VHH antibodies which bind to canine albumin

Abstract

The present invention relates generally to the field of antibodies, in particular VHH antibodies that bind to canine albumin (canine serum albumin). The invention also relates to constructs comprising such VHH antibodies. The invention also relates to compositions comprising such antibodies or constructs. The invention also relates to therapeutic uses of such VHH antibodies or constructs.

Description

[0001] VHH antibodies which bind to canine albumin

[0002] This invention relates generally to the field of antibodies, in particular VHH antibodies that bind to canine albumin (canine serum albumin). The invention also relates to constructs comprising such VHH antibodies. The invention also relates to compositions comprising such antibodies or constructs. The invention also relates to therapeutic uses of such VHH antibodies or constructs.

[0003] Albumin is an abundant protein in the blood of many animals, including canines, and may be referred to as serum albumin. Serum albumin has a long halflife in the circulation. Serum albumin is maintained at a high concentration in plasma (i.e. in the circulation) due to the neonatal Fc receptor (FcRn) recycling pathway. Recycling is facilitated by pH-dependent binding of albumin to FcRn. In this regard, albumin binds to FcRn in acidified intracellular compartments such as endosomes, and protects (or rescues) the albumin from lysosomal degradation following endocytosis (lysosomal degradation that would occur for other, non-FcRn binding, proteins). In this regard, albumin binds to FcRn in acidified intracellular compartments such as endosomes, and the albumin is then recycled back to the cell surface, where it dissociates from the FcRn at neutral pH and is released in serum, thus extending the lifetime of the albumin in the circulation.

[0004] The present inventors have generated VHH antibodies which bind to canine albumin. VHH antibodies are single domain antibodies that have only heavy chain variable domain antibody sequences. More specifically, VHH antibodies have a VHH domain that contains three heavy chain complementarity determining regions (CDRs). Unlike conventional antibodies, VHH antibodies are devoid of light chain antibody sequences. VHH antibodies may alternatively be referred to as, for example, nanobodies or single domain antibodies (sdAbs). VHH antibodies have some advantages over conventional (e.g. IgG) antibodies. For example in this regard, due to their small size and structural differences as compared to conventional antibodies, VHH antibodies can have improved tissue penetration, and they may exhibit lower immunogenicity in vivo.

[0005] VHH antibodies generated by the present inventors are able to bind to canine albumin at a neutral pH (e.g. pH7.4; plasma I circulatory pH) and also at endosomal pH (e.g. pH5.5 or pH6.0). This is advantageous, as this property can endow the anti-canine albumin VHH antibodies with an extended half-life. This makes the anti-canine albumin VHH antibodies particularly useful as half-life extending moieties, to extend the half-life of an agent (e.g. a therapeutic agent) linked (e.g. fused) to such an anti-canine albumin VHH antibody. In this regard, an anti-canine albumin VHH antibody that can bind to canine albumin at a neutral pH (e.g. pH7.4; plasma I circulatory pH) and also at endosomal pH (e.g. pH5.5 or pH6.0) can also be protected from degradation and recycled back to the cell surface (and thus be endowed with an extended half-life). In this regard, such a VHH antibody can bind to albumin at neutral pH and bind (or remain bound) to albumin in acidified intracellular compartments such as endosomes (the albumin in turn being bound to FcRn), thus protecting the anti-canine VHH antibody from degradation. An agent linked (e.g. fused) to the anti-canine VHH antibody of the invention would also be protected from lysosomal degradation (and thus can be endowed with an extended half-life).

[0006] Thus, it is evident that VHH antibodies which can bind to canine albumin are useful as half-life extending agents (or moieties) to extend the half-life in the circulation of other agents linked (e.g. fused) thereto. Such agents linked thereto may include, for example, therapeutic agents. Such therapeutic agents may be, for example, other VHH antibodies against therapeutic targets, for which having a longer half-life would be therapeutically advantageous. Such therapeutic agents may be biologies such as therapeutic polypeptides or peptides, for example ligands or receptors, for which having a longer half-life would be therapeutically advantageous.

[0007] Although some strategies aimed at extending the half-life of therapeutic agents in the circulation are known, there clearly remains a need for alternative, and preferably advantageous or improved, molecules for use in extending the half-life of agents (e.g. therapeutic agents) in the circulation (e.g. of canines). The present inventors have provided such molecules in the form of VHH antibodies that bind to canine albumin.

[0008] Although VHH antibodies have only a single antigen binding domain (unlike for example IgG antibodies, which have two), the present inventors have generated VHH antibodies which bind to canine albumin with excellent binding characteristics (e.g. affinity) at a neutral pH (e.g. pH7.4; plasma I circulatory pH) and also at endosomal pH (e.g. pH5.5 or pH6.0). The inventors have generated VHH antibodies that have an excellent half-life in vivo (in dogs). Of particular note, the inventors have generated VHH antibodies which bind to canine albumin but which do not bind significantly to human albumin. This is advantageous, for example because if such a VHH antibody (or construct comprising such an antibody) accidentally enters the blood stream of an animal handler (e.g. by needlestick injury), minimal (or no) human albumin binding means that the VHH antibody (or construct comprising such an antibody) would be quickly be eliminated, minimising risk to the animal handler.

[0009] Due to their small size, anti-canine albumin VHH antibodies of the invention are well suited to being included in more complex, multi-component, constructs (e.g. that comprise multiple different VHH antibodies). Indeed, the present inventors have generated such constructs and shown that these have excellent activity.

[0010] Thus, in one aspect, the present invention provides a VHH antibody which binds to canine albumin.

[0011] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises

[0012] (i) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto;

[0013] (ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto;

[0014] (iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto;

[0015] (iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto; or

[0016] (v) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:50 or SEQ ID NO:91 or a sequence substantially homologous thereto.

[0017] Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0018] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises

[0019] (i) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto;

[0020] (ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto;

[0021] (iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto;

[0022] (iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto; or

[0023] (v) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91 or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence. In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:10 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0024] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:57 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:10 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0025] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0026] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence. In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0027] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0028] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:91 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0029] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises

[0030] (i) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto; (ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto;

[0031] (iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto;

[0032] (iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto; or

[0033] (v) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91 or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence that is caninized (or that has been caninized) compared to the given CDR sequence. In some such embodiments, such a caninized CDR sequence may be a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0034] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises

[0035] (i) a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 and a CDR3 that has the amino acid sequence of SEQ ID NO: 10;

[0036] (ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24, a CDR2 that has the amino acid sequence of SEQ ID NO:25 and a CDR3 that has the amino acid sequence of SEQ ID NO:26; (iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32, a CDR2 that has the amino acid sequence of SEQ ID NO:33 and a CDR3 that has the amino acid sequence of SEQ ID NO:34;

[0037] (iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40, a CDR2 that has the amino acid sequence of SEQ ID NO:41 and a CDR3 that has the amino acid sequence of SEQ ID NO:42; or

[0038] (v) a CDR1 that has the amino acid sequence of SEQ ID NO:48, a CDR2 that has the amino acid sequence of SEQ ID NO:49 and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91.

[0039] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:9 and a CDR3 that has the amino acid sequence of SEQ ID NQ:10.

[0040] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:57 and a CDR3 that has the amino acid sequence of SEQ ID NQ:10.

[0041] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:24, a CDR2 that has the amino acid sequence of SEQ ID NO:25 and a CDR3 that has the amino acid sequence of SEQ ID NO:26.

[0042] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:32, a CDR2 that has the amino acid sequence of SEQ ID NO:33 and a CDR3 that has the amino acid sequence of SEQ ID NO:34.

[0043] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NQ:40, a CDR2 that has the amino acid sequence of SEQ ID NO:41 and a CDR3 that has the amino acid sequence of SEQ ID NO:42. In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48, a CDR2 that has the amino acid sequence of SEQ ID NO:49 and a CDR3 that has the amino acid sequence of SEQ ID NQ:50.

[0044] In some embodiments, a VHH antibody of the present invention comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48, a CDR2 that has the amino acid sequence of SEQ ID NO:49 and a CDR3 that has the amino acid sequence of SEQ ID NO:91.

[0045] In some embodiments, the invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody comprises a VHH domain that has an amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:23 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:31 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:39 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:47 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:55 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:63 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:71 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:79 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NQ:80 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:81 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:82 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:83 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:84 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:85 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:86 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:87 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NQ:104 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NQ:105 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO:106 or a sequence substantially homologous thereto, or that has an amino acid sequence of SEQ ID NO: 107 or a sequence substantially homologous thereto. Substantially homologous sequences may be as described elsewhere herein (e.g. a sequence having at least 80% sequence identity thereto, for example at least 85%, 90%, 95% or 98% sequence identity thereto).

[0046] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:7 or a sequence having at least 80% sequence identity thereto.

[0047] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:23 or a sequence having at least 80% sequence identity thereto.

[0048] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:31 or a sequence having at least 80% sequence identity thereto.

[0049] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:39 or a sequence having at least 80% sequence identity thereto.

[0050] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:47 or a sequence having at least 80% sequence identity thereto.

[0051] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:55 or a sequence having at least 80% sequence identity thereto.

[0052] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:63 or a sequence having at least 80% sequence identity thereto.

[0053] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:71 or a sequence having at least 80% sequence identity thereto.

[0054] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:79 or a sequence having at least 80% sequence identity thereto. In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:80 or a sequence having at least 80% sequence identity thereto.

[0055] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:81 or a sequence having at least 80% sequence identity thereto.

[0056] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:82 or a sequence having at least 80% sequence identity thereto.

[0057] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:83 or a sequence having at least 80% sequence identity thereto.

[0058] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:84 or a sequence having at least 80% sequence identity thereto.

[0059] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:85 or a sequence having at least 80% sequence identity thereto.

[0060] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:86 or a sequence having at least 80% sequence identity thereto.

[0061] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:87 or a sequence having at least 80% sequence identity thereto.

[0062] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:104 or a sequence having at least 80% sequence identity thereto.

[0063] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:105 or a sequence having at least 80% sequence identity thereto.

[0064] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:106 or a sequence having at least 80% sequence identity thereto. In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:107 or a sequence having at least 80% sequence identity thereto.

[0065] In some embodiments, the invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody comprises a VHH domain having (i) an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106, and SEQ ID NQ:107, or (ii) an amino acid sequence that is sequence substantially homologous to an amino acid sequence of (i), wherein said substantially homologous sequence is a sequence that is caninized (or that has been caninized) compared to the given VHH domain sequence. In some such embodiments, such a caninized VHH domain sequence may be a sequence having at least 70% sequence identity, or preferably at least 80% sequence identity, thereto.

[0066] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106 and SEQ ID NQ:107.

[0067] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:7.

[0068] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:23.

[0069] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:31.

[0070] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:39.

[0071] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:47. In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:55.

[0072] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:63.

[0073] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:71.

[0074] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:79.

[0075] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:80.

[0076] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:81.

[0077] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:82.

[0078] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:83.

[0079] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:84.

[0080] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:85.

[0081] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:86.

[0082] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NO:87.

[0083] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:104.

[0084] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:105.

[0085] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:106.

[0086] In some embodiments, a VHH antibody of the present invention comprises a VHH domain having an amino acid sequence of SEQ ID NQ:107. The CDRs of VHH antibodies of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and / or effective engineered antibodies. Thus, the CDR sequences of the invention are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and / or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions.

[0087] Appropriate sequences that can be used for framework regions are well known and documented in the art and any of these may be used. Preferred sequences for framework regions are one or more of the framework regions making up the VHH domains of the invention, i.e. one or more of the framework regions of the VHHcA8, VHHcA83, VHHcA159, VHHcA160, VHHcA77, VHHcA208, VHHcA209, VHHcA215 or VHHcA8(CAN) VHH antibodies, as disclosed in Tables A-H, herein, or framework regions substantially homologous thereto, and in particular framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the VHH antibody. Other sequences for framework regions are one or more of the framework regions making up the VHH domains of one or more of the VHH antibody sequences set forth herein in Tables l-N, or framework regions substantially homologous thereto.

[0088] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:11 , 12, 13 and 14, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0089] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:27, 28, 29 and 30, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0090] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:35, 36, 37 and 38, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0091] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:43, 44, 45 and 46, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention. In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:51 , 52, 53 and 54, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0092] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:59, 60, 61 and 62, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0093] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:67, 68, 69 and 70, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0094] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:75, 76, 77 and 78, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0095] In some embodiments, all four of the framework regions (FR) of SEQ ID NOs:111 , 112, 113 and 114, or FR regions substantially homologous thereto, are found in the VHH antibodies of the invention.

[0096] In another aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO: 102 (or preferably SEQ ID NO: 103), and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto. Preferably said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence. Discussion of various features of the VHH antibodies of other aspects of the invention and preferred embodiments may be applied, mutatis mutandis, to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto, or SEQ ID NO:55 or a sequence substantially homologous thereto, or SEQ ID NO:63 or a sequence substantially homologous thereto, or SEQ ID NO:71 or a sequence substantially homologous thereto, SEQ ID NO:79 or a sequence substantially homologous thereto, or SEQ ID NQ:80 or a sequence substantially homologous thereto, or SEQ ID NO:81 or a sequence substantially homologous thereto, or SEQ ID NO:82 or a sequence substantially homologous thereto, or SEQ ID NO:83 or a sequence substantially homologous thereto, or SEQ ID NO: 104 or a sequence substantially homologous thereto, or SEQ ID NO: 105 or a sequence substantially homologous thereto, or SEQ ID NO: 106 or a sequence substantially homologous thereto or SEQ ID NO: 107 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:7, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106, or SEQ ID NQ:107).

[0097] In one aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 or a sequence substantially homologous to SEQ ID NO:9 or to SEQ ID NO:57, and a CDR3 that has the amino acid sequence of SEQ ID NQ:10 or a sequence substantially homologous thereto. Preferably said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence. Preferred embodiments of this aspect of the invention include antibodies comprising one or more of the VHH antibody sequences (e.g. CDR sequences and / or VHH domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the VHH antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto, or SEQ ID NO:55 or a sequence substantially homologous thereto, or SEQ ID NO:63 or a sequence substantially homologous thereto, or SEQ ID NO:71 or a sequence substantially homologous thereto, SEQ ID NO:79 or a sequence substantially homologous thereto, or SEQ ID NQ:80 or a sequence substantially homologous thereto, or SEQ ID NO:81 or a sequence substantially homologous thereto, or SEQ ID NO:82 or a sequence substantially homologous thereto, or SEQ ID NO:83 or a sequence substantially homologous thereto, or SEQ ID NO: 104 or a sequence substantially homologous thereto, or SEQ ID NO: 105 or a sequence substantially homologous thereto, or SEQ ID NO: 106 or a sequence substantially homologous thereto, or SEQ ID NO: 107 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:7, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106 or SEQ ID NQ:107).

[0098] In one aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto. Preferably said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence. Preferred embodiments of this aspect of the invention include VHH antibodies comprising one or more of the VHH antibody sequences (e.g. CDR sequences and / or VHH domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:23 or a sequence substantially homologous thereto, or SEQ ID NO:84 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:23 or SEQ ID NO:84).

[0099] In one aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto. Preferably said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence. Preferred embodiments of this aspect of the invention include VHH antibodies comprising one or more of the VHH antibody sequences (e.g. CDR sequences and / or VHH domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:31 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:31).

[0100] In one aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NQ:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto. Preferably said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence. Preferred embodiments of this aspect of the invention include VHH antibodies comprising one or more of the VHH antibody sequences (e.g. CDR sequences and / or VHH domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:39 or a sequence substantially homologous thereto, or SEQ ID NO:85 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:39 or SEQ ID NO:85).

[0101] In one aspect, the present invention provides a VHH antibody, for example an isolated VHH antibody, that binds to (or specifically binds to) canine albumin and that comprises a VHH domain that comprises three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91 or a sequence substantially homologous to SEQ ID NQ:50 or to SEQ ID NO:91. Preferably said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence. Preferred embodiments of this aspect of the invention include antibodies comprising one or more of the VHH antibody sequences (e.g. CDR sequences and / or VHH domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the antibodies of other aspects of the invention and preferred embodiments apply mutatis mutandis to this aspect of the invention. In some preferred embodiments of this aspect of the invention, the invention provides a VHH antibody comprising a VHH domain that has the amino acid sequence of SEQ ID NO:47 or a sequence substantially homologous thereto, or SEQ ID NO:86 or a sequence substantially homologous thereto, or SEQ ID NO:87 or a sequence substantially homologous thereto (e.g. a sequence having at least 80% sequence identity to SEQ ID NO:47 or SEQ ID NO:86 or SEQ ID NO:87).

[0102] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:7 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0103] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:55 (or VH CDR1, VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0104] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:23 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention. In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:31 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0105] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:39 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0106] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:47 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0107] In another aspect, the present invention provides a VHH antibody that binds to (or specifically binds to) canine albumin, said VHH antibody comprising a VHH domain that comprises three CDRs, wherein said VHH domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 of (or from or from within) the VHH domain that has the sequence of SEQ ID NO:88 (or VH CDR1 , VH CDR2 and VH CDR3 sequences substantially homologous thereto). Discussion of various features of the antibodies of other aspects of the invention and preferred embodiments may be applied mutatis mutandis to this aspect of the invention.

[0108] In some embodiments, the CDR sequences of (or from or from within) a VHH domain are CDR sequences as set forth in Tables A-H, M or N herein. CDR sequences of (or from or from within) a VHH domain (or heavy chain variable domain) may be CDR sequences as identified using the IMGT numbering scheme (e.g. Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imgt.org)), e.g. as shown in Tables A-H and M herein. In some other embodiments, CDR sequences of (or from or from within) a VHH domain are CDR sequences as identified using any suitable method (or tool), for example as identified according to the well-known methods of Kabat (e.g. Kabat, et al., "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669, 1991) or Chothia (e.g. Chothia C, et al. (1989) Nature, 342:877-883, or Al- Lazikani et al., (1997) JMB 273,927-948).

[0109] Alternatively viewed, in some embodiments the invention provides a VHH antibody that binds to canine albumin, wherein said VHH antibody comprises a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0110] Alternatively viewed, in some embodiments the invention provides a VHH antibody that binds to canine albumin, wherein said VHH antibody comprises a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0111] Alternatively viewed, in some embodiments the invention provides a VHH antibody that binds to canine albumin, wherein said VHH antibody comprises a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0112] Alternatively viewed, in some embodiments the invention provides a VHH antibody that binds to canine albumin, wherein said VHH antibody comprises a CDR1 that has the amino acid sequence of SEQ ID NO:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0113] Alternatively viewed, in some embodiments the invention provides a VHH antibody that binds to canine albumin, wherein said VHH antibody comprises a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91 or a sequence substantially homologous thereto. Substantially homologous sequences are described elsewhere herein. Preferably, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0114] In some embodiments, the invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody comprises (or consists of) (i) an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID

[0115] NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81, SEQ ID

[0116] NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID

[0117] NO:87, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106 and SEQ ID NQ:107, or

[0118] (ii) an amino acid sequence that is sequence substantially homologous to an amino acid sequence of (i). Said sequence substantially homologous thereto may, for example, be a sequence having at least 80% sequence identity thereto.

[0119] In some embodiments, a VHH antibody of the present invention does not comprise a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO: 9 or a sequence substantially homologous thereto, and a CDR3 that has the amino acid sequence of SEQ ID NO:10, or a sequence substantially homologous thereto. Said sequence substantially homologous thereto may, for example, be a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

[0120] In some embodiments, a VHH antibody of the present invention does not comprise a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO: 9, and a CDR3 that has the amino acid sequence of SEQ ID NQ:10.

[0121] In some embodiments, a VHH antibody of the present invention does not comprise a VHH domain having an amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto. Said sequence substantially homologous thereto may, for example, be a sequence having at least 80% sequence identity thereto.

[0122] In some embodiments, a VHH antibody of the present invention does not comprise a VHH domain having an amino acid sequence of SEQ ID NO:7.

[0123] The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 65%, 70% or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid or nucleic acid sequence disclosed. Substantially homologous sequences of the invention thus include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level preferred substantially homologous sequences contain up to 5, e.g. only 1 , 2, 3, 4 or 5, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in one or more of the framework regions and / or one or more of the CDRs making up the sequences of the invention.

[0124] Certain examples of substantially homologous sequences are sequences that have at least 65% identity to the amino acid sequences disclosed. In certain embodiments, the VHH antibodies of the invention comprise a VHH domain having an amino acid sequence of at least about 65%, 70% or 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% or 95% and most preferably at least about 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NQ:104, SEQ ID NO:105, SEQ ID NQ:106 or SEQ ID NQ:107.

[0125] Alterations in amino acid sequences can be with conservative or nonconservative amino acids. Preferably, said alterations are conservative amino acid substitutions.

[0126] Other preferred examples of substantially homologous sequences are sequences containing conservative amino acid substitutions of the amino acid sequences disclosed.

[0127] Other preferred examples of substantially homologous sequences are sequences containing 1 , 2 or 3, preferably 1 or 2 (more preferably 1), altered amino acids in one or more of the CDR regions disclosed. Alterations may be additions, substitutions, insertions or deletions. Such alterations might be conserved or nonconserved amino acid substitutions, or a mixture thereof. In some such embodiments, preferred alterations are conservative amino acid substitutions.

[0128] Other preferred examples of substantially homologous sequences are sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to one or more of the CDR sequences disclosed. Thus, in some embodiments, a “substantially homologous” CDR sequence may be a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a given CDR sequence described herein. Altered residues might be conserved or non-conserved amino acid substitutions, or a mixture thereof. In some embodiments, preferred alterations are conservative amino acid substitutions.

[0129] In some embodiments, in VHH antibodies having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) are not in a CDR region. For example, in antibodies having a VHH domain sequence that has a certain degree of sequence identity to a given VHH domain sequence of a particular antibody of the invention (e.g. VHHcA8, VHH208, VHH209, VHH215, VHHcA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN), or other VHH antibody of the invention), the altered (or variant) residue(s) are not in a CDR region. Thus, in some embodiments, in VHH antibodies having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) are in one or more framework regions.

[0130] As is evident from elsewhere herein, in other embodiments, in VHH antibodies having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) may be in a CDR region.

[0131] In some embodiments, in a VHH antibody having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the three CDR amino acid sequences (i.e. all three CDR sequences taken together) are considered together to be the whole (or entire) CDR complement of the VHH antibody, and the amino acid sequence of said whole CDR complement of said antibody is at least 70%, preferably at least 80%, or at least 90%, or at least 95% identical to the corresponding whole (or entire) CDR complement of a given starting (or reference) VHH antibody. The starting (or reference) antibody may have the CDR sequences of a particular VHH antibody of the invention. Thus, the starting (or reference) VHH antibody may have the CDR sequences of the VHHcA8, VHH208, VHH209, VHH215, VHHCA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN) VHH antibodies of the present invention, or other VHH antibodies of the invention.

[0132] In some embodiments, a VHH antibody which binds to canine albumin of the present invention (or other VHH antibody as described herein) has a D (Asp) residue as the first (i.e. N-terminal-most) amino acid in the framework 1 (FR1) region. A VHH antibody defined herein as having an amino acid other than D (e.g. a Q (Gin) or an E (Glu)) residue as the first (i.e. N-terminal-most) amino acid in the FR1 region may, in some alternative embodiments, have a D residue as the first (i.e. N-terminal-most) amino acid residue in the framework 1 (FR1) region instead of said amino acid other than D (i.e. in some embodiments the non-D residue that is the first (i.e. N-terminal-most) amino acid residue in the FR1 region is replaced by a D residue). In some embodiments of constructs of the invention, a VHH antibody which binds to canine albumin of the present invention (or other VHH antibody as described herein) that has a D (Asp) residue as the first (i.e. N-terminal-most) amino acid in the FR1 region may be positioned as the N-terminal-most element or agent (e.g. N-terminal-most VHH antibody) in the construct. In some embodiments of constructs of the invention, the construct has a D (Asp) residue as the first (i.e. N- terminal-most) amino acid residue in the construct. In some embodiments, a VHH antibody which binds to canine albumin of the present invention (or other VHH antibody as described herein) has an A (Ala) residue as the final (i.e. C-terminal-most) amino acid in the framework 4 (FR4) region. A VHH antibody defined herein as having an S (Ser) residue as the final (i.e. C-terminal-most) amino acid in the FR4 region may, in some alternative embodiments, have an additional amino acid residue, preferably an A (Ala) residue, positioned immediately C-terminal to (i.e. as the next residue to) the S residue (that would otherwise be the C-terminal residue, e.g. such that said additional residue, preferably an A residue, is the final (C-terminal-most) amino acid in the FR4 region). In some embodiments of constructs of the invention, a VHH antibody which binds to canine albumin of the present invention (or other VHH antibody as described herein) that has such an additional residue, preferably an additional A (Ala) residue, may be positioned as the C-terminal-most element or agent (e.g. C-terminal-most VHH antibody) in the construct. In some embodiments of constructs of the invention, the construct has such an additional residue, preferably an additional A residue, as the final (i.e. C-terminal-most) amino acid residue in the construct. In some embodiments, a VHH antibody defined herein as having an A (Ala) residue as the final (i.e. C-terminal-most) amino acid residue in the FR4 region may, in some alternative embodiments, have said A residue omitted (or alternatively viewed in some alternative embodiments said A residue may be absent).

