Nanobody for nipah virus

Abstract

The present disclosure relates to nanobodies, therapeutic agents, compositions and methods for the diagnosis, prevention, amelioration and treatment of henipaviral infections. Herein disclosed are camelid-derived nanobodies that specifically bind the F protein of both Nipah virus (NiV) and Hendra virus (HeV). The disclose nanobodies effectively bind and potently neutralise both of these viruses, making these nanobodies suitable therapeutic agents for the prevention or treatment of henipaviral infections.

Description

"Nanobody for Nipah virus" Technical field

[0001] The present disclosure relates to the field of nanobodies, therapeutic agents, compositions and methods for the diagnosis, prevention, amelioration and treatment of henipaviral infections. Background

[0002] Hendra virus (HeV) and Nipah virus (NiV), belonging to the Henipavirus genus in the Paramyxoviridae family, are zoonotic pathogens that cause severe viral disease in humans characterized by serious respiratory illness and encephalitis with high mortality. These viruses have high potential to cause significant human epidemics following their spillover from wildlife reservoirs to humans and domestic animals due to their wide host tropism and high pathogenicity. In this regard, HeV transmission to humans can occur indirectly from fruit bats following direct human contact with infected horses, whilst the transmission of NiV to humans may occur directly from fruit bats, infected pigs or infected humans.

[0003] Currently, a variety of candidate vaccines and therapeutic antibodies for Nipah virus are under development, including recombinant subunit vaccines prepared using Hendra virus G Protein and recombinant viral vector vaccines carrying NiV-G Protein genes. The m102.4 monoclonal antibody obtained by screening through a phage surface display library technology shows good neutralizing activity on Nipah virus in vitro, but has only demonstrated limited or no therapeutic efficacy in in vivo studies (see, e.g., Mire et al., 2016). Antibody therapies are also typically associated with a high cost and therefore may not be a tenable solution for a large scale outbreak of NiV in developing countries, such as India and Bangladesh.

[0004] Accordingly, there remains an unmet clinical need for effective drug therapies for the prevention and treatment of henipaviral infections caused by NiV and HeV. Summary

[0005] The present disclosure is based on the surprising finding of a number of camelid-derived nanobodies that specifically bind the F protein of both Nipah virus (NiV) and Hendra virus (HeV). Additionally, their demonstrated ability to effectively bind and potently neutralise both of these viruses in vitro makes these nanobodies suitable therapeutic agents for the prevention or treatment of henipaviral infections.

[0006] In a first aspect, the present disclosure provides a single domain antibody that is directed against an F protein of a Nipah virus, said single domain antibody comprising: (a) a CDR1 that comprises an amino acid sequence of: AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1is R or F, X2is G or S and X3is Y or V; AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1is A or S; GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0007] In some examples, the single domain antibody comprises:(a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0008] In other examples, the CDR3 comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10), AKPSTYSGSWLTDFGS (SEQ ID NO: 13), or a variant thereof.

[0009] In certain examples, the single domain antibody comprises: (i) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A, or a variant thereof; or (ii) the CDR1 that comprises the amino acid sequence of AASGFTFDDYGMSWVRQ (SEQ ID NO: 11) or a variant thereof, the CDR2 that comprises the amino acid sequence of GLEWVSAISWNGGGTYY (SEQ ID NO: 12) or a variant thereof and the CDR3 that comprises the amino acid sequence of AKPSTYSGSWLTDFGS (SEQ ID NO: 13) or a variant thereof.

[0010] For some examples, the single domain antibody comprises the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or a variant thereof.

[0011] Suitably, the single domain antibody of the present aspect comprises: (i) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof; (ii) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9) or a variant thereof; (iii) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or a variant thereof;(iv) the CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWVRQ (SEQ ID NO: 26) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof; or (v) the CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWYRQ (SEQ ID NO: 28) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof.

[0012] According to particular examples, the single domain antibody, in a monovalent format, has a KD for the F protein of the Nipah virus of lower than about 600 nM, lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM or lower than about 10 nM.

[0013] In various examples, the single domain antibody further binds an F protein of a Hendra virus.

[0014] Referring to certain examples, the single domain antibody has been at least partly humanized.

[0015] Suitably, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 1 to 4 and 22 to 25, or a fragment, variant or derivative thereof.

[0016] In a second aspect, the present disclosure provides an antigen binding molecule comprising the single domain antibody of the first aspect.

[0017] Suitably, the antigen binding molecule is or comprises a monovalent single domain antibody, a multivalent single domain antibody, or a multispecific single domain antibody comprising one or more of the single domain antibodies of the first aspect. More particularly, the antigen binding molecule can be a multispecific single domain antibody comprising a further single domain antibody that is directed to a receptor binding glycoprotein (G protein) of a Nipah virus and / or a Hendra virus.

[0018] Suitably, the antigen binding molecule is or comprises an immunoconjugate. For some examples, the immunoconjugate comprises one or more of a detectable label, a therapeutic agent, a half-life extender and a nanocarrier.

[0019] In a third aspect, the present disclosure provides a composition comprising the single domain antibody of the first aspect or the antigen binding molecule of the second aspect and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0020] In some examples, the composition further comprises an antibody, an antibody fragment or a single domain antibody that is directed to a receptor binding glycoprotein (G protein) of a Nipah virus and / or a Hendra virus.

[0021] In a fourth aspect, the present disclosure provides a method of diagnosing or monitoring a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the step of contacting the subject and / or a biological sample from the subject with the single domain antibody of the first aspect, the antigen binding molecule of the second aspect or the composition of the third aspect.

[0022] Suitably, the present method further includes the step of detecting and / or measuring a level of antigen binding to the single domain antibody or the antigen binding molecule.

[0023] In a fifth aspect, the present disclosure provides a method of treating and / or preventing a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of the single domain antibody of the first aspect, the antigen binding molecule of the second aspect or the composition of the third aspect.

[0024] In a sixth aspect, the present disclosure provides for the use of the single domain antibody of the first aspect, the antigen binding molecule of the second aspect or the composition of the third aspect for therapy.

[0025] In a seventh aspect, the present disclosure provides for the use of the single domain antibody of the first aspect, the antigen binding molecule of the second aspect or the composition of the third aspect in the manufacture of a medicament for the treatment and / or prevention of a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject.

[0026] In an eighth aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the single domain antibody of the first aspect or a fragment, variant or derivative thereof or the antigen binding molecule of any one of the second aspect.

[0027] In a ninth aspect, the present disclosure provides a genetic construct comprising: (i) the isolated nucleic acid of the eighth aspect; or (ii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

[0028] In a tenth aspect, the present disclosure provides a host cell transformed with a nucleic acid molecule according to the eighth aspect or the genetic construct of the ninth aspect.

[0029] In an eleventh aspect, the present disclosure provides a method of producing the single domain antibody of the first aspect or the antigen binding molecule of the second aspect, including the steps of; (i) culturing the previously transformed host cell of the tenth aspect: and (ii) isolating the single domain antibody or the antigen binding molecule from said host cell cultured in step (i). Brief description of the drawings

[0030] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0031] Figure 1: NiV F foldon antigen characterisation and immunisation to generate camelid nanobody library. (A) Initial antigen design of NiV F foldon FcM, with F ectodomain coloured black, foldon domain coloured white and FcM coloured grey. Coomassie stained SDS-PAGE ran under reducing conditions of NiV F foldon FcM and cleaved NiV F foldon antigens is also shown. (B) Indirect ELISA of biotinylated 5B3 against NiV F foldon. Mean of duplicate values are shown with standard deviation. (C) Immunisation of alpaca with NiV F foldon and subsequent steps undertaken to generate bacterial nanobody display library and screen for NiV F specific clones.

[0032] Figure 2: Sequence alignment of camelid-derived nanobodies. (A) Amino acid sequence alignment of camelid-derived nanobodies. Conservation of residues is displayed as a colour gradient where white is conserved and grey is divergent. This is also displayed graphically as a conservation percentage bar graph. Coomassie stained SDS- PAGEs of nanobodies expressed either as Fc dimers or Fc monomers (FcM). SDS-PAGEs ran under reducing conditions (B) with heating and DTT or under non-reducing conditions (C).

[0033] Figure 3: Nanobodies bind NiV F and HeV F with nanomolar affinities. (A) Indirect ELISAs affinities (Kd) of nanobodies expressed as Fc dimers or Fc monomers (FcM) against prefusion NiV and HeV F proteins. Non-specific nanobody included as negative control. Kdanalysis were calculated from a one-site specific model fitted on Graphpad Prism 9 software. Values shown in nM with standard error. NA denotes “not applicable” and dashed line denotes undetected binding.

[0034] Figure 4: Nanobodies potently neutralise pseudotyped NiV. NiV pseudovirus neutralisation assays conducted with nanobody dimers (A,D) or monomers (B). Panels A and B are against wild- type NiV pseudovirus and panel D is against the NiV F- P52N / K55E mutant. Nanobody W25 was included as a non-specific control. In all assays, values normalised to virus only control as relative light units (RLU) and datapoints represents average of triplicate (A,B) or duplicate (D) values with standard deviation. Data was fit with three parameter inhibitor vs response modelin Graphpad Prism 9 andused to calculated IC50values in (C), with standard error shown. NA indicates not- applicable and a dashed line indicates not detected.

[0035] Figure 5: DS90 binds a novel quaternary pocket in the NiV F trimer. NiV F and DS90 complexes were generated and imaged on cryoARM300. Final resolution was 3.6 Å. Model was built using ModelAngelo and refined in ISOLDE, Phenix & Coot. (A) Three DS90 monomers (dark grey) bind to a single NiV F trimer (light grey). (B) DS90 model shown in ribbon form, with paratope residues highlighted and annotated. (C) Sequence of DS90 with paratope residues highlighted and annotated.

[0036] Figure 6: Design and sequence of DS90-m102.4 fusion antibody. (A) Proposed structure of the DS90- m102.4 mAb with gene schematic shown below. (B) Annotated amino acid sequence of DS90-m102.4 fusion antibody.

[0037] Figure 7: Characterization of DS90-m102.4 fusion antibody. (A) Coomassie stained SDS-PAGE of antibody proteins run under reducing (+DTT) or non-reducing conditions. (B) Indirect ELISAs of antibody binding to NiV F or G glycoproteins. (C) Surface-plasmon resonance plots of antibody binding to NiV F or G proteins. (D) Affinity summary of capture antibody binding to analyte from SPR.

[0038] Figure 8: Neutralization of DS90-m102.4 fusion antibody. (A) Live NiV neutralization of antibodies. Values displayed are the endpoint titers (nM) at which CPE is observed. Non-specific antibody negative control included. (B) Antibody neutralization of pseudotyped wild-type NiV (left) or m102.4 knockout mutant bearing a N557A mutation in G (right). Below are IC50 values.

[0039] Figure 9: Live virus NiV CPE analysis of antibodies at different timepoints and concentrations. Black arrows indicate evidence of CPE such as gaps in monolayer and cell rounding.

[0040] Figure 10: Humanization of DS90. (A) Alignment of DS90 with IGHV3-23*04 human allele performed via IgBlast. Resulting humanized DS90 (hDS90) sequence is shown, with matching residues shown as dots and incorporated residues from IGHV3-23*04 highlighted in grey. Preliminary ELISA binding data displayed reducedbinding of hDS90, and so three residues close to binding interface where mutated back to the original sequence, highlighted by black box in (A). (B) ELISA binding data of hDS90 monomeric constructs against prefusion stabilized NiV F. (C) Nipah pseudovirus neutralization of monomeric hDS90 constructs. Key to the Sequence ListingDetailed description General Techniques and Definitions

[0041] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0042] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0043] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0044] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0045] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0046] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0047] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0048] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0049] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0050] By “consisting essentially of” in the context of an amino acid sequence, such as a VHH chain, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus thereof. By “consisting essentially of” in the context of a nucleotide sequence is meant the recited nucleotide sequence together with an additional one, two or three nucleotides at the 5’ or 3’ end thereof.

[0051] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0052] For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0053] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims. Single domain antibodies

[0054] The inventors have surprisingly shown for the first time that particular variable heavy chain domains (VHHs) derived from camelid-based heavy chain only antibodies (HCAbs) can selectively bind to a particular epitope (e.g., an F pocket epitope; see Figure 5) of the F protein of NiV and optionally HeV. These single domain antibodies were also demonstrated to potently inhibit or neutralise NiV and HeV in in vitro assays.

