a non-human sequence to a counterpart residue occupying the equivalent position in a corresponding human homolog (‘back-mutation’).
  • Preferably only the structural (e.g. framework) regions are humanised such that binding capabilities are not affected.
  • back-mutations in humanised embodiments of the invention are shown in Table 1, denoted in bold and underlined type.
  • the single domain antibodies of the invention can be modified to reduce immunogenicity in other species, such as non-human primates, cats, dogs, horses, mice, cows, birds and pigs.
  • the single domain antibodies of the invention are humanised.
  • the single domain antibodies comprise humanising back-mutations in one or more framework regions, preferably two or more framework regions.
  • ‘parental’ or ‘non-humanised’ (‘nh ’) VHHs do not comprise any humanising back-mutations within the VHH sequence.
  • a parental VHH sequence can be considered to be a ‘wild-type’ sequence.
  • ‘Lightly humanised’ (‘light’) or ‘partially humanised’ (‘ph ’) VHHs comprise at least 1 humanising back-mutation within the FRs, typically between 1 and 12, optionally between 2 and 11, optionally between 3 and 10 back- mutations.
  • ‘Lightly’ humanised single domain antibodies comprise humanising back- mutations in one or more framework regions, such as two or more framework regions, preferably three framework regions. In some embodiments said three framework regions are FR1, FR3 and FR4.
  • the single domain antibodies comprise at least one humanising back-mutation in FR1, at least one humanising back-mutation in FR3 and at least one humanising back-mutation in FR4.
  • the single domain antibodies comprise at least one humanising back-mutation in one, two or three framework region(s), preferably in three framework regions.
  • the three framework regions which have been humanised by back-mutation are FR1, FR3 and FR4.
  • ‘lightly’ humanised single domain antibodies comprise at least 1 humanising back-mutation, optionally at least 2 or at least 3 humanising back-mutations, preferably at least 4 humanising back mutations.
  • ‘lightly’ humanised single domain antibodies comprise amino acid L at the residue corresponding to residue 5 of SEQ ID NO: 154, and/or amino acid S at the residue corresponding to residue 75 of SEQ ID NO: 154, and/or amino acid A at the residue corresponding to residue 88 of SEQ ID NO: 154, and/or amino acid L at the residue corresponding to residue 110 of SEQ ID NO: 154.
  • the humanised single domain antibodies comprise:
  • humanised single domain antibodies comprise:
  • ‘Aggressively humanised 9 (‘agg’J VHHs comprise said lightly humanising mutations and additionally comprise further humanising back-mutations as compared to a lightly humanised VHH.
  • aggressively humanised VHHs described herein comprise 1 additional back-mutation, 2 additional back-mutations or at least 2 additional back- mutations.
  • Aggressively humanised VHHs may comprise, for example, at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 back-mutations in total, more typically between 7 and 15, between 7 and 14, between 7 and 13 or between 7 and 12 back-mutations in total.
  • ‘Aggressively’ humanised single domain antibodies comprise humanising back-mutations in all four framework regions.
  • the single domain antibodies comprise at least one humanising back-mutation in FR1, at least one humanising back- mutation in FR2, at least one humanising back-mutation in FR3 and at least one humanising back-mutation in FR4.
  • the single domain antibodies comprise at least one humanising back-mutation in all four framework regions.
  • the amino acid sequences of the single domain antibodies have been humanised by back- mutation in FR1 , FR2, FR3 and FR4.
  • ‘aggressively’ humanised single domain antibodies comprise at least 1 humanising back -mutation additional to the back- mutations of the corresponding ‘lightly’ humanised single domain antibody. In some embodiments ‘aggressively’ humanised single domain antibodies comprise at least 5, at least 6 or at least 7 humanising back-mutations. In some embodiments ‘aggressively’ humanised single domain antibodies comprise amino acid L at the residue corresponding to residue 45 of SEQ ID NO: 154.
  • ‘aggressively’ humanised single domain antibodies comprise amino acid L at the residue corresponding to residue 45 of SEQ ID NO: 154 and further comprise one or more of the following: amino acid L at the residue corresponding to residue 5 of SEQ ID NO: 154, and/or amino acid S at the residue corresponding to residue 75 of SEQ ID NO: 154, and/or amino acid A at the residue corresponding to residue 88 of SEQ ID NO: 154, and/or amino acid L at the residue corresponding to residue 110 of SEQ ID NO: 154.
  • humanised single domain antibodies comprise:
  • humanised single domain antibodies comprise:
  • sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • homology or similarity or homology
  • NCBI Basic Local Alignment Search Tool (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet (along with a description of how to determine sequence identity using this program).
  • Homologs of a VHH that specifically binds a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologs When a portion of the full-length sequence is being compared for the purposes of determining sequence identity, homologs will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence.
  • sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • Binding affinity is a measure of the tightness of binding of a ligand to its binding partner (or "receptor’'). Binding affinity can be readily assayed via standard techniques in the art, for example but not limited to surface plasmon resonance (SPR), bio-layer interferometry (BLI), and enzyme-linked immunosorbent assay (ELISA). Binding affinity may be expressed in terms of equilibrium dissociation constant (KD). A KD of less than 10' 5 M indicates tight binding, and antibodies and single domain antibodies may generally bind with nanomolar affinity to their ligand (KD in the order 10' 7 M and below). Unless otherwise specified, the KDS values reported herein are obtained via SPR.
  • ECso is the concentration of antibody required to give half-maximal binding, determined herein by ELISA unless otherwise specified (although other techniques may be used and will be readily apparent to one skilled in the art).
  • the ECso values referred to herein result from assays carried out at pH 7.4, unless stated otherwise.
  • Half-life represents the dwelling time of a substance, being the time taken for the amount of substance to reduce by half. Effective half-life is the time taken for the amount of a biological molecule to be reduced by half in vivo. In biological systems and as used herein (unless otherwise specified or apparent from the context) effective half-life and halflife are used interchangeably. Half-life of biological molecules is reduced by metabolic and physiological clearance mechanisms or increased by counter-acting metabolic and physiological methods of retention, sequestration or recycling.
  • Specific binding when referring to an antibody or single domain antibody, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biologies.
  • an antibody or single domain antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as albumin) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject.
  • Specific binding can be determined by methods known in the art.
  • a consensus sequence is a sequence representative of all sequences within an assigned grouping. Various notations may be used in the art and will be understood by the skilled person. For example, as used herein, a consensus amino acid sequence containing (Y/R) indicates that the residue may be either tyrosine or arginine. An amino acid consensus sequence containing (N/-) or (N/none) indicates that the residue may be asparagine or absent. As a further example, the consensus amino acid sequence (Y/R)G(N/-) represents the sequences YGN or RGN or YG or RG, i.e. each of sequences YGN, RGN, YG and RG fall within the consensus sequence.
  • a recombinant protein or nucleic acid is one that has an amino acid and/or nucleic acid sequence that is not naturally occurring or has an amino acid and/or nucleic acid sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a binding molecule herein refers to a protein comprising one or more single domain antibodies and one or more agents, optionally wherein the agents are conjugated to said one or more single domain antibodies, optionally via a linker.
  • the one or more agents may comprise a polypeptide and therefore the term binding molecule encompasses fusion proteins.
  • Single domain antibodies and binding molecules herein may additionally comprise tags for the purposes of protein detection and/or purification, for example but not limited to His tags, biotin, fluorescent markers and radiolabels.
  • compositions described herein may comprise a pharmaceutically acceptable carrier and/or excipient.
  • Remington s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, which include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the present invention provides single domain antibodies, optionally humanised single domain antibodies, that bind to serum albumin with nanomolar affinity maintained across a wide pH range, including acidic endosomal pHs between pH 4.0 and 7.5, between 4.5 and 7.5, and/or between 5.0 and 7.4.
  • Single domain antibodies of the present invention do not interfere with albumin-FcRn binding (Example 14) since they bind specifically to Domain II of albumin.
  • Said tight, acid-resistant binding of the single domain antibodies to Domain II of serum albumin advantageously enables said antibodies to ‘piggyback’ through the endocytic recycling compartment.
  • the single domain antibodies of the invention are thereby protected from renal clearance and lysosomal degradation and half-life is significantly increased.
  • One or more agents may be easily combined to the single domain antibodies of the invention, creating mono-, bi- and or multi-specific biotherapeutics with a significantly extended halflife.
  • Binding molecules comprising the single domain antibodies conjugated to one or more agents are able to exploit the serum albumi FcRn binding interaction and piggyback through the FcRn endocytic recycling pathway in the same way, such that the half-life of said agents is significantly extended.
  • Agents may be conjugated at any location within the single domain antibodies of the invention that does not disrupt serum albumin binding, but are preferably conjugated at the N-terminus or at the C-terminus.
  • Binding molecules may further comprise a linker between the single domain antibody and the one or more agents.