[0133] In some embodiments, a “substantially homologous” sequence may be a caninized sequence, i.e. a sequence that has been caninized compared to the given sequence (or a caninized version or caninized variant of the given sequence). Thus, in some embodiments, a “substantially homologous” CDR sequence may be a caninized CDR sequence, i.e. a CDR sequence that has been caninized compared to the given sequence (or a caninized version or caninized variant of the given sequence). In some embodiments, a “substantially homologous” VHH domain sequence may be a caninized VHH domain sequence, i.e. a VHH domain sequence that has been caninized compared to the given sequence (or a caninized version or caninized variant of the given sequence). In caninized VHH domains, the caninized sequences (or caninized residues) may be in one or more of the CDRs and / or in one or more of the framework regions. Preferably, the caninized sequences (or caninized residues) are in one or more (or all) of the framework regions. In some embodiments, the caninized sequences (or caninized residues) are not in the CDRs. In all embodiments, VHH antibodies containing substantially homologous sequences retain the ability to bind to canine albumin. Preferably, antibodies containing substantially homologous sequences retain one or more (preferably all) of the properties of (e.g. described in relation to) the VHHcA8, VHH208, VHH209, VHH215, VHHCA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN) VHH antibodies of the invention.

[0134] A "conservative amino acid substitution", as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

[0135] Methods of carrying out the above described manipulation of amino acids (e.g. to generate “substantially homologous” sequences) are well known to a person skilled in the art.

[0136] Homology (e.g. sequence identity) may be assessed by any convenient method. However, for determining the degree of homology (e.g. identity) between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSLIM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1, so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appt. Math., 2:482, 1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

[0137] Generally, computer programs will be employed for such calculations. Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

[0138] By way of providing a reference point, sequences according to the present invention having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, Montpellier, France).

[0139] In certain embodiments, if a given starting sequence is relatively short (e.g. five amino acids in length), then fewer amino acid substitutions may be present in sequences substantially homologous thereto as compared with the number of amino acid substitutions that might optionally be made in a sequence substantially homologous to a longer starting sequence. For example, in certain embodiments, a sequence substantially homologous to a starting CDR2 sequence in accordance with the present invention, e.g. a starting CDR2 sequence of seven amino acid residues in length, preferably has 1 or 2 (more preferably 1) altered amino acids in comparison with the starting sequence. Accordingly, in some embodiments the number of altered amino acids in substantially homologous sequences (e.g. in substantially homologous CDR sequences) can be tailored to the length of a given starting CDR sequence. For example, different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence such as to achieve a particular % sequence identity in the CDRs, for example a sequence identity of at least 50%, 60%, 75%, 80%, 85%, 90% or 95%.

[0140] Routine methods in the art such as alanine scanning mutagenesis and / or analysis of crystal structure of the antigen-antibody complex can be used in order to determine which amino acid residues of the CDRs do not contribute or do not contribute significantly to antigen binding and therefore are good candidates for alteration or substitution in the embodiments of the invention involving substantially homologous sequences.

[0141] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the invention in substantially the same way.

[0142] Substantially homologous sequences of antibodies of the invention also include, without limitation, for example alterations that do not affect the VHH or CDR domains of the antibodies, e.g. VHH antibodies where tag sequences or other components are added that do not contribute to the binding of antigen.

[0143] Preferably, any substantially homologous antibody should retain the ability to bind (or specifically bind) to the same (or substantially the same) epitope as recognized by the VHH antibody in question (e.g. a reference VHH antibody in question). Thus, preferably, any substantially homologous antibody should retain the ability to compete with one or more of the various VHH antibodies of the invention (e.g. one of the described VHH antibodies VHHcA8, VHH208, VHH209, VHH215, VHHCA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN)) for binding to canine albumin. Binding to the same epitope / antigen can be readily tested by methods well known and described in the art, e.g. using binding assays, e.g. a competition assay. Retention of other functional properties can also readily be tested by methods well known and described in the art or herein.

[0144] Thus, a person skilled in the art will appreciate that binding assays can be used to test whether "substantially homologous" antibodies have the same binding specificities as the VHH antibodies of the invention, for example, binding assays such as competition assays or ELISA assays as described elsewhere herein. Surface Plasmon Resonance (e.g. BIAcore) assays could also be used to establish whether "substantially homologous" antibodies bind to canine albumin. The skilled person will be aware of other suitable methods and variations.

[0145] As outlined below, a competition binding assay can be used to test whether "substantially homologous" VHH antibodies retain the ability to bind (or specifically bind) to substantially the same epitope (or the same epitope) of canine albumin as recognized by a VHH antibody of the invention (e.g. antibody VHHcA8, VHH208, VHH209, VHH215, VHHcA83, VHHcA159, VHHcA160, VHHcA77, or VHHcA8(CAN), or antibodies based on these antibodies), or have the ability to compete with a VHH antibody of the invention (e.g. VHHcA8, VHH208, VHH209, VHH215, VHHCA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN), or antibodies based on these antibodies). The method described below is only one example of a suitable competition assay. The skilled person will be aware of other suitable methods and variations.

[0146] An exemplary competition assay involves assessing the binding of various effective concentrations of a VHH antibody of the invention to canine albumin in the presence of varying concentrations of a test VHH antibody (e.g. a substantially homologous VHH antibody). The amount of inhibition of binding induced by the test VHH antibody can then be assessed. A test VHH antibody that shows increased competition with a VHH antibody of the invention at increasing concentrations (i.e. increasing concentrations of the test VHH antibody result in a corresponding reduction in the amount of VHH antibody of the invention binding to canine albumin) is evidence of binding to substantially the same epitope. Preferably, the test VHH antibody significantly reduces the amount of VHH antibody of the invention that binds to canine albumin. Preferably, the test VHH antibody reduces the amount of VHH antibody of the invention that binds to canine albumin by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. ELISA assays may be used for assessing inhibition of binding in such a competition assay but other suitable techniques would be well known to a person skilled in the art.

[0147] In some embodiments, “substantially homologous” antibodies which retain the ability to bind (or specifically bind) to substantially the same (or the same) epitope of canine albumin as recognized by an antibody of the invention (e.g. VHHcA8, VHH208, VHH209, VHH215, VHHcA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN), or antibodies based on these antibodies) or which have the ability to compete with one or more of the various antibodies of the invention (e.g. VHHcA8, VHH208, VHH209, VHH215, VHHcA83, VHHcA159, VHHcA160, VHHcA77 or VHHcA8(CAN), or antibodies based on these antibodies) are preferred.

[0148] The term "competing antibodies", as used herein, refers to VHH antibodies that bind to about, substantially or essentially the same, or even the same, epitope as a "reference antibody". "Competing antibodies" include VHH antibodies with overlapping epitope specificities. Competing antibodies are thus able to effectively compete with a reference VHH antibody for binding to canine albumin. Preferably, the competing antibody can bind to the same epitope as the reference antibody. Alternatively viewed, a competing antibody preferably has the same epitope specificity as the reference antibody. "Reference antibodies" as used herein include VHH antibodies of the present invention. “Reference antibodies” include antibodies which have a VHH domain as defined herein, for example a VHH domain of SEQ ID NO:7, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 , SEQ ID NO:23, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:47 or SEQ ID NQ:107). Certain preferred reference antibodies are selected from antibodies e.g. VHHcA8, VHH208, VHH209, VHH215, VHHcA83, VHHCA159, VHHCA160, VHHcA77, VHHcA8(CAN)).

[0149] As the identification of competing VHH antibodies is determined in comparison to a reference antibody, it will be understood that actually determining the epitope to which either or both VHH antibodies bind is not in any way required in order to identify a competing antibody. However, epitope mapping can be performed using standard techniques, if desired.

[0150] In one aspect, and in some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody as defined elsewhere herein (e.g. a reference antibody described herein). Other features and properties of other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0151] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:7.

[0152] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:55.

[0153] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:63.

[0154] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:71. In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:23.

[0155] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:31.

[0156] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:39.

[0157] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:47.

[0158] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NO:88.

[0159] In some embodiments, the present invention provides a VHH antibody which binds to canine albumin, wherein said VHH antibody binds to the same (or substantially the same) epitope on canine albumin as a VHH antibody that has a VHH domain of SEQ ID NQ:107.

[0160] A yet further aspect of the invention provides a VHH antibody, e.g. an isolated antibody, which binds to canine albumin and which has the ability to compete with (i.e. bind to the same or substantially the same epitope as) an antibody of the invention for binding to canine albumin. For example, VHH antibodies that can compete with VHH antibodies (e.g. VHH antibodies such as those described in the Example section herein) represent a further aspect of the invention. Other features and properties of other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0161] Binding to the same (or substantially the same) epitope / antigen can be readily tested by methods well known and described in the art, e.g. using binding assays such as a competition assay, e.g. as described elsewhere herein (e.g. in relation to “test” or “substantially homologous” antibodies).

[0162] In the following descriptions of the VHH antibodies, constructs, polypeptides, compositions, pharmaceuticals, combinations, kits, first and second medical uses and all methods in accordance with this invention, the terms "VHH antibody", unless otherwise specifically stated or made clear from the context or scientific terminology, refer to a range of VHH antibodies that bind to canine albumin in accordance with the invention as well as to the specific VHH antibodies described in the Example section herein.

[0163] Antibodies in accordance with the present invention are VHH antibodies. “VHH” is an acronym for Variable Heavy domain of Heavy chain. VHH antibodies are also known as VHH antibodies, nanobodies, single domain antibodies, sdAbs, DABs or dAbs. A VHH antibody may also be referred to simply as a VHH.

[0164] VHH antibodies comprise (or consist of) a single (or only a single, or one, or only one) antigen binding domain, which may be referred to as a VHH domain (or a heavy chain variable region). Thus, VHH antibodies comprise (or consist of) a single (or only a single, or one, or only one) monomeric variable antibody domain, which may be referred to as a VHH domain (or a heavy chain variable region) which can bind to antigen. Put another way, VHH antibodies are characterized by having a single (only a single) monomeric variable antibody domain (a single VHH domain or a single heavy chain variable region or single heavy chain variable domain).

[0165] Thus, VHH antibodies have only three CDRs (typically together with four FR regions in the standard or usual order). Alternatively viewed, VHH antibodies have only three heavy chain CDRs (typically together with four heavy chain FR (framework) regions in the standard or usual order).

[0166] VHH antibodies do not contain light chain antibody sequences. Thus, VHH antibodies do not contain a light chain variable region (or VL domain) and do not contain light chain CDRs. Put another way, in VHH antibodies no VL regions or domains are present. Furthermore, in VHH antibodies, no antibody constant regions or hinge regions are present. Thus, in VHH antibodies, the VHH domain is the only part of the antibody that is antibody-derived.

[0167] Thus, in some embodiments a VHH antibody in accordance with the present invention may consist of a VHH domain.

[0168] However, in some embodiments, a VHH antibody of the invention may contain one or more additional (non-antibody derived) sequences or (non-antibody derived) moieties. For example, in some embodiments a VHH antibody may additionally comprise (i.e. in addition to the VHH domain), one or more affinity tags (e.g. a His-tag and / or a myc-tag) that may facilitate detection and / or purification.

[0169] The monomeric variable antibody domain (VHH domain) of VHH antibodies in accordance with the present invention typically corresponds to (or corresponds essentially to or is derived from or is based on or is substantially homologous to) a heavy chain variable domain (or variable heavy domain) of a heavy chain (or heavy chain only) antibody (e.g. a camelid heavy chain antibody such as llama antibody). However, as is evident from discussion elsewhere herein, VHH antibodies in accordance with the present invention include antibodies having sequences (e.g. CDR sequences) that may have a VHH domain that does not correspond exactly to a VHH domain of a given heavy chain antibody, e.g. the VHH domain may have a sequence substantially homologous to a VHH domain of a heavy chain antibody (e.g. it may have substantially homologous CDR sequences and / or be a caninized version of a VHH domain).

[0170] Although VHH antibodies are characterized by having a single (only a single) monomeric variable antibody domain (a single VHH domain), as described elsewhere herein, in some aspects of the present invention (e.g. constructs of the invention) more than one VHH antibody of the present invention with the same or different sequences can be present together in the same construct or molecule.

[0171] VHH antibodies can be obtained or prepared using standard techniques which are well known and described in the art. For example, such VHH antibodies can be obtained by immunizing appropriate animals, e.g. camelids such as llamas, with the desired antigen and then cloning the VHH domains of the antibodies generated into appropriate expression vectors and selecting for binders. Libraries of VHH domains are also available or can be generated and can then be screened.

[0172] In some embodiments, monoclonal VHH antibodies are preferred (e.g. llama monoclonal VHH antibodies or caninized versions thereof).

[0173] As will be understood by those in the art, the term “VHH antibody” includes or extends to recombinant and engineered forms of these antibodies.

[0174] The techniques for preparing and using the VHH antibodies and constructs are well known in the art.

[0175] In some embodiments, the VHH antibodies of the present invention are camelid VHH antibodies (i.e. derived from or based on the VHH domain of a camelid heavy chain (heavy chain only) antibody).

[0176] In some embodiments, the VHH antibodies of the present invention are llama VHH antibodies (i.e. derived from or based on the VHH domain of a llama heavy chain (heavy chain only) antibody). The term "camelid" (e.g. llama) as used herein in connection with VHH antibodies first refers to VHH antibodies having a VHH domain or CDRs isolated or derived from a camelid (e.g. llama) repertoire or derived from or corresponding to sequences found in a camelid (e.g. llama) or a camelid (e.g. llama) repertoire, e.g., in the camelid (e.g. llama) germline or somatic cells.

[0177] "Camelid" VHH antibodies further include amino acid residues not encoded by camelid sequences, e.g., mutations introduced by random or site directed mutations in vitro, for example mutations introduced by in vitro cloning or PCR. Particular examples of such mutations are mutations that involve conservative substitutions or other mutations in a small number of residues of the VHH antibody, e.g., in up to 5, 4, 3, 2 or 1 of the residues of the VHH antibody, preferably e.g., in up to 5, 4, 3, 2 or 1 of the residues making up one or more of the CDRs of the antibody. Certain examples of such "camelid" antibodies include VHH antibodies that have been subjected to standard modification techniques to reduce the amount of potentially immunogenic sites.

[0178] Thus, "camelid" VHH antibodies include sequences derived from and related to sequences found in camelids, but which may not naturally exist within the camelid antibody germline repertoire in vivo.

[0179] In addition, camelid VHH antibodies are not limited to combinations of CDR or FR regions that are themselves found in combination in camelid antibody molecules. Thus, camelid antibodies can include or correspond to combinations of such regions that do not necessarily exist naturally in camelids (e.g. are not naturally occurring antibodies).

[0180] In some embodiments, VHH antibodies of the invention are caninized VHH antibodies. “Caninized" VHH antibodies, which are based on a (or a substantially) non-canine variable region domain, are VHH antibodies in which certain amino acids have been changed to better correspond with the amino acids typically present in canine antibodies. Methods for generating caninized antibodies are well known in the art. In some cases, one or more CDR residues may be changed to better correspond with the amino acids typically present in canine antibodies. In some cases, one or more framework residues may be changed to better correspond with the amino acids typically present in canine antibodies. The amino acid sequences of certain exemplary caninized VHH antibodies of the present invention are set forth in Tables F, G, H and N herein. In the caninized VHH antibodies set forth in Tables F, G, H and N herein, certain framework residues have been changed to better correspond with the amino acids typically present in canine antibodies. In some embodiments, a caninized anti-canine albumin VHH antibody of the invention may be characterized in that it comprises:

[0181] (i) a framework 1 (FR1) region in which amino acid residue 13 thereof is (or has been changed to) a K (Lys), and / or residue 23 thereof is (or has been changed to) a V (Vai); and / or

[0182] (ii) a framework 2 (FR2) region in which amino acid residue 11 thereof is (or has been changed to) a G (Gly); and / or

[0183] (iii) a framework 3 (FR3) region in which amino acid residue 5 thereof is (or has been changed to) an A (Ala), and / or residue 21 thereof is (or has been changed to) a L (Leu), and / or residue 29 thereof is (or has been changed to) a R (Arg).

[0184] In one aspect, the present invention provides a caninized version of a VHH antibody of the present invention.

[0185] The term "complementarity determining region" ("CDR") as used herein refers to a region of hypervariability within an antibody chain variable region (or variable domain) of an antibody molecule.

[0186] The term "heavy chain complementarity determining region" ("heavy chain CDR" or “VH CDR”) as used herein refers to regions of hypervariability within the heavy chain variable region of an antibody molecule, or within a VHH antibody molecule or VHH domain. The heavy chain variable region has three CDRs termed heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. The heavy chain variable region also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

[0187] The term "heavy chain variable region" as used herein refers to the variable region of a heavy chain of an antibody molecule.

[0188] VHH antibodies comprise a VHH domain. A VHH domain may be a considered a heavy chain variable region. As VHH antibodies comprise a VHH domain, a CDR of a VHH antibody (i.e. of a VHH domain) may be considered or referred to as a "heavy chain complementarity determining region" ("heavy chain CDR"). In VHH antibodies, this refers to a region of hypervariability within the VHH domain. The VHH domain has three CDRs termed heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. Such CDRs may also be referred to herein simply as CDR1 , CDR2 and CDR3. The VHH domain also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs. The term "light chain complementarity determining region" ("light chain CDR") as used herein refers to regions of hypervariability within the light chain variable region (VL domain) of an antibody molecule. Light chain variable regions have three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

[0189] The term "light chain variable region" (VL domain) as used herein refers to the variable region of a light chain of an antibody molecule.

[0190] The CDRs of the VHH antibodies of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and / or effective engineered antibodies. Thus, individual CDR sequences are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and / or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, e.g. T cell receptor frameworks can be used. Appropriate sequences that can be used for framework regions are well known and documented in the art and any of these may be used. In particular, framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the antibody.

[0191] CDR sequences of certain VHH antibodies of the invention are set forth herein in Tables A to H, M and N. These CDR sequences are identified using the IMGT numbering scheme (e.g. Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imgt.org)). In some other embodiments, CDR sequences of (or from or from within) a VHH domain may be CDR sequences as identified using any suitable method (or tool), for example as identified according to the well-known methods of Kabat (e.g. Kabat, et al., "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669, 1991) or Chothia (e.g. Chothia C, et al. (1989) Nature, 342:877-883, or Al- Lazikani et al., (1997) JMB 273,927-948).

[0192] The VHH antibodies of the invention can be produced naturally or can be wholly or partially synthetically produced. Thus, the VHH antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants, or using a microbial (e.g. bacterial) expression system or a mammalian cell expression system. Thus, the VHH antibody molecules (or constructs or polypeptides) can be produced in vitro or in vivo.

[0193] In some aspects of the present invention, one or more VHH antibody of the present invention may be present in a construct. Such a construct may comprise one or more additional components (or one or more additional elements), i.e. in addition to one or more VHH antibody of the present invention. Constructs of the present invention bind to (or are capable of binding to) canine albumin.

[0194] Thus, in one aspect, the present invention provides a construct comprising at least one VHH antibody of the present invention.

[0195] In some embodiments, the present invention provides a construct comprising at least two VHH antibodies of the present invention (e.g. at least 2, at least 3, at least 4 or at least 5).

[0196] In some embodiments, the present invention provides a construct in which there is one (only one), or two (only two), or three (only 3), or four (only 4), or 5 (only 5) VHH antibodies of the present invention.

[0197] In one aspect, and in some embodiments, the present invention provides a construct comprising:

[0198] (a) at least one VHH antibody which binds to canine albumin; and

[0199] (b) at least one agent which binds to a protein other than albumin.

[0200] Preferably, said at least one VHH antibody which binds to canine albumin of (a) does not bind significantly (or does not bind) to human albumin.

[0201] In one aspect, and in some embodiments, the present invention provides a construct comprising:

[0202] (a) at least one VHH antibody which binds to canine albumin; and

[0203] (b) at least one agent which binds to a mammalian protein other than albumin.

[0204] Preferably, said at least one VHH antibody which binds to canine albumin of (a) does not bind significantly (or does not bind) to human albumin.

[0205] In some embodiments, the present invention provides a construct comprising: (a) at least one VHH antibody which binds to canine albumin; and

[0206] (b) at least one agent which binds to a mammalian protein other than canine albumin.

[0207] Preferably, said at least one VHH antibody which binds to canine albumin of (a) does not bind significantly (or does not bind) to human albumin.

[0208] Preferred VHH antibodies which bind to canine albumin for inclusion in constructs in accordance with the invention (e.g. as referred to in (a) above) are VHH antibodies of the present invention as described elsewhere herein.

[0209] In preferred embodiments, the mammalian protein other than albumin (referred to in (b) above) is a canine protein. Thus, preferably the mammalian protein other than albumin (referred to in (b) above) is canine protein other than canine albumin (i.e. a canine protein that is not canine albumin).

[0210] As described elsewhere herein, constructs of the invention typically comprise at least one (e.g. 1 , 2, 3, 4 or 5) agent which binds to a protein other than albumin (referred to in (b) above).

[0211] In some embodiments of constructs of the invention, there is a single copy of (or a single unit of) a single agent (referred to in (b) above) which binds to a protein other than albumin.

[0212] In some embodiments of constructs of the invention, there is more than one copy of (or more than one unit of) a single agent (referred to in (b) above) which binds to a protein other than albumin.

[0213] In some embodiments of constructs of the present invention, there may be one (or only one, or a single) copy or (unit) of a given agent which binds to a protein other than albumin (referred to in (b) above). In some embodiments, there may be more than one (e.g. 2, 3, 4 or 5, or at least 2, at least 3, at least 4 or at least 5) copy (or unit) of a given (or of the same) agent which binds to protein other than albumin (referred to in (b) above).

[0214] In some other embodiments of constructs of the present invention, there may be at least two (e.g. 2, 3, 4, or 5, or at least 2, at least 3, at least 4 or at least 5) different agents (referred to in (b) above). In some embodiments, at least two (e.g. 2, 3, 4, or 5, or at least 2, at least 3, at least 4 or at least 5) different agents (referred to in (b) above) bind to the same protein other than albumin (i.e. bind to the same protein that is not albumin). In some embodiments, at least two (e.g. 2, 3, 4, or 5, or at least 2, at least 3, at least 4 or at least 5) different agents (referred to in (b) above) bind to different proteins other than albumin (i.e. in some embodiments different agents bind to different proteins that are not albumin). In embodiments of constructs of the invention in which there are at least two different agents (i.e. at least two different agents of (b)), there may, in some embodiments be one (or only one, or a single) copy or (unit) of each given agent (or of each different agent) in the construct. In other embodiments of constructs of the invention in which there are at least two different agents (i.e. at least two different agents of (b)), there may, in some embodiments be more than one (e.g. 2, 3, 4 or 5, or at least 2, at least 3, at least 4 or at least 5) copy or (unit) of each given agent (or of each different agent). In other embodiments of constructs of the invention in which there are at least two different agents (i.e. at least two different agents of (b)), there may, in some embodiments be one (or only one, or a single) copy or (unit) of at least one of the given agents (or of at least one of the different agents), and more than one (e.g. 2, 3, 4 or 5, or at least 2, at least 3, at least 4 or at least 5) copy or (unit) of at least one of the other given agents (or of at least one of the other different agents).

[0215] In constructs of the invention, the at least one agent (referred to in (b) above) is typically composed of amino acids (i.e. is itself a protein or polypeptide) and, typically and preferably, is in the same (single) polypeptide chain construct as the at least one VHH antibody which binds to canine albumin. This is discussed further elsewhere herein.

[0216] Preferably, at least one agent which binds to a protein other than albumin (referred to in (b) above) is an agent for (or suitable for) the treatment or prevention of a disease in a subject. Thus, at least one agent which binds to a protein other than albumin (referred to in (b) above) is preferably a therapeutic agent (or therapeutically active agent) or a therapeutic moiety (e.g. a therapeutic agent or moiety that is suitable for the treatment or prevention of a disease or condition in canines).

[0217] In preferred embodiments of the invention, at least one agent which binds to a protein (preferably a mammalian protein, more preferably canine protein) other than canine albumin (referred to in (b) above) is a VHH antibody. Of course, such a VHH antibody is not an antibody of the present invention which binds to canine albumin, but rather is a VHH antibody which binds to a different protein (different antigen).

[0218] Thus, in some embodiments, the present invention provides a construct comprising: (a) at least one VHH antibody which binds to canine albumin (preferably which does not bind significantly to human albumin); and

[0219] (b) at least one VHH antibody which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (preferably other than canine albumin).