[0055] Accordingly, the present disclosure provides a single domain antibody that is directed against an F protein of a Nipah virus and / or a Hendra virus. In one broad form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1is R or F, X2is G or S and X3is Y or V; AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S; GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and(c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0056] In another form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); EREFVSRISWSGGHTHS (SEQ ID NO: 27); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0057] In further form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); EREFVSRISWSGGHTHS (SEQ ID NO: 27); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8); NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9); NAEPTATGSWFTPGLQSY (SEQ ID NO: 10); AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0058] In a particular form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0059] In a related form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); EREFVSRISWSGGHTHS (SEQ ID NO: 27); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0060] In a further form, said single domain antibody comprises:(a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S; GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8); NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9); NAEPTATGSWFTPGLQSY (SEQ ID NO: 10); AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0061] In yet still another form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1 is R or F, X2 is G or S and X3 is Y or V; or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S; or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; or a variant thereof.

[0062] In another form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); EREFVSRISWSGGHTHS (SEQ ID NO: 27); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8); NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9); NAEPTATGSWFTPGLQSY (SEQ ID NO: 10); or a variant thereof.

[0063] In one form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; or a variant thereof.

[0064] In other forms, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8);NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9); NAEPTATGSWFTPGLQSY (SEQ ID NO: 10); or a variant thereof.

[0065] In some forms, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVSRISWSGGHTHS (SEQ ID NO: 27); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; or a variant thereof.

[0066] In a certain form, said single domain antibody comprises: (a) a CDR1 that comprises an amino acid sequence of: AASGFTFSSYVMSWVRQ (SEQ ID NO: 26); AASGFTFSSYVMSWYRQ (SEQ ID NO: 28); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVSRISWSGGHTHS (SEQ ID NO: 27); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8); NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9); NAEPTATGSWFTPGLQSY (SEQ ID NO: 10); or a variant thereof.

[0067] In another form, the present disclosure provides a single domain antibody that is directed against an F protein of a Nipah virus, said single domain antibody comprising: (a) a CDR1 that comprises an amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5), AASGFTFDDYGMSWVRQ (SEQ ID NO: 11), or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6), GLEWVSAISWNGGGTYY (SEQ ID NO: 12), or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10), AKPSTYSGSWLTDFGS (SEQ ID NO: 13), or a variant thereof.

[0068] In the context of the present disclosure, the terms “single domain antibody”, “VHH”, “VHH antibody fragment”, “VHH chain” and “nanobody” can be used interchangeably herein and denote the variable domain or region of the single heavy chain of antibodies of the type of those found in camelids, which are naturally devoid of light chains. It is noted that the terms “Nanobody” and “Nanobodies” are registered trademarks and thus may also be referred to as Nanobody® and / or Nanobodies®.

[0069] In the absence of a light chain, single domain antibodies generally each have three CDRs, denoted CDR1, CDR2 and CDR3 respectively. Additionally, single domain antibodies typically include three or four framework regions (FRs; FR1, FR2, FR3 and optionally FR4). The single domain antibodies described herein can be derived from camel, dromedary, llama or alpaca HCAbs. In particular examples, the single domain antibodies according to the present disclosure are derived from alpaca HCAbs.

[0070] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody (e.g., the VHH chain of a camelid-derived antibody) as defined herein that specifically binds to an antigen and, for example,includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. As used herein, the term “complementarity determining regions” (i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region (e.g., a VHH chain) the presence of which are major contributors to specific antigen binding. Each VHH chain of a camelid-derived antibody typically has three CDR regions identified as CDR1, CDR2 and CDR3. “Framework regions” are those variable domain residues other than the CDR residues.

[0071] There are multiple conventions to define and describe the CDRs of an immunoglobulin or antibody, such as a VHH chain or single domain antibody. Exemplary conventions to define CDRs include the Kabat definition (which is based on sequence variability and is the most commonly used; See, e.g., Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No.91-3242 (1991)), the Chothia definition (which is based on the location of the structural loop regions; See, e.g., Chothia, et al., (1987) J Mol. Biol. 196:901-917), the AbM definition (which is a compromise between the Kabat and Chothia definitions and is based on Oxford Molecular's AbM antibody modelling software), and the method described by Kontermann and Diibel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp.33-51, 2010). In particular examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by the Kabat definition. In other examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by the Chothia definition.

[0072] Like other paramyxoviruses and henipaviruses, NiV and HeV possess two major membrane-anchored glycoproteins in the envelope of the viral particle: a fusion glycoprotein (an F protein) and a receptor binding glycoprotein (a G protein). The receptor binding glycoprotein is required for host cell receptor recognition and attachment. In contrast, the fusion glycoprotein is a trimeric class I fusogenic envelope glycoprotein containing two heptad repeat (HR) regions and a hydrophobic fusion peptide (Fp) that mediate fusion of the virus particle with a host cell.

[0073] The term “Fusion glycoprotein” or “F protein” as used herein includes any of the recombinant or naturally- occurring forms of a paramyxoviral, a henipaviral, a NiV or a HeV F protein, or variants or homologs thereof that at least partly maintain or retain F protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to a wildtype or naturally occurring F protein sequence). In some examples, the variants or homologs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a wildtype or naturally occurring F protein sequence (e.g., SEQ ID NOs: 18 and 19). According to some examples, the F protein is substantially identical to the protein identified by the UniProt accession number Q9IH63 (Nipah virus F protein) or O89342 (Hendra virus F protein).

[0074] The sequence of an F protein of a NiV is publicly available (e.g., UniProt accession number Q9IH63). An exemplary amino acid sequence is set forth in SEQ ID NO: 18. Thus, the F protein amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 18 or a fragment or derivative thereof.

[0075] The sequence of an F protein of a HeV is also publicly available (e.g., UniProt accession number O89342). An exemplary amino acid sequence is set forth in SEQ ID NO: 19. Thus, the F protein amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 19 or a fragment or derivative thereof.

[0076] The expression “directed against an F protein of a Nipah virus” is intended to mean that a single domain antibody or an antigen binding molecule of the present disclosure is capable of selectively or specifically binding to and / or has been raised against an epitope of the F protein of a NiV. Given the sequence similarity between the respective F proteins of Nipah viruses and Hendra viruses, the single domain antibody or antigen binding molecule of the present disclosure may also be capable of selectively or specifically binding to the F protein of a Hendra virus. Accordingly, the single domain antibody or antigen binding molecule may be described as specific or selective for the F protein of Nipah and Hendra viruses, that is to say that it binds to the F protein of these two viruses to the exclusion of any other molecule or protein. More particularly, the single domain antibody or antigen binding molecule may be described as specific or selective for the prefusion form of an F protein of Nipah and Hendra viruses.

[0077] In view of the above, the single domain antibody or the antigen binding molecule provided herein can specifically or selectively bind to an F protein of a NiV, such as a prefusion form thereof, and optionally an F protein of a HeV. The terms “specifically binds” or “selectively binds” can be used interchangeably herein and shall be taken to mean that the binding interaction between a binding agent disclosed herein (e.g., a single domain antibody, an antigen binding molecule) and a target molecule described herein (e.g., an F protein, such as those set forth in SEQ ID NOs: 18 and 19) is dependent on detection of the target molecule by the binding agent. Accordingly, the binding molecule preferentially binds or recognizes the target molecule even when present in a mixture of other molecules or organisms. The formation of a complex with a target molecule (e.g., a F protein) that is relatively stable under physiologic conditions can also be a characteristic of such selective or specific binding by a VHH chain of a single domain antibody or an antigen binding molecule.

[0078] As used herein, the term “binds” refers to the interaction of a binding agent, such as a single domain antibody or an antigen binding molecule, with a target molecule (e.g., an F protein) and means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule. For example, a single domain antibody or an antigen binding molecule of the present disclosure recognizes and binds to a specific structural element of an F protein of NiV and / or HeV rather than to molecules generally.

[0079] Specific or selective binding can be characterized by a KDof about 5×10−2M or less (e.g., less than 5×10−2M, less than 10−2M, less than 5×10−3M, less than 10−3M, less than 5×10−4M, less than 10−4M, less than 5×10−5M, less than 10−5M, less than 5×10−6M, less than 10−6M, less than 5×10−7M, less than 10−7M, less than 5×10−8M, less than 10−8M, less than 5×10−9M, less than 10−9M, or less than 10−10M). Methods for determining the binding affinity of a single domain antibody or an antigen binding molecule (e.g., a multi-specific antigen binding molecule) to a target molecule or an effector molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (SPR; e.g., Biacore assays), fluorescent-activated cell sorting (FACS) binding assays and the like.

[0080] According to certain examples, the single domain antibody binds to an F protein, and more particularly a prefusion form of an F protein, of a NiV and / or a HeV with high affinity or relatively high affinity. As used herein, theterms “high affinity” and “relatively high affinity” are used interchangeably herein and refer to a binding affinity between a binding agent and the target molecule of interest with a KDof at least about 10-6M, more particularly at least about 10-7M, even more particularly at least about 10-7M and still even more particularly between about 10-8M to about 10-10M. Again, the determination of such affinity may be conducted under standard competitive binding immunoassay procedures, such as those provided herein.

[0081] In particular examples, the single domain antibody provided herein, or a conjugate thereof, in a monovalent format, has a KD for the F protein of a Nipah virus (e.g., SEQ ID NO: 18) of lower than about 600 nM (e.g., lower than about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 5 nM or any range therein), lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM or lower than about 5 nM.

[0082] In certain examples, the single domain antibody provided herein, or a conjugate thereof, in a divalent format, has a KD for the F protein of a Nipah virus (e.g., SEQ ID NO: 18) of lower than about 300 nM (e.g., lower than about 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5 nM or any range therein), lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM, lower than about 5 nM or lower than about 1 nM.

[0083] In various examples, the single domain antibody provided herein, or a conjugate thereof, in a monovalent format, has a KDfor the F protein of a Hendra virus (e.g., SEQ ID NO: 19) of lower than about 1500 nM (e.g., lower than about 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 5 nM or any range therein), lower than about 1000 nM, lower than about 600 nM, lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM or lower than about 5 nM.

[0084] In some examples, the single domain antibody provided herein, or a conjugate thereof, in a divalent format, has a KD for the F protein of a Hendra virus (e.g., SEQ ID NO: 19) of lower than about 600 nM (e.g., lower than about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 5 nM or any range therein), lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM or lower than about 5 nM.

[0085] According to certain examples, the single domain antibody or antigen binding molecule provided herein does not significantly or substantially bind to a molecule other than the target molecule (e.g., an F protein of a NiV and / or a HeV). The phrase “does not significantly bind to” can mean, for example, that the single domain antibody or antigen binding molecule provided herein binds to a molecule other than the target molecule (or to any molecule other than the target molecule) with a binding affinity (e.g., KD) that is at most 50% (e.g., 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less or any range therein) of the binding affinity of said single domain antibody or antigen binding molecule for the target molecule, such as an F protein expressed by a NiV and / or a HeV, under the same physiological conditions.

[0086] The current disclosure describes a single domain antibody that selectively binds to a NiV and / or a HeV F protein epitope. According to various examples, the single domain antibody or antigen binding molecule provided hereininhibits a Nipah virus infection or a Hendra virus infection by at least partly inhibiting, disrupting or preventing virus- host cell membrane fusion.

[0087] As such, the single domain antibodies or the antigen binding molecules of the present disclosure are capable of neutralizing a NiV in a virus neutralization assay. In particular examples, the single domain antibodies (e.g., in a monovalent or bivalent format) or the antigen binding molecules are capable of neutralizing a NiV in a neutralization assay at an IC50 of less than about 50 nM (e.g., less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 nM or any range therein), less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM or less than about 0.01 nM. According to some examples, the single domain antibodies or the antigen binding molecules are further capable of neutralizing a HeV in a virus neutralization assay, such as at an IC50 of less than about 50 nM (e.g., less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 nM or any range therein), less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM or less than about 0.01 nM. Suitable virus neutralisation assays include, for example, plaque reduction assays, pseudovirus neutralisation assays and microneutralisation assays.

[0088] Suitably, the single domain antibody includes the CDR1 that comprises the amino acid sequence of AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1is R or F, X2is G or S and X3is Y or V, or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S, or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A, or a variant thereof. For such examples, the CDR1 suitably comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5), AASGFTFSSYVMSWVRQ (SEQ ID NO: 26), AASGFTFSSYVMSWYRQ (SEQ ID NO: 28), or a variant thereof. Referring to such examples, the CDR2 suitably comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6), EREFVSRISWSGGHTHS (SEQ ID NO: 27), or a variant thereof. In such examples, the CDR3 suitably comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or a variant thereof.

[0089] Accordingly, in some examples, the single domain antibody includes the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof. In these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.

[0090] In other examples, the single domain antibody includes the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9) or a variant thereof. For such examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2.