  • the single domain antibodies of the invention may be provided as a complex comprising said single domain antibody bound to serum albumin, e.g. recombinant HSA, wherein the serum albumin molecule is conjugated to one or more agents.
  • serum albumin e.g. recombinant HSA
  • Such complexes may be formed in vitro and administered to a patient in order to facilitate delivery of a therapeutic agent which has been conjugated to albumin.
  • the single domain antibodies of the invention are VHHs.
  • VHHs are versatile, clinically validated monomers benefitting from high binding affinities, high stability and solubility, low immunogenicity, small size (typically around 15 kDa, less than 400 bp in length), a lack of VH-VL chain mispairing, and a lack of Fc receptor binding.
  • the serum albumin-specific VHHs are cross-reactive for serum albumin from multiple species, including humans, mice and cynomolgus monkeys (Example 4), essential for e.g. pre-clinical pharmacokinetic (PK) and toxicology studies and/or veterinary applications.
  • PK pharmacokinetic
  • the invention provides single domain antibodies comprising a CDR1 comprising the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the single domain antibodies of the invention comprise a CDR1 comprising variants of SEQ ID NOs: 1, 2 or 3 wherein the variants comprise one modified amino acid.
  • the single domain antibodies of the invention comprise variants of SEQ ID NOs: 1, 2 or 3 wherein the variants comprise two or three modified amino acids.
  • the single domain antibodies of the invention comprise a CDR1 consisting of the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the single domain antibodies of the invention consist of variants of SEQ ID NOs: 1, 2 or 3 wherein the variants comprise one modified amino acid. In some embodiments the single domain antibodies of the invention consist of variants of SEQ ID NOs: 1, 2 or 3 wherein the variants comprise two or three modified amino acids.
  • the single domain antibody comprises a CDR1 comprising the amino acid sequence SEQ ID NO: 13 or 10.
  • the invention provides single domain antibodies comprising a CDR1 having consensus amino acid sequence SEQ ID NO: 1. More preferably, the single domain antibody comprises a CDR1 comprising the amino acid sequence SEQ ID NO: 13.
  • the single domain antibodies may comprise variants of said preferred CDR1 sequences, for example comprising a modified amino acid, wherein the single domain antibodies retain serum albumin binding activity.
  • the single domain antibodies of the invention comprise a CDR2 comprising the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments the single domain antibodies of the invention comprise a CDR2 comprising variants of SEQ ID NOs: 4, 5 or 6 wherein the variants comprise one modified amino acid. In some embodiments the single domain antibodies of the invention comprise variants of SEQ ID NOs: 4, 5 or 6 wherein the variants comprise two or three modified amino acids. In some embodiments the single domain antibodies of the invention comprise a CDR2 consisting of the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the single domain antibodies of the invention consist of variants of SEQ ID NOs: 4, 5 or 6 wherein the variants comprise one modified amino acid. In some embodiments the single domain antibodies of the invention consist of variants of SEQ ID NOs: 4, 5 or 6 wherein the variants comprise two or three modified amino acids.
  • the single domain antibody comprises a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, 23 or 24, preferably SEQ ID NO: 23 or 24.
  • the invention provides single domain antibodies comprising a CDR2 comprising consensus amino acid sequence of SEQ ID NO: 190, preferably SEQ ID NO: 189. More preferably, the single domain antibody comprises a CDR2 comprising the amino acid sequence of SEQ ID NO: 23.
  • the single domain antibodies may comprise variants of said preferred CDR2 sequences, for example comprising a modified amino acid, wherein the single domain antibodies retain serum albumin binding activity.
  • the single domain antibodies of the invention comprise a CDR3 comprising the amino acid sequence SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments the single domain antibodies of the invention comprise a CDR3 comprising variants of SEQ ID NOs: 7, 8 or 9 wherein the variants comprise one modified amino acid. In some embodiments the single domain antibodies of the invention comprise variants of SEQ ID NOs: 7, 8 or 9 wherein the variants comprise two or three modified amino acids. In some embodiments the single domain antibodies of the invention comprise a CDR3 consisting of the amino acid sequence SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the single domain antibodies of the invention consist of variants of SEQ ID NOs: 7, 8 or 9 wherein the variants comprise one modified amino acid. In some embodiments the single domain antibodies of the invention consist of variants of SEQ ID NOs: 7, 8 or 9 wherein the variants comprise two or three modified amino acids.
  • the single domain antibody comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or 30.
  • the invention provides single domain antibodies comprising a CDR3 comprising consensus amino acid sequence of SEQ ID NO: 191. More preferably, the single domain antibody comprises a CDR3 comprising the amino acid sequence of SEQ ID NO: 33.
  • the single domain antibodies may comprise variants of said preferred CDR3 sequences, for example comprising a modified amino acid, wherein the single domain antibodies retain serum albumin binding activity.
  • the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof
  • the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4 or a variant thereof
  • the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or a variant thereof.
  • the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or a variant thereof
  • the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or a variant thereof
  • the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or a variant thereof.
  • the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or a variant thereof
  • the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or a variant thereof
  • the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 9 or a variant thereof.
  • a single domain antibody may have a CDR1, CDR2 and/or CDR3 selected from the CDRs set out in Table 1. In some embodiments, the single domain antibodies may have a CDR1, CDR2 and CDR3 selected from those set out in Table 1.
  • the single domain antibody comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 190 or a functional variant thereof, and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 191 or a functional variant thereof.
  • the single domain antibody comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a functional variant thereof, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 189 or a functional variant thereof, and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 33 or a functional variant thereof.
  • the single domain antibody comprises any of the following:
  • a CDR1 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or a functional variant thereof
  • a CDR2 comprising or consisting of the amino acid sequence SEQ ID NO: 23 or a functional variant thereof
  • a CDR3 comprising or consisting of the sequence SEQ ID NO: 33 or a functional variant thereof
  • a CDR1 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or a functional variant thereof
  • a CDR2 comprising or consisting of the amino acid sequence SEQ ID NO: 24 or a functional variant thereof
  • a CDR3 comprising or consisting of the sequence SEQ ID NO: 33 or a functional variant thereof
  • a functional variant may comprise, for example, one, two or three modifications in each of CDR1, CDR2 and/or CDR3 without loss of serum-albumin binding, cross-reactivity and protein A binding.
  • a functional variant comprises one or two modifications in CDR1, one or two modifications in CDR2, and/or one or two modifications in CDR3.
  • a functional variant comprises one modification in CDR1, one modification in CDR2, and/or one modification in CDR3.
  • the single domain antibody comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 consisting of the sequence of SEQ ID NO: 30.
  • Such a single domain antibody may be referred to as an ‘F8 ’ single domain antibody (including ‘F8 Light’ or ‘F8L’ and ‘F8 agg’ single domain antibodies).
  • the single domain antibody comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 consisting of the sequence of SEQ ID NO: 33.
  • Such a single domain antibody may be referred to as a ‘B7’ single domain antibody (including ‘B7 Light’ or ‘B7L’ and ‘B7 agg’ single domain antibodies).
  • the single domain antibody comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 23, and a CDR3 consisting of the sequence of SEQ ID NO: 33.
  • Such a single domain antibody may be referred to as a ‘C8’ single domain antibody (including ‘C8 Light’ or ‘C8L’ and ‘C8 agg’ single domain antibodies).
  • the single domain antibodies may further comprise one or more framework regions selected from those set out in Table 1, optionally wherein the framework region(s) is a humanised framework region(s) set out in Table 1.
  • the CD1, CDR2 and/or CDR3 sequences are each from the same single domain antibody indicated in Table 1.
  • the framework regions i.e. FR1, FR2, FR3 and/or FR4 are each from the same single domain antibody indicated in Table 1.
  • the single domain antibody may comprise the CDR1, CDR2 and/or CDR3 sequences of the single domain antibody named Fl 1.
  • the single domain antibody may have the CDR1, CDR2, CDR3, FR1, FR2, FR3 and/or FR4 sequences of the single domain antibody named Fl l as indicated in Table 1.
  • the single domain antibody may have the CDR1, CDR2 and/or CDR3 sequences of Fl l and the FR1, FR2, FR3, and/or FR4 sequences of Fl 1 Light.
  • the single domain antibody is a VHH comprising a sequence selected from the group consisting of SEQ ID NOs: 153 to 163 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 153 to 163.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 159.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 160.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 156.
  • the VHHs are partially humanised and comprise a sequence selected from the group consisting of SEQ ID NOs: 124 to 133 and 174 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 124 to 133 and 174.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 131.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 132.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 124.
  • the VHHs are aggressively humanised and comprise a sequence selected from the group consisting of SEQ ID NOs: 134 to 141 and 175 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 134 to 141 and 175.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 139.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 140.
  • the single domain antibody is a VHH comprising the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 134.
  • the single domain antibody of the invention comprises the sequence SEQ ID NO: 126. In some embodiments the single domain antibody of the invention comprises a sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity to the sequence SEQ ID NO: 126.