[0220] In some embodiments, the present invention provides a construct comprising:

[0221] (a) at least one VHH antibody which binds to a first canine protein; and

[0222] (b) at least one VHH antibody which binds to a second canine protein, wherein said first canine protein is canine albumin and wherein said second canine protein is different from said first canine protein.

[0223] In preferred embodiments of the invention, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is a VHH antibody, wherein said VHH antibody binds to a protein (preferably a mammalian protein, more preferably a canine protein) that is implicated in a disease or condition (e.g. a protein that is associated with a disease, or a protein that is a mediator of a disease, or a protein that causes a disease or condition). In preferred embodiments, said disease or condition may be a disease or condition of a canine.

[0224] For example, in some embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is an agent which binds to IL-31 (interleukin- 31), and preferably inhibits IL-31 (IL-31 signalling, e.g. IL-31 mediated STAT3 signalling). In some embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is a VHH antibody which binds to IL-31 (interleukin-31), and preferably inhibits IL-31 (IL-31 signalling, e.g. IL-31 mediated STAT3 signalling). In preferred embodiments, the IL-31 is canine IL-31. IL-31 has been identified as an important mediator in pruritic conditions, e.g. pruritus (itch). Pruritus the most prominent symptom of atopic dermatitis (AD). Binding to, and inhibiting, IL-31 (or IL-31 signalling) is an established strategy for treating pruritus and atopic dermatitis, e.g. in canines. Thus, constructs of the invention that comprise a VHH antibody which binds to IL-31 may be used for the treatment of pruritus and atopic dermatitis. In some embodiments, constructs in accordance with the invention do not comprise an agent which binds to IL-31. In some embodiments, constructs in accordance with the invention do not comprise a VHH antibody which binds to IL- 31.

[0225] In some embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is an agent which binds to TN Fa (tumor necrosis factor alpha), and preferably inhibits TNFa (TNFa signalling). In some embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is a VHH antibody which binds to TNFa, and preferably inhibits TNFa (TNFa signalling). In preferred embodiments, the TNFa is canine TNFa. TNFa has been identified as an important mediator in certain conditions (e.g. inflammatory conditions and autoimmune diseases). Binding to, and inhibiting, TNFa (or TNFa signalling) is an established strategy for treating certain conditions (e.g. inflammatory conditions and autoimmune diseases). Thus, constructs of the invention that comprise a VHH antibody which binds to TNFa may be used for the treatment of inflammatory condition or an autoimmune disease. Constructs of the invention that comprise a VHH antibody which binds to TNFa may be used for the treatment of, for example, inflammatory bowel disease, rheumatoid arthritis or dry eye (preferably in canines).

[0226] In some embodiments of constructs of the invention which comprise a VHH antibody which binds to canine TNFa, the VHH antibody which binds to canine TNFa may comprise a VHH domain comprising three CDRs, wherein said VHH domain comprises a CDR1 that has the amino acid sequence of SEQ ID NO:117 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:118 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:119 or a sequence substantially homologous thereto. In some embodiments of constructs of the invention which comprise a VHH antibody which binds to TNFa, the VHH antibody which binds to TNFa may comprise a VHH domain having (i) an amino acid sequence of SEQ ID NO:116 or a sequence substantially homologous thereto, or (ii) an amino acid sequence of SEQ ID NO:124 or a sequence substantially homologous thereto.

[0227] In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising, in sequence (or order), in the N-terminal to C-terminal direction, a first VHH antibody which binds to TNFa (preferably canine TNFa) e.g. as described above, a VHH antibody which binds to canine albumin in accordance with the present invention (e.g. a VHH antibody having the 3 CDR sequences of, or the VHH domain sequence of, the cA8 or cA8(CAN) VHH antibodies of the invention or sequences substantially homologous thereto), and a second VHH antibody which binds to TNFa (preferably canine TNFa) e.g. as described above. Said first and second VHH antibodies which binds to TNFa may be the same or different (i.e. have the same or different amino acid sequences).

[0228] In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising, in sequence (or order), in the N-terminal to C-terminal direction, a first VHH antibody which binds to TNFa, a VHH antibody which binds to canine albumin in accordance with the present invention (e.g. a VHH antibody having the 3 CDR sequences of, or the VHH domain sequence of, the cA8 or cA8(CAN) VHH antibodies of the invention or sequences substantially homologous thereto), and a second VHH antibody which binds to TNFa, wherein said first and second VHH antibodies each comprise a VHH domain comprising a CDR1 that has the amino acid sequence of SEQ ID NO:117 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:118 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:119 or a sequence substantially homologous thereto. In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising, in sequence (or order), in the N-terminal to C-terminal direction, a first VHH antibody which binds to TNFa, a VHH antibody which binds to canine albumin in accordance with the present invention (e.g. a VHH antibody having the 3 CDR sequences of, or the VHH domain sequence of, the cA8 or cA8(CAN) VHH antibodies of the invention or sequences substantially homologous thereto), and a second VHH antibody which binds to TNFa, wherein said first and second VHH antibodies each comprise a VHH domain having the amino acid sequence of SEQ ID NO: 116 or a sequence substantially homologous thereto. In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising, in sequence (or order), in the N-terminal to C-terminal direction, a first VHH antibody which binds to TNFa, a VHH antibody which binds to canine albumin in accordance with the present invention (e.g. a VHH antibody having the 3 CDR sequences of, or the VHH domain sequence of, the cA8 or cA8(CAN) VHH antibodies of the invention or sequences substantially homologous thereto), and a second VHH antibody which binds to TNFa, wherein said first and second VHH antibodies each comprise a VHH domain having the amino acid sequence of SEQ ID NO: 124 or a sequence substantially homologous thereto. In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising, in sequence (or order), in the N-terminal to C-terminal direction, a first VHH antibody which binds to canine TNFa, a VHH antibody which binds to canine albumin in accordance with the present invention (e.g. a VHH antibody having the 3 CDR sequences of, or the VHH domain sequence of, the cA8 or cA8(CAN) VHH antibodies of the invention or sequences substantially homologous thereto), and a second VHH antibody which binds to canine TNFa, wherein said first antibody comprises a VHH domain having the amino acid sequence of SEQ ID NO:124 in which Xi is a D (Asp) residue and Xne is no amino acid (or a sequence substantially homologous thereto), and wherein said second VHH antibody comprises a VHH domain having the amino acid sequence of SEQ ID NO: 124 in which Xi is an E (Glu) residue and Xne is an A (Ala) residue (or a sequence substantially homologous thereto). In some embodiments, the present invention provides a construct (e.g. a polypeptide construct or a single chain polypeptide construct or a fusion protein) comprising (or consisting of) (i) the amino acid sequence of SEQ ID NO: 115, or a sequence substantially homologous thereto, or (ii) the amino acid sequence of SEQ ID NO: 132, or a sequence substantially homologous thereto. Substantially homologous sequences may be, mutatis mutandis, as described elsewhere herein.

[0229] In some embodiments, at least one agent which binds to a protein other than albumin (referred to in (b) above) is an agent which binds to GnRH (gonadotropinreleasing hormone), and preferably inhibits GnRH (GnRH signalling). In some embodiments, at least one agent which binds to a protein other than albumin (referred to in (b) above) is a VHH antibody which binds to GnRH, and preferably inhibits GnRH (GnRH signalling). In preferred embodiments, the GnRH is canine GnRH. GnRH has been identified as an important factor for reproduction / fertility. Binding to, and inhibiting, GnRH (or GnRH) signalling) can reduce reproductive ability, or prevent reproduction, or reduce testicular function, or reduce fertility. Thus, constructs of the invention that comprise a VHH antibody which binds to GnRH may be used for reducing reproductive ability, preventing reproduction, reducing testicular function or reducing fertility, preferably in canines, (e.g. in males).

[0230] In some embodiments of the invention, at least one agent which binds to a protein (preferably a mammalian protein, more preferably canine protein) other than canine albumin (referred to in (b) above) is not a VHH antibody.

[0231] In other embodiments of the invention, at least one agent which binds to a protein (preferably a mammalian protein, more preferably canine protein) other than canine albumin (referred to in (b) above) is a protein or polypeptide or peptide that binds to a protein (preferably a mammalian protein, more preferably a canine protein) that is implicated in a disease or condition (e.g. a protein that is associated with a disease, or a protein that is a mediator of a disease, or a protein that causes a disease or condition). In preferred embodiments, said disease or condition may be a disease or condition of a canine. In some embodiments, said protein or polypeptide or peptide is not an antibody or antigen-binding fragment thereof. In some embodiments, said polypeptide is a ligand (e.g. a canine ligand). In some embodiments, said polypeptide is a receptor (e.g. a canine receptor).

[0232] For example, in some embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is (i) p75NTR (p75 neurotrophin receptor, e.g. SEQ ID NO: 133), or (ii) a fragment (or functional fragment) of p75NTR, or (iii) an analogue (or functional analogue) of (i) or (ii). In some embodiments, a fragment of p75NTR may be at least 100, at least 150, at least 200, at least 250, at least 300, at least 350 or at least 400 amino acid residues in length. In some embodiments, a fragment of p75NTR may be 100-400, 100-300, 100-200, 150-400, 150-300 or 150- 200 amino acid residues in length. In some embodiments, a fragment of p75NTR may be up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, or up to 428 amino acid residues in length. An analogue may comprise (or consist of) an amino acid sequence that is substantially homologous to the amino acid sequence of p75NTR or fragment thereof. Substantially homologous sequences may be as described elsewhere herein, e.g. substantially homologous sequences may contain 1 , 2, or 3 amino acid substitutions, deletions or additions as compared to the amino acid sequence of p75NTR or fragment thereof, or e.g. may have at least 80% sequence identity to the amino acid sequence of p75NTR or fragment thereof. Fragments and analogues of p75NTR should retain ability to bind to one or more neurotrophins (e.g. canine neurotrophins). Preferably, the p75NTR is canine p75NTR (SEQ ID NO:133). p75NTR has been identified as being useful in the treatment of certain diseases, such as pain (e.g. chronic pain) and osteoarthritis. Thus, constructs of the invention that comprise p75NTR (or fragments or analogues thereof) may be used for the treatment of, for example chronic pain or osteoarthritis (e.g. in canines).

[0233] In some other embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is (i) insulin, or (ii) a fragment (or functional fragment) of insulin, or (iii) an analogue (or functional analogue) of (i) or (ii). In some embodiments, the insulin is a long-acting insulin (or a fragment of a long acting insulin, or a long acting insulin analogue). Insulin comprises an A-chain (e.g. of SEQ ID NO:134) and a B-chain (e.g. of SEQ ID NO:135). In some embodiments, an insulin A-chain (or a fragment or analogue thereof) is connected to an insulin B- chain (or a fragment or analogue thereof) by one or more disulphide bonds.

[0234] In some embodiments, at least one agent which binds to a protein other than albumin (referred to in (b) above) is (i) the insulin A-chain (e.g. SEQ ID NO: 134), or (ii) a fragment (or functional fragment) of the insulin A-chain, or (iii) an analogue (or functional analogue) of (i) or (ii). In embodiments of constructs of the invention that comprise (i) an insulin A-chain, or (ii) a fragment (or functional fragment) of the insulin A-chain, or (iii) an analogue (or functional analogue) of (i) or (ii), an insulin B- chain (or fragment or functional fragment or analogue or functional analogue thereof) is also present in the construct. In some embodiments, the insulin B-chain (or fragment or functional fragment or analogue or functional analogue thereof) may be connected to the insulin A-chain (or a fragment or analogue thereof) by one or more disulphide bonds.

[0235] In some embodiments, at least one agent which binds to a protein other than albumin (referred to in (b) above) is (i) the insulin B-chain (e.g. SEQ ID NO: 135), or (ii) a fragment (or functional fragment) of the insulin B-chain, or (iii) an analogue (or functional analogue) of (i) or (ii). In embodiments of constructs of the invention that comprise (i) an insulin B-chain, or (ii) a fragment (or functional fragment) of the insulin B-chain, or (iii) an analogue (or functional analogue) of (i) or (ii), an insulin A- chain (or fragment or functional fragment or analogue or functional analogue thereof) is also present in the construct. In some embodiments, the insulin A-chain (or fragment or functional fragment or analogue or functional analogue thereof) may be connected to the insulin B-chain (or a fragment or analogue thereof) by one or more disulphide bonds. In some embodiments, a fragment of the insulin A-chain may be at least 10, at least 11 , at least 12, at least 13, at least 14 or at least 15 amino acids in length. In some embodiments, a fragment of the insulin A-chain may be 10-15 or 10-20 or 15 to 20 amino acid residues in length. In some embodiments, a fragment of the insulin A-chain may be up to 15 or up to 20 amino acid residues in length. In some embodiments, a fragment of the insulin B-chain may be at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20 or at least 25 amino acids in length. In some embodiments, a fragment of the insulin B-chain may be 10-15, IQ- 20, 10-25, 10-29, 15 to 20, 15 to 25 or 15 to 29 amino acid residues in length. In some embodiments, a fragment of the insulin B-chain may be up to 15, up to 20 amino acid, up to 25 or up to 29 amino acid residues in length.

[0236] In some embodiments, an analogue may comprise (or consist of) an amino acid sequence that is substantially homologous to the amino acid sequence of insulin or fragment thereof. Substantially homologous sequences may be as described elsewhere herein, e.g. substantially homologous sequences may contain 1 , 2, or 3 amino acid substitutions, deletions or additions as compared to the amino acid sequence of insulin or fragment thereof, or e.g. may have at least 80% sequence identity to the amino acid sequence of insulin or fragment thereof. In some embodiments, an analogue may comprise (or consist of) an amino acid sequence that is substantially homologous to the amino acid sequence of the insulin A-chain (or fragment thereof) and / or an amino acid sequence that is substantially homologous to the amino acid sequence of the insulin B-chain (or fragment thereof). Sequences that are substantially homologous to the insulin A chain (or fragment thereof) and / or to the insulin B-chain (or fragment thereof) may be as described elsewhere herein, e.g. substantially homologous sequences may contain 1 , 2, or 3 amino acid substitutions, deletions or additions as compared to the amino acid sequence of the insulin A-chain (or fragment thereof) and / or insulin B-chain (or fragment thereof), or e.g. may have at least 80% sequence identity to the amino acid sequence of the insulin A-chain (or fragment thereof) and / or insulin B-chain (or fragment thereof).

[0237] Fragments and analogues of insulin should retain (or have an) ability to exert glycaemic control (e.g. in canines). In some embodiments, an analogue of insulin is an insulin derivative (or modified form of insulin) that retains (or has an) ability to exert glycaemic control (e.g. in canines), wherein said analogue comprises an insulin A-chain (or fragment or analogue thereof) and an insulin B-chain (or fragment or analogue thereof). Preferably, the insulin is canine insulin. The amino acid sequence of the A- chain of canine insulin is set forth herein as SEQ ID NO:134. The amino acid sequence of the B-chain of canine insulin is set forth herein as SEQ ID NO:135. Insulin is useful in the treatment of certain diseases, such as Type I or Type II diabetes. Thus, constructs of the invention that comprise insulin (or fragments or analogues thereof) may be used for the treatment of, for example, Type I or Type II diabetes (e.g. in canines).

[0238] In some other embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is a Glucagon-like peptide-1 (GLP-1) receptor agonist.

[0239] In some other embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is (i) GLP-1 (Glucagon-like peptide-1, e.g. SEQ ID NO: 136), or (ii) a fragment (or functional fragment) of GLP-1 , or (iii) an analogue (or functional analogue) of (i) or (ii). In some embodiments, the GLP-1 analogue is a long-acting GLP-1 or a fragment of a long acting GLP-1 , (or a long acting GLP-1 analogue). In some embodiments, a fragment of GLP-1 may be at least 20, at least 25 amino acid residues or at least 30 amino acid residues in length. In some embodiments, a fragment of GLP-1 may be 30 or 31 amino acid residues in length. In some embodiments, a fragment of GLP-1 may be 20-30, 20-31 , 25 to 30 or 25 to 31 amino acid residues in length. In some embodiments, a fragment of GLP-1 may be up to 25, up to 30, up to 31 or up to 36 amino acid residues in length. In some embodiments, the fragment of GLP-1 comprises or consists of an amino acid sequence as set forth as SEQ ID NO: 137 (GLP-1 (7-37)) or SEQ ID NO: 138 (GLP- 1(7-36)). An analogue may comprise (or consist of) an amino acid sequence that is substantially homologous to the amino acid sequence of GLP-1 or fragment thereof. Substantially homologous sequences may be as described elsewhere herein, e.g. substantially homologous sequences may contain 1, 2, or 3 amino acid substitutions, deletions or additions as compared to the amino acid sequence of GLP-1 or fragment thereof, or e.g. may have at least 80% sequence identity to the amino acid sequence of GLP-1 or fragment thereof. In some embodiments, the analogue of GLP-1 comprises or consists of an amino acid sequence as set forth as SEQ ID NO:139 (GLP-1 (7-37)A8G) or SEQ ID NQ:140 (GLP-1(7-36)A8G). Fragments and analogues of GLP-1 should retain (or have) ability to bind to, and preferably agonise, GLP-1 receptor (e.g. in canines). Preferably, the GLP-1 is canine GLP-1 (SEQ ID NO:136). GLP-1 (and fragments and analogues thereof) is useful in the treatment of certain diseases, such as obesity, kidney disease (e.g. chronic kidney disease or diabetic kidney disease), and Type II diabetes. Thus, constructs of the invention that comprise GLP-1 (or fragments or analogues thereof) may be used for the treatment of, for example, obesity, kidney disease (e.g. chronic kidney disease or diabetic kidney disease), and Type II diabetes, for example in canines.

[0240] In some other embodiments, at least one agent which binds to a protein (preferably a mammalian protein, more preferably a canine protein) other than albumin (referred to in (b) above) is (i) Exendin-4, or (ii) a fragment (or functional fragment) of Exendin-4, or (iii) an analogue (or functional analogue) of (i) or (ii). In some embodiments, a fragment of Exendin-4 may be at least 20, at least 25 amino acid residues, at least 30 amino acid or at least 35 amino acid residues in length. In some embodiments, a fragment of Exendin-4 may be 20-30, 20-35, 20-38, 25-30, 25-35, 25-38, 30-35 or 30-38 amino acid residues in length. In some embodiments, a fragment of GLP-1 may be up to 25, up to 30, up to 35 or up to 38 amino acid residues in length. An analogue may comprise (or consist of) an amino acid sequence that is substantially homologous to the amino acid sequence of Exendin-4 or fragment thereof. Substantially homologous sequences may be as described elsewhere herein, e.g. substantially homologous sequences may contain 1 , 2, or 3 amino acid substitutions, deletions or additions as compared to the amino acid sequence of Exendin-4 or fragment thereof, or e.g. may have at least 80% sequence identity to the amino acid sequence of Exendin-4 or fragment thereof. Exendin-4 has the amino acid sequence set forth herein as SEQ ID NO:141. Fragments and analogues of Exendin-4 should retain (or have) ability to bind to, and preferably agonise, GLP-1 receptor (e.g. in canines). Exendin-4 is useful in the treatment of certain diseases, such as obesity and kidney disease (e.g. chronic kidney disease or diabetic kidney disease) and Type II diabetes. Thus, constructs of the invention that comprise Exendin-4 (or fragments or analogues thereof) may be used for the treatment of, for example, obesity and kidney disease (e.g. chronic kidney disease or diabetic kidney disease) and Type II diabetes, for example in canines.

[0241] Typically and preferably, constructs of the invention are polypeptides (polypeptide constructs). Thus, typically and preferably, all of the components of the construct are on (or present on) the same (single) polypeptide chain. Thus, the construct may be a single polypeptide chain construct. Thus, constructs described herein which comprise at least one VHH antibody which binds to canine albumin and at least one (other) agent, preferably all of the components are on the same (single) polypeptide chain. Such polypeptides may be referred to as fusion polypeptides (or fusion proteins or single polypeptide chain constructs). This may be considered a “beads on a string” type arrangement. Thus, for example, in constructs of the invention defined herein as comprising (a) at least one VHH antibody which binds to canine albumin and (b) at least one agent which binds to a protein other than albumin (e.g. at least one VHH antibody which binds to a protein other than albumin), components (a) and (b) are typically and preferably on (or present on) the same (single) polypeptide chain.

[0242] In embodiments of constructs of the invention in which the construct comprises more than one component, the individual components are typically linked (or linked together or connected) by appropriate linker(s). Such linkers are typically amino acid (or peptide) linkers (i.e. comprise or consist of amino acids), e.g. nonnative peptide or artificial linkers. In some embodiments, an amino acid (or peptide) linker may be 1-50, 1-25, 1-20, 1-15, 1-10 1-5, 5-50, 5-25, 5-20, 5-15, 5-10, 10-50, 10-25, 10-20, 10-15, 20-50 or 20-25 amino acids in length. In some embodiments, an amino acid (or peptide) linker may be at least 5, or at least 10, or at least 15, or at least 20, or at least 25 amino acids in length. In some embodiments, an amino acid (or peptide) linker may be up to 10, up to 15, up to 20, up to 25 amino acids, or up to 50 amino acids in length. In some embodiments, an amino acid (or peptide) linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In some embodiments, the linker may comprise (or consist of) the amino acid sequence GGGGS. Thus, the linker may be a (GGGGS)nlinker, wherein “n” is, for example, 1 to 5 (e.g. 1, 2, 3, 4 or 5, preferably 2 or 3). In some embodiments, the linker may comprise (or consist of) the amino acid sequence GGGS. Thus, the linker may be a (GGGS)nlinker, wherein “n” is, for example, 1 to 5 (e.g. 1, 2, 3, 4 or 5, preferably 3). Typically and preferably of course, the various (individual) components of the construct are linked (or connected) together using appropriate techniques, e.g. spacing, such that each component can exert its respective effect.

[0243] In constructs of the invention that comprise (a) at least one VHH antibody which binds to canine albumin (an antibody of the invention) and (b) at least one agent which binds to a protein other than albumin, a (or the) agent of (b) may be positioned anywhere (i.e. be at any position) within the construct. Of course, the positioning of an (or the) agent which binds to a protein other than albumin in the construct should preferably be such that activity (e.g. binding ability) of the a (or the) VHH antibody of the invention and the activity of an (or the) agent which binds to a protein other than albumin are maintained (or substantially maintained). In some embodiments, a (or the) agent of (b) may be positioned N-terminally with respect to a (or the) VHH antibody of the invention in the construct, or may be positioned N- terminally with respect the N-terminal-most VHH antibody of the invention in the construct. In some embodiments, a (or the) agent of (b) may be the N-terminal most component in the construct. In some embodiments, a (or the) agent of (b) may be positioned C-terminally with respect to a (or the) VHH antibody of the invention in the construct, or may be positioned C-terminally with respect the C-terminal-most VHH antibody of the invention in the construct. In some embodiments, a (or the) agent of (b) may be the C-terminal most component in the construct. In some embodiments, in constructs comprising more than one agent of (b) (e.g. more than one copy (or more than one unit) of the same agent, or more than one different agent), a VHH antibody of the present invention (i.e. VHH antibody of (a)) may be positioned in the construct between two such agents. In some embodiments, constructs comprising more than one VHH antibody of the invention of (a) (e.g. more than one copy or more than one unit of the same VHH antibody of the invention, or more than one different VHH antibody of the invention), an agent of (b) may be positioned in the construct between such VHH antibodies.

[0244] By way of certain examples, and where (a) is a VHH antibody which binds to canine albumin (a VHH antibody of the present invention) and (b) is an agent (e.g. a VHH antibody) that binds to a protein other than albumin, the construct may in some embodiments, in sequence (or order), in the N-terminal to C-terminal direction, have one of the following arrangements: (a)-(b); (b)-(a); (a)-(b)-(b); (b)-(b)-(a); (b)-(a)-(b); (a)-(b)-(b)-(b); (b)-(a)-(b)-(b); (b)-(b)-(a)-(b); (b)-(b)-(b)-(a). In such arrangements, the agents of (b) may be the same or different. Other arrangements are of course also encompassed. Typically and preferably, there is a linker between the adjacent (a) and (b) components of such constructs.

[0245] Preferably, constructs in accordance with the present invention do not comprise any antibody light chain sequences (e.g. light chain CDRs) and do not comprise any antibody constant domain (or antibody constant region) sequences and do not comprise any antibody hinge region sequences.

[0246] In one aspect, and in some embodiments, the present invention provides a construct comprising: (a) at least one VHH antibody which binds to canine albumin; and

[0247] (b) at least one therapeutic agent (e.g. a therapeutic agent suitable for use in treating a disease or a condition in canines).