[0091] In various examples, the single domain antibody includes the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or a variant thereof. In relation to these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.

[0092] Suitably, the single domain antibody includes the CDR1 that comprises the amino acid sequence of AASGFTFDDYGMSWVRQ (SEQ ID NO: 11) or a variant thereof, the CDR2 that comprises the amino acid sequence of GLEWVSAISWNGGGTYY (SEQ ID NO: 12) or a variant thereof and the CDR3 that comprises the amino acid sequence of AKPSTYSGSWLTDFGS (SEQ ID NO: 13) or a variant thereof. For these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0093] Suitably, the single domain antibody includes a CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWVRQ (SEQ ID NO: 26) or a variant thereof, a CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and a CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof. For these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 22, 24 or 25.

[0094] Suitably, the single domain antibody includes a CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWYRQ (SEQ ID NO: 28) or a variant thereof, a CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and a CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof. For these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23.

[0095] Suitably, the single domain antibody disclosed herein, comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 1 to 4 and 22 to 25, or a fragment, variant or derivative thereof. More particularly, the single domain antibody disclosed herein suitably comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 1 to 3 and 22 to 25, or a fragment, variant or derivative thereof. Even more particularly, the single domain antibody disclosed herein suitably comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 1 and 22 to 25, or a fragment, variant or derivative thereof.

[0096] In some examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. For other examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2. According to certain examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. In various examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4. Referring to other examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22. For some examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23. In particular examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24. According to certain examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25.

[0097] The single domain antibodies, antigen binding molecules and nucleic acids described herein may be considered to be isolated. For the purposes of the present disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0098] By “protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.

[0099] The term “protein” includes and encompasses “peptide”, which is typically used to describe a protein having no more than fifty (50) amino acids (e.g., no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids and any range therein) and “polypeptide”, which is typically used to describe a protein having more than fifty (50) amino acids.

[0100] As used herein, a protein, polypeptide or peptide “variant” shares a definable amino acid sequence relationship with a reference amino acid sequence. In particular examples, the reference amino acid sequence is that of a CDR1, CDR2 or CDR3 sequence. As such, the reference amino acid sequence may be the amino acid sequence of any one of SEQ ID NOs: 5 to 13 and 26 to 30. According to other examples, the reference amino acid sequence is that of aVHH chain sequence, such as that of any one of SEQ ID NOs: 1 to 4 and 22 to 25. For some examples, the reference amino acid sequence is that of a FR1, FR2, FR3 or FR4 sequence.

[0101] The “variant” protein, polypeptide or peptide may have one or a plurality of amino acids of the reference amino acid sequence deleted, inserted / added or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted, inserted / added or deleted without changing the activity of the single domain antibody (i.e., conservative substitutions). Accordingly, one or more of the residues of a single domain antibody, such as of a CDR (e.g., those defined by SEQ ID NOs: 5 to 13 and 26 to 30) and / or a FR, may be conservatively modified (e.g., by amino acid substitution or deletion) without altering the biological activity, function, or other desired property of the single domain antibody, such as its affinity or its specificity for an antigen like an F protein. Exemplary variant proteins, such as variant CDRs and VHH chain sequences, are provided in SEQ ID NOs: 22 to 30.

[0102] In some examples, a variant of the single domain antibody provided herein substantially retains the antigen binding ability (i.e., F protein binding ability) of the unmodified or reference single domain antibody. Thus, one or more amino acid residues within the CDR and / or FR regions of a single domain antibody of the present disclosure can be deleted or replaced with other amino acid residues, such as those from the same side chain family, and the variant single domain antibody can be tested for retained function (e.g., the ability to specifically bind an F protein of NiV and / or HeV at high affinity) using the functional assays described herein.

[0103] According to some examples, modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to modify one or more FR residues to that respective FR residue of the corresponding human germline sequence. Another type of framework modification involves modifying one or more residues within the FR and / or CDR regions to remove T cell epitopes to thereby reduce the potential immunogenicity of the single domain antibody. Exemplary humanized FR and / or CDR regions are provided in SEQ ID NOs: 22 to 30.

[0104] Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in, for example, Bowie et al., Science 247:1306-1310 (1990).

[0105] Suitably, protein, polypeptide or peptide variants provided herein share at least 70% or 75%, more particularly at least 80% or 85% or even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference amino acid sequence, such as those set forth in SEQ ID NOs: 1 to 13 and 18 to 30. To this end, variants of the single domain antibodies described herein and their respective CDRs are contemplated for the present disclosure. Exemplary variants of the single domain antibodies are provided in SEQ ID NOs: 22 to 25, whilst exemplary variants of CDR sequences are provided in 26 to 30.

[0106] Accordingly, modifications to the CDR sequences disclosed herein are envisaged. In particular examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.5 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In other examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.11 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In some examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.26 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In certain examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.28 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0107] In certain examples, said CDR2 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.6 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In particular examples, said CDR2 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.12 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In various examples, said CDR2 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.27 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0108] According to further examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.8 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. For some examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.9 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In other examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.10 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In various examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence SEQ ID NO.13 or a sequence that is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0109] Larger peptides and isolated proteins comprising a plurality of single domain antibodies (e.g., multivalent or multispecific antigen binding molecules) or conjugates thereof are also contemplated by the present disclosure and are described in more detail hereinafter.

[0110] Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids / proteins may each comprise (1) only one or more portions of a complete nucleic acid / protein sequence that are shared by the nucleic acids / proteins, and (2) one or more portions which are divergent between the nucleic acids / proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of, for example, 6, 9, 12 or 20 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program byIntelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1991, Nucl. Acids Res.253389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0111] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0112] The present disclosure also provides fragments of the single domain antibodies. As used herein, a “fragment” is a segment, domain, portion or region of a protein or peptide (such as those set forth in SEQ ID NOs: 1 to 13 and 18 to 30) which constitutes less than 100% of the amino acid sequence of the protein or peptide.

[0113] In general, fragments may comprise, consist essentially of or consist of up to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 or 124 contiguous amino acids of a single domain antibody (such as of SEQ ID NOs: 1 to 4 and 22 to 25). In a particular example, the protein fragment is or comprises a conserved region or one or more conserved amino acids, such as the CDR1-CDR3, of a single domain antibody. In this regard, one or more residues of FR1 and / or FR4 at an N- and / or C- terminus of a single domain antibody may not be present in the protein fragment. In various examples, the fragment comprises, or is contained within, a single domain antibody, such as those set forth in SEQ ID NOs: 1 to 4 and 22 to 25.

[0114] Suitably, the fragment substantially retains the antigenic binding ability of the single domain antibody from which the fragment is derived. In this regard, fragments of the present disclosure may retain the CDR1, CDR2 and CDR3 sequences of the single domain antibody. Additionally, fragments of the present disclosure suitably retain at least partly a natural structure and / or conformation of the full length peptide or protein.

[0115] The present disclosure also contemplates derivatives of the single domain antibodies described herein. As used herein, “derivatives” are molecules such as proteins, fragments or variants thereof that have been altered, for example, by conjugation or complexing with other chemical moieties, by post-translational modification (e.g., phosphorylation, acetylation and the like), modification of glycosylation (e.g., adding, removing or altering glycosylation), lipidation and / or inclusion of additional amino acid sequences as would be understood in the art.

[0116] Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and / or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g., polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP), polylysine, epitope tags, such as myc, FLAG and haemagglutinin tags. In one particular example, an additional amino acid sequence may comprise one or a plurality of histidine residues at an N and / or C-terminus thereof. The plurality of histidine residues (e.g., polyhistidine) may be a linear sequence of histidine residues or may be branched chain sequences of histidine residues. These additional histidine residues may facilitate purification of the single domain antibody.

[0117] Other derivatives contemplated by the disclosure include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and / or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the single domain antibodies, fragments and variants of the disclosure. In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

[0118] Further derivatives may include conjugates of the single domain antibodies. The term “conjugated” can be used in the context of the present disclosure to describe single domain antibodies disclosed herein that are conjugated to another compound or structure, such as a label or carrier molecule or protein. Accordingly, in one example, the single domain antibodies of the present disclosure are “conjugated”. Single domain antibodies of the disclosure may be modified via conjugation or complexing with other chemical moieties, by post-translational modification (e.g., phosphorylation, ubiquitination, glycosylation), chemical modification (e.g., cross-linking, acetylation, biotinylation, oxidation or reduction) and / or conjugation with labels (e.g., fluorophores, enzymes, radioactive isotopes) and / or other functional elements (e.g., a half-life extender, a therapeutic agent), as described in more detail below.

[0119] The present disclosure also contemplates “naked” single domain antibodies. The term “naked” can be used to describe single domain antibodies of the present disclosure that are not conjugated to another compound or incorporated into a broader structure. Put another way, the single domain antibodies of the present disclosure can be un-conjugated.

[0120] Conjugated single domain antibodies of the disclosure suitably retain their ability to bind an F protein of a NiV and / or a HeV. In an example, a single domain antibody disclosed herein is conjugated to a label, such as biotin, so as to facilitate coupling to a substrate. Additional C- or N-terminal residues may be used as linkers to conjugate the single domain antibodies of the present disclosure to another moiety, or tags that aid the detection of the molecule. Such linkers and tags are well known in the art and include, for example, linker His tags, e.g., hexa-His (HHHHHH, SEQ ID NO: 20) or myc tags.

[0121] In various examples, a conjugate of the single domain antibody provided herein comprises a human rhinovirus 3C protease cleavage site (HRV3C) (e.g., LEVLFQGP, SEQ ID NO: 21) as a linker. In such examples, the single domain antibody is suitably conjugated to an Fc region or Fc domain by way of the human rhinovirus 3C protease cleavage site. The Fc domain or Fc region may be useful for extending the half-life of the single domain antibody, orotherwise to provide a desired functionality conferred by the Fc domain or Fc region, such as recognition by a secondary reagent or binding to a solid support for purification.

[0122] By “Fc” or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. By “Fc domain” as used herein is meant a polypeptide that comprises all or part of an Fc region.

[0123] The single domain antibodies of the present disclosure, inclusive of variants, fragments and / or derivatives thereof, may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.

[0124] Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99 / 02550 and International Publication WO 97 / 45444.

[0125] Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6. Typically, recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell.

[0126] The present disclosure further contemplates humanized or at least partly humanized (also referred to as “humaneered”) versions of the single domain antibodies provided herein. Accordingly, in some examples, the single domain antibody provided herein is a humanized or substantially humanized single domain antibody.

[0127] By “humanized” is meant the amino acid sequence of the single domain antibody is mutated or modified so that immunogenicity upon administration in human patients is reduced, minor or non-existent (e.g., a single domain antibody that originated from a species other than human that has had immunogenic or potentially immunogenic amino acid residues replaced with amino acids that are less immunogenic or not immunogenic in the context of a single domain antibody administered to a human subject). Accordingly, humanized single domain antibodies should be substantially non-immunogenic in humans, but retain the affinity and activity of the wild-type or camelid single domain antibody (e.g., the single domain antibodies of SEQ ID NOs:1-4).

[0128] Any method known in the art for creating humanized antibodies are envisaged herein, including but not limited to the humanizing technology of KaloBios Pharmaceuticals and that described in Vincke et al. (JBC 2008). By way of example, humanising a single domain antibody generally comprises a step of replacing one or more of the Camelidae- or alpaca-derived amino acid residues with their human counterpart as found in a corresponding human consensus sequence, without that single domain antibody losing its typical character or biological function. Suitably, humanizationdoes not significantly affect the antigen binding capacity of the resulting humanized single domain antibody. Notwithstanding this, humanizing modifications or mutations may be made in one or more CDRs and / or FRs of the single domain antibodies. In particular examples, the humanized single domain antibody may contain one or more fully human FR sequences, as are known in the art.

[0129] Suitably, a humanised single domain antibody of the present disclosure includes one or more modifications or variants to a wild-type, unmodified or native version thereof, such as that set forth in one of SEQ ID NOs: 1-4. Accordingly, in one form, the present disclosure provides a humanised single domain antibody comprising an amino acid sequence, such as that set forth in SEQ ID NOs: 1 to 4 or more particularly SEQ ID NOs: 1 to 3, wherein one or more amino acid residues thereof is modified. In some examples, the one or more amino acid residues are modified at a position selected from the group consisting of 14, 27, 35, 37, 49, 75, 79, 87, 88, 89, 93 and any combination thereof, of a full-length single domain antibody amino acid sequence (e.g., SEQ ID NOs: 1 to 4). More particularly, the one or more amino acid residues can be modified at a position selected from the group consisting of A14, R27, G35, Y37, V37, A49, A75, V79, K87, P88, D89, I93 and any combination thereof, of a full-length single domain antibody amino acid sequence (e.g., SEQ ID NOs: 1 to 4).