  • the single domain antibody of the invention comprises the sequence SEQ ID NO: 142. In some embodiments the single domain antibody of the invention comprises a sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity to the sequence SEQ ID NO:142.
  • sequences are at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % identical to the recited VHH sequence
  • said at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % identical sequences are functional variants of the recited VHH sequence and as such retain the serum-albumin binding, crossreactivity and protein A binding properties of the parental VHH sequence.
  • the single domain antibody is a lightly humanised VHH comprising the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 124.
  • a single domain antibody may be referred to herein for example as a ‘F8 Light’, ‘F8L’ or ‘partially humanised F8’ single domain antibody.
  • the single domain antibody is a lightly humanised VHH comprising the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 132.
  • a single domain antibody may be referred to herein for example as a ‘B7 Light’, ‘B7L’ or ‘partially humanised B7’ single domain antibody.
  • the single domain antibody is a lightly humanised VHH comprising the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to the amino acid sequence of SEQ ID NO: 131.
  • a single domain antibody may be referred to herein for example as a ‘C8 Light’, ‘C8L’ or ‘partially humanised C8’ single domain antibody.
  • the single domain antibodies comprise further optimising mutations.
  • a functional variant of the VHH having the amino acid sequence of SEQ ID NO: 131 has the amino acid sequence of SEQ ID NO: 142, wherein the residue arginine residue at position 100 in CDR2 has been substituted with a glutamine residue (i.e. R100Q).
  • SEQ ID NO: 142 corresponds to the variant VHH designated ‘C8 Light R100Q’.
  • R100 of SEQ ID NO: 131 corresponds to the amino acid residue position 3 (‘R3’) of the CDR2 sequence of SEQ ID NO: 23.
  • CDRs and FRs may be mixed and matched to form single domain antibodies of the invention. Mixing and matching of CDR sequences can be tolerated in VHHs of the invention.
  • the single domain antibody comprises: a CDR1 sequence SEQ
  • the single domain antibodies bind serum albumin so as to increase the half-life of agents conjugated thereto.
  • the single domain antibodies are cross-reactive to serum albumin homologs from different animal species.
  • the single domain antibodies bind Protein A and are suitable for pull-down using Protein A affinity chromatography.
  • the single domain antibodies of the invention may increase the half-life of agents conjugated thereto at least about 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times or at least about 500 times relative to the half-life of said agent in the absence of the single domain antibody.
  • the extent of half-life extension may be measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 5 times relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey. In some embodiments the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 10 times relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 1 .5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times or at least about 500 times relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of agents conjugated thereto by at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 800%, at least 1000%, at least 2500%, at least 5000%, at least 7500%, at least 10,000%, at least 20,000%, at least 30,000%, at least 40,000%, or at least 50,000% relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 400% relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey. In some embodiments the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 1000% relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of an agent conjugated thereto approximately 1200% relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life of an agent conjugated thereto at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 800%, at least 1000%, at least 2500%, at least 5000%, at least 7500%, at least 10,000%, at least 20,000%, at least 30,000%, at least 40,000%, or at least 50,000% relative to the half-life of said agent in the absence of the single domain antibody, as measured in humans or in a suitable animal model such as a mouse or cynomolgus monkey.
  • the single domain antibodies of the invention increase the half-life in a human subject of antibody-based agents or antibody-based fragments (for example but not limited to immunoglobulins, VHHs, such as but not limited to T cell engager (‘TcE’) or tumour associated antigen (‘TAA’) (VHHs, VNARS and scFvs) conjugated thereto to a halflife of at least five days, at least six days, at least seven, eight, nine, ten or more days, preferably at least fifteen, such as sixteen, seventeen, eighteen or nineteen days.
  • VHHs such as but not limited to T cell engager (‘TcE’) or tumour associated antigen (‘TAA’) conjugated thereto
  • TAA tumour associated antigen
  • the single domain antibodies of the invention may increase the half-life in a human subject of antibodybased agents or antibody-based fragments (for example but not limited to immunoglobulins, VHHs and scFvs) conjugated thereto to a half-life of at least one week.
  • the single domain antibodies of the invention may increase the half-life in a human subject of antibody-based agents or antibody-based fragments (for example but not limited to immunoglobulins, VHHs and scFvs) conjugated thereto to a half-life of at least two weeks.
  • a single domain antibody of the invention binds HSA with a KD less than 500 nM, less than 300 nM, preferably less than 200 nM or less than 100 nM.
  • a single domain antibody of the invention binds MSA with a KD less than 500 nM, less than 300 nM, preferably less than 200 nM or less than 100 nM. In some embodiments, a single domain antibody of the invention binds cynomolgus monkey serum albumin (CSA) with a KD less than 500 nM, less than 300 nM, preferably less than 200 nM or less than 100 nM. In some embodiments, a single domain antibody of the invention binds both HSA and MSA with a KD less than 500 nM, less than 300 nM, preferably less than 200 nM or less than 100 nM.
  • CSA cynomolgus monkey serum albumin
  • a single domain antibody of the invention binds both HSA and CSA with a KD less than 500 nM, less than 300 nM, preferably less than 200 nM or less than 100 nM. In some embodiments, a single domain antibody of the invention binds each of HSA, MSA and CSA with a KD less than 500 nM or less than 300 nM.
  • a single domain antibody of the invention binds HSA with a KD less than 200 nM. In some embodiments, a single domain antibody of the invention binds HSA with a KD less than 100 nM.
  • a single domain antibody of the invention binds to HSA with higher affinity than other anti-albumin single domain antibodies. In some embodiments, a single domain antibody of the invention binds HSA with lower ECso than other anti-albumin single domain antibodies.
  • single domain antibodies comprising the amino acid sequences of SEQ IDNOs: 10, 20 or 30 as CDR1, CDR2 and CDR3, respectively, bind HSA with higher affinity than Alb23 (SEQ ID NO: 127).
  • a single domain antibody comprising the amino acid sequence of SEQ ID NO: 124 or variants thereof bind HSA with higher affinity than Alb23 (SEQ ID NO: 127).
  • single domain antibodies comprising the amino acid sequences of SEQ ID NOs: 10, 20 or 30 as CDR1, CDR2 and CDR3, respectively bind HSA with a lower ECso than Alb23 (SEQ ID NO: 127).
  • a single domain antibody comprising the amino acid sequence of SEQ ID NO: 124 or variants thereof binds HSA with a lower ECso than Alb23 (SEQ ID NO: 127).
  • a single domain antibody of the invention binds HSA with a KD less than 200 nM and binds MSA with a KD less than 500 nM. In some embodiments, a single domain antibody of the invention binds HSA with a KD less than 100 nM and binds MSA with a KD less than 300 nM.
  • a single domain antibody of the invention binds to HSA with an ECso of less than 10 nM, optionally less than 8 nM, less than 7 nM, less than 6 nM, preferably less than 2 nM. In some embodiments, a single domain antibody of the invention binds to HSA with an ECso of less than 1 nM.
  • a single domain antibody of the invention binds to CSA with an ECso of less than 10 nM, optionally less than 8 nM, less than 7 nM, less than 6 nM, preferably less than 2 nM. In some embodiments, a single domain antibody of the invention binds to CSA with an ECso of less than 1 nM.
  • a single domain antibody of the invention binds to MSA with an ECso of less than 10 nM, optionally less than 8 nM, less than 7 nM, less than 6 nM, preferably less than 2 nM. In some embodiments, a single domain antibody of the invention binds to HSA with an ECso of less than 1 nM.
  • the ECso of a single domain antibody of the invention for example a single domain antibody comprising amino acid sequences of SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, or SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3 respectively
  • a single domain antibody of the invention for example a single domain antibody comprising amino acid sequences of SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, or SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3 respectively
  • MSA and CSA is less than 3 nM, optionally less than 2 nM.
  • the ECso of a single domain antibody of the invention for example a single domain antibody having amino acid sequence SEQ ID NO: 124, 131, 132 or 142, or variants thereof) for binding to each of HSA, MSA and CSA is less than 3 nM, optionally less than 2 nM.
  • the ECso of a single domain antibody of the invention for example a single domain antibody comprising amino acid sequences of SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, SEQ ID NOs: 13, 24 and 33 as CDR1, CDR2 and CDR3 respectively, or SEQ ID NOs: 10, 20 and 30 as CDR1, CDR2 and CDR3 respectively) for binding to each of HSA, MSA and CSA is less than InM.
  • the EC50 of a single domain antibody of the invention (for example a single domain antibody having amino acid sequence SEQ ID NO: 124) for binding to each of HSA, MSA and CSA is less than 1 nM, In some embodiments, the EC50 of a single domain antibody of the invention for binding to canine serum albumin is less than 10 nM, optionally less than 5 nM. In some embodiments, the EC50 of a single domain antibody of the invention for binding to feline serum albumin is less than 10 nM, optionally less than 5 nM, less than 2 nM, preferably less than 1 nM. In some embodiments, the EC50 of a single domain antibody of the invention for binding to bovine serum albumin is less than 10 nM.