[0248] Embodiments of other aspects of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0249] As described herein, in one aspect the present invention provides a construct comprising at least one VHH antibody which binds to canine albumin (a VHH antibody of the present invention). In another aspect, and in some embodiments, the present invention provides a construct comprising at least one VHH domain sequence in accordance with the present invention. Embodiments of other aspects of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0250] As described elsewhere herein, constructs of the invention are preferably single chain polypeptides (or single chain polypeptide constructs or fusion polypeptides or fusion polypeptide constructs). Thus, in multi-component constructs the various (individual) components of the constructs are preferably on a single polypeptide chain (or present in a single fusion polypeptide or single fusion protein).

[0251] However, in some other embodiments, various (individual) components of a multi-component construct may be linked (or conjugated) together by chemical means. For example, various (individual) components could be linked (or conjugated or coupled) using heterobifunctional thiol-containing linkers, N- succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl- 5 thioacetate.

[0252] In another aspect, the present invention provides a polypeptide comprising (or consisting of) at least one VHH antibody in accordance with the present invention, or at least one VHH domain sequence in accordance with the present invention or at least one construct in accordance with the present invention. Embodiments of other aspects of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0253] As indicated above, VHH antibodies in accordance with the present invention bind to canine albumin (or are capable of binding or specifically binding to canine albumin). Constructs and polypeptides of the present invention also bind to canine albumin.

[0254] Canine albumin may also be referred to as canine serum albumin (CSA) or cAlb” or “caAlbumin”. Exemplary forms of canine albumin include native canine albumin and recombinant canine albumin. In some embodiments, the canine albumin is canine albumin in (or from) canine (dog) serum (e.g. heat inactivated dog serum).

[0255] Exemplary forms of canine albumin (e.g. native canine albumin) include full length canine albumin (with or without its signal peptide and propeptide, preferably without its signal peptide and propeptide). Canine albumin without its signal peptide and propeptide may be considered full-length mature (or full-length processed) canine albumin. Thus, full-length mature (or full-length processed) canine albumin is canine albumin lacking its signal peptide and propeptide.

[0256] Sequences of canine albumin are well known and described in the art and can be obtained for example from various sequence databases, e.g. Uniprot. Canine albumin (e.g. full-length mature (or full-length processed) canine albumin) is commercially available.

[0257] A preferred and exemplary canine albumin molecule has the amino acid sequence of SEQ ID NO:1. The amino acid sequence of SEQ ID NO:1 is a canine albumin amino acid sequence that does not include the signal peptide or propeptide sequences. Thus, the amino acid sequence of SEQ ID NO:1 is a full-length mature (or full-length processed) canine albumin amino acid sequence. Thus, preferred VHH antibodies, constructs and polypeptides of the invention bind to or are capable of binding (or specifically binding) to SEQ ID NO:1.

[0258] Methods of assessing binding to (or ability to bind to) appropriate forms of canine albumin would be well-known to a person skilled in the art and any appropriate method can be used.

[0259] A convenient and appropriate method for assessing binding would include in vitro binding assays such as ELISA assays to assess binding of VHH antibodies, constructs and polypeptides to immobilised antigen, such as immobilised forms of canine albumin as described above, e.g. canine albumin of SEQ ID NO:1. Thus, in certain embodiments, VHH antibodies, constructs and polypeptides of the present invention bind to canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay. The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of VHH antibodies, constructs and polypeptides to bind to canine albumin in such an assay. For example, in certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay when a VHH antibody (or construct or polypeptide) is used at a concentration of at least 10nM, or at least 20nM, or at least 50nM, or at least 100nM, or at least 150nM, or at least 200nM, or a concentration (or concentrations) in the range of 10nM to 50nM, or 10nM to 100nM, or 10nM to 150nM, or 10nM to 200nM, or 20nM to 50nM, or 20nM to 100nM, or 20nM to 150nM, or 20nM to 200nM, or 50nM to 100nM, or 75nM to 100nM, or 50nM to 150nM, or 50nM to 200nM, or 75nM to 150nM, or 75nM to 200nM. Particularly preferred ELISA assays are described elsewhere herein, e.g. in the Examples section (e.g. in Example 3 herein).

[0260] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) a Surface Plasmon Resonance (SPR) assay (or, alternatively viewed, by SPR). An SPR assay may, for example, be a BIACore assay (e.g. performed on a T200 Biacore (Cytvia)). Suitable SPR assays are known in the art. In some embodiments, the SPR assay is a multi-cycle kinetics (MCK) SPR assay. A Surface Plasmon Resonance (SPR) assay may be performed, for example, at 25°C. In preferred embodiments, a Surface Plasmon Resonance (SPR) assay is a SPR assay performed at 25°C. In some embodiments, the SPR assay is a multi-cycle kinetics (MCK) SPR assay performed at 25°C. In certain preferred SPR assays, canine albumin, is captured (or immobilised) on a solid support (e.g. a sensor chip), for example via amine coupling (e.g. with EDC / NHS and / or e.g. approximately 500 or approximately 1000 Resonance Units (RU) canine albumin is immobilised) and various concentrations (e.g. a dilution series, e.g. a doubling or trebling or five-fold dilution series, e.g. 2- fold serial dilutions from 200nM to 12.5nM) of the VHH antibody (or construct or polypeptide) to be tested is then injected. VHH antibody (or construct or polypeptide) concentrations may be selected at a range such that the chip is not saturated and which allow robust fitting by the SPR / Biacore software. Preferred concentrations and flow-rates for injection are described in the Examples section (see e.g. Example 3 and Example 6). Particularly preferred SPR assays are described elsewhere herein, e.g. in the Examples section (e.g. in Example 3 and in Example 6 herein).

[0261] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at pH7.4. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at endosomal pH (e.g. endosomal pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to canine albumin at pH5.5 or pH6.0.

[0262] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines) and bind to (or are capable of binding to) canine albumin at endosomal pH (e.g. endosomal pH of canines). In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at pH7.4 and at pH5.5. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at pH7.4 and at pH6.0. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin at pH7.4 and at pH6.0 and at pH5.5.

[0263] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) to canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). Performance of an ELISA at a given (or desired) pH may be done by performing the ELISA with all steps (or all incubation steps and washing steps) in a buffer of the given pH (e.g. PBS (or e.g. PBS-T for washing steps), pH7.4; or e.g. CPB (or e.g. CPB-T for washing steps), pH5.5 or pH6.0). For example, in certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) an ELISA assay when a VHH antibody (or construct or polypeptide) is used at a concentration of at least 10nM, or at least 20nM, or at least 50nM, or at least 100nM, or at least 150nM, or at least 200nM, or a concentration (or concentrations) in the range of 10nM to 50nM, or 10nM to 100nM, or 10nM to 150nM, or 10nM to 200nM, or 20nM to 50nM, or 20nM to 100nM, or 20nM to 150nM, or 20nM to 200nM or 50nM to 100nM, or 50nM to 150nM, or 50nM to 200nM or 75nM to 100nM, or 75nM to 150nM, or 75nM to 200nM. A particularly preferred ELISA assay is described in the Examples section (e.g. in Example 3 herein).

[0264] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). Performance of a SPR assay at a given pH may be done running the VHH antibodies (or constructs or polypeptides) in a buffer (e.g. HBS-P+ buffer; 10 mM HEPES, 150 mM NaCI, 0.005% v / v surfactant P20), adjusted to the desired pH (e.g. HBS-P+, adjusted to pH7.4 or 5.5 or 6.0). Particularly preferred SPR assays are described elsewhere herein, e.g. in the Examples section (e.g. in Example 3 and Example 6 herein).

[0265] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1) in the nanomolar (nM) or picomolar range (pM), for example when determined in a SPR assay (e.g. a SPR assay as described herein). Such a SPR assay may be performed (or as performed) at endosomal pH (e.g. p6.0 or pH5.5, preferably 6.0) or may be performed (or as performed) at circulatory (e.g. bloodstream) pH (e.g. 7.4). Thus, such affinities may, for example, be at (or as determined at) in a SPR assay at pH6.0 or pH7.4. Such a SPR assay may, for example, be performed (or as performed) at 25°C.

[0266] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1) in the range of 10pM to 100nM, or lower (better / stronger), for example when determined in a SPR assay (e.g. a SPR assay as described herein). Such a SPR assay may be performed (or as performed) at endosomal pH (e.g. p6.0 or pH5.5, preferably 6.0) or may be performed (or as performed) at circulatory (e.g. bloodstream) pH (e.g. 7.4). Thus, such affinities may, for example, be at (or as determined at) in a SPR assay at pH6.0 or pH7.4. Such a SPR assay may, for example, be performed (or as performed) at 25°C.

[0267] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined at pH6.0 and / or at pH7.4 in a SPR assay, in the range of 10pM to 75nM, 10pM to 50nM, 10pM to 25nM, 10pM to 10nM, 10pM to 5nM, 10pM to 1nM, 10pM to 500pM, 100pM to 100nM, 100pM to 75nM, 100pM to 50nM, 100pM to 25nM, 100pM to 10nM, 100pM to 5nM, 100pM to 1nM, 100pM to 500pM, 500pM to 100nM, 500pM to 75nM, 500pM to 50nM, 500pM to 25nM, 500pM to 10nM, 500pM to 5nM, 500pM to 1nM, 1nM to 100nM, 1nM to 75nM, 1nM to 50nM, 1nM to 25nM, 1nM to 10nM, or 1nM to 5nM.

[0268] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined at pH6.0 and / or pH7.4 in a SPR assay, in the range of 500pM to 50nM, 500pM to 40nM, 500pM to 30nM, 100pM to 50nM, 100pM to 40nM, or 100pM to 30nM.

[0269] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in an SPR assay, in the range of 100pM to 50nM at pH6.0 and in the range of 100pM to 50nM at pH7.4.

[0270] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in an SPR assay, in the range of 500pM to 50nM at pH6.0 and in the range of 500pM to 50nM at pH7.4.

[0271] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in an SPR assay, in the range of 100pM to 50nM at pH6.0 and in the range of 100pM to 50nM at pH7.4.

[0272] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in an SPR assay, in the range of 500pM to 10nM at pH6.0 and in the range of 500pM to 50nM at pH7.4.

[0273] In some embodiments, VHH antibodies of the present invention have a binding affinity for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Koof less than 100 nM, less than 50nM, less than 10nM, less than 5nM, less than 1nM (or less than 1000pM), less than 900pM, less than 800pM, less than 700pM, less than 600pM or less than 500pM, for example when determined in an SPR assay (e.g. at pH6.0 and / or at pH7.4). Particular exemplary binding affinities are disclosed in the Examples. By way of example, the VHH cA8 antibody exemplified herein shows a binding affinity of 613pM at pH6.0 and a binding affinity of 28nM at pH7.4. Thus, some preferred VHH antibodies of the invention bind to canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) with a Koof 50nM or less (or less than 50nM), for example when determined in an SPR assay (e.g. at pH6.0 and / or at pH7.4).

[0274] In some embodiments, VHH antibodies of the present invention have a binding affinity canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Koof less than less than 10nM, less than 5nM, less than 1nM (or less than 1000pM), less than 900pM, less than 800pM, less than 700pM, less than 600pM or less than 500pM, for example when determined in an SPR assay at pH6.0.

[0275] In some embodiments, VHH antibodies of the present invention have a binding affinity canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Koof less than 50nM, less than 10nM, less than 5nM, less than 1nM (or less than 1000pM), less than 900pM, less than 800pM, less than 700pM, less than 600pM or less than 500pM, for example when determined in an SPR assay at pH7.4.

[0276] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that corresponds to a Koof less than 50nM at pH6.0 and less than 50nM at pH7.4.

[0277] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that corresponds to a Koof less than 10nM at pH6.0 and less than 50nM at pH7.4.

[0278] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that corresponds to a Koof less than 5nM at pH6.0 and less than 50nM at pH7.4.

[0279] In some embodiments, VHH antibodies of the present invention have an affinity (KD (equilibrium dissociation constant)) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that corresponds to a Koof less than 1nM at pH6.0 and less than 50nM at pH7.4.

[0280] In some embodiments, VHH antibodies of the present invention have an affinity for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is stronger (or higher) at endosomal pH than at circulatory pH (e.g. bloodstream pH). Thus, in some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is lower at endosomal pH than at circulatory pH (e.g. bloodstream pH).

[0281] Thus, in some embodiments, VHH antibodies of the present invention have an affinity for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is stronger (or higher) at pH5.5 or pH6.0 (preferably at pH6.0) than at pH7.4. Thus, in some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is lower at pH5.5 or pH6.0 (preferably at pH6.0) than at pH7.4.

[0282] In some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is at least 1.5-fold lower, at least 2-fold lower, at least 4-fold lower, at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, at least 30-fold lower, at least 40-fold lower or at least 50-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0283] In some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is at least 10-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0284] In some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is at least 40-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0285] In some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is 1.5-fold to 50-fold lower, 2-fold to 50-fold lower, 3-fold to 50-fold lower, 4-fold to 50-fold lower, 5-fold to 50-fold lower, 10-fold to 50-fold, 1.5-fold to 100-fold lower, 2-fold to 100-fold lower, 3-fold to 100-fold lower, 4-fold to 100-fold lower, 5- fold to 100-fold lower, 10-fold to 100-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0286] In some embodiments, VHH antibodies of the present invention have an affinity value (KD value, e.g. in nM or pM) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is at least one order of magnitude lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4. In some preferred embodiments, VHH antibodies of the present invention have an affinity value (KD value) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is <1nM at pH5.5 or pH6.0 (preferably at pH6.0) and >10nM (or >20nM) at pH7.4.

[0287] In some preferred embodiments, VHH antibodies of the present invention have an affinity value (KD value) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is <1nM at pH5.5 or pH6.0 (preferably at pH6.0) and that is in the range of 10nM to 50nM, 10nM to 100nM, 20nM to 50nM or 20nM to 100nM at pH7.4.

[0288] Binding affinity (or binding affinity values, e.g. KD) and / or other kinetic parameters (e.g. Ka(or “on-rate” or “association rate constant” or Kon) and Kd (or “off-rate” or “dissociation rate constant” or KOff)) may be determined by SPR, for example as described elsewhere herein.

[0289] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1), e.g. with an affinity or affinity value (e.g. KD) described elsewhere herein, in (as determined in) a (multi-cycle kinetics) SPR assay (e.g. a BIACore assay, e.g. Biacore T200 assay) in which

[0290] • EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS ( / V- hydroxysuccinimide) are used to activate the sensor chip (matrix) surface (e.g. a CM5 sensor chip surface (matrix surface));

[0291] • Native full-length mature canine albumin (SEQ ID NO:1), preferably diluted in pH5.0 acetate buffer (preferably in 10mM Sodium Acetate buffer, pH5.0), is flowed over the activated chip surface to immobilise canine albumin (e.g. with the aim to reach 1,000 Rll, or e.g. achieving about 500 (e.g. 524) Rll);

[0292] • Remaining activated surface groups are blocked (preferably with ethanolamine, e.g. with 1M ethanolamine-HCI);

[0293] • Doubling dilutions (e.g. a concentration series, e.g. from 200nM to 12.5nM) of VHH antibody (or construct or polypeptide) in running buffer (e.g. HBS-P+) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4) are injected at a flow rate of 10pl / minute.

[0294] • Association is measured over 200 seconds (i.e. 200 second association phase) and dissociation is measured over 1800 seconds (i.e. 1800 second dissociation phase) and preferably all measurements are performed at 25°C in buffer (e.g. HBS-P+) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4), and chip regeneration (preferably 20 seconds regeneration) is preferably performed with Glycine pH 1.5 buffer (e.g. 20mM Glycine pH 1.5 buffer, e.g. from Cytvia); and

[0295] • Kinetic parameters are determined by fitting curves using a 1 :1 binding model (e.g. using Biacore T200 evaluation software).

[0296] This type of SPR assay may also be used, mutatis mutandis, to determine the binding (or absence of binding) to albumin from other (non-canine) species (e.g. feline, porcine or human albumin). Of course, in such cases feline, porcine or human albumin would be immobilised, instead of canine albumin.

[0297] In some embodiments, a 300 second association phase may be used instead of the 200 second association phase described for the assay above.

[0298] In some embodiments, a 3600 second association phase may be used instead of the 1800 second dissociation phase described for the assay above.

[0299] In some embodiments, the chip regeneration (or chip regeneration step) may be for 20 seconds or 30 seconds (preferably performed with 20mM Glycine pH1 .5 buffer).

[0300] In some embodiments, a five-fold dilution series (e.g. a concentration series, e.g. from 250nM to 10nM, or from 40nM to 0.32nM) of VHH antibody (or construct or polypeptide) in running buffer (e.g. HBS-P+) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4) is used instead of the doubling dilution series described for the assay above.

[0301] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1), e.g. with an affinity or affinity value (e.g. KD) described elsewhere herein, in (as determined in) a (multi-cycle kinetics) SPR assay (e.g. a BIACore assay, e.g. 1 K+ Biacore assay) in which

[0302] • EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS ( / V- hydroxysuccinimide) are used to activate the sensor chip (matrix) surface (e.g. a CM5 sensor chip surface (matrix surface));

[0303] • Native full-length mature canine albumin (SEQ ID NO:1), preferably diluted in pH4.5 acetate buffer (preferably in 10mM Sodium Acetate buffer, pH4.5), is flowed over the activated chip surface to immobilise canine albumin (e.g. with the aim to reach approximately 1 ,000 Rll);

[0304] • Remaining activated surface groups are blocked (preferably with ethanolamine, e.g. with 1M ethanolamine-HCI);

[0305] • A five-fold dilution series (e.g. a concentration series, e.g. from 250nM to 10nM) of VHH antibody (or construct or polypeptide) in running buffer (e.g. HBS-EP+ buffer; 10mM HEPES, 150mM NaCI, 3nM EDTA, 0.005% v / v surfactant P20) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4) are injected, e.g. at a flow rate of 30pl / minute.

[0306] • Association is measured over 180 seconds (i.e. 180 second association phase) and dissociation is measured over 600 seconds (i.e. 600 second dissociation phase) and preferably all measurements are performed at 25°C in buffer (e.g. HBS-EP+) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4), and chip regeneration (preferably 30 seconds regeneration) is preferably performed with Glycine pH 1.5 buffer (e.g. 10mM Glycine pH 1.5 buffer, e.g. from Cytiva); and

[0307] • Kinetic parameters are determined by fitting curves using a 1 :1 binding model (e.g. using Biacore 1 K+ evaluation software (Cytiva)).

[0308] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention bind to (or are capable of binding to) canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1), e.g. with an affinity or affinity value (e.g. KD) described elsewhere herein, in (or as determined in) a (multi-cycle kinetics) SPR assay (e.g. a BIACore assay, e.g. Biacore 1 K+ assay) in which

[0309] (a) EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS ( / V-hydroxysuccinimide) are used to activate the sensor chip (matrix) surface (e.g. a CM5 sensor chip surface (matrix surface));

[0310] (b) Canine albumin (e.g. native canine albumin, e.g. with SEQ ID NO:1), preferably diluted in pH4.5 acetate buffer (preferably in 10mM Sodium Acetate buffer, pH4.5), is flowed over the activated chip surface to immobilise canine albumin (e.g. with the aim to reach e.g. approximately 600 Rll, or e.g. achieving about 600 Rll);

[0311] (c) Remaining activated surface groups are blocked (or deactivated) (preferably with ethanolamine, e.g. with 1 M ethanolamine-HCI, pH8.5); (d) Dilutions (e.g. serial dilutions or a concentration series, e.g. concentrations of 250nM and 25nM or e.g. five-fold serial dilutions, e.g. from 250nM down to 0.4nM or down to 0.2nM) of VHH antibody (or construct or polypeptide) in running buffer (e.g. HBS-EP+ buffer; 10mM HEPES, 150mM NaCI, 3nM EDTA, 0.005% v / v surfactant P20) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4) are injected, e.g. at a flow rate of 30pl / minute.

[0312] (e) Association is measured, e.g. over 300 seconds (i.e. 300 second association phase) and dissociation is measured over 10 minutes (i.e. 10 minute dissociation phase), and preferably all measurements are performed at 25°C in buffer (e.g. HBS-EP+) adjusted to the pH under investigation (e.g. pH6.0 or pH5.5 or pH7.4), and chip regeneration (preferably 30 second regeneration at e.g. a flow rate of 30pl / min) is preferably performed with Glycine pH1.5 buffer (e.g. 10mM Glycine pH1.5 buffer); and

[0313] (f) Kinetic parameters are determined by fitting curves using a 1 :1 binding model (e.g. using Biacore 1 K+ evaluation software).

[0314] In addition to being useful to determine KD (equilibrium dissociation constant), this type of SPR assay (or other SPR assay described herein) may also be used to determine other kinetic parameters (e.g. Ka(or “on-rate” or “association rate constant” or Kon) and Kd (or “off-rate” or “dissociation rate constant” or KOff)) .

[0315] Particularly preferred SPR assays are described herein in Examples 3, 7 and 8.

[0316] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an on-rate (Ka, “association rate constant” or Kon) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kain the range of 1x1051 / Ms to 5x1081 / Ms, 1x1061 / Ms to 5x1081 / Ms, 5x1051 / Ms to 5x1081 / Ms, 1x1051 / Ms to 1x1071 / Ms, 5x1051 / Ms to 1x1071 / Ms, 1x1061 / Ms to 1x1071 / Ms or 5x1061 / Ms to 1x1071 / Ms (1 / Ms is the unit of on- rate), for example when determined in a SPR assay (e.g. a SPR assay as described herein). Such a SPR assay may be performed (or as performed) at endosomal pH (e.g. p6.0 or pH5.5, preferably 6.0) or may be performed (or as performed) at circulatory (e.g. bloodstream) pH (e.g. 7.4). Thus, such on-rates may, for example, be at (or as determined at) in a SPR assay at pH6.0 and / or in a SPR assay at pH7.4. Such a SPR assay may, for example, be performed (or as performed) at 25°C.

[0317] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an off-rate (Kd, “dissociation rate constant” or KOff) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kd in the range of 1x1 O'41 / s to 10x1 O'11 / s, or in the range of 1x1 O'41 / s to 5x10'11 / s, or in the range of 5x10'41 / s to 1x1 O'11 / s, or in the range of 1x1 O'31 / s to 5x10'21 / s, or in the range of 2x10'31 / s to 3x10'21 / s (1 / s is the unit of off-rate), for example when determined in a SPR assay (e.g. a SPR assay as described herein). Such a SPR assay may be performed (or as performed) at endosomal pH (e.g. p6.0 or pH5.5, preferably pH6.0) or may be performed (or as performed) at circulatory (e.g. bloodstream) pH (e.g. pH7.4). Thus, such off-rates may, for example, be at (or as determined at) in a SPR assay at pH6.0 or at pH7.4. Such a SPR assay may, for example, be performed (or as performed) at 25°C.

[0318] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an off-rate (Kd, “dissociation rate constant” or KOff) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kd of less than 5x1 O'1, less than 1x1 O'1, less than 5x1 O'2, less than 1x1 O'21 / s or less than 5x10'3, for example when determined in a SPR assay (e.g. a SPR assay as described herein) at pH6.0 and / or when determined in a SPR assay at pH7.4. In some preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an off-rate (Kd, “dissociation rate constant” or KOff) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kd of less than 9x10'31 / s, less than 8x10'31 / s, less than 7x10'31 / s, less than 6x10'31 / s, less than 5x10'31 / s, less than 4x10'31 / s, less than 3x10'31 / s, less than 2x10'31 / s or less than 1x1 O'31 / s, for example when determined in an SPR assay at pH6.0 and / or when determined in an SPR assay at pH7.4.

[0319] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an off-rate (Kd, “dissociation rate constant” or KOff) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kd of at least 1x1 O'41 / s, for example when determined in a SPR assay (e.g. a SPR assay as described herein) at pH6.0. In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention have an off- rate (Kd, “dissociation rate constant” or KOff) for canine albumin (e.g. native full- length mature canine albumin, e.g. of SEQ ID NO:1) that corresponds to a Kd of at least 1x1 O'31 / s, or at least 5x1 O'31 / s, or at least 1x1 O'21 / s, for example when determined in an SPR assay at pH7.4.

[0320] Thus, in some embodiments, VHH antibodies of the present invention have an off-rate for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is slower at pH5.5 or pH6.0 (preferably at pH6.0) than at pH7.4. Thus, in some embodiments, VHH antibodies of the present invention have an off-rate value (Kd value, e.g. in 1 / s) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is lower at pH5.5 or pH6.0 (preferably at pH6.0) than at pH7.4.

[0321] In some embodiments, VHH antibodies of the present invention have an off- rate value (Kd value, e.g. in 1 / s) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is at least 1.5-fold lower, at least 2-fold lower, at least 3-fold lower, at least 4- fold lower or at least 5-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0322] In some embodiments, VHH antibodies of the present invention have an off- rate value (Kd value, e.g. in 1 / s) for canine albumin (e.g. native full-length mature canine albumin, e.g. of SEQ ID NO:1), for example as determined in a SPR assay, that is 1.5-fold to 5-fold lower, 2-fold to 5-fold lower, 3-fold to 5-fold lower or 4-fold to 5-fold lower at pH5.5 or pH6.0 (preferably at pH6.0) than it is at pH7.4.