[0130] More particularly, the modifications to the one or more amino acid residues of the humanised single domain antibody provided herein are suitably selected from the group consisting of: (a) P at position 14; (b) F at position 27; (c) S at position 35; (d) V or Y at position 37; (e) S at position 49; (f) S at position 75; (g) L or V at position 79; (h) R at position 87; (i) A at position 88; (j) E at position 89; (k) V or I at position 93; and any combination thereof of a full length single domain antibody amino acid sequence (e.g., as set out in SEQ ID NOs: 1 to 4). Even more particularly, in some examples, the modifications to the one or more amino acid residues of the humanised single domain antibody are selected from the group consisting of A14P, R27F, G35S, Y37V, V37Y, A49S, A75S, V79L, K87R, P88A, D89E, I93V and any combination thereof, of a full length H2 amino acid sequence (e.g., as set out in SEQ ID NOs: 1-4).

[0131] According to certain examples, a humanised single domain antibody described herein suitably includes a Y residue at position 37 of a wild-type, unmodified or native version thereof, such as that provided in SEQ ID NOs: 1 to 4. To this end, the wild-type sequence of the single domain antibody may be modified to include a Y residue at position 37 thereof (e.g., position 37 of SEQ ID NO: 4 may be modified to include a Y residue) or alternatively a Y residue may already be present in the wild-type sequence thereof (e.g., SEQ ID NOs: 1-3), such that this position is not modified. By way of example, the Y37 residue of SEQ ID NOs: 1-3 is suitably not modified to include a V residue.

[0132] Exemplary amino acid and encoding nucleic acid sequences for humanised single domain antibodies and CDRs of the present disclosure are also provided in SEQ ID NOs: 22 to 34. In particular examples, a humanised single domain antibody comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 22 to 25. For some examples, a humanised single domain antibody of the present disclosure includes a CDR1 comprising, consisting of or consisting essentially of an amino acid sequence of AASGFTFSSYVMSWVRQ (SEQ ID NO: 26). In alternative examples, a humanised single domain antibody of the present disclosure includes a CDR1 comprising, consisting of or consisting essentially of an amino acid sequence of AASGFTFSSYVMSWYRQ (SEQ ID NO: 28). In some examples, a humanised single domain antibody of the present disclosure includes a CDR2 comprising, consisting of or consisting essentially of an amino acid sequence of EREFVSRISWSGGHTHS (SEQ IDNO: 27). For such examples, the humanised single domain antibody described herein may further include a CDR3 comprising, consisting of or consisting essentially of an amino acid sequence of NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A or AKPSTYSGSWLTDFGS (SEQ ID NO: 13) or a variant thereof. More particularly, the humanised single domain antibody described herein may further comprise a CDR3 comprising, consisting of or consisting essentially of an amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or AKPSTYSGSWLTDFGS (SEQ ID NO: 13) or a variant thereof. Antigen binding molecules

[0133] The present disclosure further provides an antigen binding molecule that includes the single domain antibody described herein.

[0134] An antigen binding molecule is one which includes a single domain antibody disclosed herein (e.g., any of SEQ ID NOs: 1 to 4 and 22 to 25 or a variant thereof) and one or more functional moieties and / or one or more further antigen binding moieties, domains or units in addition to the single domain antibody.

[0135] As such, in a particular form, the antigen binding molecule comprises a single domain antibody disclosed herein and one or more further antigen binding moieties, domains or units.

[0136] Exemplary antigen binding moieties, domains or units include single domain antibodies (inclusive of those described herein and further single domain antibodies, such as those directed against an antigen of a NiV or a HeV as are known in the art), monoclonal antibodies (mAbs) (e.g., m102.4 mAb, 5B3 mAb and mAb66), antibody mimetic proteins, aptamers and antibody fragments, such as Fc, Fab or F(ab)2 fragments and / or may comprise single chain Fv antibodies (scFvs). Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293. Antigen binding moieties, domains or units may also include multivalent recombinant antibody fragments, such as diabodies, triabodies and / or tetrabodies, comprising a plurality of scFvs, as well as dimerisation- activated demibodies (e.g. WO / 2007 / 062466). By way of example, such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 906444; or in Kipriyanov, 2009 Methods Mol Biol 562177. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991- 1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbour, Cold Spring Harbour Laboratory, 1988.

[0137] Suitably, the antigen binding molecule is a monospecific antigen binding molecule. A monospecific antigen binding molecule binds a single antigen (e.g., an F protein of a NiV and / or a HeV). In some examples, the monospecific antigen binding molecule includes one or more of the single domain antibodies provided herein. More particularly, the monospecific antigen binding molecule may comprise two or more (e.g., 2, 3, 4, 5, 6) of the single domain antibodies provided herein. In this regard, the antigen binding molecule may comprise, for example, a dimer, trimer, quadramer, tetramer, hexamer etc of a single domain antibody described herein. For such examples, each of the single domain antibodies of the antigen binding molecule can comprise the same or substantially the same amino acid sequence connected by a linker, such as a polypeptide linker as are known in the art.

[0138] Suitably, the antigen binding molecule is a multi-specific antigen binding molecule, such as one that comprises one or more of the single domain antibodies provided herein and one or more (e.g., 1, 2, 3, 4, 5) further binding moieties, domains or units (e.g., one or more further single domain antibodies) that bind to one or more respective epitopes other than to which the single domain antibody disclosed herein is raised against or binds (e.g., an epitope other than that of an F protein of a NiV and / or a HeV, such as an antigen of a NiV and / or a HeV other than an F protein thereof). In particular examples, the antigen binding molecule comprises a single domain antibody provided herein that is directed to an F protein of a NiV and / or a HeV and a further antigen binding moiety, domain or unit that is directed to a G protein of a NiV and / or a HeV. To this end, the further antigen binding moiety, domain or unit that is directed to the G protein of the NiV and / or a HeV may be any as is known in the art, such as the m102.4 monoclonal antibody (see Zhu et al., J Infect Dis 2008) or functional fragments or derivatives thereof. In various examples, the antigen binding molecule comprises a single domain antibody provided herein that is directed to an F protein of a NiV and / or a HeV and a further single domain antibody that is directed to a G protein of a NiV and / or a HeV.

[0139] In certain examples, the antigen binding molecule is or comprises a monovalent antigen binding molecule. A monovalent antigen binding molecule is one that comprises a single antigen-binding moiety, domain or unit (e.g., a single nanobody or VHH chain), such as those provided herein.

[0140] In other examples, the antigen binding molecule is or comprises a multivalent antigen binding molecule, such as a bivalent antigen binding molecule. A multivalent antigen binding molecule comprises two or more antigen binding moieties, domains or units (e.g., a single domain antibody dimer, trimer, quadramer, pentamer, hexamer etc). In some examples, the antigen binding molecule is or comprises a bivalent antigen binding molecule. A bivalent antigen binding molecule comprises two antigen-binding moieties, domains or units (i.e., 2 single domain antibodies, such as one or two of those provided herein).

[0141] Accordingly, if one single domain antibody described herein is linked to another single domain antibody, such as one of the present disclosure, the single domain antibody has a bivalent format, and if, for example, three single domain antibodies, such as those of the present disclosure, are linked together, the antigen binding molecule has a trivalent format. Such formats can be desirable to enhance or otherwise modify the effectiveness of the antigen binding molecules or single domain antibodies of the present disclosure. To generate multivalent antigen binding molecules as described herein, respective or adjacent antigen-binding moieties, domains or units may be connected by a linker, such as a polypeptide linker.

[0142] According to particular examples, the antigen binding molecule provided herein is multiparatopic. A multiparatopic antigen binding molecule binds the same antigen, but the respective antigen binding moieties, domains or units thereof bind to more than one epitope (i.e., different epitopes) in said antigen (e.g., an F protein of a NiV and / or a HeV). In certain examples, the antigen binding molecule is a biparatopic antigen binding molecule. A biparatopic antigen binding molecule is monospecific, but the respective binding moieties, domains or units thereof bind to two different epitopes of the same antigen (e.g., an F protein of a NiV and / or a HeV). In such examples, the antigen binding molecule can comprise a single domain antibody provided herein and a further antigen binding moiety, domain or unit, such as a further single domain antibody, that binds to an F protein of a NiV and / or a HeV, but at a different or overlapping epitope to that of said single domain antibody.

[0143] According to various examples, the antigen binding molecule comprises a single domain antibody disclosed herein conjugated or otherwise linked to one or more functional moieties. To this end, the antigen binding molecule may be described as an immunoconjugate. As used herein, the term “immunoconjugate” refers to a polypeptide molecule that includes at least one functional moiety and an antigen binding moiety, such as a single domain antibody disclosed herein.

[0144] Exemplary functional moieties include a detectable marker or label, a therapeutic agent, a half-life extender and a nanocarrier.

[0145] In certain examples, the functional moiety is or comprises a half-life extender that may serve to prolong the half- life of the antigen binding molecule or single domain antibody in vivo following administration to a subject. Such half- life extenders may comprise, for example, an antibody, or part thereof, or a protein, or part thereof, that binds or is derived from a serum albumin (e.g., a serum albumin protein, an albumin binding domain). In particular examples, the half-life extender is or comprises an Fc domain. Other half-life extenders that may be utilised for the present disclosure include polymers, such as polyethylene glycol (PEG) and starch. Half-life may be increased by at least 1.5 times, more particularly at least 2 times, even more particularly at least 5 times, yet even more particularly at least 10 times or still even more particularly at least 20 times, greater than the half-life of the corresponding single domain antibodies or antigen binding molecules of the present disclosure that do not include such a half-life extender. For example, the half- life may be increased by more than 1 hour, more particularly more than 2 hours, even more particularly more than 6 hours, yet even more particularly more than 12 hours, or still even more particularly more than 24, 48 or 72 hours, compared to the single domain antibodies or antigen binding molecules of the present disclosure that do not include such a half-life extender.

[0146] It is further envisaged that the single domain antibodies or nanobodies of the present disclosure may be conjugated or otherwise linked to an E3 ubiquitin ligase ligand. To this end, the single domain antibody may be considered to form part of a Proteolysis Targeting Chimeric (PROTAC) molecule. PROTACs are heterobifunctional compounds composed of a target protein-binding ligand and an E3 ubiquitin ligase ligand or substrate that induce proteasome-mediated degradation of the target protein (e.g., an F protein of a NiV and / or HeV) via their recruitment of E3 ubiquitin ligase and subsequent ubiquitination. Such compounds can be capable of inducing the inactivation of a target protein upon addition to cells or administration to an animal or human, and therefore have been proposed for the treatment of disease by removing pathogenic or oncogenic target proteins.

[0147] Single domain antibodies have been shown previously to work functionally as PROTAC components. When a single domain antibody is fused to a ubiquitin E3 ligase substrate, receptors trigger protein degradation of ectopic and endogenous targets. For instance, the ADprom system optimizes the fusion of nanobodies against a target protein to the VHL receptor and depletes endogenous targets (13, 14). Nanobodies have also been engineered and fused directly to the active domains of Ubiquitin E3 ligases, such as the antibody RING-mediated destruction system (ARMeD), which uses the RING finger domain of the Ubiquitin E3 ligase RNF4 fused to a nanobody. A valuable feature of the ARMeD system is its independence of the endogenous ubiquitin E3 ligases (15). Accordingly, it is further contemplated that a single domain antibody of the present disclosure can be conjugated or otherwise linked to a ubiquitin E3 ligase or functional portion or domain thereof, as are known in the art.

[0148] According to other examples, the single domain antibodies or antigen binding molecules of the present disclosure are labelled with a detectable or functional marker or label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescent labels, radiolabels, enzymes, chemiluminescent labels, a nuclear magnetic resonance active label or photosensitizers, as described in more detail herein. Thus, binding of the labelled single domain antibody or antigen binding molecule to an F protein of a NiV and / or a HeV may be detected and / or measured by detecting fluorescence, luminescence, radioactivity, positron emission tomography, enzyme activity or light absorbance thereof.

[0149] For some examples, the single domain antibodies or antigen binding molecules of the present disclosure are coupled to a therapeutic agent or moiety (e.g., a small molecule, a protein, a nucleic acid, such as an siRNA), such as a drug, an enzyme, a cytokine (e.g., IL-2, IL-12, and TNF), a radionuclide or a toxin (e.g., Enterobacter cloacae β- Lactamase, Pseudomonas Exotoxin A, TRAIL and granzyme B). In particular examples, the therapeutic agent is an antiviral agent, as are known in the art. Exemplary antiviral agents include remdesivir, ribavirin, favipiravir, 4'azidocytidine (R1479) and 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (ALS-8112).