  • a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 30 nM, less than 25 nM, or less than 20 nM. In some embodiments, a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 15 nM. In some embodiments, a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 10 nM.
  • a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 5 nM, 4 nM, 3 nM, or less than 2 nM. In some embodiments, a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 1.9 nM, 1 ,8 nM, 1.7 nM, 1.6 nM, 1.5 nM, 1.4 nM, 1.3 nM, less than 1.2 nM or less than 1.1 nM. In some embodiments, a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody binds to the serum albumin with an EC50 of less than 1 nM.
  • a single domain antibody is considered to bind to a serum albumin homolog if the single domain antibody can selectively pull-down said serum albumin homolog from crude serum.
  • the present invention provides a binding molecule comprising a single domain antibody of the invention and one or more agents.
  • the binding molecule comprises a single domain antibody of the invention conjugated to one or more agents.
  • the one or more agent comprises a therapeutic agent.
  • the one or more agent comprises a diagnostic agent.
  • the one or more agent is conjugated to the single domain antibody via a linker.
  • the linker is a poly-glycine or poly-glycine/serine linker.
  • Suitable poly- glycine/serine linkers may comprise or consist of the amino acid sequence GGGS (‘G4S’; SEQ ID NO: 186) or GGGSGGS (‘(G 4 S) 2; SEQ ID NO: 123).
  • the linker may comprise or consist of an amino acid sequence (GGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably n is 2.
  • the linker consists of the amino acid sequence of SEQ ID NO: 123.
  • the linker has a length adjusted for optimal positioning of the single domain antibody and/or the agent to their binding target(s).
  • a binding molecule of the invention comprises a single domain antibody of the invention genetically or post-translationally conjugated to one or more molecules with binding capabilities such as antibodies or fragments thereof, single domain antibodies including single domain antibodies of the invention, VHHs including VHHs of the invention and/or other VHHs (e.g. TcE or TAA), VNARS, SCFVS, receptors and/or canonical binding partners, optionally via polymeric linkers, to produce dimeric or multimeric binding molecules.
  • a binding molecule of the invention is a fusion protein comprising a single domain antibody of the invention fused to one or more molecules with binding capabilities such as antibodies or fragments thereof, single domain antibodies including single domain antibodies of the invention, VHHs including VHHs of the invention and/or other VHHs (e.g. TcE or TAA), VNARS, scFvs, receptors and/or canonical binding partners, optionally via polymeric linkers, to produce dimeric or multimeric binding molecules.
  • VHHs including VHHs of the invention and/or other VHHs e.g. TcE or TAA
  • VNARS scFvs
  • receptors and/or canonical binding partners optionally via polymeric linkers, to produce dimeric or multimeric binding molecules.
  • Said dimeric or multimeric binding molecules may be mono- specific in that they bind two or more of the same epitope, bi-specific in that they bind two different epitopes, or multi-specific (multimeric binding molecules only) in that they bind multiple different epitopes.
  • a binding molecule of the invention comprises a single domain antibody of the invention which has been genetically or post- translationally conjugated to one or more moieties. Said moieties may have biological activity and/or may modify the properties of the single domain antibody.
  • said moieties may modify properties of the single domain antibody in terms of enzymatic cleavage, degradation, sequestration, folding or unfolding, esterification, phosphorylation, glycosylation, PEGylation, post-translation modification, and combinations thereof.
  • Moieties conjugated to the single domain antibodies of the invention may include, but are not limited to, single chain variable fragments (scFvs), small molecule drugs, radioactive labels, nanoparticles, viral particles, cytokines, fluorescent labels, cells, receptors, nucleic acids, liposomes, peptides, and/or toxins.
  • a binding molecule of the invention has a half-life in a human subject of at least one day, at least two, three or four days, at least five days, at least six days, at least seven, eight, nine, ten or more days, preferably at least fifteen, such as sixteen, seventeen, eighteen or nineteen days. In some embodiments a binding molecule of the invention has a half-life in a human subject of at least one week. In some embodiments a binding molecule of the invention has a half-life in a human subject of at least two weeks.
  • the present invention further provides one or more polynucleotides encoding a single domain antibody described herein. Also provided by the present invention are one or more polynucleotides encoding a binding molecule of the invention.
  • the polynucleotides may comprise or consist of DNA.
  • a single domain antibody of the invention is encoded by one polynucleotide.
  • a binding molecule of the invention is encoded by one polynucleotide, and in others a binding molecule of the invention is encoded by more than one nucleotide whereby assembly of the resulting translated polypeptides into the binding molecule occurs post-translationally. Said assembly may occur in the subject in need of an agent comprised within the binding molecule, or may occur in another organism in which the binding molecule is expressed for later extraction, purification, formulation and administration.
  • Polynucleotides of the invention may be introduced into a host cell for expression in vivo or ex vivo. Introduction may occur by transformation, transduction and/or transfection.
  • One or more polynucleotides may be contained within one or more DNA-based constructs, for example but not limited to a viral vector polynucleotide, a plasmid, a cosmid or an artificial chromosome.
  • Said one or more polynucleotides, optionally said one or more vector molecules may be introduced into a cell as naked DNA or by means of a vector, such as a viral vector or a non- viral vector.
  • Viral vectors include but are not limited to adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, bacteriophages.
  • Non-viral vectors include but are not limited to liposomes, nanoparticles, quantum dots, polymer-based vectors.
  • the invention further provides a vector comprising one or more polynucleotides of the invention.
  • the invention also provides a cell comprising the one or more polynucleotides and/or one or more vectors of the invention.
  • the invention provides a pharmaceutical composition comprising one or more single domain antibodies of the invention, one or more binding molecules of the invention, one or more polynucleotides of the invention and/or one or more vectors of the invention, optionally also comprising one or more pharmaceutically acceptable carriers or excipients.
  • the invention provides a method of extending the half-life of an agent, comprising administering a single domain antibody of the invention conjugated to an agent or a binding molecule of the invention comprising an agent to a patient.
  • the invention also provides a method of extending the half-life of an agent comprising administering a single domain antibody of the invention and an agent to a patient, wherein the agent is administered simultaneously or sequentially with the single domain antibody.
  • the invention also provides a method of extending the half-life of an agent comprising administering a single domain antibody of the invention to a patient simultaneously or sequentially with an agent, optionally wherein the single domain antibodies associates with or couples to the agent in vivo.
  • the invention provides a use of a single domain antibody of the invention conjugated to an agent or a binding molecule of the invention in a method of extending the half-life of an agent, wherein the method comprises administering an agent to a patient.
  • the invention also provides a use of a single domain antibody of the invention in a method of extending the half-life of an agent comprising administering to a patient the single domain antibody and an agent, wherein the agent is administered simultaneously or sequentially with the single domain antibody.
  • the invention also provides a use of a single domain antibody of the invention in a method of extending the half-life of an agent comprising administering the single domain antibody to a patient simultaneously or sequentially with an agent, optionally wherein the single domain antibody is administered to a patient together with (e.g. coupled to or associated with or fused to) the agent and wherein the antibody and agent dissociate (e.g. via cleavage of a cleavable linker) in vivo.
  • Single domain antibodies, binding molecules, polynucleotides and/or vectors of the invention may be administered to a subject, human or another mammal (such as but not limited to a mouse, horse, cat, dog, cow, llama, cynomolgus monkey) by any routine method known in the art, but preferably via intravenous or subcutaneous injection.
  • the single domain antibodies and/or vectors may be administered orally.
  • Said single domain molecules, binding molecules, polynucleotides and/or vectors will be formulated appropriately according to their route of administration and the properties of the subject (mass, age, species, and so on) and such as to preserve the pharmacokinetic and pharmacodynamic - and particularly binding - properties of the single domain molecules or binding molecules.
  • Table 1 -Amino acid sequences. Back-mutated residues in humanised sequences are shown in bold and underlined type. Residues altered for optimisation are shown in underlined and italicised type.
  • Two llamas received 6 intra-muscular injections (on days 0, 14, 28, 35, 42, and 49) of soluble recombinant human serum albumin (lOOpg on days 0 and 14; 50pg on days 35 and 49) or soluble recombinant murine serum albumin (50pg on days 28 and 42) after mixing 1:1 with Stimune® (oil in water) adjuvant (ThermoFisher 7925000, Generic name ‘Specol’). Serum samples of days 0, 28, 43, 51, and 56 were collected. Immune responses to immunization were assessed at days 51 and 56.
  • RNA samples were isolated from a large bleed of each llama at day 51 and 56 from which RNA was isolated, precipitated in ethanol and sodium acetate and dissolved in RNAse-free milli-Q.
  • the RNA of day 51 and 56 was pooled for library construction. The integrity of the RNA was confirmed by the clear visibility of intact 28S and 18S rRNA on an 1% agarose gel.