[0323] As is evident from discussion elsewhere herein, in embodiments of the present invention which refer to a SPR assay, preferably the SPR assay (e.g. a multi-cycle kinetic SPR assay) is performed at 25°C.

[0324] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind to, or do not bind significantly to, human albumin. Human albumin may also be referred to as human serum albumin (HSA) or “hAlb”. Exemplary forms of human albumin include native human albumin and recombinant human albumin.

[0325] Exemplary forms of human albumin (e.g. native human albumin) include full length human albumin (with or without its signal peptide and propeptide, preferably without its signal peptide and propeptide). Human albumin without its signal peptide and propeptide may be considered full-length mature (or full-length processed) human albumin. Thus, full-length mature (or full-length processed) human albumin is human albumin lacking its signal peptide and propeptide. Sequences of human albumin are well known and described in the art and can be obtained for example from various sequence databases, e.g. Uniprot. Human albumin (e.g. full-length mature (or full-length processed) human albumin) is commercially available. A preferred and exemplary human albumin molecule has the amino acid sequence of SEQ ID NO:2. The amino acid sequence of SEQ ID NO:2 is a human albumin amino acid sequence that does not include the signal peptide or propeptide sequences. Thus, the amino acid sequence of SEQ ID NO:2 is a full-length mature (or full-length processed) human albumin amino acid sequence

[0326] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the invention do not bind to, or do not bind significantly to, SEQ ID NO:2.

[0327] Negligible binding (or de minimis binding) to human albumin is not considered binding to, or significant binding to, human albumin. By way of example, any negligible I de minimis amount (or level) of binding that may be observed (or measured or detected) for a VHH antibody of the invention that is equivalent to (or that is essentially equivalent to), or that corresponds to (or that essentially corresponds to), or that is less than or equal to, or that is not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin and does not bind to human albumin), is not considered binding, or significant binding, to human albumin. Thus, for example, in embodiments in which VHH antibodies of the present invention do not bind to, or do not bind significantly to, human albumin, the amount (or level) of binding (if any) observed (or measured) may be equivalent to (or may be essentially equivalent to), or may correspond to (or may essentially correspond to), or may be less than or equal to, or may be not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin and does not bind to human albumin).

[0328] Methods of assessing binding to (or ability to bind to) appropriate forms of human albumin would be well-known to a person skilled in the art and any appropriate method can be used.

[0329] A convenient and appropriate method for assessing binding would include in vitro binding assays such as ELISA assays to assess binding of VHH antibodies, constructs and polypeptides to immobilised antigen, such as immobilised forms of human albumin as described above, e.g. human albumin of SEQ ID NO:2. Thus, in certain embodiments, VHH antibodies, constructs and polypeptides of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay. The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of VHH antibodies (or constructs or polypeptides) to bind to human albumin in such an assay. A particularly preferred ELISA assay is described in the Examples section (e.g. in Example 3 herein).

[0330] In certain embodiments, VHH antibodies, constructs and polypeptides of the present invention do not bind, or do not bind significantly to, human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) a Surface Plasmon Resonance (SPR) assay (or, alternatively viewed, by SPR), e.g. a multicycle SPR assay. An SPR assay may, for example, be a BIACore assay. Suitable SPR assays are known in the art and are described elsewhere herein. A Surface Plasmon Resonance (SPR) assay may be performed, for example, at 25°C. In certain preferred SPR assays, human albumin, is captured (or immobilised) on a solid support (e.g. a sensor chip), for example via amine coupling (e.g. with EDC / NHS and / or e.g. approximately 625 Resonance Units (RU) canine albumin is immobilised) and various concentrations (e.g. a dilution series, 5-fold serial dilutions from 250nM to 10nM) of the VHH antibody (or construct or polypeptide) to be tested is then injected. VHH antibody (or construct or polypeptide) concentrations may be selected at a range such that the chip is not saturated and which allow robust fitting by the SPR / Biacore software. Preferred concentrations and flow-rates for injection are described in the Examples section (see e.g. Example 3). Preferred SPR assays are described elsewhere herein, for example a particularly preferred SPR assay is described in the Examples section (e.g. in Example 3 herein).

[0331] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly to, human albumin at pH7.4.

[0332] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly to, human albumin at endosomal pH (e.g. endosomal pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at pH5.5 or pH6.0. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines) or at endosomal pH (e.g. endosomal pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at pH7.4 or at pH5.5. Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at pH7.4 or at pH6.0. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to human albumin at pH7.4 or at pH5.5 or at pH6.0.

[0333] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0) (i.e. no binding, or significant binding, at either neutral or acidic pH). Performance of an ELISA at a given (or desired) pH may be done by performing the ELISA with all steps (or all incubation steps and washing steps) in a buffer of the given pH (e.g. PBS (or e.g. PBS-T for washing steps), pH7.4; or e.g. CPB (or e.g. CPB-T for washing steps), pH5.5 or pH6.0). For example, in certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) an ELISA assay when a VHH antibody (or construct or polypeptide) is used at a concentration of at least 50nM, or at least 100nM, or at a concentration (or concentrations) in the range of 50nM to 100nM or 75nM to 100nM. A particularly preferred ELISA assay is described in the Examples section (e.g. in Example 3 herein).

[0334] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and do not bind, or do not significantly bind, to human albumin (e.g. native human albumin, e.g. with SEQ ID NO:2) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0) (i.e. no binding, or significant binding, at either neutral or acidic pH). Performance of a SPR assay at a given pH may be done running the VHH antibodies (or constructs or polypeptides) in a buffer (e.g. HBS-P+ buffer; 10 mM HEPES, 150 mM NaCI, 0.005% v / v surfactant P20), adjusted to the desired pH (e.g. HBS-P+, adjusted to pH7.4 or 5.5 or 6.0). Preferred SPR assays are described elsewhere herein, for example a particularly preferred SPR assay is described in the Examples section (e.g. in Example 3 herein).

[0335] In some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind to, or do not bind significantly to, feline albumin, and / or do not bind to, or do not bind significantly to, porcine albumin. Feline albumin may also be referred to as feline serum albumin (FSA). Porcine albumin may also be referred to as porcine serum albumin (PSA). Exemplary forms of feline albumin include native feline albumin and recombinant canine albumin. Exemplary forms of porcine albumin include native porcine albumin and recombinant porcine albumin.

[0336] Exemplary forms of feline and porcine albumin (e.g. native feline and porcine albumin) include full length native feline or porcine albumin (with or without their signal peptide and propeptide, preferably without their signal peptide and propeptide). Feline and porcine albumin without their signal peptide and propeptide may be considered full-length mature (or full-length processed) feline or porcine albumin. Thus, full-length mature (or full-length processed) feline albumin is feline albumin lacking its signal peptide and propeptide. Full-length mature (or full-length processed) porcine albumin is porcine albumin lacking its signal peptide and propeptide.

[0337] Sequences of feline and porcine albumin are well known and described in the art and can be obtained for example from various sequence databases, e.g. Uniprot. Feline and porcine albumin (e.g. full-length mature (or full-length processed) feline or porcine albumin) are commercially available.

[0338] A preferred and exemplary feline albumin molecule has the amino acid sequence of SEQ ID NO:100. The amino acid sequence of SEQ ID NO:100 is a feline albumin amino acid sequence that does not include the signal peptide or propeptide sequences. Thus, the amino acid sequence of SEQ ID NQ:100 is a full- length mature (or full-length processed) feline albumin amino acid sequence.

[0339] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the invention do not bind to, or do not bind significantly to, SEQ ID NO: 100.

[0340] A preferred and exemplary porcine albumin molecule has the amino acid sequence of SEQ ID NO: 101. The amino acid sequence of SEQ ID NO: 101 is a porcine albumin amino acid sequence that does not include the signal peptide or propeptide sequences. Thus, the amino acid sequence of SEQ ID NO: 101 is a full- length mature (or full-length processed) porcine albumin amino acid sequence.

[0341] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the invention do not bind to, or do not bind significantly to, SEQ I D NO: 101.

[0342] Negligible binding (or de minimis binding) to feline or porcine albumin is not considered binding to, or significant binding to feline or porcine albumin. By way of example, any negligible I de minimis amount (or level) of binding that may be observed (or measured or detected) for a VHH antibody of the invention that is equivalent to (or that is essentially equivalent to), or that corresponds to (or that essentially corresponds to), or that is less than or equal to, or that is not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin and does not bind to feline albumin and does not bind to porcine albumin), is not considered binding, or significant binding, to feline or porcine albumin. Thus, for example, in embodiments in which VHH antibodies of the present invention do not bind to, or do not bind significantly to, feline or porcine albumin, the amount (or level) of binding (if any) observed (or measured) may be equivalent to (or may be essentially equivalent to), or may correspond to (or may essentially correspond to), or may be less than or equal to, or may be not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin and does not bind to feline albumin and does not bind to porcine albumin).

[0343] Methods of assessing binding to (or ability to bind to) appropriate forms of feline or porcine albumin would be well-known to a person skilled in the art and any appropriate method can be used.

[0344] A convenient and appropriate method for assessing binding would include in vitro binding assays such as ELISA assays to assess binding of VHH antibodies, constructs and polypeptides to immobilised antigen, such as immobilised forms of feline or porcine albumin as described above. Thus, in certain embodiments, VHH antibodies, constructs and polypeptides of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native feline and / or porcine albumin) in (as determined in) an ELISA assay. The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of VHH antibodies, constructs and polypeptides to bind to feline or porcine albumin in such an assay.

[0345] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly to, feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) a Surface Plasmon Resonance (SPR) assay (or, alternatively viewed, by SPR), e.g. a multi-cycle SPR assay. A Surface Plasmon Resonance (SPR) assay may be performed, for example, at 25°C. An SPR assay may, for example, be a BIACore assay. Suitable SPR assays are known in the art and are described elsewhere herein. In certain preferred SPR assays, feline or porcine albumin is captured (or immobilised) on a solid support (e.g. a sensor chip), for example via amine coupling (e.g. with EDC / NHS and / or e.g. approximately 1140 Resonance Units (RU) feline albumin is immobilised, and / or e.g. approximately 4741 RU porcine albumin immobilised) and various concentrations (e.g. a dilution series, 2-fold serial dilutions from 200nM to 12.5nM) of the VHH antibody, construct or polypeptide to be tested is then injected. VHH antibody (or construct or polypeptide) concentrations may be selected at a range such that the chip is not saturated and which allow robust fitting by the SPR / Biacore software. Preferred concentrations and flow-rates for injection are described in the Examples section (see e.g. Example 3). Preferred SPR assays are described elsewhere herein, for example a particularly preferred SPR assay is described in the Examples section (e.g. in Example 3 herein).

[0346] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly to, feline and / or porcine albumin at circulatory (e.g. bloodstream) pH7.4.

[0347] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly to, feline and / or porcine albumin at endosomal pH (e.g. endosomal pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at pH5.5 or pH6.0.

[0348] In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at circulatory (e.g. bloodstream) pH (e.g. circulatory (e.g. bloodstream) pH of canines) or at endosomal pH (e.g. endosomal pH of canines). Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at pH7.4 or at pH5.5. Thus, in preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at pH7.4 or at pH6.0. In preferred embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not bind significantly, to feline and / or porcine albumin at pH7.4 or at pH5.5 or at pH6.0.

[0349] Thus, in certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) an ELISA assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) an ELISA assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0) (i.e. no binding, or significant binding, at either neutral or acidic pH). Performance of an ELISA at a given (or desired) pH may be done by performing the ELISA with all steps (or all incubation steps and washing steps) in a buffer of the given pH (e.g. PBS (or e.g. PBS-T (e.g. PBS containing 0.05% Tween-20) for washing steps), pH7.4; or e.g. CPB (or e.g. CPB-T (e.g. CPB containing 0.05% Tween-20) for washing steps), pH5.5 or pH6.0).

[0350] In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0). In certain embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) a SPR assay that is performed at a circulatory (e.g. bloodstream) pH (e.g. pH7.4), and do not bind, or do not significantly bind, to feline and / or porcine albumin (e.g. native full-length mature feline and / or porcine albumin) in (as determined in) a SPR assay that is performed at endosomal pH (e.g. pH5.5 or pH6.0) (i.e. no binding, or significant binding, at either neutral or acidic pH). Performance of a SPR assay at a given pH may be done running the VHH antibodies (or constructs or polypeptides) in a buffer (e.g. HBS-P+ buffer; 10 mM HEPES, 150 mM NaCI, 0.005% v / v surfactant P20), adjusted to the desired pH (e.g. HBS-P+, adjusted to pH7.4 or 5.5 or 6.0). Preferred SPR assays are described elsewhere herein, for example a particularly preferred SPR assay is described in the Examples section (e.g. in Example 3 herein). As is evident from discussion elsewhere herein, FcRn-mediated recycling of albumin relies on the ability of albumin to bind to FcRn. Thus, in preferred embodiments, VHH antibodies of the present invention (or constructs or polypeptides of the invention), do not interfere with (or do not impair or do not inhibit), or do not significantly interfere with (or do not significantly impair or do not significantly inhibit) the ability of canine albumin to bind to FcRn (canine FcRn). Put another way, in preferred embodiments, when a VHH antibody of the invention is bound to canine albumin, said VHH antibody does not interfere (or does not impair or does not inhibit), or does not significantly interfere (or does not significantly impair or does not significantly inhibit) the ability of canine albumin to bind to FcRn (canine FcRn). Alternatively viewed, in some preferred embodiments of the present invention, when a VHH antibody of the invention is bound to canine albumin, said canine albumin maintains (or substantially maintains) the ability to bind to FcRn (canine FcRn). Thus, in some embodiments, a VHH antibody of the invention and a FcRn (canine FcRn) may simultaneously bind to canine albumin. In some embodiments, a VHH antibody of the invention does not prevent FcRn binding to canine albumin.

[0351] Preferably, VHH antibodies of the present invention (or constructs or polypeptides of the invention) bind to canine albumin at endosomal pH (e.g. pH5.5 or pH6.0, preferably pH5.5) and do not interfere with, or do not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn) at endosomal pH (e.g. pH5.5 or pH6.0, preferably pH5.5).

[0352] Methods of assessing interference with FcRn binding to albumin would be well-known to a person skilled in the art, and any appropriate method can be used. For example, an ELISA (e.g. a competition ELISA) may be used. Thus, in some embodiments, VHH antibodies (or constructs or polypeptides) of the present invention do not interfere with (or do not impair or do not inhibit), or do not significantly interfere with (or do not significantly impair or do not significantly inhibit), the ability of canine albumin to bind to FcRn (canine FcRn) in (or as determined by) an ELISA assay (for example at endosomal pH, e.g. pH5.5).

[0353] In some embodiments, the ability of a VHH antibody of the present invention to interfere with canine FcRn binding to canine albumin (canine FcRn-canine albumin interaction) may be determined in (or be as determined in) an ELISA assay (or competition ELISA assay) in which the VHH antibody (test antibody) is tested (or assayed) for its ability to compete with canine FcRn for binding to canine albumin. Absence (or substantial absence) of competition would be indicative that a VHH antibody of the invention does not interfere (or does not significantly interfere) with canine FcRn binding to canine albumin.

[0354] In some embodiments, the ability of a VHH antibody of the present invention to interfere with canine FcRn binding to canine albumin (canine FcRn-canine albumin interaction) may be determined in (or be as determined in) an ELISA assay (or competition ELISA assay), for example performed at pH5.5, in which (i) canine albumin immobilised in (or on) wells of an ELISA plate is incubated with canine FcRn (FcRn polypeptide) to achieve a 1 :1 molar ratio of canine FcRn:canine albumin (canine FcRn:canine albumin complexes), and (ii) a dilution series of the VHH antibody that will achieve a range of molar excesses of VHH antibody:canine albumin (e.g. a two-fold dilution series, e.g. to achieve molar excesses of 40:1 to 0.31 :1 of VHH:canine albumin) when added to the wells of the ELISA plate is added to wells of the ELISA plate containing immobilised canine FcRn:canine albumin complexes, wherein no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn).

[0355] In some embodiments, the ability of a VHH antibody of the present invention to interfere with canine FcRn binding to canine albumin (canine FcRn-canine albumin interaction) may be determined in (or be as determined in) an ELISA assay (or competition ELISA assay or FcRn interference assay) that comprises:

[0356] (a) coating wells of an ELISA (e.g. MaxiSorb) plate with canine albumin (e.g. 10.72nM canine albumin and / or e.g. overnight and / or e.g. at 4°C), and preferably then blocking the wells (e.g. with 1 % casein, e.g. 1 % casein in a pH5.5 buffer, e.g. in citrate-phosphate buffer (CPB), pH5.5);

[0357] (b) adding to the wells (e.g. 10.72nM) biotinylated canine FcRn in a buffer at pH5.5 (e.g. CPB, pH5.5) to the coated (and preferably blocked) wells and incubating (e.g. for 2 hours and / or e.g. at room temperature) in order to form canine FcRmcanine albumin complexes (1 :1 molar ratio of canine FcRmcanine albumin);

[0358] (c) adding to the wells a two-fold dilution series, in a pH5.5 buffer (e.g. CPB, pH5.5), of a c-myc tagged (preferably C-terminally c-myc tagged) form of the VHH antibody to achieve molar excesses of 40:1 to 0.31 :1 of VHH:canine albumin, and incubating (e.g. for 2 hours and / or e.g. at room temperature); (d) adding, to parallel wells, either

[0359] (i) streptavidin-HRP in a pH5.5 buffer (e.g. in CPB, pH5.5), e.g.

[0360] 0.5pg / ml streptavidin-HRP in 0.25% casein in pH5.5 buffer (e.g. in 0.25% casein in CPB, pH5.5), and incubating (e.g. for 1 hour and / or e.g. at room temperature), or

[0361] (ii) a mouse-anti-c-myc tag (primary) antibody in a pH5.5 buffer (e.g. CPB, pH5.5), e.g. 0.2pg / ml mouse-anti-c-myc tag (primary) antibody (e.g. mouse THE™ c-myc Tag Antibody (Genscript)) in 0.25% casein in a pH5.5 buffer (e.g. in 0.25% casein in CPB, pH5.5), and incubating (e.g. for 1 hour and / or e.g. at room temperature), and subsequently (typically after washing, e.g. 3x with CPB-T), incubating with an anti-mouse-HRP (secondary) antibody in a pH5.5 buffer (e.g. in CPB, pH5.5), e.g. 0.08pg / ml anti-mouse-HRP (secondary) antibody in 0.25% casein in pH5.5 buffer (e.g. in 0.25% casein in CPB, pH5.5), and incubating (e.g. for 1 hour and / or e.g. at room temperature);

[0362] (e) adding a chromogenic substrate (e.g. 3,3',5,5'-tetramentylbenzidine, TMB) for HRP (horseradish peroxidase) to the wells (e.g. for 15 minutes for the wells of step (d)(i), or for 7 minutes for the wells of (d)(ii)), and preferably then stopping the reaction (e.g. with 0.5M H2SO4);

[0363] (f) detecting (or measuring or quantifying) the signal produced from the chromogenic substrate / HRP reactions (e.g. with a spectrophotometer, e.g. measuring absorbance at 450nm (OD450), wherein detected signal from the wells processed in accordance with step (d)(i) is indicative of binding of canine FcRn to canine albumin, and wherein detected signal from the wells processed in accordance with step (d)(ii) is indicative of binding of the VHH antibody to canine albumin. Typically, and thus in preferred embodiments, no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn). In some embodiments, no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin from 0.31 :1 to 2.5:1 is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn). In some embodiments, no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin from 0.31:1 to 5:1 is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn). In some embodiments, no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin from 0.31 :1 to 10:1 is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn). In some embodiments, no reduction, or no significant reduction, in canine FcRn binding to canine albumin at increasing molar excesses of VHH antibody:canine albumin from 0.31 :1 to 40:1 is indicative that the VHH antibody does not interfere with, or does not significantly interfere with, the ability of canine albumin to bind to FcRn (canine FcRn). Typically, washing steps (e.g. with a buffer e.g. at the desired pH) may be performed between one or more (or all) steps of ELISA assays, e.g. one or more (or all) steps (a) to (f) in the ELISA assay described above. As indicated above, such ELISAs may typically be performed (or be as performed) at an endosomal pH (e.g. pH5.5). A particularly preferred FcRn interference assay (ELISA) is described in Example 3 herein. When processing the results / data / detected (or measured) signals (e.g. absorbance at 450nm (OD450)) of FcRn interference assays (ELISAs), background signal (if any) from an appropriate negative control (e.g. “no canine albumin” negative control) may, in some embodiments, be subtracted.

[0364] In preferred embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain III (Dill) of canine albumin. Dill of albumin is the key domain of albumin that is responsible for the albumin binding to FcRn. Thus, not binding to, or not significantly binding to, Dill of FcRn is advantageous as is allows Dill to be available for binding to FcRn which is important for FcRn-mediated recycling, as described elsewhere herein. An exemplary amino acid sequence of canine albumin Dill is set forth herein as SEQ ID NO:5. A recombinant canine albumin Dill amino acid sequence (including an additional N-terminal M residue and a C-terminal Avi-His tag) is set forth herein as SEQ ID NO:6. Thus, preferably VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain III (Dill) of canine albumin, wherein Domain III has the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Thus, preferably VHH antibodies of the present invention do not bind to, or do not bind significantly to, an isolated polypeptide consisting of an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Negligible binding (or de minimis binding) to Dill of canine albumin is not considered binding to, or significant binding to, Dill of canine albumin. By way of example, any negligible I de minimis amount (or level) of binding that may be observed (or measured or detected) for a VHH antibody of the invention that is equivalent to (or that is essentially equivalent to), or that corresponds to (or that essentially corresponds to), or that is less than or equal to, or that is not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin), is not considered binding, or significant binding, to Dill of canine albumin. Thus, for example, in embodiments in which VHH antibodies of the present invention do not bind to, or do not bind significantly to, Dill of canine albumin, the amount (or level) of binding (if any) observed (or measured) may be equivalent to (or may be essentially equivalent to), or may correspond to (or may essentially correspond to), or may be less than or equal to, or may be not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin).

[0365] Preferably, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Dill of canine albumin at an endosomal pH (e.g. pH5.5 or pH6.0). Preferably, VHH antibodies of the present invention do not bind to Dill of canine albumin at a circulatory (e.g. blood stream) pH (e.g. pH7.4). Preferably, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Dill of canine albumin at an endosomal pH (e.g. pH5.5 or pH6.0) or at a circulatory (e.g. blood stream) pH (e.g. pH7.4).

[0366] Methods of assessing binding to (or ability to bind to) Dill of canine albumin would be well-known to a person skilled in the art, and any appropriate method can be used. For example, an ELISA may be used. Thus, in some embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain III (Dill) of canine albumin, as determined by an ELISA assay.

[0367] In some embodiments, the ability of a VHH antibody of the present invention to bind to Dill of canine albumin may be determined in (or be as determined in) an ELISA assay in which a canine albumin Dill polypeptide (e.g. of SEQ ID NO:5 or SEQ ID NO:6) is immobilised in (or coated on) wells of an ELISA plate, the VHH antibody (test VHH antibody) is subsequently added to wells containing immobilised canine albumin Dill polypeptide, and binding of the VHH antibody to the canine albumin Dill polypeptide is detected (or measured or quantified). The absence of a detectable (or measurable) signal, or only a negligible (or de minimis) signal (e.g. a signal is that is equivalent to or corresponds to (or essentially equivalent to or essentially corresponds to) binding observed (or measured) with a negative control VHH antibody that does not bind to canine albumin) is indicative that the VHH antibody does not bind to, or significantly bind to, Dill of canine albumin.

[0368] For example, in some embodiments, the ability of a VHH antibody of the present invention to bind to canine albumin Dill may be determined in (or be as determined in) an ELISA assay that comprises:

[0369] (a) coating wells of an ELISA (e.g. MaxiSorp) plate with (e.g. 10.72nM of) a canine albumin Dill polypeptide (e.g. SEQ ID NO:6) (e.g. for 1 hour at room temperature), and preferably then blocking the wells (e.g. with milk, e.g. 4% milk);

[0370] (b) adding a c-myc tagged (preferably C-terminally c-myc tagged) form of the VHH antibody (test VHH antibody) (e.g. at a (or a range of) concentration(s) of between 1.34nM to 171.52nM, e.g. in 0.25% milk) to the coated (and preferably blocked) wells and incubating (e.g. for 2 hours, e.g. at room temperature);

[0371] (c) adding a mouse-anti-c-myc tag (primary) antibody, e.g. in 1% milk at e.g.