[0150] In some examples, the single domain antibodies or antigen binding molecules of the present disclosure are coupled to a nanocarrier, such as a liposome, a micelle, a lipid nanoparticle, albumin-based nanoparticles and polymer- based nanoparticles. Because of the binding specificity of single domain antibodies, they may be broadly used in such drug delivery platforms to deliver their cargo (e.g., a therapeutic payload) to its specific location (e.g., a viral particle).

[0151] It is envisaged that all combinations of the antigen binding molecule, in particular those combinations specifically listed herein, can be used in any therapeutic, diagnostic, or prognostic method or use, such as those hereinafter described. Encoding nucleic acids

[0152] The present disclosure also provides an isolated nucleic acid encoding the single domain antibody or the antigen binding molecule described herein.

[0153] The term “nucleic acid” as used herein designates single- or double- stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as modified purines (for example inosine, methylinosine and methyladenosine) and modified pyrimidines (for example thiouridine and methylcytosine).

[0154] As used herein, a “polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides. A “primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid “template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM. A “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

[0155] Suitably, the isolated nucleic acid encodes a single domain antibody described herein, inclusive of humanized versions thereof. For example, the isolated nucleic acid of the present disclosure encodes a single domain antibody comprising: (a) a CDR1 that comprises an amino acid sequence of: AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1is R or F, X2is G or S and X3is Y or V; AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S; GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0156] More particularly, the isolated nucleic acid of the present disclosure suitably encodes a single domain antibody comprising: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

[0157] In some examples, the isolated nucleic acid encodes the single domain antibody comprising, consisting essentially of or consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1 to 4 or 22 to 25, or a fragment, derivative or variant thereof.

[0158] Suitably, the isolated nucleic acid encoding the single domain antibody comprises, consists essentially of or consists of the nucleotide sequence set forth in any of SEQ ID NOs: 14 to 17 and 31 to 34, or a fragment, derivative or variant thereof.

[0159] Also contemplated are fragments and variants of the isolated nucleic acid. Variants may comprise a nucleotide sequence at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any nucleotide sequence encoding the single domain antibody or the antigen binding molecule of the present disclosure (e.g., SEQ ID NOs: 14 to 17 and 31 to 34).

[0160] Fragments of the isolated nucleic acid may comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95-99% of the contiguous nucleotides present in any nucleotide sequence encoding the single domain antibody or antigen binding molecule of the present disclosure, such that they encode at least a portion of the single domain antibody or antigen binding molecule. In general, fragments may comprise, consist essentially of or consist of up to 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 372, contiguous nucleic acids that encode a portion of a single domain antibody or an antigen binding molecule described herein (e.g., SEQ ID NOs: 1 to 4 or 22 to 25).

[0161] The present disclosure also provides nucleic acids that have been modified such as by taking advantage of codon sequence redundancy. In a more particular example, codon usage may be modified to optimize expression of a nucleic acid in a particular organism or cell type.

[0162] The isolated nucleic acids disclosed herein can be conveniently prepared using standard protocols such as those described in Chapter 2 and Chapter 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

[0163] Nucleic acids of the present disclosure may be isolated, detected and / or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques.

[0164] Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to the skilled addressee, and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification, recombinase polymerase amplification (RPA) and helicase-dependent amplification, although without limitation thereto. Genetic constructs

[0165] The present disclosure also provides a genetic construct comprising the isolated nucleic acid hereinbefore described. The genetic construct may be a vector.

[0166] In particular examples, the genetic construct comprises the isolated nucleic acid operably linked or connected to one or more other genetic components. A genetic construct may be suitable for therapeutic delivery of the isolated nucleic acid or for recombinant production of the single domain antibody or the antigen binding molecule of the disclosure in a host cell.

[0167] Broadly, the genetic construct can be in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and / or expression of the nucleic acid or an encoded protein of the present disclosure.

[0168] For the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the present disclosure operably linked to one or more additional sequences in an expression vector. An “expression vector” may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

[0169] By “operably linked” is meant that said additional nucleotide sequence(s) is / are positioned relative to the nucleic acid of the present disclosure preferably to initiate, regulate or otherwise control transcription.

[0170] Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0171] Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, polyadenylation sequences, transcriptional start andtermination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive, repressible or inducible promoters as known in the art are contemplated by the present disclosure.

[0172] The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein is expressed as a fusion protein.

[0173] The expression construct may also include an additional nucleotide sequence encoding a selection marker such as ampR, neoRor kanR, although without limitation thereto. Host cells

[0174] The present disclosure also provides a host cell transformed with a nucleic acid molecule or a genetic construct described herein. The host cell may be an isolated host cell or a cell in vitro or alternatively a host cell or cell in vivo or in situ in a subject.

[0175] Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells (e.g. HeLa, Cos, NIH-3T3, HEK293T, Jurkat, CHO cells), yeast cells (e.g. Saccharomyces cerevisiae, Pichia pastoris), insect cells (e.g., Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc.1995-2015), in particular Chapters 9 and 16. Methods of production

[0176] Related aspects of the present disclosure provide a method of producing the single domain antibody or the antigen binding molecule described herein, including the steps of; (i) culturing the host cell disclosed herein; and (ii) isolating the single domain antibody or the antigen binding molecule from said host cell cultured in step (i).

[0177] In this regard, the recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols, such as those hereinbefore provided.

[0178] The present disclosure further provides a single domain antibody or an antigen binding molecule produced by the method described herein. Pharmaceutical compositions

[0179] Further aspects of the present disclosure provide a composition comprising the single domain antibody and / or the antigen binding molecule described herein, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0180] By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, syntihetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

[0181] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991), which is incorporated herein by reference.

[0182] In some examples, the present composition is in the form of a diagnostic composition.

[0183] In other examples, the present composition is in the form of a therapeutic composition.

[0184] A therapeutically effective amount of a composition comprising a single domain antibody and / or an antigen binding molecule may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity of the viral infection, and the manner of administration of the therapy or composition.

[0185] Any safe route of administration may be employed for administering the single domain antibodies and antigen binding molecules described herein. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra- articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids arid certain cellulose derivatives such as hydroxypropylmethyl cellulose, in addition, the controlled release may be achieved by using other polymer matrices, liposomes and / or microspheres. In particular examples, the composition is capable of being or configured or adapted to be aerosolised so as to be capable of being administered by inhalation to a subject in need thereof.

[0186] Compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an οil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the therapeutic agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0187] The above compositions may be administered in a manner compatible with the dosage formulation, and in such an amount as is effective to prophylactically and / or therapeutically treat henipaviral infections, such as Nipah virus infections and Hendra virus infections, and / or diseases, disorders or conditions associated therewith and / or alleviate symptoms associated therewith. The dose administered to a patient, in the context of the present disclosure, should be sufficient to achieve a beneficial response in a patient over time such as a reduction in a level of viral shedding in their bodily fluids (e.g., blood, urine, saliva). The quantity of the therapeutic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the therapeutic agent(s) required to be administered will depend on the judgement of the clinician. The total dose required for each treatment may be administered by multiple doses or in a single dose. In any event, suitabledosages of the therapeutic agents described herein may be readily determined by those skilled in the art. Such dosages may be in the order of nanograms to milligrams of the therapeutic agents of the disclosure.

[0188] Suitably, the composition further comprises one or more further or additional therapeutic agents, such as an anti-inflammatory agent (e.g., NSAIDs, corticosteroids) and / or an antiviral agent (e.g., m102.4 mAb, remdesivir, ribavirin, favipiravir, 4'azidocytidine (R1479) and 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (ALS-8112)). In particular examples, the composition further includes a further single domain antibody that binds to a receptor binding glycoprotein (G protein) of a NiV and / or a HeV. In various examples, the composition further comprises an m102.4 mAb or a functional fragment or derivative thereof. In some examples, the composition further comprises an 5B3 mAb or a functional fragment or derivative thereof. In some examples, the composition further comprises an mAb66 mAb or a functional fragment or derivative thereof. Methods for preventing and treating henipaviral infections

[0189] In view of the foregoing, the single domain antibodies, antigen binding molecules and compositions described herein can be utilised to prevent, ameliorate and / or treat henipaviral infections, such as Nipah virus infections and Hendra virus infections, and / or diseases, disorders or conditions associated therewith.

[0190] In one broad form, the present disclosure relates to the use of a single domain antibody, an antigen binding molecule, a composition, an isolated nucleic acid or a genetic construct described herein for therapy.

[0191] Accordingly, there is provided herein a method of treating and / or preventing a henipaviral infection, such as a Nipah virus infection and / or a Hendra virus infection, and / or a disease, disorder or condition associated therewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of the single domain antibody, the antigen binding molecule or the composition provided herein.

[0192] In a related form, the present disclosure provides for the use of the single domain antibody, the antigen binding molecule or the composition disclosed herein for therapy.

[0193] In another form, there is provided the use of the single domain antibody, the antigen binding molecule or the composition disclosed herein in the manufacture of a medicament for the treatment and / or prevention of a henipaviral infection (e.g., a Nipah virus infection and / or a Hendra virus infection) and / or a disease, disorder or condition associated therewith in a subject.

[0194] In yet a further related form, the present disclosure relates to the single domain antibody, the antigen binding molecule or the composition disclosed herein for use in the treatment and / or prevention of a henipaviral infection (e.g., a Nipah virus infection and / or a Hendra virus infection) and / or a disease, disorder or condition associated therewith in a subject.

[0195] In relation to the above aspects of the present disclosure, the henipaviral infection can be a Nipah virus infection and / or a disease, disorder or condition associated therewith. In alternative examples, the henipaviral infection can be a Hendra virus infection and / or a disease, disorder or condition associated therewith.

[0196] As used herein, “treating” (or “treat” or “treatment”) refers to a therapeutic intervention that ameliorates a sign or symptom of a disease, disorder or condition characterized by henipaviral infection, after it has begun to develop. The term “ameliorating”, with, reference to such diseases, disorders or conditions, refers to any observable beneficialeffect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0197] As used herein, “preventing” (or “prevent” or “prevention”) refers to a course of action (such as administering a therapeutically effective amount of the single domain antibody) initiated prior to the onset of a symptom, aspect, or characteristic of a henipaviral infection so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject.

[0198] Prophylactic administration of the single domain antibodies, antigen binding molecules and compositions described herein is also envisaged for the present disclosure, particularly in respect of patients who have had prior exposure or contact with an animal or human known or suspected to have been infected with a henipavirus, such as a NiV or a HeV. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition or exhibits only early signs for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic thereof.

[0199] By “administration” is meant the introduction of a composition (e.g., a composition comprising a single domain antibody) into a subject by a chosen route. In some examples, the therapeutically effective amount of the single domain antibody is administered subcutaneously. In other examples, the therapeutically effective amount of the single domain antibody is administered intramuscularly. In various examples, the therapeutically effective amount of the single domain antibody is administered intravenously. In some examples, the therapeutically effective amount of the single domain antibody is administered by inhalation. In particular examples, the therapeutically effective amount of the single domain antibody is administered by inhalation by lumbar puncture (spinal tap).

[0200] The term “therapeutically effective amount” describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising the single domain antibody or antigen binding molecule necessary to reduce, alleviate and / or prevent a henipaviral infection. In some examples, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of the henipaviral infection. In other examples, a “therapeutically effective amount” is an amount sufficient, to achieve a desired biological effect, for example, an amount that is effective to decrease a symptom associated with a henipaviral infection.

[0201] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent, such as a single domain antibody, useful for reducing, alleviating and / or preventing henipaviral infection will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition, and the manner of administration of the therapeutic composition.

[0202] With respect to the aspects described herein, the term “subject” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as pigs, cows, sheep, horses) and companion animals (such as cats and dogs). In certain examples, the subject is a human. In other examples, the subject is a pig. In various examples, the subject is a horse. In some examples, the subject is a primate or more particularly a non-human primate.

[0203] As used herein, the terms “a disease, disorder or condition associated with NiV” and “a disease, disorder or condition associated with HeV” refer to diseases, disorders or conditions caused, directly or indirectly, by infection with a NiV and a HeV respectively. These viruses can be amplified and cause severe disease in large animals, such as pigs and horses. NiV and HeV may then be subsequently transmitted to humans (i.e., a zoonotic disease), where the infection is typically manifested as a severe respiratory illness and / or febrile encephalitis, which can be fatal. Methods for diagnosing henipaviral infections

[0204] The single domain antibodies and antigen binding molecules disclosed herein may be used to detect viral proteins or antigens, such as an F protein of a NiV and / or a HeV, in vitro or in vivo. Such in vitro testing may involve obtaining a biological sample, such as blood, plasma or serum, from the subject. The detection of viral protein or elevated levels of viral protein in the biological sample from a subject may be indicative of a henipaviral infection (e.g., a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith) in said subject.