  • IG-H cDNA fragments (both conventional and heavy chain) were amplified using primers annealing at the IGH leader sequence region and the CH2 region. This PCR resulted in the amplification of two DNA fragments ( ⁇ 700bp and ⁇ 900bp), which represents the VHH and VH cDNA respectively.
  • the 700bp fragment was excised and purified from gel and subsequently used as a template for a nested PCR to introduce flanking Sfil and BstEII restriction sites.
  • the purified PCR product was digested with Sfil and BstEII (400 bp fragment) and ligated into the phagemid vector pQ81.
  • the ligated libraries were transformed into TGI E. coli, which was stored as a glycerol stock. The transformation was also serially diluted to determine the number of transformants, which is indicative of the library size. Libraries were of excellent size with titres >10 n /ml.
  • Sequence alignment of the 172 double binders revealed good diversity with at least 12 different epitope family sequences, which originated from three germline families (‘KEREF’, ‘KQREL’ and ‘KGLEW’). From these, a panel of individual clones were picked such that diversity was properly represented considering CDR3 length, residues’ hydrophobicity and families with VH characteristics. 12 VHHs were selected as hits to proceed onto further characterisation, having SEQ ID NOs: 153 to 163 and 176.
  • VHHs were produced in TGI E. coli and purified by immobilized metal affinity chromatography (IMAC) using Cobalt beads and the C-terminal His tag. Integrity of VHHs was confirmed by SDS-PAGE and PageBlue staining.
  • IMAC immobilized metal affinity chromatography
  • Relative potencies of the purified VHHs to recombinant MSA and HSA was determined in an ELISA.
  • White Maxisorp plates were coated with 50 pl per well of 2 pg/ml of recombinant antigen HSA (HSA-H5220) or MSA (MSA-M52H8), in PBS, and incubated at 4°C overnight. Plates were washed 3x in PBST and blocked with 200 pl 4% Marvel milk + PBS + 0.1% Tween for 1 hour with agitation. Serial dilutions of each VHH were prepared starting at lOOOnM and following a lOx dilution down to O.OlnM in lx PBS with 1% Marvel milk.
  • Blocking buffer was removed and 50 pl of diluted VHH solutions were added (single well/clone/dilution) to respective wells. Plate was incubated for 1 hour at room temperature with agitation. Plate was washed as before and 50 pl of 1 :2000 dilution of Rabbit anti VHH (QE19) in lx PBS with 1% Marvel milk were added to each well. Plate was incubated for lh at room temperature with agitation. Plate was washed as before and 50 pl of 1:5000 dilution of donkey-anti-rabbit-HRP, in lx PBS with 1% Marvel milk, were added to each well. Plate was incubated for lh at room temperature with agitation.
  • ELISA plates were washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plates were incubated for 1 hour at room temperature with agitation. Plates were washed as before and 50 pl of 1:10 000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plates were incubated for lh at room temperature with agitation. Plates were washed and 50 pl per well of POD substrate added. Luminescence was read and RLU/min retrieved for analysis. No significant binding was observed against the off-target streptavidin. All 11 VHHs showed binding to all surfaces albeit with different affinities. Binding to Domain II indicates no interference with albumin binding to FcRn ( Figure 2 and Table 2).
  • Example 4 Selected hits bind native serum albumin
  • MSE02SRMP2N MSE02SRMP2Nserum were prepared, starting at 1:10 and following a lOx dilution down to 1:1000000 in 1% soya milk + PBS + 0.1% Tween and 1 lOpl of each dilution transferred into a shallow U-bottomed 96- well plate. To these wells, the appropriate volume of each clone was transferred so that the final concentration was equal to that obtained for the ECso for the recombinant albumin of the respective species. Plates were incubated for 1 hour at room temperature with agitation. The ELISA plates were washed, as before, and 50 pl of serum+VHH solutions were added and plates incubated for Ih, r.t. with agitation.
  • Example 6 Selected monomers accept N- and C- terminal linker formatting
  • Multimeric VHH constructs were created using PCR of individual clones with extended primers and Bsal restriction sites. Briefly, each VHH was amplified with primers specific to either the N-terminal or C-terminal position, digested with Bsal, and simultaneously ligated into a digested acceptor vector containing C-terminal Flag and His tags using complementary sticky ends. Ligation mixtures were transformed into E. coli T7 SHuffle Express cells and correct assemblies were confirmed by Sanger sequencing. Multimeric constructs were expressed cytosolically and purified using Ni affinity chromatography followed by a desalting step.
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant antigen HSA (HSA-H5220) or MSA (MSA-M52H8) or 5 pg/ml streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 200 pl 3% soya milk + PBS + 0.1% Tween for 1 hour. Serial dilutions of each N- or C- terminal link VHH were prepared starting at 200nM and following a 4x dilution down to 0.195nM in 1% soya milk + PBS + 0.1% Tween. ELISA plate was washed as before and 50 pl of diluted VHH solutions were added to respective wells.
  • N- or C- terminal linker engineered clones (wherein the linker is a G4S poly-glycine/serine linker) bind recombinant human and murine albumin in a dose dependent manner with different affinities ( Figure 4). In some instances, insertion of linker resulted in different binding characteristics in comparison to parental clones. No significant binding was observed against the off-target streptavidin. 10 clones were selected to be humanized.
  • ELISA plate was washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation. Plate was washed as before and 50 pl of 1:10 000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plate was incubated for Ih at room temperature with agitation. Plate was washed and 50 pl per well of POD substrate added. Luminescence was read and RLU/min retrieved for analysis. No significant binding was observed against the off-target streptavidin (n.s.). Most clones displayed no significant decrease in binding when lightly or aggressively humanized ( Figure 5 and Table 4).
  • Table 4 Summary of ECso values for dose response binding of humanized VHHs to recombinant HSA and MSA. (* ambiguous values)
  • Example 10 Selected humanised clones bind native serum albumin
  • Tm Melting temperature vas evaluated for all humanized and parental clones.
  • Samples were mixed with SYPRO Orange dye in a MicroAmp Fast Optical 96-well reaction plate and heated to 100°C using a ramp rate of 0.5 "CmirT in a StepOne Plus qPCR machine (Applied Biosystems) using the Melt Curve program. Melting temperatures were determined from the nadir of the derivative reporter plotted against temperature.
  • All parental and humanized clones were expressed from 5 ml E. coli autoinduction cultures with C- terminal Flag-His tags and purified using Ni affinity chromatography and an ion exchange polishing step. Purified VHHs were analysed by SDS-PAGE and demonstrated expected sizes. SEC analysis was conducted for all light humanization clones.
  • Fl 1 light SEQ ID NO: 126)
  • C8 light SEQ ID NO: 131
  • A8 light SEQ ID NO: 125
  • F8 light SEQ ID NO:124
  • B7 SEQ ID NO: 160 was also selected for humanisation (B7 light; SEQ ID NO: 132) and further investigations (see Examples 24 to 28).
  • C8Light R100>Q A mutant version of C8Light termed C8Light R100>Q was designed such that the nucleotides encoding R100 were modified to code for a glutamine residue. This gene was synthesised and cloned into a cytoplasmic expression vector with C-terminal Flag and His tags using Bsal digestion and ligation. The correct sequence was confirmed by Sanger sequencing and the construct was expressed cytosolically from E. coli T7 SHuffle Express cells. Purified material was isolated from the cell pellet using Ni affinity chromatography followed by an ion exchange polishing step. C81ight R100>Q has sequence SEQ ID NO: 142.
  • Example 14 C8 light R100>Q - ELISA ECso binding to recombinant HSA, MSA and specificity to Domain II
  • White Maxisorp plates were coated with 50 pl per well of 1 jrg/ml of recombinant antigen HSA (HSA-H5220), CSA (CSA-C52H4), MSA (MSA-M52H8), Domain II (Albumin Bioscience 1002), Domain I (Albumin Bioscience 1001) or Domain III (Albumin Bioscience 1003) or 5 pg/ml streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 200 pl 3% soya milk + PBS + 0.1% Tween for 1 hour.
  • VHH Serial dilutions of each VHH were prepared starting at 500nM and following a 3x dilution down to 0.008nM in 1% soya milk + PBS + 0.1% Tween.
  • ELISA plate was washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation. Plate was washed as before and 50 pl of 1:10 000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plate was incubated for Ih at room temperature with agitation. Plate was washed and 50 pl per well of POD substrate added. Luminescence was read and RLU/min retrieved for analysis.
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant antigen HSA (HSA-H5220), 50 pg/ml of Cardiolipin (C0563), 5 pg/ml of KLH(H8283), 10 pg/ml of LPS (tlrl-eblps-InvivoGen ), 1 pg/ml of ssDNA (D8899) or dsDNA (D4522) and 5 pg/ml insulin (19278) or streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 300 pl 0.5% soya milk + PBS + 0.1% Tween for 1 hour.