[0372] 0.2pg / ml (e.g. mouse THE™ c-myc Tag Antibody (Genscript)), and incubating (e.g. for 2 hours, e.g. at room temperature);

[0373] (d) adding an anti-mouse-HRP (secondary) antibody, e.g. in 1% milk at e.g. 0.08 pg / ml, and incubating (e.g. for 2 hours, e.g. at room temperature);

[0374] (e) adding a chromogenic substrate (e.g. 3,3',5,5'-tetramentylbenzidine, TMB) for HRP (horseradish peroxidase) to the wells (e.g. for 7 minutes), preferably then stopping the reaction (e.g. with 0.5M H2SO4);

[0375] (f) detecting (or measuring or quantifying) the signal produced from the chromogenic substrate / HRP reaction (e.g. with a spectrophotometer, e.g. measuring absorbance at 450nm), wherein detected signal is indicative of binding of the VHH antibody to Dill of canine albumin. The absence of a detectable (or measurable) signal, or only a negligible (or de minimis) signal (e.g. a signal is that is equivalent to or corresponds to (or essentially equivalent to or essentially corresponds to) binding observed (or measured) with a negative control VHH antibody that does not bind to canine albumin) is indicative that the VHH antibody does not bind to, or significantly bind to, Dill of canine albumin. Typically, washing steps (e.g. with a buffer e.g. at the desired pH) may be performed between one or more of steps (a), (b), (c), (d), (e) and (f) above. ELISA may be performed at pH5.5 or pH7.4. Thus, ELISAs may be run with steps (and preferably washes) performed at pH7.4 or pH5.5, depending on the pH under investigation (e.g. steps run in PBS (pH7.4) or CPB (pH5.5), and washes in PBS-T (pH7.4) or CPB-T (pH5.5)). A particularly preferred ELISA is described in Example 3 herein.

[0376] Preferably, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain III (Dill) of canine albumin when the VHH antibody is used at a concentration in the range of 1nM to 175nM (e.g. 1.34 nM to 171.52nM), or 1nM to 50nM, or 1nM to 10nM, 10nM to 20nM or 10nM to 50nM (e.g. at a concentration of 1nM, 1.34nM, 2.68nM, 3nM, 5nM, 5.36nM, 10nM or 10.72nM), for example in an ELISA assay as described herein. In some embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Dill of canine albumin when the VHH antibody is used at a concentration of about 10nM (e.g. 10.72nM), for example in an ELISA assay as described herein.

[0377] In preferred embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain I (DI) of canine albumin. DI of canine albumin has a minor contribution to albumin binding to FcRn. An exemplary amino acid sequence of canine albumin DI is set forth herein as SEQ ID NO:3. A recombinant canine albumin DI amino acid sequence (including an additional N- terminal M residue and a C-terminal Avi-His tag) is set forth herein as SEQ ID NO:4. Thus, preferably VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain I (DI) of canine albumin, wherein Domain I has the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. Thus, preferably VHH antibodies of the present invention do not bind to, or do not bind significantly to, an isolated polypeptide consisting of an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. Negligible binding (or de minimis binding, or background binding) to DI of canine albumin is not considered binding to, or significant binding to, DI of canine albumin. By way of example, any negligible I de minimis amount (or level) of binding that may be observed (or measured or detected) for a VHH antibody of the invention that is equivalent to (or that is essentially equivalent to), or that corresponds to (or that essentially corresponds to), or that is less than or equal to, or that is not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin), is not considered binding, or significant binding, to DI of canine albumin. Thus, for example, in embodiments in which VHH antibodies of the present invention do not bind to, or do not bind significantly to, DI of canine albumin, the amount (or level) of binding (if any) observed (or measured) may be equivalent to (or may be essentially equivalent to), or may correspond to (or may essentially correspond to), or may be less than or equal to, or may be not greater than (or not significantly greater than) binding observed (or measured) with an appropriate negative control (e.g. with a negative control VHH antibody that does not bind to canine albumin).

[0378] Preferably, VHH antibodies of the present invention do not bind to DI, or do not bind significantly to DI, of canine albumin at an endosomal pH (e.g. pH5.5 or pH6.0). Preferably, VHH antibodies of the present invention do not bind to DI of canine albumin at a circulatory (e.g. blood stream) pH (e.g. pH7.4). Preferably, VHH antibodies of the present invention do not bind to DI of canine albumin at an endosomal pH (e.g. pH5.5 or pH6.0) or at a circulatory (e.g. blood stream) pH (e.g. pH7.4).

[0379] Methods of assessing binding to (or ability to bind to) DI of canine albumin would be well-known to a person skilled in the art, and any appropriate method can be used. For example, an ELISA may be used. Thus, in some embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain I (DI) of canine albumin, as determined by an ELISA assay.

[0380] In some embodiments, the ability of a VHH antibody of the present invention to bind to Dill of canine albumin may be determined in (or be as determined in) an ELISA assay in which a canine albumin DI polypeptide (e.g. of SEQ ID NO:5 or SEQ ID NO:6) is immobilised in (or coated on) wells of an ELISA plate, the VHH antibody (test VHH antibody) is subsequently added to wells containing immobilised canine albumin DI polypeptide, and binding of the VHH antibody to the canine albumin DI polypeptide is detected (or measured or quantified). The absence of a detectable (or measurable) signal, or only a negligible (or de minimis) signal (e.g. a signal is that is equivalent to or corresponds to (or essentially equivalent to or essentially corresponds to) binding observed (or measured) with a negative control VHH antibody that does not bind to canine albumin) is indicative that the VHH antibody does not bind to, or significantly bind to, DI of canine albumin.

[0381] For example, in some embodiments, the ability of a VHH antibody of the present invention to bind to canine albumin DI may be determined in (or be as determined in) an ELISA assay that comprises: (a) coating wells of an ELISA (e.g. MaxiSorp) plate with (e.g. 10.72nM of) a canine albumin DI polypeptide (e.g. SEQ ID NO:4) (e.g. for 1 hour at room temperature), and preferably then blocking the wells (e.g. with milk, e.g. 4% milk);

[0382] (b) adding a c-myc tagged (preferably C-terminally c-myc tagged) form of the VHH antibody (test VHH antibody) (e.g. at a (or a range of) concentration(s) of between 1.34nM to 171.52nM, e.g. in 0.25% milk) to the coated (and preferably blocked) wells and incubating (e.g. for 2 hours, e.g. at room temperature);

[0383] (c) adding a mouse-anti-c-myc tag (primary) antibody, e.g. in 1% milk at e.g. 0.2pg / ml (e.g. mouse THE™ c-myc Tag Antibody (Genscript)), and incubating (e.g. for 2 hours, e.g. at room temperature);

[0384] (d) adding an anti-mouse-HRP (secondary) antibody, e.g. in 1% milk at e.g. 0.08 pg / ml, and incubating (e.g. for 2 hours, e.g. at room temperature);

[0385] (e) adding a chromogenic substrate (e.g. 3,3',5,5'-tetramentylbenzidine, TMB) for HRP (horseradish peroxidase) to the wells (e.g. for 7 minutes), preferably then stopping the reaction (e.g. with 0.5M H2SO4);

[0386] (f) detecting (or measuring or quantifying) the signal produced from the chromogenic substrate / HRP reaction (e.g. with a spectrophotometer, e.g. measuring absorbance at 450nm), wherein detected signal is indicative of binding of the VHH antibody to DI of canine albumin. The absence of a detectable (or measurable) signal, or only a negligible (or de minimis) signal (e.g. a signal is that is equivalent to or corresponds to (or essentially equivalent to or essentially corresponds to) binding observed (or measured) with a negative control VHH antibody that does not bind to canine albumin) is indicative that the VHH antibody does not bind to, or significantly bind to, DI of canine albumin. Typically, washing steps (e.g. with a buffer e.g. at the desired pH) may be performed between one or more of steps (a), (b), (c), (d), (e) and (f) above. ELISA may be performed at pH5.5 or pH7.4. Thus, ELISAs may be run with steps (and preferably washes) performed at pH7.4 or pH5.5, depending on the pH under investigation. A particularly preferred ELISA is described in Example 3 herein.

[0387] Preferably, VHH antibodies of the present invention do not bind to, or do not bind significantly to, Domain I (DI) of canine albumin when the VHH antibody is used at a concentration in the range of 1nM to 175nM (e.g. 1.34 nM to 171.52nM), or 1nM to 50nM, or 1nM to 10nM, 10nM to 20nM or 10nM to 50nM (e.g. at a concentration of 1nM, 1.34nM, 2.68nM, 3nM, 5nM, 5.36nM, 10nM or 10.72nM), for example in an ELISA assay as described herein. In some embodiments, VHH antibodies of the present invention do not bind to, or do not bind significantly to, DI of canine albumin when the VHH antibody is used at a concentration of about 10nM (e.g. 10.72nM), for example in an ELISA assay as described herein.

[0388] In some preferred embodiments, VHH antibodies of the present invention (i) do not bind to, or do not bind significantly to, Dill of canine albumin, and (ii) do not bind to, or do not bind significantly to, DI of canine albumin.

[0389] Typically and preferably, the half-life of a VHH antibody in accordance with the present invention in dogs (i.e. in vivo in dogs) is longer than (or extended in comparison to), preferably significantly longer than, the half-life of a (preferably similarly sized) VHH antibody (a control VHH antibody) which does not bind to canine albumin (e.g. a VHH antibody that binds to green fluorescent protein, GFP). Put another way, preferably a VHH antibody in accordance with the present invention has a longer serum half-life in dogs than a VHH antibody (a control VHH antibody) which does not bind to canine albumin. The dogs may be beagles (e.g. male beagles). Methods of assessing the half-life of molecules (e.g. VHH antibodies) in dogs (e.g. in dog serum) would be well-known to a person skilled in the art and any appropriate method can be used (e.g. by ELISA assay performed on samples (e.g. serum samples) taken from dogs at different time points post administration of the VHH antibody to the dogs). A particularly preferred method is described in Example 4 herein.

[0390] For example, a VHH antibody in accordance with the invention may be detectable or quantifiable (e.g. above an established lower limit of quantification, LLQQ) in a dog(s) (or in samples, e.g. serum samples, obtained from a dog(s)) to which the VHH antibody had been administered (e.g. intravenously and / or e.g. at 1 mg / kg) for at least 24 hours, at least 2 days, at least 5 days, at least 10 days, at least 15 days, at least 25 days or at least 28 days after the VHH antibody of the invention was administered to the dog(s). The day of administration of the VHH antibody to the dog(s) may be considered day 0. A particularly preferred method is described in Example 4 herein.

[0391] Constructs of the invention comprising a VHH antibody or a VHH domain of the present invention typically and preferably have a half-life in dogs (i.e. in vivo in dogs) that is longer than (or extended in comparison to), preferably significantly longer than, the half-life of a corresponding (e.g. control) construct that does not comprise a VHH antibody or a VHH domain of the present invention. The discussion above in relation to the extended half-life of VHH antibodies of the present invention may be applied, mutatis mutandis, to constructs (and polypeptides) of the invention.

[0392] In some embodiments, the half-life (or serum half-life) of a VHH antibody or construct or polypeptide of the present invention in dogs (e.g. beagles) may be >5 days, >6 days, >7 days, >8 days, >9 days or >10 days. In some embodiments, the half-life (or serum half-life) of a VHH antibody or construct or polypeptide of the present invention in dogs (e.g. beagles) may be 5 to 15 days, e.g. 5 to 12 days or 8 to 15 days or 8 to 12 days. In some embodiments, the half-life (or serum half-life) of a VHH antibody or construct or polypeptide of the present invention in dogs (e.g. beagles) is about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 days (preferably about 10 days). The half-life (or serum half-life) of a VHH antibody or construct or polypeptide of the present invention in dogs may be determined (or be as determined) by any suitable means, and the skilled person is familiar with appropriate means for half-life determination. In some embodiments, half-life (or serum half-life) of a VHH antibody or construct or polypeptide of the present invention in dogs may be determined (or be as determined) by an ELISA assay. For example, following administration of a VHH antibody or construct or polypeptide to a dog, samples (e.g. serum samples) taken from the dog at a series of time points may be assessed to by ELISA to determine the level (or concentration) of the VHH antibody (or construct or polypeptide) therein. The half-life can then be determined (e.g. as described in Example 8). An anti-VHH antibody may be used as a primary detection reagent in such an ELISA. A particularly suitable ELISA is described in Example 8 herein. In some embodiments, the half-life is the terminal half-life.

[0393] In some embodiments, VHH antibodies of the present invention bind to (or are capable of binding to) Protein A. Protein A is well-known in the antibody field as being a reagent useful for the purification of IgG antibodies, by virtue of its ability to bind to the Fc region (fragment crystallizable region) of IgG antibodies. VHH antibodies do not have a Fc region. The ability of VHH antibodies of the present invention of to bind to Protein A is surprising, and is advantageous as it means that such VHH antibodies of the invention could be purified by using Protein A.

[0394] Methods of determining (or assessing) whether or not a VHH antibody is able to bind to Protein A would be well-known to a person skilled in the art and any appropriate method can be used (e.g. an ELISA assay). The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of VHH antibodies to bind to Protein A in such an assay.

[0395] In some embodiments, the ability of a VHH antibody of the present invention to bind to Protein A may be determined in (or be as determined in) an ELISA assay in which a VHH antibody of the invention is immobilised in (or coated on) wells of an ELISA plate, Protein A (e.g. in the form of Protein A-HRP) is subsequently added to wells containing immobilised VHH antibody, and the binding of Protein A to the VHH antibody is detected (or measured or quantified).

[0396] For example, in some embodiments, the ability of a VHH antibody of the present invention to bind to Protein A may be determined in (or be as determined in) an ELISA assay that comprises:

[0397] (a) coating wells of an ELISA (e.g. MaxiSorp) plate with (e.g. 56nM of) the VHH antibody (test VHH antibody), and preferably then blocking the wells (e.g. with milk, e.g. 4% milk);

[0398] (b) adding Protein A-HRP (e.g. at a dilution of 1 :10,000, e.g. in 1% milk) to the coated (and preferably blocked) wells and incubating (e.g. for 2 hours);

[0399] (c) adding a chromogenic substrate (e.g. 3,3',5,5'-tetramentylbenzidine, TMB) for HRP (horseradish peroxidase) to the wells (e.g. for 7 minutes), preferably then stopping the reaction (e.g. with 0.5M H2SO4);

[0400] (d) detecting (or measuring or quantifying) the signal produced from the chromogenic substrate / HRP reaction (e.g. with a spectrophotometer, e.g. measuring absorbance at 450nm), wherein the detected signal is indicative of binding of the VHH antibody to Protein A. Typically and preferably, a detected signal that is higher (e.g. at least 2x, 3x, 4x or 5x higher) than the signal obtained in control (negative control) ELISA wells (e.g. control wells that were not coated with a VHH, or control wells that were coated with a VHH that does not bind to albumin (e.g. a VHH that binds to GFP)) is indicative of binding of the VHH antibody to Protein A. In some embodiments, washing steps (e.g. with a buffer) may be performed between one or more of steps (a), (b), (c) and (d) above. A particularly preferred ELISA is described in Example 3 herein.

[0401] In some embodiments, constructs and polypeptides of the invention comprising a VHH antibody or a VHH domain of the present invention may also bind to (or be capable of binding to Protein A). The discussion above in relation to Protein A binding in connection with VHH antibodies of the present invention may be applied, mutatis mutandis, to constructs (and polypeptides) of the invention.

[0402] Canine albumin (e.g. native canine albumin) may form oligomers.

[0403] Preferably, VHH antibodies of the present invention are bind to (or are capable of binding to) a monomeric form of canine albumin and to an oligomeric form of canine albumin (e.g. at pH5.5 and / or pH7.4). Canine albumin monomers (i.e. monomeric form of canine albumin) and canine albumin oligomers (i.e. oligomeric form of canine albumin) may be obtained by size exclusion chromatography of native canine albumin. Thus, canine albumin monomers (i.e. monomeric form of canine albumin) and canine albumin oligomers (i.e. oligomeric form of canine albumin) may be separated from each other by size exclusion chromatography of native canine albumin. Thus, preferably, VHH antibodies in accordance with the present invention bind to (or are capable of binding to) monomeric canine albumin in a sample (or fraction) of monomeric canine albumin that has been obtained by size exclusion chromatography of native canine albumin and bind to (or are capable of binding to) oligomeric canine albumin in a sample (or fraction) of oligomeric canine albumin that has been obtained by size exclusion chromatography of native canine albumin. Methods of determining (or assessing) whether or not a VHH antibody is able to bind to monomeric and oligomeric canine albumin would be known to person skilled in the art and any appropriate method can be used (e.g. an ELISA assay). The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of VHH antibodies to bind to monomeric and oligomeric canine albumin in such an assay. A particularly preferred method is described herein in Example 3.

[0404] Discussion of binding and functional properties described herein in relation to VHH antibodies of the present invention may be applied, mutatis mutandis, to constructs and polypeptides of the present invention.

[0405] Nucleic acid molecules comprising nucleotide sequences that encode the VHH antibodies or VHH domains or constructs of polypeptides of the present invention, or nucleic acid molecules substantially homologous thereto, form yet further aspects of the invention.

[0406] Thus, one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a VHH antibody or VHH domain of the present invention or that encodes a construct of the present invention or that encodes a polypeptide of the present invention. Preferred nucleic acid molecules include those encoding a VHH antibody comprising a VHH domain that has an amino acid sequence of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID

[0407] NO:63, SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID

[0408] NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID

[0409] NO:87, SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106 or SEQ ID NQ:107 (or a sequence substantially homologous thereto, e.g. a sequence having at least 80% identity thereto).

[0410] Thus, preferred nucleic acid molecules comprise sequences which encode a VHH domain that has the amino acid sequence of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63,

[0411] SEQ ID NO:71 , SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82,

[0412] SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87,

[0413] SEQ ID NQ:104, SEQ ID NQ:105, SEQ ID NQ:106, SEQ ID NQ:107 (or a sequence substantially homologous thereto, e.g. a sequence having at least 80% identity thereto). Substantially homologous sequences are described elsewhere herein.

[0414] The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecules may be double stranded or single stranded. The nucleic acid molecules may be wholly or partially synthetic or recombinant.

[0415] The VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules of the invention may be isolated VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules. The VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules of the invention are generally "isolated" or "purified" molecules insofar as they are distinguished from any such components that may be present in situ within an animal body or a tissue sample derived from an animal body. The sequences may, however, correspond to or be substantially homologous to sequences as found in an animal body. Thus, the term "isolated" or "purified" as used herein in reference VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules refers to such molecules when isolated from, purified from, or substantially free of their natural environment, e.g. isolated from or purified from an animal body (if indeed they occur naturally), or refers to such molecules when produced by a technical process, i.e. includes recombinant and synthetically produced molecules.

[0416] Thus, when used in connection with VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules, the term "isolated" or "purified" typically refers to such a molecule that is substantially free of cellular material or other proteins from the source from which it is derived. In some embodiments, particularly where the molecule is to be administered to subjects (or animals), such isolated or purified molecules are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

[0417] A person skilled in the art will appreciate that the VHH antibodies, constructs, polypeptides, proteins and nucleic acid molecules of the invention may be prepared in any of several ways well known and described in the art.

[0418] In some embodiments, VHH antibodies, constructs and polypeptides of the invention may be prepared by recombinant methods.

[0419] Nucleic acid molecules encoding VHH antibodies, constructs and polypeptides of the invention can be derived or produced by any appropriate method, e.g. by cloning or synthesis.

[0420] Once nucleic acid molecules of the invention have been obtained, these can be further manipulated by standard recombinant DNA techniques. As part of this further manipulation procedure, the nucleic acid molecules may be incorporated into one or more appropriate expression vectors in order to facilitate production of the VHH antibodies or constructs or polypeptides of the invention.

[0421] Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.

[0422] The invention therefore contemplates a recombinant expression vector containing a nucleic acid molecule of the invention and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

[0423] Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes and are well known in the art. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.

[0424] The recombinant expression vectors of the invention may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.

[0425] The recombinant expression vectors may also contain genes that encode a fusion moiety that provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification (for example appropriate "tags" to enable purification and / or identification may be present, e.g., His tags and / or myc tags).

[0426] Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.

[0427] Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the VHH antibodies or constructs or polypeptides of the invention may be expressed in yeast cells or mammalian cells. In addition, the proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli. Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.

[0428] Alternatively, the VHH antibodies or constructs or polypeptides of the invention may also be expressed in non-human transgenic animals.

[0429] The VHH antibodies or constructs or polypeptides of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.

[0430] A yet further aspect provides an expression construct or expression vector comprising one or more of the nucleic acid molecules of the invention. Preferably the expression constructs or vectors are recombinant.

[0431] Preferably said constructs or vectors further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

[0432] A yet further aspect provides a host cell or virus comprising one or more expression constructs of the invention. Also provided are host cells or viruses comprising one or more of the nucleic acid molecules of the invention. A host cell (e.g. a mammalian host cell or a prokaryotic host cell) or virus expressing an VHH antibody or construct or polypeptide of the invention forms a yet further aspect.

[0433] A yet further aspect of the invention provides a method of producing (or manufacturing) a VHH antibody or construct or polypeptide of the present invention comprising a step of culturing the host cells of the invention. Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid molecules of the invention under conditions suitable for the expression of the encoded VHH antibody or construct or polypeptide; and optionally (ii) isolating or obtaining the VHH antibody or construct or polypeptide from the host cell or from the growth medium / supernatant.

[0434] In some embodiments, methods of producing (or manufacturing or isolating or identifying or generating) a VHH antibody or construct or polypeptide in accordance with the invention may also comprise a step of purification of the antibody or construct or polypeptide product and / or formulating the antibody or construct or polypeptide product into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

[0435] In another aspect, the invention provides a method of binding canine albumin, said method comprising contacting a composition comprising canine albumin with a VHH antibody or construct or polypeptide of the invention. In yet another aspect, the invention provides a method of detecting canine albumin, comprising contacting a composition or sample suspected of containing canine albumin with a VHH antibody or construct or polypeptide of the invention, under conditions effective to allow the formation of canine albumin / VHH antibody complexes, or canine albumin / construct complexes or canine albumin / polypeptide complexes, and detecting the complexes so formed.

[0436] Testing the ability of a VHH antibody or construct or polypeptide of the invention to bind to canine albumin can be carried out by any appropriate method, which are well known and described in the art.

[0437] Compositions comprising at least one VHH antibody of the invention or at least one construct of the invention or at least one polypeptide of the invention constitute a further aspect of the present invention. Formulations (compositions) comprising at least one VHH antibody of the invention or at least one construct of the invention or at least one polypeptide of the invention in admixture with a suitable diluent, carrier or excipient constitute a preferred embodiment of the present invention. Such formulations may be for pharmaceutical use and thus compositions of the invention are preferably pharmaceutically acceptable. Suitable diluents, excipients and carriers are known to the skilled person.

[0438] Thus, in one aspect, the present invention provides a composition comprising a VHH antibody of the invention or a construct of the invention or a polypeptide of the invention, wherein said composition comprises a diluent, carrier or excipient, preferably a pharmaceutically acceptable diluent, carrier or excipient.

[0439] In some embodiments, compositions of the invention may comprise more than one different antibody of the invention (e.g. 2 or 3 or more), or more than one construct of the invention or more than one polypeptide of the invention.

[0440] The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral, intraperitoneal, intravenous, intratumoral, topical or rectal administration.

[0441] The active compounds (e.g. VHH antibodies and constructs) defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p- hydroxybenzoates (para-hydroxybenzoates), or stabilizers, such as EDTA. The solutions may then be filled into injection vials or ampoules.

[0442] Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers, either with an aerosol propellant or provided with means for manual compression.

[0443] The pharmaceutical compositions (formulations) of the present invention are preferably administered parenterally. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the VHH antibody or construct or polypeptide in the form of a nasal or pulmonal spray. As a still further option, the VHH antibodies of the invention can also be administered transdermally , e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.

[0444] Suitable dosage units can be determined by a person skilled in the art.

[0445] The pharmaceutical compositions may additionally comprise further active ingredients in the context of co-administration regimens or combined regimens.

[0446] A further aspect of the present invention provides a VHH antibody or construct or polypeptide of the invention for use in therapy (i.e. for use in therapy of a subject).

[0447] In particular, the present invention provides a construct or polypeptide of the invention that comprises a therapeutic agent for (or active against) a given disease or condition, for use in the treatment or prevention of said disease or condition.

[0448] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31) for use in the treatment or prevention of a disease or condition (e.g. a skin disease or skin condition) that is associated with (or caused by, or mediated by) IL-31 (i.e. for use in the treatment or prevention of a disease or condition that is associated with (or caused by, or mediated by) IL-31 in a subject). Preferably the IL-31 is canine IL-31.

[0449] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31) for use in therapy of a pruritic condition (i.e. for use in the treatment or prevention of a pruritic condition in a subject). Preferably the IL-31 is canine IL-31.

[0450] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31) for use in the treatment or prevention of pruritus (i.e. for use in the treatment or prevention of pruritus in a subject). Preferably the IL-31 is canine IL-31.