[0205] Accordingly, in one broad form, the present disclosure provides a method of diagnosing or monitoring a henipaviral infection, such as a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith, in a subject, said method including the step of contacting the subject and / or a biological sample from the subject with a single domain antibody, an antigen binding molecule or a composition described herein.

[0206] In particular examples, the present method is for diagnosing or monitoring a Nipah virus infection and / or a disease, disorder or condition associated therewith in the subject. In alternative examples, the present method is for diagnosing or monitoring a Hendra virus infection and / or a disease, disorder or condition associated therewith in the subject.

[0207] The present methods may include the step of determining a presence or absence of henipaviral infection in said subject, wherein the presence of a viral protein, such as an F protein, of a Henipavirus, indicates a current or previous henipaviral infection. As such, the subject and / or a biological sample therefrom may be contacted with the single domain antibody, the antigen binding molecule or the composition for a time and under conditions sufficient to detect antigen-specific binding thereof. Suitably, the method of this aspect is for determining a relative or absolute level of the viral protein in the biological sample.

[0208] The present method may further include the earlier step of collecting the biological sample from the subject. Such a sample may be obtained by freshly collecting a sample or may be obtained from a previously collected and stored sample. By way of example, a sample may be obtained from a previously collected and stored (e.g., refrigerated or frozen) blood or serum sample. Suitably, a sample is obtained by freshly collecting a sample from the subject. Alternatively, a sample can be obtained from a previously collected and stored sample from the subject. Once collected the sample may be processed in a way, such as purifying, concentrating or solubilising, to make it more suitable for the subsequent contacting and / or detection steps. Such assays may include immunoassays, such as western blot and ELISA, or immunochromatographic assays, such as a rapid antigen test (RAT) or lateral flow test. It should be understood, however, that this disclosure is not limited by reference to the specific methods of detection or immunoassays disclosed herein.

[0209] The methods of the present disclosure can be performed on various biological samples. As used herein, the term “biological sample” is suitably a sample obtained from a subject. For example, the biological sample can be a bodily fluid of the subject. In certain examples, the biological sample is selected from a group consisting of blood, serum, plasma, urine, saliva, faeces, tears, broncho-alveolar lavage fluid (BALF), cerebrospinal fluid (CSF) and seminal fluid. In some examples, the biological sample is blood. In other examples, the biological sample is plasma. In various examples, the biological sample is serum.

[0210] Suitably, the single domain antibody or antigen binding molecules for use in the present method are labelled, such as by way of conjugation to a detectable or functional label or marker. Various methods of labelling proteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionucleotides (such as35S,11C,13N,15O,18F,19F,99TC,131I,3H,14C,15N,90Y,177Lu,99Tc,111In and125I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents (such as gadolinium chelates). In some examples, labels are conjugated or operably linked to the single domain antibody or antigen binding molecules by one or more linkers of various lengths to reduce potential steric hindrance for the binding of the single domain antibody or antigen binding molecules disclosed herein to a viral protein.

[0211] Suitably, the present method further includes the step of detecting and / or measuring a level of antigen binding to the single domain antibody or the antigen binding molecule. Any method or assay known in the field can be employed in the diagnostic and prognostic methods of the invention, e.g. ligand binding assays, immunoassays, competition binding assays, etc., for detecting and / or measuring antigen determining the presence of a Henipavirus or a viral protein or antigen derived therefrom.

[0212] It is envisaged that the single domain antibody or antigen binding molecules disclosed herein can be used in vitro and in vivo to monitor the course of a henipaviral infection, such as during therapy thereof. Thus, for example, by measuring the increase or decrease in a level of a viral protein / antigen or the number of cells infected with a Henipavirus or changes in the subject or in a biological sample therefrom, it would be possible to determine whether a particular therapeutic regimen aimed at treating or ameliorating a henipaviral infection, such as a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith, is effective. Nucleic acid delivery

[0213] The present disclosure also provides certain aspects relating to the administration of one or more nucleic acids encoding the single domain antibodies or antigen binding molecules described herein (e.g., a single domain antibody comprising, consisting essentially of or consisting of an amino acid sequence set forth in any of SEQ ID NOs:1 to -4 and 22 to 25), or fragments, variants or derivatives thereof, for: (i) passively immunizing a subject against a henipaviral infection; and / or (ii) treating, ameliorating or preventing a henipaviral infection in a subject.

[0214] Accordingly, in one form, there is provided herein a method of treating and / or preventing a henipaviral infection, such as a Nipah virus infection and / or a Hendra virus infection, and / or a disease, disorder or condition associatedtherewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of an isolated nucleic acid encoding a single domain antibody or an antigen binding molecule provided herein.

[0215] In some examples, the isolated nucleic acid encoding the single domain antibody or the antigen binding molecule is in the form of a genetic construct suitable for administration to a mammal such as a human. More particularly, the genetic construct may be suitable for DNA delivery of the single domain antibodies or antigen binding molecules to a mammal such as a human. A useful reference describing DNA delivery of peptides is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

[0216] According to certain examples, the isolated nucleic acid encoding the single domain antibody or antigen binding molecule is in the form of RNA, such as mRNA, suitable for administration to a mammal, such as a human. In various examples, the isolated nucleic acid is or comprises an mRNA having an open reading frame encoding a single domain antibody or an antigen binding molecule provided herein. mRNA vaccines are described, for example, in International Patent Application Nos. PCT / US2015 / 027400 and PCT / US2016 / 044918, herein incorporated by reference in their entirety. The mRNA delivery of antibodies, such as single domain antibodies, is further described in PCT / US2018 / 037918, which is also incorporated by reference herein.

[0217] It will be appreciated that mRNA delivery of single domain antibodies (and antigen binding molecules comprising such single domain antibodies) provides a unique therapeutic alternative to peptide-based or DNA-based methods of administering such agents. When the mRNA is delivered to a cell, the mRNA will be processed into a polypeptide or peptide by the intracellular machinery which can then process the polypeptide or peptide into the single domain antibodies or antigen binding molecules capable of binding a viral protein or antigen of a Henipavirus (e.g., a NiV or a HeV) on or in a virally-infected cell or biological fluid of the subject.

[0218] Compositions comprising isolated nucleic acids or polynucleotides that encode the single domain antibodies or antigen binding molecules described herein, or fragments, variants or derivatives thereof that may be used for such methods, are also contemplated by the present disclosure. For such examples, the composition suitably comprises a delivery agent, such as a nanoparticle, as are known in the art. In various examples, the nanoparticle has a mean diameter of 50-200 nm. In some examples, the composition comprising the mRNA polynucleotide (e.g., an mRNA polynucleotide having an open reading frame that encodes a single domain antibody or an antigen binding molecule described herein) is formulated in a lipid nanoparticle.

[0219] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples. Examples

[0220] The aim of the present example was to generate and characterise NiV F-protein-based nanobodies. Materials and Methods Alpaca immunisation

[0221] The alpaca immunisation process was conducted as previously described (1, 2). An alpaca was immunised twice two weeks apart with 100 μg of NiV F stabilised by the foldon domain (NiV F foldon). The antigen was dissolved in 2 mL of sterile water and mixed with 2 mL of adjuvant (Veterinary Vaccine Adjuvant, GERBU FAMA) and injectedsubcutaneously at four different location in a male alpaca. A 120 mL blood sample was taken from the immunised alpaca 15 days after the prime immunisation, and PBMCs were isolated. PBMC RNA was then extracted and cDNA was generated. The variable antigen-binding (VHH) domain was amplified and ligated into pNae2 vector, for transformation into DH10B-T1 R E. coli cells. These cells were plated on LB agar plates containing chloramphenicol and 2% glucose, which after an overnight incubation, was used to create a glycerol stock stored at -80 °C until required. Bacterial cells were grown and induced with IPTG to induce expression of cell surface displayed nanobody VHH domains. Individual cells expressing VHH domains with specificity to NiV F foldon antigen were isolated by density gradient separation, using sepharose beads conjugated with the antigen. Isolated individual colonies were sequenced, and the VHH domains were amplified from the pNae2 vector for subsequent cloning and expression in ExpiCHOs. Protein Expression

[0222] ExpiCHO cells were transfected with pNBF plasmid encoding nanobodies genetically fused to either Fc or FcM. ExpiCHO cells (ThermoFisher Scientific) were seeded at 3-4 x 106viable cells / mL and allowed to grow overnight. On the day of transfection, cells were diluted to a final density of 6 x 106cells / mL in 25mL of pre-warmed ExpiCHO Expression Medium in 125 mL vented flasks. To set up transfections, 1 μg of plasmid DNA per 1 mL of cell culture was diluted in 1 mL OptiPRO media. In the case of mAb transfections, a ratio of 3:1 of LC to HC vectors was used. A volume of 80 μL of ExpiFectamine™ transfection reagent was diluted in 1 mL OptiPRO per transfection before addition to diluted DNA and incubation at room temperature for 1-5 mins. The transfection solution was then slowly added to cells, and cells were allowed to incubate rotating at 37 C with 8% CO2for 24 hours. The next day, cultures were supplemented with 6 mL ExpiCHO Feed and 150 μL of ExpiCHO Enhancer before returning to the incubator for further culturing. One week after transfection, culture supernatant was harvested by centrifugation at 4800 x g for 30 mins at 4 C before filter sterilisation with a 0.22 μm filter (Merck Millipore). Protein Purification

[0223] All protein purification procedures were performed at 4 °C. Fc-tagged nanobodies were purified from ExpiCHO supernatant using Protein A purification method. Here, supernatant was loaded onto a 50 or 150 mL super-loop and passed through an equilibrated HiTrap Protein A HP purification column (GE Healthcare Life Sciences), using an ÄKTA fast protein liquid chromatography or ÄKTA pure system (GE Healthcare Life Sciences). After loading the sample, the column was washed with 10 column volumes (CV) of Protein A wash buffer (25 mM Tris, 25 mM NaCl, pH 7.4). Protein was then eluted from the column by passing through Protein A elution buffer (100 mM sodium citrate, 150 mM NaCl, pH 3.0) and collecting 3 mL fractions. Fractions displaying high absorbance values at 280 nm were neutralised with a 1:1 v / v ratio of 1.5 M Tris-HCl pH 8.8 and filter sterilised using a 0.22 µm filter. Filter-sterilised fractions containing eluted proteins were concentrated to the desired volume and buffer exchanged using Amicon 30 kDa MWCO centrifugal filter units as outlined by the manufacturer (Sigma Aldrich). Briefly, fractions of interest were pooled in the centrifugal units and centrifuged at 4000 × g at 15 mins intervals, until the volume reached ~500 µL. Concentrated protein was then buffer exchanged by adding PBS, centrifuging and repeating this process three times. Protein concentration was determined by using the Nanodrop One system (ThermoFisher Scientific). HRV3C Cleavage of Nanobodies or Antigen

[0224] Purified HRV3C-site containing proteins (mAb, nanobody or antigen) were incubated with the HRV3C protease at a 20:1 w / w ratio in cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) with 1 mM EDTA for 24 hrs at 37 °C. Protein A agarose slurry (Sigma Aldrich) and Pierce glutathione agarose beads (ThermoFisher Scientific) were used to purify away GST-tagged HRV3C and cleaved Fc as per manufacture’s guidelines. Briefly, 1 mL of each slurry was first equilibrated in PBS and added to reactions, bringing the total volume to 10 mL in PBS. Reactions were incubated rotating at 4 °C for 1 hour before passing through a gravity column and collecting flow-through, which contained the cleaved proteins. Beads were washed with ~10 mL of PBS, which was collected and kept separately for downstream analyses. Cleaved Fc domains were eluted from Protein A beads by adding 10 mL of 0.1 M glycine pH 2.7. GST- tagged HRV3C was eluted from glutathione beads by adding 10 mL of 10 mM reduced glutathione in PBS. All collected fractions were concentrated in centrifugal Amicon units and buffer exchanged to PBS. SDS-PAGE