  • Affinity constants were determined by surface plasmon resonance (SPR) on a Biacore T200 instrument.
  • SPR surface plasmon resonance
  • biotinylated HSA Sigma # H5667-20ML
  • MSA Sigma # M5905-10ML
  • Biotin CAPture reagent #29423383
  • Lead clones were then tested at 5 concentrations lOOOnM, 200nM, 40nM, 8nM, 1 ,6nM with 2 min association followed by 5 min dissociation using Single Cycle Kinetics.
  • the surface of the chip was regenerated after the last dissociation in the cycle using regeneration solution (3 parts of 8M guanidine -HC1 mixed with 1 part of IM NaOH). Binding curves at different concentrations were used to calculate the kinetic parameters k on -values (k a ), koff -values(kd) and KD. Kinetic parameters were determined using 1:1 binding model. The obtained results are presented in Table54.
  • a mammalian expression vector was constructed containing the scFv-His6 sequence in a C-terminal position, with a Bsal cloning site followed by a G4S linker at the N-terminal position.
  • the VHHs were codon optimized for mammalian (human) expression and cloned into the N-terminal position of the acceptor vector using Bsal digestion and ligation. Correct assemblies of VHH-G4S-His6 were confirmed by Sanger sequencing and clean vector preps were produced.
  • Expression vectors were transiently transfected and expressed from Expi293 HEK cells, and purified from cell culture supernatants by Ni affinity chromatography. All molecules demonstrated the expected size when analysed by SDS-PAGE.
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant antigen HSA (HSA-H5220) or CD3 (CDD-H82W1) or 5 pg/ml streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer with IxPBS-T and blocked with 200 pl 3% soya milk + PBS + 0.1% Tween for 1 hour. Serial dilutions of each VHH were prepared starting at lOOnM and following a 4x dilution down to 9.5x10-5nM in 1% soya milk + PBS + 0.1% Tween.
  • ELISA plate was washed as before in IxPBS-T and 50 pl of diluted VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation. Plate was washed as before with IxPBS-T and 50 pl of 1:10,000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plate was incubated for Ih at room temperature with agitation. Plate was washed with IxPBS-T and 50 pl per well of POD substrate added. Luminescence was read and RLU/min retrieved for analysis. No significant binding was observed against the off-target streptavidin. All VHHs showed binding to both surfaces, with bi-specifics C8Light-scFv and F8Light-scFv being the best performers ( Figure 10).
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant antigen HSA (HSA-H5220), CSA (CSA-C52H4), MSA (MSA-M52H8), Domain II (Albumin Bioscience 1002), Domain I (Albumin Bioscience 1001) or Domain III (Albumin Bioscience 1003) or 5 pg/ml streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 200 pl 3% soya milk + PBS + 0.1% Tween for 1 hour.
  • VHH Serial dilutions of each VHH were prepared starting at lOOnM and following a 3x dilution down to 0.002nM in 1% soya milk + PBS + 0.1% Tween.
  • ELISA plate was washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation. Plate was washed as before and 50 pl of 1:10 000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plate was incubated for Ih at room temperature with agitation. Plate was washed and 50 pl per well of POD substrate added.
  • Luminescence was read and RLU/min retrieved for analysis. No significant binding was observed against the off-target streptavidin. C8Light- scFv bi-specific showed binding to all surfaces albeit with different affinities. Binding to Domain II but not Domains I and III indicates no interference with albumin binding to FcRn ( Figure 11).
  • VHHirr / TRR
  • the lead clones were gene synthesized as bi-specifics with an irrelevant VHH arm (‘VHHirr’ / TRR’), which binds a non-human target (lysozyme) and has the amino acid sequence of SEQ ID NO: 150.
  • the lead clones were linked at the N-terminal with a 2(G4S) linker to the C- terminal of the irrelevant VHH, with a final C-terminal His6 tag. Sequences of VHHlead- 2(G4S)-VHHirr-His6 constructs were confirmed and vector preps were produced and transiently transfected into HEK293 cells. Secreted VHH bi-specifics were purified from culture supernatants and purified using Ni affinity chromatography and desalting. All clones demonstrated high purity and expected molecular weights.
  • Bi-specific VHH:VHH constructs were analysed by ELISA to determine binding to HSA, MSA, CSA, and domain specificity, and the retention of HSA and MSA binding across the pH range required for endosomal recycling. This demonstrates that the lead VHH clones were tolerant of the bi-specific VHH:VHH format.
  • Example 21 VHH:VHH bi-specific molecules - ELISA ECso binding to recombinant HSA, MSA and specificity to Domain II
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant antigen HSA (HSA-H5220), CSA (CSA-C52H4), MSA (MSA-M52H8), Domain II (Albumin Bioscience 1002), Domain I (Albumin Bioscience 1001), Domain III (Albumin Bioscience 1003) or 5 pg/ml streptavidin, in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 300 pl 3% soya milk + PBS + 0.1% Tween for 1 hour.
  • VHH Serial dilutions of each VHH were prepared starting at 500nM and following a 3x dilution down to 0.008nM in 1% soya milk + PBS + 0.1% Tween.
  • ELISA plates were washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plates were incubated for 1 hour at room temperature with agitation. Plates were washed as before and 50 pl of 1 : 10000 dilution of anti-FLAG-HRP (A8591) in 1% soya milk + PBS + 0.1% Tween added to each well. Plate was incubated for Ih at room temperature with agitation. Plates were washed and 50 pl per well of POD substrate added.
  • Luminescence was read and RLU/min retrieved for analysis. No significant binding was observed against the off-target streptavidin. Both bi-specific molecules retained binding to all relevant surfaces with Fl l light bi-specific presenting the best curves ( Figure 12). Binding to Domain II but not Domains I and III indicates no interference with albumin binding to FcRn.
  • VHH Serial dilutions of each VHH were prepared starting at 500nM and following a 3x dilution down to 0.0085nM in 1% soya milk + PBS + 0.1% Tween (pH7.4 or pH5.0).
  • ELISA plates were washed as before in IxPBS-T (pH7.4 or pH5.0) and 50 pl of diluted VHH solutions were added to respective wells. Plates were incubated for 1 hour at room temperature with agitation.
  • Plates were washed as before with IxPBS-T (pH7.4 or pH5.0) and 50 pl of 1:250 dilution of rabbit anti-llama VHH (EG-VHH001-0025) in 1% soya milk + PBS + 0.1% Tween (pH7.4 or pH5.0) added to each well and plates incubated for Ih at room temperature with agitation. Plates were washed as before and 50 pl of 1:120 000 dilution of goat anti-rabbit IgG (ab6721) in 1% soya milk + PBS + 0.1% Tween (pH 7.4 or pH 5.0) added to each well and plates incubated for Ih at room temperature with agitation.
  • VHHirr-2(G4S)-Fl lLight-His bi-specific molecule (IRR:F1 ILight) was produced in HEK cells. Protein was purified and stored, in aliquots, in PBS (pH7.5) at a concentration of 0.5mg/ml. Purity >95% was determined by SDS-PAGE under reducing conditions and SEC-HPLC test and endotoxin levels ⁇ lEU/mg confirmed by LAL test. Protein was stored at -70°C and aliquots thawed whenever experimentally necessary. A PK study was conducted in BALB/c female mice treated with the VHH:VHH bi-specific molecule (IRR:F1 ILight) following animal welfare and humane treatment of animal policy.
  • mice 21 female BALB/c mice at the age of 10-12 weeks were enrolled in the study after 2 weeks of quarantine with facilitated access to food. Animals were randomized into 7 groups of 3 animals each, based on bodyweight, and the bi-specific administered intravenously (bolus), in a single dose at 2mg/Kg/day at time zero. Blood samples (130pl max) were taken retrobulbar from cohorts of mice, or terminal bleed was performed, at designated timepoints.
  • Group 1 was sampled before dosing and terminal at 168h; Group 2 was sampled at 5 min and terminal at 8h; Group 3 was sampled at 15 min or terminal at 24h; Group 4 was sampled at 30 min and terminal at 48h; Group 5 was sampled at Ih and terminal at 72h; Group 6 was sampled at 2h and terminal at 96h and Group 7 was sampled at 4h and terminal at 120h.
  • White Maxisorp plates were coated with 100 pl per well of 1 pg/ml of recombinant irrelevant antigen (Sigma L6876), in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 200 pl/well of 1% casein (USB Corporation #12840), Img/ml heparin (Thermo Scientific # 10551944), O.lmg/ml herring sperm DNA (Promega #D1816) in lx PBS (blocking buffer), for 1 hour.
  • casein USB Corporation #12840
  • Img/ml heparin Thermo Scientific # 10551944
  • O.lmg/ml herring sperm DNA Promega #D1816
  • Standard curve for interpolation was prepared by doing sequential dilutions of stock purified bi-specific, in 1% casein, Img/ml heparin, O.lmg/ml herring sperm DNA in lx PBS (sample dilution buffer), starting at 200nM and following a 2x dilution down to 0.021nM.