[0451] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31) for use in the treatment or prevention of atopic dermatitis (i.e. for use in the treatment or prevention of atopic dermatitis in a subject). Preferably the IL-31 is canine IL-31.

[0452] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa) for use in the treatment or prevention of a disease or condition (e.g. an inflammatory disease or autoimmune disease) that is associated with (or caused by, or mediated by) TNFa (i.e. for use in the treatment or prevention of a disease or condition that is associated with (or caused by, or mediated by) TNFa in a subject). Preferably the TNFa is canine TNFa.

[0453] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa) for use in therapy of an inflammatory disease or autoimmune disease (i.e. for use in the treatment or prevention of an inflammatory disease or autoimmune disease in a subject). Preferably the TNFa is canine TNFa.

[0454] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa) for use in the treatment or prevention of inflammatory bowel disease, dry eye or rheumatoid arthritis (i.e. for use in the treatment or prevention of inflammatory bowel disease, dry eye or rheumatoid arthritis in a subject). Preferably the TNFa is canine TNFa.

[0455] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises (i) p75NTR or (ii) a fragment of p75NTR, or (iii) an analogue of (i) or (ii), for use in the treatment or prevention of pain (e.g. chronic pain) or osteoarthritis (i.e. for use in the treatment or prevention of pain (e.g. chronic pain) or osteoarthritis in a subject). Preferably the p75NTR is canine p75NTR.

[0456] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises (i) insulin or (ii) a fragment of insulin, or (iii) an analogue of (i) or (ii), for use in the treatment or prevention of Type II diabetes (i.e. for use in the treatment or prevention of Type I or Type II diabetes in a subject). Preferably, the insulin is a long acting insulin (or a fragment of a long acting insulin, or a long acting insulin analogue). The insulin may be canine insulin.

[0457] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises a GLP-1 receptor agonist for use in the treatment or prevention of obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes, i.e. for use in the treatment or prevention obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes in a subject.

[0458] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises (i) GLP-1 or (ii) a fragment of GLP-1 , or (iii) an analogue of (i) or (ii), for use in the treatment or prevention of obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes, i.e. for use in the treatment or prevention obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes in a subject. Preferably, the GLP-1 is a long acting GLP-1 (or a fragment of a long acting GLP-1 , or a long acting GLP-1 analogue). Preferably the GLP-1 is canine GLP-1.

[0459] In some embodiments, the present invention provides a construct or polypeptide of the invention that comprises (i) Exendin-4 or (ii) a fragment of Exendin-4, or (iii) an analogue of (i) or (ii), for use in the treatment or prevention of obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes, i.e. for use in the treatment or prevention obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease) or Type II diabetes in a subject.

[0460] “Therapy” includes treatment and prophylaxis, i.e. in includes both treatment and preventative uses.

[0461] Thus, in some embodiments, the present invention provides a construct or polypeptide of the invention for use in the treatment of diseases or conditions described herein. In some embodiments, the present invention provides a construct or polypeptide of the invention for use in the prevention of diseases or conditions described herein.

[0462] The in vivo methods and uses as described herein are generally carried out in a mammal. Preferably, however, the mammal is a canine. Thus, preferably the mammal is a dog (Canis lupus familiaris). Thus, subjects or patients treated in accordance with the present invention are preferably canines. Thus, subjects or patients treated in accordance with the present invention are preferably dogs.

[0463] In some embodiments, subjects or patients will be those having a disease or a condition described herein, or those at risk of having or developing a condition described herein, or those suspected of having a condition described herein. In some embodiments, the present invention provides a construct or polypeptide of the invention for use in the treatment of a disease or condition described herein in a subject that has been diagnosed with said disease or condition.

[0464] Alternatively viewed, the present invention provides a method of treating or preventing a (or a given) disease or condition, which method comprises administering to a patient in need thereof a therapeutically effective amount of construct or polypeptide of the invention that comprises a therapeutic agent for (or active against) said disease or condition. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0465] In another aspect, the present invention provides a method of treating or preventing a disease or condition (e.g. a skin disease or skin condition) that is associated with (or caused by, or mediated by) IL-31 (preferably canine IL-31), which method comprises administering to a patient in need thereof a therapeutically effective amount of construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0466] In another aspect, the present invention provides a method of treating or preventing a pruritic condition (e.g. pruritus or atopic dermatitis) which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention. In another aspect, the present invention provides a method of treating or preventing a disease or condition (e.g. an inflammatory disease or autoimmune disease) that is associated with (or caused by, or mediated by) TNFa (preferably canine TNFa), which method comprises administering to a patient in need thereof a therapeutically effective amount of construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0467] In another aspect, the present invention provides a method of treating or preventing an inflammatory disease or autoimmune disease, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0468] In another aspect, the present invention provides a method of treating or preventing inflammatory bowel disease, rheumatoid arthritis or dry eye, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0469] In another aspect, the present invention provides a method of treating or preventing pain (e.g. chronic pain) or osteoarthritis, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises (i) p75NTR, or (ii) a fragment of p75NTR, or (iii) an analogue of (i) or (ii). Preferably the p75NTR is canine p75NTR. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0470] In another aspect, the present invention provides a method of treating or preventing Type I or Type II diabetes, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises (i) insulin or (ii) a fragment of insulin or (iii) an analogue of (i) or (ii). Preferably, the insulin is a long acting insulin (or a fragment of a long acting insulin, or a long acting insulin analogue). Preferably the insulin is canine insulin. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0471] In another aspect, the present invention provides a method of treating or preventing obesity or kidney disease (e.g. chronic kidney disease or diabetic kidney disease) or Type II diabetes, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises (i) GLP-1, or (ii) a fragment of GLP-1 , or (iii) an analogue of (i) or (ii). Preferably, the GLP-1 is a long acting GLP-1 (or a fragment of a long acting GLP-1 , or a long acting GLP-1 analogue). Preferably the GLP-1 is canine GLP-1. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0472] In another aspect, the present invention provides a method of treating or preventing obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes, which method comprises administering to a patient in need thereof a therapeutically effective amount of a construct or polypeptide of the invention that comprises (i) Exendin-4, or (ii) a fragment of Exendin-4, or (iii) an analogue of (i) or (ii). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0473] A therapeutically effective amount will be determined based on the clinical assessment and can be readily monitored.

[0474] Further alternatively viewed, the present invention provides the use of a VHH antibody or construct or polypeptide of the invention as defined herein in the manufacture of a medicament for use in therapy. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0475] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises an agent that binds to IL-31 (e.g. a VHH antibody that binds to IL-31) in the manufacture of a medicament for the treatment or prevention of a disease or condition (e.g. a skin disease or skin condition) that is associated with (or caused by, or mediated by) IL-31 (i.e. for use in the treatment or prevention of a disease or condition that is associated with (or caused by, or mediated by) by IL-31 in a subject). In some such embodiments, the disease or condition is a pruritic condition (e.g. pruritus or atopic dermatitis). Preferably the IL- 31 is canine IL-31. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention. In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises an agent that binds to TNFa (e.g. a VHH antibody that binds to TNFa) in the manufacture of a medicament for the treatment or prevention of a disease or condition (e.g. an inflammatory disease or an autoimmune disease) that is associated with (or caused by, or mediated by) TNFa (i.e. for use in the treatment or prevention of a disease or condition that is associated with (or caused by, or mediated by) TNFa in a subject). In some such embodiments, the disease or condition is an inflammatory disease or an autoimmune disease. In some embodiments, the disease or condition is inflammatory bowel disease, rheumatoid arthritis or dry eye. Preferably the TNFa is canine TNFa. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0476] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises (i) p75NTR or (ii) a fragment of p75NTR or (iii) an analogue of (i) or (ii) in the manufacture of a medicament for the treatment or prevention of pain (e.g. chronic pain) or osteoarthritis. Preferably the p75NTR is canine p75NTR. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0477] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises (i) insulin or (ii) a fragment of insulin or (iii) analogue of (i) or (ii) in the manufacture of a medicament for the treatment or prevention of Type II diabetes. Preferably, the insulin is a long acting insulin (or a fragment of a long acting insulin, or a long acting insulin analogue). Preferably the insulin is canine insulin. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0478] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises (i) GLP-1 or (ii) a fragment or (iii) an analogue of (i) or (ii) in the manufacture of a medicament for the treatment or prevention of obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes. Preferably, the GLP-1 is a long acting GLP-1 (or a fragment of a long acting GLP-1, or a long acting GLP-1 analogue). Preferably the GLP-1 is canine GLP-1. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0479] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises (i) Exendin-4 or (ii) a fragment or (iii) an analogue of (i) or (ii) in the manufacture of a medicament for the treatment or prevention of obesity, or kidney disease (e.g. chronic kidney disease or diabetic kidney disease), or Type II diabetes. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0480] In another aspect, the present invention provides a construct or polypeptide of the invention that comprises an agent that binds to GnRH (e.g. a VHH antibody that binds to GnRH) for use in reducing reproductive ability, preventing reproduction, reducing testicular function or reducing fertility in a subject, preferably a canine subject. In preferred embodiments the subject is a male (preferably a male canine). Preferably the GnRH is canine GnRH. Embodiments described herein in relation to other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0481] In another aspect, the present invention provides a method of reducing reproductive ability, preventing reproduction, reducing testicular function or reducing fertility in a subject, which method comprises administering to a subject an effective amount of construct or polypeptide of the invention that comprises an agent that binds to GnRH (e.g. a VHH antibody that binds to GnRH). In preferred embodiments the subject is a male (preferably a male canine). Preferably the GnRH is canine GnRH. Embodiments described herein in relation to other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0482] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises an agent that binds to GnRH (e.g. a VHH antibody that binds to GnRH) in the manufacture of a product for reducing reproductive ability, preventing reproduction, reducing testicular function or reducing fertility in a subject. In preferred embodiments the subject is a male (preferably a male canine). Preferably the GnRH is canine GnRH. Embodiments described herein in relation to other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0483] In some embodiments, the invention provides the use of a construct or polypeptide of the invention that comprises an agent that binds to GnRH (e.g. a VHH antibody that binds to GnRH) for reducing reproductive ability, preventing reproduction, reducing testicular function or reducing fertility in a subject. In preferred embodiments the subject is a male (preferably a male canine). Preferably the GnRH is canine GnRH. Embodiments described herein in relation to other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0484] A VHH antibody or construct or polypeptide or method or uses of the present invention may be used in combination with (e.g. in the same therapeutic or prophylactic regimen as) one or more other therapeutic or prophylactic agents (e.g. one or more other agents used for the treatment or prevention of a disease or condition described herein).

[0485] In another aspect, the present invention provides a use of a VHH antibody which binds to canine albumin of the present invention to extend the half-life (e.g. serum half-life or circulatory half-life) of an agent (e.g. a therapeutic agent) in a subject (preferably a canine subject). Embodiments described herein in relation to other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention.

[0486] The invention further includes kits comprising one or more of the VHH antibodies or constructs or polypeptides of the invention, or one or more of the nucleic acid molecules encoding a VHH antibody or construct or polypeptide of the invention, or one or more recombinant expression vectors comprising a nucleic acid molecule of the invention, or one or more host cells or viruses comprising a recombinant expression vector or nucleic acid molecule of the invention. Preferably said kits are for use in the methods and uses as described herein, e.g. the therapy, methods and uses as described herein, or are for use in the in vitro assays or methods as described herein. Preferably, said kits comprise instructions for use of the kit components. Preferably, said kits are for treating or preventing diseases or conditions as described elsewhere herein, and optionally comprise instructions for use of the kit components to treat or prevent such diseases.

[0487] The VHH antibodies and constructs and polypeptides of the invention as defined herein may also be used as molecular tools for in vitro or in vivo applications and assays. As the VHH antibodies and constructs are capable of binding to an antigen (here canine albumin), these can function as members of specific binding pairs and these molecules can be used in any assay where the particular binding pair member is required.

[0488] Thus, yet further aspects of the invention provide a reagent that comprises a VHH antibody or construct or polypeptide of the invention as defined herein and the use of such VHH antibodies, constructs and polypeptides as molecular tools, for example in in vitro or in vivo assays. As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. Therefore, a "VHH antibody", as used herein, means "at least a first VHH antibody". The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

[0489] Unless otherwise clear from the context, preferably the above described abilities and properties are observed at a measurable or significant level and more preferably at a statistically significant level, when compared to appropriate control levels. In any statistical analysis referred to herein, preferably the statistically significant difference as compared to a relevant control or other comparative entity or measurement has a probability value of < 0.1 or < 0.1, preferably < 0.05 or < 0.05. If a property is described as being not significant, it is preferably not statistically significant. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.

[0490] In addition, where the terms “comprise”, “comprises”, “has” or “having”, or other equivalent terms are used herein, then in some more specific embodiments these terms include the term “consists of’ or “consists essentially of’, or other equivalent terms.

[0491] LIST AND TABLES OF AMINO ACID (aa) SEQUENCES DISCLOSED HEREIN AND THEIR SEQUENCE IDENTIFIERS (SEQ ID NOs):

[0492] SEQ ID NO:1: Amino acid sequence of canine albumin (full-length mature canine albumin)

[0493] EAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACAAE

[0494] ESGANCDKSLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDDNP

[0495] GFPPLVAPEPDALCAAFQDNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGVFA

[0496] ECCQAADKAACLGPKIEALREKVLLSSAKERFKCASLQKFGDRAFKAWSVARLSQ

[0497] RFPKADFAEISKVVTDLTKVHKECCHGDLLECADDRADLAKYMCENQDSISTKLKE

[0498] CCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLY EYARRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNLV

[0499] KTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKVGTKCCKKPES

[0500] ERMSCAEDFLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCFSGLEVDETYV PKEFNAETFTFHADLCTLPEAEKQVKKQTALVELLKHKPKATDEQLKTVMGDFGAF

[0501] VEKCCAAENKEGCFSEEGPKLVAAAQAALV

[0502] SEQ ID NO:2: Amino acid sequence of human albumin (full-length mature human albumin)

[0503] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD

[0504] ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNP

[0505] NLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFT

[0506] ECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS

[0507] QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLY

[0508] EYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLI

[0509] KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA

[0510] KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFV

[0511] EKCCKADDKETCFAEEGKKLVAASQAALGL

[0512] EAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACAAE

[0513] ESGANCDKSLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDDNP GFPPLVAPEPDALCAAFQDNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGVFA

[0514] ECCQAADKAACLGPKIEALREKVLLSSA

[0515] SEQ ID NO:4: Amino acid sequence of Domain I (DI) of canine albumin with a C-terminal Avi-His tag (the Avi-His tag is shown underlined), and an additional methionine (M) residue at the N-terminus

[0516] MEAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACA AEESGANCDKSLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDD NPGFPPLVAPEPDALCAAFQDNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGV

[0517] FAECCQAADKAACLGPKIEALREKVLLSSAGGGGSGLNDIFEAQKIEWHEGGHHH HHH

[0518] SEQ ID NO: 5: Amino acid sequence of Domain III (Dill) of canine albumin

[0519] LVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKVGT KCCKKPESERMSCAEDFLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCFSG LEVDETYVPKEFNAETFTFHADLCTLPEAEKQVKKQTALVELLKHKPKATDEQLKT

[0520] VMGDFGAFVEKCCAAENKEGCFSEEGPKLVAAAQAALV

[0521] SEQ ID NO:6: Amino acid sequence of Domain III (Dill) of canine albumin with a C-terminal Avi-His tag (the Avi-His tag is shown underlined), and an additional methionine (M) residue at the N-terminus

[0522] MLVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKV GTKCCKKPESERMSCAEDFLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCF SGLEVDETYVPKEFNAETFTFHADLCTLPEAEKQVKKQTALVELLKHKPKATDEQL

[0523] KTVMGDFGAFVEKCCAAENKEGCFSEEGPKLVAAAQAALVGGGGSGLNDIFEAQ KIEWHEGGHHHHHH

[0524] SEQ ID NO:15: An exemplary c-myc / His tag amino acid sequence

[0525] GGSAEQKLISEEDLGGHHHHHH

[0526] SEQ ID NO:16: An exemplary c-myc / His tag amino acid sequence

[0527] SAEQKLISEEDLGGHHHHHH

[0528] GGGSGGGSGGGSGGGS SEQ ID NO:99: An exemplary amino acid linker sequence

[0529] GGGSGGGSGGGSGGGSGGGS

[0530] SEQ ID NQ:100: Amino acid sequence of feline albumin (full-length mature feline albumin)

[0531] EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLVNEVTEFAKGCVA

[0532] DQSAANCEKSLHELLGDKLCTVASLRDKYGEMADCCEKKEPERNECFLQHKDDN

[0533] PGFGQLVTPEADAMCTAFHENEQRFLGKYLYEIARRHPYFYAPELLYYAEEYKGV

[0534] FTECCEAADKAACLTPKVDALREKVLASSAKERLKCASLQKFGERAFKAWSVARL

[0535] SQKFPKAEFAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYICENQDSISTKLK ECCGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVCKNYQEAKDVFLGTFLY

[0536] EYSRRHPEYSVSLLLRLAKEYEATLEKCCATDDPPACYAHVFDEFKPLVEEPHNLV KTNCELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSLGKVGSKCCTHPEA

[0537] ERLSCAEDYLSWLNRLCVLHEKTPVSERVTKCCTESLVNRRPCFSALQVDETYVP

[0538] KEFSAETFTFHADLCTLPEAEKQIKKQSALVELLKHKPKATEEQLKTVMGDFGSFV DKCCAAEDKEACFAEEGPKLVAAAQAALA

[0539] SEQ ID NO:1Q1: Amino acid sequence of porcine albumin (full-length mature porcine albumin)

[0540] DTYKSEIAHRFKDLGEQYFKGLVLIAFSQHLQQCPYEEHVKLVREVTEFAKTCVAD

[0541] ESAENCDKSIHTLFGDKLCAIPSLREHYGDLADCCEKEEPERNECFLQHKNDNPDI PKLKPDPVALCADFQEDEQKFWGKYLYEIARRHPYFYAPELLYYAIIYKDVFSECC

[0542] QAADKAACLLPKIEHLREKVLTSAAKQRLKCASIQKFGERAFKAWSLARLSQRFPK

[0543] ADFTEISKIVTDLAKVHKECCHGDLLECADDRADLAKYICENQDTISTKLKECCDKP

[0544] LLEKSHCIAEAKRDELPADLNPLEHDFVEDKEVCKNYKEAKHVFLGTFLYEYSRRH

[0545] PDYSVSLLLRIAKIYEATLEDCCAKEDPPACYATVFDKFQPLVDEPKNLIKQNCELF

[0546] EKLGEYGFQNALIVRYTKKVPQVSTPTLVEVARKLGLVGSRCCKRPEEERLSCAE DYLSLVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYKPKEFVEGT

[0547] FTFHADLCTLPEDEKQIKKQTALVELLKHKPHATEEQLRTVLGNFAAFVQKCCAAP DHEACFAVEGPKFVIEIRGILA

[0548] SEQ ID NQ:102: Amino acid sequence of a consensus CDR2 sequence

[0549] YINXGST , wherein “X” is any amino acid. SEQ ID NQ:103: Amino acid sequence of a consensus CDR2 sequence

[0550] YINXGST , wherein “X” is G or A.

[0551] SEQ ID NQ:104: Amino acid sequence of a VHH domain that is a variant of the VHH domain of VHH208

[0552] EVQLVESGGGLVQPGGSLRLSCAASGSFYSINVMGWYRQAPGKQRELVAAYINA GSTNYADSVKGRFTISRDRAKNMVYLQMNSLKPEDTAVYYCNVAGYTSNRGHLN SWGQGTLVTVSS

[0553] SEQ ID NQ:105: Amino acid sequence of a VHH domain that is a variant of the VHH domain of VHH209

[0554] EVQLVESGGGLVQPGGSLRLSCAASGSFYSINVMGWYRQAPGKQRQLVAAYING GSTNYADSVKGRFTISRDRAKNMVYLQMNSLKPEDTAVYYCNVAGYTSNRGHLN SWGQGTQVTVSS

[0555] SEQ ID NQ:106: Amino acid sequence of a VHH domain that is a variant of the VHH domain of VHH215

[0556] EVQLVESGGDLVKPGGSLRLSCVASGSFYSINVMGWVRQAPGKGRQWVAAYING GSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCNVAGYTSNRGHLNS WGQGTLVTVSS

[0557] SEQ ID NO:133: Amino acid sequence of full-length canine p75NTR

[0558] VGAGAAGCAMDGPRLLLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGV

[0559] AQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVC

[0560] RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDRQNTVCEECPDGTYSDEANH

[0561] VDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPSEDSDSTAPSTEEP

[0562] ELPPDQEI I ASTMADWTTVMGSSQPWTRGTADN LI PVYCSI LAAVWGLVAYIAFK

[0563] RWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVDSQSLHDQQPHTQTAAG

[0564] QALKGDGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEHIDSFTHEAC PARALLASWAAQDSATLDALLAALRRIQRADIVESLCSESTATSPV

[0565] SEQ ID NO:134: Amino acid sequence of canine insulin A-chain GIVEQCCTSICSLYQLENYCN

[0566] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Table A - Amino acid sequences of cA8 VHH antibody

[0567] Table B - Amino acid sequences of cA83 VHH antibody

[0568] ill

[0569] Table C - Amino acid sequences of cA159 VHH antibody

[0570] Table D - Amino acid sequences of cA160 VHH antibody

[0571] Table E - Amino acid sequences of cA77 VHH antibody

[0572] Table F - Amino acid sequences of VHH 208 antibody (also referred to as cA208)

[0573] Table G - Amino acid sequences of 209 VHH antibody (also referred to as cA209)

[0574] Table H - Amino acid sequences of 215 VHH antibody (also referred to as cA215)

[0575] Table I - Amino acid sequences of identified VHH antibodies that are highly related to (or cluster with) the cA8 VHH antibody described herein. The CDR sequences of the VHH antibodies in Table I are the same as the CDR sequences of the cA8 antibody. Table J - Amino acid sequence of identified VHH antibody that is highly related to (or clusters with) the cA83 VHH antibody described herein. The CDR sequences of the VHH antibody in Table J are the same as the CDR sequences of the cA83 antibody.

[0576] Table K - Amino acid sequence of identified VHH antibody that is highly related to (or clusters with) the cA160 VHH antibody described herein. The CDR sequences of the VHH antibody in Table K are the same as the CDR sequences of the cA160 antibody. Table L - Amino acid sequences of identified VHH antibodies that are highly related to (or cluster with) the cA77 VHH antibody described herein. The CDR sequences of the VHH antibody #MP03C04 in Table L are the same as the CDR sequences of the cA77 antibody. The CDR1 and CDR2 sequences of the VHH antibody #MP03D04 in Table L are the same as the CDR1 and CDR2 sequences of the cA77 VHH antibody. The CDR3 sequence of the VHH antibody #MP03D04 in Table L is different from the CDR3 sequence of the cA77 VHH antibody.

[0577] Table M - Amino acid sequences of 272BIOPI01MP03D04 VHH antibody

[0578] Table N - Amino acid sequences of a caninized version of cA8 (also referred to herein as cA8(CAN)

[0579] Table O - Amino acid sequences of an anti-TNFa VHH (which is part of the VBD- 0331-01 construct described herein). CDR sequences identified using the IMGT numbering scheme

[0580] Table P - Amino acid sequences of a caninized version of the anti-TNFa VHH of Table O. CDR sequences identified using the IMGT numbering scheme.

[0581] Table Q - Amino acid sequence of construct VBD-0331-01

[0582] Table R - Amino acid sequence of a caninized version of the construct VBD-0331- 01. The caninized version of the construct is named V033-0033-M01.

[0583] The invention will now be further described in the following non-limiting Examples with reference to the following drawings:

[0584] Figure 1 : Graph showing that an immune response to canine albumin was detected in post-immune Llama serum used to construct library L0041VHH.

[0585] Figure 2: Graph showing that an immune response to canine albumin was detected in post-immune Llama serums used to construct libraries L0093VHH and L0094VHH.

[0586] Figure 3: Biacore sensorgrams for candidate VHHs (cA8, cA74, cA77, cA83, cA159, cA160) binding to canine albumin at pH 6.0. GFP_NB = negative control VHH that does not bind to albumin (GFP_NB is a VHH antibody that binds to green fluorescent protein, GFP). Figure 4: Biacore sensorgrams for candidate VHHs (cA8, cA74, cA77, cA83, cA159, cA160) binding to canine albumin at pH 7.4. (Some interference / noise was observed during dissociation in the cA8 sensorgram at 25 nm concentration only.) GFP_NB = negative control VHH that does not bind to albumin.

[0587] Figure 5: Biacore sensorgrams for VHH cA8 at both pH 6.0 (A) and pH 7.4 (B) showing no binding to feline or porcine serum albumins. (Due to noise observed with cA8 at 25 nM at pH 7.4, this set of data has been omitted.)