[0225] SDS-PAGEs were run in order to determine protein purity and molecular size. SDS-PAGEs (12.5% resolving, 4% stacking) were made and cast in-house. The 12.5% w / v Bis-acrylamide resolving gel was made by combining 3.5 mL H2O, 2.5 mL 40% Bis-acrylamide, 2 mL 1.5 M Tris pH 8.8, 80 μL 10% ammonium persulphate (APS), 8 μL tetramethylethylenediamine (TEMED), and allowing to solidify in the gel casing. The 4% stacking gel was made by combining 1.5 mL H2O, 310 μL 40% acrylamide, 0.625 mL 1 M Tris pH 6.8, 50 μL 10% APS and 5 μL TEMED, and adding to the gel casing with a well comb. A total of 2-5 μg of protein was mixed with 80 mM dithiothreitol (DTT) and 6X SDS-PAGE loading dye (200 mM Tris-HCl pH 6.8, 1% SDS, 0.025% bromophenol blue and 30% glycerol) to a total volume of 24 μL. Samples were boiled for 3 mins to ensure denaturation of proteins, before loading onto SDS-PAGE submerged in running buffer (25 mM Tris-HCl, 0.01% SDS, 192 mM glycine). Gels were run initially at 100 V for 10 mins, followed by 60-85 mins at 160 V, or until the dye front has come off the gel. Gels were stained for at least 1 hr with Coomassie blue stain (0.23% Coomassie blue R-250, 50% methanol, 10% acetic acid). For band visualisation, gels were destained with a 10% acetic acid / 40% methanol solution. Gels were scanned using a Epson Perfection 4990 scanner. ELISA

[0226] Indirect ELISAs were conducted to test mAb or nanobody affinities. Nunc MaxiSorp 96-well plates were coated with 50 μL / well of antigen diluted to 2 μg / mL in PBS overnight at 4 °C. The next day, coated antigen was discarded from wells and plates were blocked in 150 μL / well of blocking buffer (1X KPL (SeraCare) in PBS with 0.1% Tween-20 (PBS.T)) for 1 hr at room temperature. Concurrently, mAbs were serially diluted in blocking buffer in a separate U- bottom 96-well plate. Primary antibodies were then added to appropriate wells at 50 μL / well and incubated for 1 hr at 37 °C. Plate / s were then washed thrice in water by submersion and tapped dry. Secondary horse radish peroxidase (HRP) linked antibodies were then added (goat anti-human or anti-mouse, SigmaAldrich) at 50 μL / well diluted 1:2500 in blocking buffer. Plate / s were then incubated for an additional hour at 37 °C. Plates were washed and dried as before. ELISAs were revealed by adding 50 μL / well of tetramethylbenzidine (TMB, Life Technologies) for ~5 mins before adding 25 μL / well of 1 M H2SO4to stop the reaction. Binding was quantified by measuring absorbance at 450 nm using a VarioSkan LUX machine (ThermoFisher Scientific). Data was analysed on GraphPad Prism 9 software, and Kdvalues were determined by plotting a one-site specific binding model after subtraction of background.NiV Pseudovirus Production and Neutralisation Assays

[0227] NiV pseudovirus particles (NiV-pps) were generated using established protocols (3, 4). Initially, 10 cm2dishes (Nunc EasYDish) were coated with 4 mL of poly-L-lysine (SigmaAldrich) for 2 hrs at room temperature. Poly-L-lysine was removed and plates were allowed to dry. HEK293T cells were plated in coated 10 cm2dishes at a density of 3- 4x106cells in 10 mL of DMEM + 10% FCS (D10) media. The following day, the media on the cells was replaced with 7 mL D10, and cells were transfected with p8.91 (encoding HIV-1 gag-pol), pCSFLW (lentivirus backbone encoding firefly luciferase reporter) and pcDNA3.1 vectors encoding NiV G or NiV F full length genes. For the transfection, 1 μg p8.91, 1.5 μg pCSFLW and 1 μg each of pcDNA3.1-NiV-G and pcDNA3.1-NiV-F were combined with 200 μL of serum- free OptiMEM (Gibco) with 4.5 μL of Lipofectamine Enhancer (ThermoFisher Scientific). In a separate tube, 18 μL of LTX lipofectamine (ThermoFisher Scientific) was combined with 200 μL of serum-free OptiMEM per transfection and incubated for 5 mins at room temperature. The LTX mix was added to the DNA mix and incubated for 20 mins at room temperature. The transfection mix was added to plated cells dropwise and transfected cells were incubated at 37 °C with 5% CO2 overnight. The following day, the media on the cells was discarded and replaced with 7 mL D10. Pseudovirus particles were harvested 48, 60 and 72 hrs post-transfection. Harvests were pooled and centrifuged at 2000 x rpm for 10 mins at 4 °C to pellet cellular debris. Virus stocks were aliquoted and stored at -80 °C for later use.

[0228] Target BHK-21 cells were seeded in a white 96 well plate at a density of 2 x 104cells / well in DMEM + 5% FCS (D5) and incubated overnight at 37 °C with 5% CO2. mAb or nanobody were serially diluted in a round-bottom 96 well plate 8-fold in duplicate or triplicate in D5 media, with a final volume of 70 μL / well. An equal volume NiV pseudovirus was added to wells at a final dilution that would yield ~2 x 106RLU (stock diluted in media to 5 x 106RLU). Inhibitor and virus mixtures were incubated for 1 hr at 37 °C with 5% CO2. Following this, media from white plate containing seeded BHK-21 cells was discarded and 100 μL of titrated virus / inhibitor was added. Plates were incubated for 3 days at 37 °C with 5% CO2. Luciferase signal output was revealed using Bio Glo reagent (Promega) and luminescence was read on VarioSkan Lux. Each plate luciferase signal was normalised to virus only wells, and serum neutralisation IC50s were calculated as the inverse of the dilution that yielded 50% inhibition of NiV pseudovirus viral entry using a 3 parameter non-linear dose-response regression model in Graphpad Prism 9.0. Cryo-EM of DS90 and NiV complex

[0229] DS90 was added to NiV F trimers at a 5x molar excess and incubated at 4 °C for 1 hr. Sample was then loaded on Superose 6 Increase 10 / 300 GL (Cytiva) column. Fractions containing DS90-bound NiV F were collected and concentrated. SEC-purified complexes were diluted to 0.04 mg / mL and 4 μL was adsorbed onto glow discharged Quantifoil 200 mesh copper grids (Q3.5 / 1). Grids were plunged frozen using EMGP2 system (Leica) and imaged on a Cryo-ARM 300 (JEOL) equipped with a K3 detector (Gatan) in counting mode. Data processing was conducted on cryoSPARC v3.1 and initial model was built using ModelAngelo. Model was then refined using ISOLDE, Phenix and Coot software (https: / / tristanic.github.io / isolde / index.html). Surface Plasmon Resonance (SPR)

[0230] SPR was performed using a Biacore 8K+ instrument (Cytiva). Purified nanobody-Fc or antibody were first captured on a Protein A Series S Sensor Chip (Cytiva). Binding of NiV F or G was tested using a Multi-Cycle High Performance Kinetics protocol. Capture antibodies were all prepared at 2 μg / mL in running buffer (HBS-P+, pH 7.4,Cytiva) and injected (30 sec at 30 μL / min) over Fc2 on all 8 channels. Serial dilutions of NiV proteins were included at several concentrations: 0 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. The proteins were injected over both Fc1 and Fc2 of Channels 1-8 in series for 180 sec at 30 μL / min, followed by a dissociation period of 1400 sec. The Protein A chip was regenerated after each cycle using 10 mM glycine pH 1.5. The data was double reference subtracted; reference cell subtraction (Fc1) from the active cell (Fc2), and zero concentration subtraction for each analyte-antibody pair. Sensorgrams for the association and dissociation phases were fitted to a 1:1 binding model, using Biacore Insight Evaluation software (Cytiva) and graphed using GraphPad Prism 9 software. Results Isolation and characterisation of NiV F protein-specific camelid nanobodies

[0231] NiV F foldon was initially expressed with a monomeric Fc (FcM) tag for purification means. The FcM tag was then cleaved by HRV3C protease, made possible through a cleavage site engineered between foldon and FcM domains (Figure 1A). The antigen was then validated for prefusion-specific 5B3 binding via an indirect ELISA (Figure 1B). The cleaved and purified NiV F foldon was used to immunise a male alpaca twice two weeks apart (Figure 1C). Following the second immunisation, P8MCs were harvested and cDNA amplicons corresponding to nanobody VHH domains were amplified. Amplified VHH domains were then used to construct a nanobody bacterial expression library, which was later used to screen for antigen-binding clones through a density gradient with antigen conjugated Sepharose beads. Individual clones were isolated and subjected to preliminary screening against cell surface expressed NiV F under pIRES-GFP. Clones exhibiting favourable binding were sequenced and subsequent cloning as either dimeric Fc or FcM fusion proteins for further expression and analysis.

[0232] A panel of nanobodies screened to bind cell surface expressed NiV F were sequenced via Sanger sequencing. Sequence analysis revealed that the nanobodies DS90, DS76 and DS34 are highly similar, however with several amino acid substitutions (Figure 2A). DS76 possesses a R19G substitution in comparison to DS90 & DS34, which constitutes a part of the nanobody scaffold, and a T101S substitution as part of CDR3. DS34 possesses a V102A substitution in CDR3 in comparison to DS90 and DS76. DS29 possesses unique CDR1-3 sequences. Notably, CDR3 of DS29 is significantly shorter than that of DS34, DS76 and DS90. This panel of nanobodies was cloned and expressed as dimeric Fc (Nb-Fc) or FcM (Nb-FcM) fusion proteins in the ExpiCHO system. When purified using protein A chromatography, these proteins appeared highly pure, with Nb-Fc dimers and Nb-FcM displaying a molecular weights of -80 kDa and 40 kDa under non-reducing conditions, respectively (Figure 2B).

[0233] Next, Nb-Fc and Nb-FcM affinities to NiV and HeV F antigens were assessed via ELISA (Figure 3). In this assay, prefusion NiV F antigen was used to confirm nanobody specificity to the F ectodomain. Here, DS90, DS76 and DS34 all bound NiV F with Kd affinities ranging from 0.82-3.9 nM as dimers.. DS29 was able to bind NiV F with a Kd of 1558 nM as a dimer. Notably, all nanobodies cross-reacted to HeV F as Fc dimers.

[0234] To assess neutralisation capacity, Nb-Fc dimers were tested in a NiV pseudovirus neutralisation assay, where all tested nanobodies neutralised pseudotyped NiV to various extents (Figure 4). It was observed that DS34 and DS90 were the most potent neutralisers with IC50s in the picomolar range (IC50 = 8 pM). In this assay, these nanobodies outperformed the 5B3 mAb which neutralised pseudotyped NiV with an IC50 of 44 pM. DS76 neutralised pseudotyped NiV with a similar potency to that of 5B3 (IC50 = 45 pM). The neutralisation capacity of DS76 when tested as a monomerwas IC50 = 260 nM, whereas the neutralisation capacity of DS34 and DS90 as monomers was IC50 = 21.2 & 14.2 nM, respectively. DS29 possesses unique CDR1-3 sequences and was able to neutralise pseudotyped NiV with an IC50 of 22 nM as a dimer. Expectedly, neutralisation by all mAbs and nanobodies was reduced when tested as monovalent versions, as seen for 5B3 when tested as a Fab fragment.

[0235] Next, the nanobodies were tested for neutralisation against a pseudotyped NiV mutant, where the 5B3 epitope was ablated by F-P52N / K55E mutations (3, 5) (Figure 4D). The NiV-FP52N / K55E mutant included the previously identified charge swap K-E escape mutation at position 55, which has been shown to confer resistance to neutralisation by the prefusion specific 5B3 mAb against live NiV (3, 5). To further reduce the binding of 5B3-like mAbs, an additional mutation at position 52 (P52N) was included, which introduced a potential N-linked glycan site due to the presence of a downstream native serine residue at position 54. Interestingly, the neutralisation offered by dimeric nanobodies DS29 & DS90 was completely abrogated against the NiV F-P52N / K55E mutant. This suggests that these nanobodies likely bind a similar 5B3-like epitope that is compromised by the P52N / K55E mutations.