  • Mouse serum BALB/c mouse serum, Alternative Research Inc. #IMSBCSER 50ml
  • sample dilution buffer was diluted in sample dilution buffer to a final concentration of 33.34% (or 1:3).
  • the sequential dilutions of stock bi-specific were then diluted 1:2 in the 33.34% murine serum to final bi-specific concentrations from lOOnM to 0.042nM and murine serum at 16.67% (or 1:6).
  • Leftover samples of dosing material were sequentially diluted in sample dilution buffer starting at 160nM and following a 3x dilution down to 1.98nM. These were then diluted 1:2 in murine serum to final concentrations from 80nM to InM.
  • Serum PK samples were sequentially diluted in murine serum (matching species to that used for PK study).
  • Samples from pre-dosing and 48h, 72h and 96h started from neat and followed a 2x dilution down to 1 in 16.
  • Samples from 120h and 168h started from neat and followed a 1 ,5x dilution down to 1 in 5.06.
  • Samples from 5min, 15min, 30min and Ih started from an initial 1:8 dilution from neat and followed a 2x dilution down to 1 in 128.
  • Samples from 2h, 4h, 8h and 24h started from an initial 1:4 dilution from neat and followed a 2x dilution down to 1 in 64. All PK serum samples were then diluted 1 :6 in sample dilution buffer.
  • PK serum samples, dosing material and stock bi-specific All diluted material (PK serum samples, dosing material and stock bi-specific) was incubated at r.t. with agitation, for Ih to allow equilibrium.
  • ELISA plates were washed as before and 50 pl of diluted PK samples, stock bi-specific or dosing material were added to respective wells and plates incubated for 1 hour at room temperature with agitation.
  • Plates were washed as before and 50 pl of 1 :250 dilution of rabbit anti-llama VHH (EG-VHH001-0025) in 0.1% casein, Img/ml heparin, O.lmg/ml herring sperm DNA in lx PBS (antibody dilution buffer) added to each well and plates incubated for Ih at room temperature with agitation. Plates were washed as before and 50 pl of 1:60 000 dilution of goat anti-rabbit IgG (ab6721) in antibody dilution buffer added to each well and plates incubated for Ih at room temperature with agitation. Plates were washed and 50 pl per well of POD substrate added. Luminescence was read and RLU/min retrieved for analysis.
  • Concentrations of the VHH:VHH bi-specific molecule (IRR:F 11 Light) in serum samples was determined by interpolation of sample RLU/min values with the corresponding standard curve (standard curves analysed with Sigmoidal, 4PL, X is log (concentration)) using GraphPad Prism. Values obtained from sample interpolation were adjusted to experimental dilution to obtain total amount of bi-specific/sample/animal/time -point ( Figure 14).
  • Noncompartmental analysis using Phoenix® WinNonlin® was applied to the composite serum concentration data to determine bi-specific half-life for the VHH:VHH bi-specific molecule (IRR:F1 ILight). A mean half-life of 26.2 hour was obtained for this bi-specific molecule containing F 11 Light.
  • Example 24 Humanised VHH clones have improved protein A binding relative to comparator clones T0023500 and Alb23
  • Protein A beads were resuspended by vortexing the vial for 1 min. 50ul beads/ sample were transferred to a new microcentrifuge tube and placed on a magnet to separate the beads from the solution. The supernatant was removed before proceeding to antigen binding. 0.5ug antigen, diluted in 20ul PBS + 0.02% Tween, was added to protein A-coated DynabeadsTM (Thermo Fisher) and incubated for Ih at RT, with rotation. The tube was placed on a magnet, the supernatant (SN) removed, and dispensed into a new tube for further analysis by SDS- PAGE.
  • SN supernatant
  • Example 25 Cross-reactivity - ELISA ECso of humanised VHH clones for binding to recombinant HSA, MSA, CSA and native purified canine, feline and bovine serum albumin at pH 7.4
  • White Maxisorp plates were coated with 50 pl per well of 1 pg/ml of recombinant HSA (Aero Biosystems, Cat. HSA-H5220), MSA (Aero Biosystems, Cat. MSA-M52H8), CSA (Aero Biosystems, Cat. CSA-C52H4) or native purified Canine (Abeam, Cat. AB119814- 100mg;, Feline (Generon, Cat. 22070068-1) and Bovine Serum Albumin (Sigma, Cat. A3059-500G) in PBS, at 4°C overnight. Plates were washed in plate washer and blocked with 200 pl 3% soya milk + PBS + 0.1% Tween for 1 hour.
  • Clones of interest were selected, namely: A8-Light, B7-Light, B9-parental, B9-Light, B9-Aggressive, C8-Light, E6-Light, F8-Light and Fl l-Light and compared against Alb23 and T0023500 ( Figure 16).
  • Serial dilutions of each VHH were prepared starting at 500nM and following a 3x dilution down to 0.008nM in 1% soya milk + PBS + 0.1% Tween.
  • ELISA plate was washed as before and 50 pl of diluted VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation.
  • Example 26 Cross-reactivity - VHH binding to rabbit, mini pig and rat serum albumin by bead pull down lug of each FLAG-tagged VHH sample was incubated in 20ul serum ( 1 : 100 dilution in PBS for rabbit (Gibco, Cat. 16120-099-100ML) and rat serum (BioIVT, Cat. RAT00SRMPZN); 1:1000 dilution in PBS for mini pig serum (BioIVT, Cat. MPG00SRMPZN)) for Ih at RT with agitation.
  • Anti -FLAG beads were resuspended by vortexing the vial for 1 min. lOul of beads/sample were used in the experiment.
  • VHH + serum samples were added to the beads and incubated for 20min at RT with agitation. Tubes were placed on a magnet, the supernatant (SN) removed, and dispensed into a new tube for further analysis by SDS-PAGE. The beads were washed twice in 1ml PBS + 0.02% Tween, separating beads from the solution between each wash. Beads were then resuspended in 20ul PBS (PD). In a new tube lug of the input samples (VHH) were prepared in 20ul PBS (I).
  • DI 16-00-2) serum was prepared at 1:5 dilution (2x final concentration) and serially diluted 10-fold in 1% Soya in PBS + 0.1% Tween. 55ul of each dilution was transferred into a shallow, round -bottom 96-well plate with 55ul of VHH dilutions according to ECsos, prepared at 2x final concentration in assay buffer. Plates were incubated at RT for 1 h with shaking. Plates were washed with 200ul of PBST and 50 pl of serum/VHH solutions were added to respective wells. Plate was incubated for 1 hour at room temperature with agitation.
  • Equal amounts of each VHH were spiked into bacterial lysate.
  • Protein A purification was performed using Protein A SuperSpin columns (Biotoolomics, Cat. 230102) according to the manufacturer’s protocol, with the following three modifications: 1) the binding step was extended to 1 hour, 2) two washes were performed, 3) 5 ul of IM Tris was added to neutralise samples. The neutralised elution fractions obtained were then quantified using Lunatic® A280 measurements
  • Alb23 represents the ‘best on the market’ or the state of the art for manufacturability, but demonstrates poor species cross-reactivity.
  • T0023500 represents ‘best on the market’ or the state of the art for species cross-reactivity, but demonstrates poor Protein A binding.
  • Neither Alb23 or T0023500 possesses both broad species cross-reactivity and the capacity for purification via protein A affinity chromatography.
  • Examples 25 to 28 show that the VHHs of the invention have improved species cross-reactivity compared to at least Alb23.
  • Examples 24 and 29 demonstrate that VHHs of the invention show improved protein A binding in comparison to at least T0023500.
  • VHH clones F8L, C8L and B7L show cross-reactivity with a significantly larger number of species than Alb23.
  • VHH clones C8L and B7L additionally show significant improvement relative to T0023500 in binding to feline and bovine serum albumin;
  • VHH clones F8L, C8L, and B7L show statistically significant (p ⁇ 0.05) improvement in Protein A binding in comparison to T0023500.
  • the protein A binding properties of F8L, C8L, and B7L shows no significant difference to that of Alb23, i.e. is at least as good as that of Alb23 (Tables 9 and 10).
  • the single domain antibodies represent the first demonstration of anti-albumin single domain antibodies that have both broad species cross-reactivity and which are suitable for purification using protein A affinity chromatography.
  • a single domain antibody that specifically binds serum albumin wherein the single domain antibody comprises a complementarity-determining region (CDR) 1 comprising the amino acid sequence of:
  • the CDR1 comprising the amino acid sequence SEQ ID NO: 2 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 15, 16, 18, 164, and variants thereof comprising up to three modifications; or
  • the CDR1 comprising the amino acid sequence SEQ ID NO: 3 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 11, 17, 19, and variants thereof comprising up to three modifications.