[0236] Based on its high neutralisation capacity, DS90 was picked for further structural analyses via cryo-EM. Here, DS90 nanobody was cleaved from the Fc tag by incubation with HRV3C, followed by purification via SEC. Purified DS90 was then complexed at a 5 times molar excess with prefusion NiV F. Complexes were run through SEC and SEC fractions containing DS90-complexed antigen were diluted to 0.04 mg / mL and plunged-frozen on to grids for data collection using Cryo-ARM 300 (JEOL) microscope. Data was analysed on cryoSPARC v3.1 to yield a final structure at 3.6 Å and model was built using ModelAngelo and refined using ISOLDE, Phenix and Coot software packages (6- 8). Cryogenic electron microscopy revealed that NiV F trimer is bound by three DS90 nanobodies within a novel, glycan-free, quaternary epitope that spans DI-III of NiV F (Figure 5A). This is largely facilitated by CDR3, which was found to extend within a novel quaternary binding pocket that forms contacts with DIII and DII of a single F protomer as well as DI of a neighboring protomer. CDR2 binds to DIII of NiV F, and forms contacts with a helical structure and beta strand central to the NiV F trimer. CDR2 also extends further and forms contacts with DI of the same NiV F protomer through residues R49-S53. Additional framework residues within DS90 contribute to the paratope including: G41, K42 & E43 in FR2, which make contacts with a beta-hairpin within DIII of NiV F apex; and Y36 & F46 in FR2, which bind a leucine residue within the beta-core sheet of NiV F (Figure 5B,C). In FR2, E43 makes a salt bridge with K167 of NiV F. CDR1 of DS90 makes minimal contact with NiV F, and only includes V32 of DS90 which coordinates with N51 of NiV F within DI. A bispecific therapeutic approach provides potent neutralisation of NiV and prevents viral escape

[0237] Henipaviruses are RNA viruses that are prone to accumulating mutations under selection pressures. As such, therapeutics that target a single epitope may be rendered ineffective by a single point mutation, as was previously shown for 5B3, mAb66 and m102.4 mAbs (3, 5, 9, 10). To overcome this, bispecific antibodies that target multiple epitopes across two antigenic targets may be more resilient to viral escape mutations. To this end, the present inventors genetically fused the DS90 nanobody (F target) with best-in-class m102.4 mAb (G target) to create a bispecific NiV therapy. DS90 was added to the N-terminus of the m102.4 HC, linked by a G2SG2linker (DS90-m102.4) (Figure 6). Recombinant expression yielded a highly pure antibody preparation (Figure 7A) that was capable of binding both NiV F and G in both ELISAs and SPR (Figure 7B-D) with high affinity. This Example demonstrated that DS90-m102.4 wasable to neutralise authentic NiV, and provided synergistic effects in comparison to DS90 or m102.4 alone (Figure 8A). Furthermore, DS90-m102.4 was capable of neutralising a prospective NiV G mutant (G-N557A) in a pseudovirus neutralisation assay, whereas m102.4 neutralisation was completely ablated (Figure 8B).

[0238] To test the capacity of DS90-m102.4 to prevent viral escape, authentic NiV was sequentially passaged in the presence of sub-neutralising concentrations of antibody. Here, NiV passaged in DS90-m102.4 did not show signs of cytopathic effects (CPE), suggesting minimal to no live NiV replication (Figure 9). In contrast, significant cytopathic effects was observed for NiV passaged in DS90 or m102.4 alone, suggesting viral escape. After sequentially passaging NiV in sub-neutralising concentrations of mAb, the ability of each mAb to neutralise this virus was tested. Here, significant escape from neutralisation offered by m102.4 and DS90 was observed, whereas DS90-m102.4 retained its neutralisation (Table 1). This demonstrated that the bispecific approach, which targets both NiV F and G proteins, limits NiV viral escape and therefore offers significant advantages over antibodies that target a single epitope.

[0239] Table 1: NiV was passaged three times in the presence of antibody at different concentrations and then subjected to a neutralization assay with mAbs to determine if escape mutants were generated. Rabbit #405 is an in- house made G specific control mAb. Data shows mAb dilution titer required for neutralization.Humanisation of DS90

[0240] The DS90 nanobody was isolated from an alpaca, and therefore may be immunogenic and have a short half- life when used as a human therapeutic. Camelid species and humans possess a high similarity (>80%) in antibody germline sequences, allowing for humanisation of camelid nanobodies (11, 12). This could increase the effectiveness and half-life of DS90 as a human therapy. Alignment of DS90 with human germline sequences revealed a high similarity to the IGHV3-23*04 germline (>91%, Figure 10a). Humanised DS90 (hDS90) was expressed as a monomer and tested in both ELISA and NiV pseudovirus neutralisation assays. Here, both binding to NiV F and neutralisation of hDS90 was observed, albeit slightly lower than compared to parental DS90. The present inventors reverted 3 residues in hDS90 back to the original sequence (V37Y, L79V, V93I). It was discovered that the hDS90 V37Y reversion demonstrated anincreased affinity to NiV F over the parental DS90 monomer and equivalent neutralisation against NiV pseudovirus (Figure 10b,c). Here, this Example demonstrated that DS90 was able to be successfully humanised. Summary

[0241] This Example describes the characterisation of four camelid-derived nanobody clones generated against the prefusion form of NiV F protein. Of these, DS90, DS34 and DS76 were 97-99% similar in amino acid sequence (>99% similarity in nucleotide sequence), suggesting a similar epitope. One clone, DS29, was found to have unique sequences with shorter CDR3 sequences in comparison to DS90. DS90 bound NiV F with sub-nanomolar affinity, which translated to a picomolar range IC50 in a NiV pseudovirus neutralisation assay, exceeding the neutralisation offered by the best- in-class 5B3 mAb and m102.4, which is currently given as a therapeutic under compassionate grounds. Without being bound by any theory, it is believed that the nanobodies confer virus neutralisation through stabilisation of the prefusion conformation of the F protein, thereby preventing significant conformational changes to the post-fusion form thereof. Furthermore, genetically fusing DS90 to m102.4 provides synergistic neutralisation against authentic NiV and limits viral escape mutations in vitro. The DS90 molecule can also be humanized to increase the half-life and reduce immunogenicity for human clinical translation. References 1. Modhiran N. et al. A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants. iScience.2023;26(7):107085. 2. Nambulli S. et al. Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses. Science Advances.2021;7(22):eabh0319. 3. Isaacs A. et al. Combinatorial F-G Immunogens as Nipah and Respiratory Syncytial Virus Vaccine Candidates. Viruses.2021;13(10). 4. Thakur N. et al. Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell–cell fusion. The Journal of general virology.2021;102(1). 5. Dang H.V. et al. An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections. Nature Structural & Molecular Biology.2019;26(10):980-7. 6. Croll T. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallographica Section D.2018;74(6):519-30. 7. Liebschner D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallographica Section D.2019;75(10):861-77. 8. Emsley P., Lohkamp B., Scott W.G., Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr.2010;66(Pt 4):486-501. 9. Borisevich V. et al. Escape from monoclonal antibody neutralization affects Henipavirus fitness in vitro and in vivo. The Journal of infectious diseases.2016;213(3):448-55. 10. Xu K. et al. Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody. PLoS pathogens.2013;9(10):e1003684. 11. Harmsen M.M., De Haard H.J. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol.2007;77(1):13-22. 12. Vincke C. et al. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem.2009;284(5):3273-84.13. Fulcher L.J., Macartney T., Bozatzi P., Hornberger A., Rojas-Fernandez A., Sapkota G.P. An affinity-directed protein missile system for targeted proteolysis. Open Biol.2016 Oct;6(10):160255. 14. Fulcher L.J., Hutchinson L.D., Macartney T.J., Turnbull C., Sapkota G.P. Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open Biol.2017 May;7(5):170066. 15. Ibrahim A.F.M., Shen L., Tatham M.H., Dickerson D., Prescott A.R., Abidi N., Xirodimas D.P., Hay R.T. Antibody RING-Mediated Destruction of Endogenous Proteins. Mol Cell.2020 Jul 2;79(1):155-166.e9.

Claims

CLAIMS:

1. A single domain antibody that is directed against an F protein of a Nipah virus, said single domain antibody comprising: (a) a CDR1 that comprises an amino acid sequence of: AASGX1TFSSYVMX2WX3RQ (SEQ ID NO: 29), wherein X1is R or F, X2is G or S and X3is Y or V; AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVX1RISWSGGHTHS (SEQ ID NO: 30), wherein X1 is A or S; GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

2. The single domain antibody of Claim 1, comprising: (a) a CDR1 that comprises an amino acid sequence of: AASGRTFSSYVMGWYRQ (SEQ ID NO: 5); AASGFTFDDYGMSWVRQ (SEQ ID NO: 11); or a variant thereof; (b) a CDR2 that comprises an amino acid sequence of: EREFVARISWSGGHTHS (SEQ ID NO: 6); GLEWVSAISWNGGGTYY (SEQ ID NO: 12); or a variant thereof; and (c) a CDR3 that comprises an amino acid sequence of: NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1is S or T and X2is V or A; AKPSTYSGSWLTDFGS (SEQ ID NO: 13); or a variant thereof.

3. The single domain antibody of Claim 1 or Claim 2, wherein the CDR3 comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8), NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9), NAEPTATGSWFTPGLQSY (SEQ ID NO: 10), AKPSTYSGSWLTDFGS (SEQ ID NO: 13), or a variant thereof.

4. The single domain antibody of any one of the preceding claims, comprising: (i) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPX1X2TGSWFTPGLQSY (SEQ ID NO: 7), wherein X1 is S or T and X2 is V or A, or a variant thereof; or (ii) the CDR1 that comprises the amino acid sequence of AASGFTFDDYGMSWVRQ (SEQ ID NO: 11) or a variant thereof, the CDR2 that comprises the amino acid sequence of GLEWVSAISWNGGGTYY (SEQ ID NO: 12) or a variant thereof and the CDR3 that comprises the amino acid sequence of AKPSTYSGSWLTDFGS (SEQ ID NO: 13) or a variant thereof.

5. The single domain antibody of any one of the preceding claims, comprising: (i) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof; (ii) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or avariant thereof and the CDR3 that comprises the amino acid sequence of NAEPSVTGSWFTPGLQSY (SEQ ID NO: 9) or a variant thereof; (iii) the CDR1 that comprises the amino acid sequence of AASGRTFSSYVMGWYRQ (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVARISWSGGHTHS (SEQ ID NO: 6) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTATGSWFTPGLQSY (SEQ ID NO: 10) or a variant thereof; (iv) the CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWVRQ (SEQ ID NO: 26) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof; or (v) the CDR1 that comprises the amino acid sequence of AASGFTFSSYVMSWYRQ (SEQ ID NO: 28) or a variant thereof, the CDR2 that comprises the amino acid sequence of EREFVSRISWSGGHTHS (SEQ ID NO: 27) or a variant thereof and the CDR3 that comprises the amino acid sequence of NAEPTVTGSWFTPGLQSY (SEQ ID NO: 8) or a variant thereof.

6. The single domain antibody of any one of the preceding claims, wherein the single domain antibody, in a monovalent format, has a KDfor the F protein of the Nipah virus of lower than about 600 nM, lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM or lower than about 10 nM.

7. The single domain antibody of any one of the preceding claims, wherein the single domain antibody further binds an F protein of a Hendra virus.

8. The single domain antibody of any one of the preceding claims, wherein the single domain antibody has been at least partly humanized.

9. The single domain antibody of any one of the preceding claims, comprising, consisting of or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 1 to 4 and 22 to 25, or a fragment, variant or derivative thereof.

10. An antigen binding molecule comprising the single domain antibody of any one of Claims 1 to 9, wherein the antigen binding molecule is or comprises a monovalent single domain antibody, a multivalent single domain antibody, or a multispecific single domain.

11. The antigen binding molecule of Claim 10, wherein the antigen binding molecule is a multispecific single domain antibody comprising a further single domain antibody that is directed to a receptor binding glycoprotein (G protein) of a Nipah virus and / or a Hendra virus.

12. The antigen binding molecule of claim 10 or claim 11, wherein the antigen binding molecule is or comprises an immunoconjugate, wherein the immunoconjugate comprises one or more of a detectable label, a therapeutic agent, a half-life extender and a nanocarrier.

13. A method of diagnosing or monitoring a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the steps of contacting the subject and / ora biological sample from the subject with the single domain antibody of any one of Claims 1 to 9, or the antigen binding molecule of claim 11 or claim 12, and detecting and / or measuring a level of antigen binding to the single domain antibody or the antigen binding molecule.

14. A method of treating and / or preventing a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of the single domain antibody of any one of Claims 1 to 9, or the antigen binding molecule of claim 11 or claim 12.

15. Use of the single domain antibody of any one of Claims 1 to 9, or the antigen binding molecule of claim 11 or claim 12 in the manufacture of a medicament for the treatment and / or prevention of a Nipah virus infection, a Hendra virus infection and / or a disease, disorder or condition associated therewith in a subject.

16. An isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the single domain antibody of any one of Claims 1 to 9, or the antigen binding molecule of claim 11 or claim 12.

17. A genetic construct comprising: (i) the isolated nucleic acid of Claim 16; or (ii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

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