  • VAA(V/I)SW(Y/none)(T/S/A)G(D/G)(R/S)(A/T) (SEQ ID NO: 4) or a variant thereof comprising up to three modifications;
  • the CDR2 comprising the amino acid sequence of SEQ ID NO: 4 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 20, 22, 23, 24, and variants thereof comprising up to three modifications;
  • the CDR2 comprising the amino acid sequence SEQ ID NO: 5 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 25, 26, 28, 166, and variants thereof comprising up to three modifications; or
  • the CDR2 comprising the amino acid sequence SEQ ID NO: 6 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 21, 27, 29, and variants thereof comprising up to three modifications.
  • the CDR3 comprising the amino acid sequence SEQ ID NO: 7 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 30, 32, 33, 34, and variants thereof comprising up to three modifications;
  • the CDR3 comprising the amino acid sequence SEQ ID NO: 8 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 35, 36, 38, 168, and variants thereof comprising up to three modifications; or
  • the CDR3 comprising the amino acid sequence SEQ ID NO: 9 or the variant thereof comprises an amino acid sequence selected from SEQ ID NOs: 31, 37, 39, and variants thereof comprising up to three modifications.
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising up to three modifications, a CDR2 comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof comprising up to three modifications, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof comprising up to three modifications;
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising up to three modifications
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: 164 or a variant thereof comprising up to three modifications
  • a CDR2 comprising the amino acid sequence of SEQ ID NO: 166 or a variant thereof comprising up to three modifications
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 168 or a variant thereof comprising up to three modifications.
  • FR 1 a framework region (FR) 1 comprising an amino acid sequence selected from SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and variants thereof comprising up to six modifications;
  • FR2 comprising an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and variants thereof comprising up to six modifications
  • FR3 comprising an amino acid sequence selected from SEQ ID NOs: 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, and variants thereof comprising up to six modifications
  • FR4 comprising an amino acid sequence selected from SEQ ID NOs: 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, and variants thereof comprising up to six modifications.
  • the single domain antibody of any one of embodiments 1 to 8 comprising:
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 80 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 102 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 81 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 103 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 82 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 104 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 85 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 107 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 86 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 108 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 87 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 109 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 88 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 110 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 89 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 111 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 90 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 112 or a variant thereof comprising up to six modifications.
  • SEQ ID NO: 153 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 154 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 155 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 156 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 157 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 158 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 159 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 160 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 161 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 162 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 163 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto.
  • FR1 having an amino acid sequence selected from SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 177, 178 and variants thereof comprising up to six modifications;
  • FR2 having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 179, and variants thereof comprising up to six modifications;
  • FR3 having an amino acid sequence selected from SEQ ID NOs: 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 180, and variants thereof comprising up to six modifications; and/or
  • FR4 having an amino acid sequence selected from SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 181, and variants thereof comprising up to six modifications.
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 61 or a variant thereof comprising up to six modifications
  • a FR3 comprising SEQ ID NO: 91 the amino acid sequence of or a variant thereof comprising up to six modifications
  • a FR4 comprising SEQ ID NO: 113 the amino acid sequence of or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 63 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 92 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 114 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 53 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 64 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 93 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 115 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 54 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 65 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 94 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 116 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 55 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 66 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 95 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 117 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 56 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 67 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 96 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 118 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 57 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 68 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 97 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 119 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 98 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 120 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 99 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 121 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 60 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 100 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 122 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 177 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 62 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 180 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 181 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 101 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 113 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 53 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 93 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 115 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 54 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 74 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 94 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 116 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 55 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 75 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 95 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 117 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 57 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 97 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 119 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 58 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 77 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 98 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 120 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO: 59 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 78 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 99 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 121 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO:60 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 79 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 100 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 122 or a variant thereof comprising up to six modifications
  • a FR1 comprising the amino acid sequence of SEQ ID NO:178 or a variant thereof comprising up to six modifications
  • a FR2 comprising the amino acid sequence of SEQ ID NO: 179 or a variant thereof comprising up to six modifications
  • a FR3 comprising the amino acid sequence of SEQ ID NO: 180 or a variant thereof comprising up to six modifications
  • a FR4 comprising the amino acid sequence of SEQ ID NO: 181 or a variant thereof comprising up to six modifications.
  • SEQ ID NO: 124 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 125 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 126 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 127 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 128 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 129 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 130 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 131 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 132 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 133 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 174 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto
  • SEQ ID NO: 135 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 137 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 138 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 141 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 143 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 146 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto;
  • SEQ ID NO: 147 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto; or (bb) SEQ ID NO: 148 or an amino acid sequence having at least 70%, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto; preferably
  • SEQ ID NO: 126 or a sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto or SEQ ID NO: 142 or a sequence having at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 %, or at least 99.9 % sequence identity thereto.
  • the serum albumin is human serum albumin, murine serum albumin, cynomolgus monkey serum albumin, bovine serum albumin, canine serum albumin, equine serum albumin, feline serum albumin, ovine serum albumin, porcine serum albumin, camelid serum albumin, rat serum albumin, rabbit serum albumin, mini pig serum albumin and/or primate serum albumin.
  • HSA human serum albumin
  • MSA murine serum albumin
  • CSA cynomolgus monkey serum albumin
  • a binding molecule comprising a single domain antibody as defined in any one of embodiments 1 to 27 and one or more agents, optionally wherein the one or more agents comprises a therapeutic agent or a diagnostic agent.
  • the binding molecule of embodiment 28, wherein the one or more agents is one or more antibodies or fragments thereof, one or more enzymes, one or more single domain antibodies, one or more single domain antibodies as defined in any one of embodiments 1 to 23, one or more single chain variable fragments (scFvs), one or more chemical moieties, one or more small molecule drugs, one or more radioactive moieties, one or more nanoparticles, one or more viral particles, one or more cytokines, one or more fluorescent moieties, one or more cells, one or more receptors, one or more nucleic acids, one or more liposomes, one or more peptides, and/or one or more toxins.
  • the one or more agents is one or more antibodies or fragments thereof, one or more enzymes, one or more single domain antibodies, one or more single domain antibodies as defined in any one of embodiments 1 to 23, one or more single chain variable fragments (scFvs), one or more chemical moieties, one or more small molecule drugs, one or more radioactive moieties
  • the linker is located at the N-terminus of the single domain antibody
  • the linker is located at the C-terminus of the single domain antibody.
  • a vector comprising the one or more polynucleotides of embodiment 33.
  • a cell comprising the one or more polynucleotides of embodiment 33 and/or the vector of embodiment 34.
  • a pharmaceutical composition comprising a single domain antibody as defined in any one of embodiments 1 to 27, a binding molecule as defined in any one of embodiments 28 to 32, the one or more polynucleotides of embodiment 33, the vector of embodiment 34, and/or the cell of embodiment 35, optionally wherein the composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • a method of extending the half-life of an agent in vivo comprising administering a single domain antibody as defined in any one of embodiments 1 to 27, a binding molecule as defined in any one of embodiments 28 to 32, the one or more polynucleotides of embodiment 33, and/or the vector of embodiment 34 to a patient simultaneously or sequentially with the agent.

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Abstract

La présente invention concerne des anticorps à domaine unique se liant à l'albumine sérique qui ont une réactivité croisée multi-espèce et qui se lient à une protéine A et des utilisations de ceux-ci. La présente invention concerne en outre des polynucléotides et des vecteurs codant pour lesdits anticorps à domaine unique ainsi que des cellules et des compositions pharmaceutiques comprenant lesdits polynucléotides, vecteurs et/ou anticorps à domaine unique.
PCT/GB2023/052694 2022-10-18 2023-10-17 Anticorps à domaine unique se liant à l'albumine WO2024084203A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB2215365.4A GB202215365D0 (en) 2022-10-18 2022-10-18 Single domain antibodies
GB2215365.4 2022-10-18
GBGB2312183.3A GB202312183D0 (en) 2023-08-09 2023-08-09 Single domain antibodies
GB2312183.3 2023-08-09

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WO2024084203A1 true WO2024084203A1 (fr) 2024-04-25

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Citations (5)

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WO2006040153A2 (fr) * 2004-10-13 2006-04-20 Ablynx N.V. Nanocorps™ contre la proteine beta-amyloide et polypeptides les renfermant pour le traitement de maladies degeneratives neurales, telles que la maladie d'alzheimer
WO2008096158A2 (fr) * 2007-02-08 2008-08-14 Domantis Limited Ligand
WO2012175400A1 (fr) 2011-06-23 2012-12-27 Ablynx Nv Protéines se liant à la sérumalbumine
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WO2008096158A2 (fr) * 2007-02-08 2008-08-14 Domantis Limited Ligand
WO2012175400A1 (fr) 2011-06-23 2012-12-27 Ablynx Nv Protéines se liant à la sérumalbumine
WO2014111550A1 (fr) * 2013-01-17 2014-07-24 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison modifiées anti-albumine sérique
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