[0588] Figure 6: Graphs showing the results of_FcRn interference ELISA. The anti-canine albumin VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159 do not significantly reduce canine FcRn binding to canine albumin. (+) Control VHH = positive control (a strong albumin binding VHH previously determined to interfere with FcRn-albumin binding). The positive control VHH potently inhibits the interaction between canine FcRn and canine albumin. (-) Control VHH = negative control (a non-albumin binding VHH; GFP_NB).

[0589] Figure 7: Graphs showing results of an ELISA experiment to assess canine albumin (CSA) domain binding of the VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159 at pH5.5. cA8, cA77, cA74, cA83, cA160 and cA159 each bind to CSA (i.e. full-length mature CSA), but not individual domain I (DI; CSA-DI) or domain III (Dill; CSA-DI 11) . cA74 was able to bind to domain I (CSA-DI). (-) control VHH = a negative control VHH antibody (GFP_NB) that does not bind to albumin. (+) control, VHH = a strong albumin binding VHH previously determined to interfere with FcRn-albumin binding, that bound Domain III.

[0590] Figure 8: Graphs showing results of an ELISA experiment to assess canine albumin (CSA) domain binding of the VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159 at pH7.4. cA8, cA77, cA74, cA83, cA160 and cA159 each bind to CSA (i.e. full-length mature CSA), but not individual domain I (DI; CSA-DI) or domain III (Dill; CSA-DI 11) . cA74 was able to bind to domain I (CSA-DI). (-) control VHH = a negative control VHH antibody (GFP_NB) that does not bind to albumin. (+) control, VHH = a strong albumin binding VHH previously determined to interfere with FcRn-albumin binding, that bound Domain III. Figure 9: Graphs showing results of an ELISA experiment to assess human serum albumin (HSA) binding of the VHH antibodies cA8, cA77, cA74, cA160 and cA159 at pH5.5. CSA = canine serum albumin.

[0591] Figure 10: Graphs showing results of an ELISA experiment to assess human serum albumin (HSA) binding of the VHH antibodies cA8, cA77, cA74, cA160 and cA159 at pH7.4. CSA = canine serum albumin.

[0592] Figure 11 : Graph showing results of an ELISA experiment to assess protein A binding and anti-VHH binding to the VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159. (-) control = a negative control VHH antibody (GFP_NB) that does not bind to albumin.

[0593] Figure 12: Graph showing results of an ELISA experiment_to assess monomeric CSA and oligomeric CSA binding by the VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159. (-) control = a negative control VHH antibody (GFP_NB) that does not bind to albumin.

[0594] Figure 13: Graph showing results of an ELISA experiment_to assess ability of the VHH antibodies cA8, cA77, cA74, cA83, cA160 and cA159 to bind to canine albumin directly from serum (10% Canine Serum). “Canine Serum Albumin” = native purified canine serum albumin. “No serum or albumin” = uncoated control wells. (-) control = a negative control VHH antibody (GFP_NB) that does not bind to albumin.

[0595] Figure 14: Time course of VHH cA8 and anti-GFP VHH (GFP_NB) concentrations in dog serum following single dose i.v. administration of either VHH to dogs. Data points depicts mean ± SD VHH concentration in n=2 replicates in the respective ELISA. The profile is shown for each individual animal. Data points with hollow symbols are values detected below LLOQ. The data points were plotted with (A) having a logarithmic scale for time and a linear scale for VHH concentration, and (B) having a linear scale for time and logarithmic scale for VHH concentration.

[0596] Figure 15: Graphs showing the results of FcRn interference ELISA. The anticanine albumin VHH antibodies cA208, cA209, cA215 and cA8 do not significantly reduce canine FcRn binding to canine albumin. cA208, cA209, cA215 are caninised variants of cA8. Control (+) VHH = positive control (a strong albumin binding VHH previously determined to interfere with FcRn-albumin binding). The positive control VHH (Control (+) VHH) potently inhibits the interaction between canine FcRn and canine albumin. Control (-) VHH = negative control (a non-albumin binding VHH, GFP_NB).

[0597] Figure 16: Biacore sensorgrams for VHH cA8 at both pH 6.0 and pH 7.4 showing no binding to human serum albumin.

[0598] EXAMPLES

[0599] EXAMPLE 1 : Immunisations and

[0600] Immunisations

[0601] Single domain antibodies were obtained from llamas immunised with native protein. Llamas were injected with native caAlb (Abbexa, cat nr. Abx655874; SEQ ID NO:1) antigen formulated in Incomplete Freund’s Adjuvant. Animals were immunised with seven subcutaneous injections (two injections with 80 pg / dose followed by five injections with 40 pg / dose) at weekly intervals. caAlb is an abbreviation for canine albumin. One week after the last boost, sera were collected to define antibody titres against canine albumin.

[0602] Antigen specific serum titres were assessed via ELISA and compared to pre- immune serum samples. 96-well plates (Maxisorp; Nunc) were coated with the native caAlb (Abbexa, cat nr. Abx655874), before being blocked. The plates were then incubated with diluted sera samples, with the presence of anti-caAlb antibodies demonstrated by incubating mouse anti-llama IgG followed by an anti-mouse immunoglobulin peroxidase conjugate (JIR, cat nr. 715-035-150). The substrate solution (TMB solution) was added before the reaction was stopped with H2SO4 and plates read in the plate reader at OD 450 nm.

[0603] In each of three immunisations, OD 450 nm values vs. log serum dilutions of pre- and post-immune serum llama plotted using GraphPad Prism 7™, applying a nonlinear regression (curve fit) of a log (agonist) vs. response - variable slope (four parameters), shown Figure 1 and Figure 2. A clear immune response to canine albumin could be detected from all three serums.

[0604] Library build

[0605] RNA was extracted from PBMC (peripheral blood mononuclear cells) of 3 immunised llamas (400 ml each), with 40 pg of RNA used for cDNA synthesis using random primers. The cDNA was used in a primary PCR amplification using nontagged primers annealing at the Leader sequence and Hinge CH1 regions, followed by a secondary PCR amplification introducing restriction endonuclease sites for cloning of VHH genes in pDCL1 phagemid vector. This phage display vector permits the inducible periplasmic expression of VHH as soluble C-terminally His and c-Myc- tagged proteins in E. coli strain TG1. The libraries were electroporated into TG1 E.coli cells and bacterial glycerol stock of the immune libraries were stored at - 80°C (L0041VHH; L0093VHH; L0094VHH).

[0606] Selections

[0607] Phage production from the llama VHH library pool were used in three consecutive rounds of phage display selection using native canine albumin protein (Abbexa, cat nr. abx655874). Selection rounds were performed using 10, 1 , 0.1 and 0.01 pg / ml of native canine albumin (PBS buffer) with washing to remove nonspecific phage, followed by specific phage elution with trypsin (total elution).

[0608] Serial dilutions of the eluted phages were performed and used to infect exponentially growing TG1 E. coli. Infected TG1 was plated on LBCarb100Glu2% plates and enrichment values calculated over the background (without antigen for selection).

[0609] EXAMPLE 2: Periplasmic extract primary screening

[0610] Screening for binding by ELISA

[0611] Individual clones from the second and third rounds of selection condition outputs were picked into 96-well Master Plates and tested as Periplasmic Extract (P.E.) for binding to canine albumin at pH 7.4 and 5.5 by ELISA. For P.E. binding ELISA, MaxiSorp™ high protein-binding capacity 96 well ELISA plates, were coated with 1 pg / ml of native canine albumin (Abbexa, cat nr. abx655874), diluted in PBS, overnight at 4°C. Wash steps were performed between each incubation and were 3x Phosphate-Buffered Saline (PBS, pH 7.4) or Citrate-Phosphate Buffer (CPB, pH

[0612] 5.5) containing 0.05 % Tween. The next day, plates were blocked for 1 hour at room temperature with 250 pl / well of 4 % Skimmed-milk / PBS. After blocking, plates were incubated with 20 pl of P.E. + 80 pl in 1 % Skimmed-milk / PBS (pH 7.4) or 1% Skimmed-milk / CPB (pH 5.5) per well, for 1 hour at RT with shaking. Plates were incubated with 100 pl of mouse anti-c-Myc antibody (Roche, cat nr. 11667203001) followed by secondary antibody anti-mouse IgG peroxidase conjugate (JI R, cat nr. 715-035-150) in 1 % Skimmed-milk / PBS (pH 7.4) or 1 % Skimmed-milk / CPB (pH

[0613] 5.5), for 1 hour at RT with shaking. Rabbit anti-canine albumin antibody (Fitzgerald, cat nr. 70R-15096) at 1 pg / ml followed by anti-rabbit IgG peroxidase conjugate (JIR, cat nr. 111-035-144) at 0.16 pg / ml was used as coating control. An irrelevant VHH and two blanks (no phage) were also included as controls. After a final wash step, substrate solution (TMB solution) was added to the plates. Reaction was stopped with H2SO4 and plates read at OD 450 nm.

[0614] A large number of clones showed binding above background at either pH 5.5 and / or pH 7.4. Clones not exhibiting binding at either pH were triaged. However, before selecting which clones to express as VHHs (i.e. as isolated VHH antibodies), additional characterisation via off-rate determination was required.

[0615] Screening for off-rate by SPR

[0616] Clones were further screened and selected for their off-rate when binding to native canine albumin, by SPR (surface plasmon resonance) at room temperature (about 22°C). Native canine albumin (Abbexa, cat nr. abx655874) was immobilised on CM5 sensor chip in 10 mM of Sodium Acetate pH 4.5 at approximately 700 Rll (Resonance Units) by amine coupling. P.E. of selected clones were diluted 1:5 in HBS-EP+ pH 7.4 buffer or HBS-EP+ pH 5.5 buffer and injected over the immobilised protein at 30 pl / min for 2 minutes. (HBS-EP+ stands for Hepes Buffer Saline - Ethylenediamine tetra-acetic acid (EDTA) and Surfactant P20; 0.01 M HEPES pH 7.4, 0.15 M NaCI, 3 mM EDTA, 0.005% v / v Surfactant P20. Note: HBS- EP+ buffer was adjusted to pH5.5 for the experiment at pH5.5.) After this binding injection, samples were left to dissociate for 5 minutes. The chip was regenerated between runs with one injection of 1 M NaCI / 1 mM glycine pH 1.5. Controls included an anti-canine albumin antibody (Fitzgerald, cat nr. 70 R 15096) at 3 pg / ml in the same buffer and an irrelevant VHH P.E.

[0617] Table 1 : Determined off-rates for selected Periplasmic Extracts binding to

[0618] Canine Serum albumin at both pH7.4 and 5.5 Overall, the results of clones at pH 5.5 and 7.4 for off-rate (kd) and RU max were generally similar, with a large number exhibiting binding. Selected clones are shown in Table 1 above.

[0619] Sequencing and clustering

[0620] The positive binders were sequenced. Clones were classified by families according to the different HCDR3 sequence, with a total of 60 unique HCDR3 sequences identified from all masterplates. These were further clustered into groups of similar CDR sequences, and clusters and unique sequences, alongside off-rates (<1 E-02) and PE ELISA results were used to select clones to be expressed as VHHs.

[0621] The clones named (i.e. with 272Bio IDs) cA8, cA74, cA83, cA159, cA160, cA77 were selected for further investigation (i.e. selected as candidate VHH antibodies of interest). The amino acid sequences (CDR sequences and full VHH domain sequences) of cA8, cA83, cA159, cA160, cA77 are set forth herein in Tables A, B, C, D and E. cA74 had CDR and full VHH sequences that are different from each of cA8, cA83, cA159, cA160 and cA77. The amino sequences of other VHHs clustered in the same groups as certain of the selected candidates are set forth herein in Tables I, J, K, L and M.

[0622] EXAMPLE 3: Expression of candidate VHHs by E. coli or HEK293 and characterisation of VHHs

[0623] After selecting the VHH based on PE extract binding and off rate results, the synthetic genes coding for the VHH variable domains with c-myc and His tags were cloned into pET15b bacterial expression vector with a periplasmic secretion leader sequence. The c-myc and His tags were positioned C-terminal to the VHH sequence. The sequence of the c-myc and His-tag motif used in the E.coli expressed VHH antibodies was GGSAEQKLISEEDLGGHHHHHH (SEQ ID NO:15). E.coli strain BL21 DE3 were transformed and grown in ZYP-5052 autoinduction media for 68 h at 18°C. Produced VHH antibodies were captured from clarified supernatants using Ni-NTA beads (Qiagen) on gravity fed columns. Eluted VHHs were buffer exchanged to 1x PBS pH 7.4 and concentrated using 10k cutoff spin concentrators (Amicon, cat nr. UFC801096D). Purified VHH protein was analysed by SDS-PAGE and SEC-HPLC for the presence of correct protein size. E.coli expressed VHHs were utilised in all subsequent experiments unless otherwise stated.

[0624] In some experiments, cA8, GFP_NB ((-) control; GFP_NB is a VHH antibody that binds to green fluorescent protein, GFP), and (+) control VHH (the latter for FcRn interference assay purposes only) were expressed with a myc-his tag through HEK293 mammalian expression. The c-myc and His tags were positioned C- terminal to the VHH sequence. The sequence of the c-myc and His-tag motif used in the HEK293 expressed VHH antibodies was SAEQKLISEEDLGGHHHHHH (SEQ ID NO:16). These were used instead of E.coli expressed VHHs. cA8 and GFP_NB were also re-expressed in HEK293 later for the PK study in Example 4.

[0625] Binding of VHH candidates to canine albumin

[0626] Binding of candidate VHHs to canine serum albumin (CSA, Abeam, cat nr. ab119814; SEQ ID NO:1) at both pH 5.5 and pH 7.4 was initially determined via ELISAs. The ELISAs were run with all steps in PBS (pH 7.4) or citrate-phosphate buffer (CPB) at pH 5.5, for 1 hour incubation at room temperature unless otherwise stated. 3x PBS or CPB containing 0.05 % tween (PBS-T or CPB-T respectively) washes were done between each incubation. A non-binding negative control ((-) control) antibody was also included alongside tested VHHs. The (-) control VHH, as well as cA8 were HEK293 expressed VHHs.

[0627] CSA at 10.72 nM was coated overnight onto Maxisorp 96 well plates, at 4°C. The wells were blocked in 4 % skimmed milk. The VHHs were diluted down to 53.6 nM (a 5:1 ratio of VHH to CSA) and were then incubated for 2 hours. Detection was performed using mouse THE™ c-Myc Tag Antibody (Genscript, cat nr. A00704) at 2-fold serial dilutions 0.4-0.025 pg / ml, followed by anti-mouse-HRP secondary antibody (JI R, cat nr. 715-035-150) at 0.08 pg / ml.

[0628] Rabbit anti canine albumin antibody (Fitzgerald, cat nr. 70R-15096) at 0.1 pg / ml followed by anti-rabbit IgG peroxidase conjugate (JIR, cat nr. 111-035-144) at 0.08 pg / ml was used as coating control. A non-albumin binding VHH ((-) control; GFP_NB), no VHH, no primary, no secondary and no albumin coated wells were also included as controls.

[0629] The plates were developed with TMB (eBioscience, cat nr. 00-4201-56) for 7 minutes before the reaction was stopped using 0.5M H2SO4 (Fisher Chemical, cat nr. J / 8430 / 15). The absorbance at OD 450 nm was then measured with a spectrophotometer. The different anti-myc concentration were used to confirm optimum antibody concentration, with 0.2 pg / ml chosen going forward at both pHs.

[0630] Background was low, and the maximum ODs observed are shown in Table 2 and Table 3 below. All VHHs bound highly to albumin at both pHs, and so all could be suitable for half-life extension (e.g. suitable for linking to a second agent in order to extend the in vivo half-life of said second agent - as is evident from discussion elsewhere herein).

[0631] Table 2: A450 OP binding of cA8 to canine serum albumin at both pH5.5 and

[0632] 7.4

[0633] Table 3: A450 OP binding of other candidate VHHs to canine serum albumin at both pH5.5 and pH7.4. Where multiple values are listed, these are the OP values from the same control on different plates used in the same experiment

[0634] Without wishing to be bound by theory, and as described elsewhere herein, the ability of the anti-canine albumin VHH antibodies to bind to canine albumin at a neutral pH (e.g. pH7.4; plasma I circulatory pH) and also at endosomal pH (e.g. pH5.5) is advantageous, as this property can endow the anti-canine albumin VHH antibodies with an extended half-life. This makes the anti-canine albumin VHH antibodies particularly useful as half-life extending moieties, to extend the half-life of an agent linked (e.g. fused) to such an anti-canine albumin VHH antibody. Serum albumin is maintained at a high concentration in plasma due to the neonatal Fc receptor (FcRn) recycling pathway. Recycling is facilitated by pH-dependent binding of albumin to FcRn. In this regard, albumin binds to FcRn in acidified intracellular compartments such as endosomes, and the albumin is then recycled back to the cell surface, where it dissociates from the FcRn at neutral pH and is released, thus extending the lifetime of the albumin in the circulation by protecting albumin from lysosomal degradation. Again without wishing to be bound be theory, an anticanine albumin VHH antibody that can bind to canine albumin at a neutral pH (e.g. pH7.4; plasma I circulatory pH) and also at endosomal pH (e.g. pH5.5) can also be protected from degradation and recycled back to the cell surface (and thus be endowed with an extended half-life). In this regard, such a VHH antibody can bind to albumin at neutral pH and remain bound to albumin in acidified intracellular compartments such as endosomes (the albumin in turn being bound to FcRn), thus protecting the anti-canine VHH antibody from degradation. It follows that an agent linked (e.g. fused) to the anti-canine VHH antibody would also be protected from lysosomal degradation (and thus can be endowed with an extended half-life).

[0635] Affinity determination of candidates

[0636] After confirming that candidates bind CSA at both high and low pH (pH7.4 and pH5.5 - see above), affinity was determined via T200 Biacore (Cytiva) in MultiCycle Kinetics, utilising a CM5 sensor chip (Cytiva) at pH 7.4 and pH 6.0. SPR was carried out at 25°C. The T200 software’s immobilisation wizard using equal mix (equal volume mix) of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.1 M N-hydroxysuccinimide (EDC / NHS) (Cytiva) was utilised to coat no albumin (buffer only), Canine albumin (Abeam, cat nr. ab119814), Feline albumin (BioWorld, Cat nr. 22070068-1 ; SEQ ID NO: 100) and Porcine albumin (Sigma-Aldrich, cat nr. A1830; SEQ ID NO: 101) onto separate flow cells, whereby an equal mix of 0.4 M 1 -ethyl-3- (3-dimethylaminopropyl)-carbodiimide and 0.1 M N-hydroxysuccinimide (EDC / NHS) was used to activate the CM5 chip matrix surface, before the albumin (diluted in 10 mM Sodium Acetate buffer, pH 5.0 (Cytiva)) was flowed over with the aim to reach approximately 1 ,000 Rll, with 1 M ethanolamine-HCI, pH 8.5, being used to block remaining activated surface groups. The no albumin flow cell had a background of 93.4 RU, with canine, feline and porcine albumins exhibiting 524, 1,141 and 4,741 Rll respectively. The VHHs were run in either pH 7.4 or 6.0 in HBS-P+ buffer (10 mM HEPES, 150 mM NaCI, 0.005% v / v surfactant P20, adjusted to the correct pH).

[0637] VHH candidates ((-) control VHH (GFP_NB) and cA8 were HEK293 expressed VHHs) were buffer exchanged into HBS-P+ buffer, and candidate VHHs were diluted 2-fold serial dilutions from 200 nM to 12.5 nM. Alongside candidate VHHs, VHH binders of feline albumin and porcine albumin, as well as a non-albumin binding VHH (GFP_NB), were used as controls. More specifically, the GFP_NB VHH was used as a negative control ((-) control), a VHH that binds to feline albumin was used as a positive control for the experiments assessing ability to bind to feline albumin, a VHH that binds to porcine albumin was used as a positive control for the experiments assessing ability to bind to porcine albumin.

[0638] These VHHs were run with the following method on the Biacore:

[0639] • 200 seconds association phase

[0640] • 1 ,800 seconds dissociation phase

[0641] • 20 seconds regeneration with 20 mM Glycine, pH 1.5 buffer (cytiva)

[0642] • Running buffer HBS-P+ at the required pH with a flow rate of 10 pl / minute

[0643] Resulting data was analysed utilising Biacore T200 evaluation software (Cytiva), fitting curves using a 1:1 binding model to determine VHH kinetics. The sensorgrams for candidate VHHs and (-) control VHH binding to canine albumin are shown at both pHs in Figure 3 (pH 6.0) and Figure 4 (pH7.4). Sensorgrams showing data for cA8 when tested for ability to bind to feline albumin and porcine albumin are shown in Figure 5 (pH 6.0 in Figure 5A; pH7.4 in Figure 5B). Kinetics for candidate VHHs to each species albumin are shown in Table 4 (canine; pH 6.0), Table 5 (canine; pH7.4), Table 6 (feline; pH6.0), Table 7 (feline; pH7.4), Table 8 (porcine; pH6.0) and Table 9 (porcine; pH7.4) below.

[0644] Table 4: Determined kinetics for candidate VHHs binding to Canine Serum albumin pH 6.0

[0645] Table 5: Determined kinetics for candidate VHH binding Canine Serum albumin at pH7.4 n.d. = not determined (as no binding was observed via SPR).

[0646] Table 6: Determined kinetics for candidate VHH binding Feline Serum albumin at pH6.0 n.d. = not determined (as no binding was observed via SPR). Table 7: Determined kinetics for candidate VHH binding Feline Serum albumin at pH7.4 n.d. = not determined (as no binding was observed via SPR).

[0647] Table 8: Determined kinetics for candidate VHH binding Porcine Serum albumin at pH6.0. The positive control had too higher affinity to accurately measure with this instrument. *N.B. in Table 8 kinetic constants were outside limits measurable by instrument and cannot be uniquely determined. n.d. = not determined (as no binding was observed via SPR). Table 9: Determined kinetics for candidate VHH binding Porcine Serum albumin at pH7.4. The positive control had too higher affinity to accurately measure with this instrument.

[0648] *N.B. in Table 9 kinetic constants were outside limits measurable by instrument and cannot be uniquely determined. n.d. = not determined (as no binding was observed via SPR).

[0649] The candidate VHHs cA8, cA77, cA83, cA159 and cA160 bound to canine albumin, with no observed binding to feline or porcine albumin. All had affinities to canine albumin between the 10’s nM and 100’s pM over both pHs, with cA83 appearing to have pH independent binding with 879 and 663 pM affinities, at pH 6.0 and 7.4, respectively. cA8 exhibited stronger affinity and binding at pH 6.0 than 7.4 (613 pM compared to 28 nM). Without wishing to be bound by theory, having a stronger affinity for albumin at endosomal pH (e.g. pH6.0) than at neutral pH (e.g. circulatory pH, e.g. pH7.4) could potentially be advantageous, as if a construct comprising a canine albumin binding VHH antibody is endocytosed in an unbound state (i.e. not already bound to canine albumin) it could readily bind to albumin within the acidic endosome and be recycled back into the bloodstream through FcRn-mediated recycling of albumin, and when in the bloodstream having a weaker affinity for canine albumin could allow the construct (which as described elsewhere herein may contain additionally a therapeutic agent) to dissociate from the albumin, which could potentially enhance its activity (e.g. therapeutic activity). Overall, these VHHs also exhibited a variety of on and off rates, at high and low pHs, allowing the choice of appropriate VHHs for specific requirements.

[0650] FcRn interference assay

[0651] A key characteristic of half-life extension by binding to albumin, is how a candidate VHH affects albumin binding to the FcRn-p2M heterodimer (FcRn). FcRn- albumin binding at low pH is required for recycling of albumin from the endosome back out of the cell, resulting in albumin’s high half-life, meaning that this a key interaction to avoid interference with. Confirmation of a lack of FcRn interference (i.e. that the anti-canine VHH antibodies do not interfere with the ability of canine albumin to bind to FcRn) was performed by ELISA, with all steps in CPB at pH 5.5, with incubation steps at 1 hour at room temperature unless otherwise stated. 3x CPB-T washes were done between each incubation. The ELISA was done at pH 5.5 as this is a pH that reflects acidified endosomal pH. A negative control (a nonalbumin binding VHH ((-) Control VHH; GFP_NB)) and a positive control for interference ((+)Control VHH, a strong albumin binding VHH previously determined to interfere with FcRn-albumin binding) were included. Both (-) control and (+) control, as well as cA8 were HEK293 expressed VHHs.

[0652] CSA (Abeam, cat nr. ab119814) at 10.72 nM was coated overnight onto Maxisorp plates, at 4°C. The wells were blocked in 1 % casein. 10.72 nM biotinylated canine FcRn (Immunitrack, cat nr. ITF11) in CPB was added for 2 h, to achieve a 1:1 molar ratio of FcRn to Albumin. VHHs were added using two-fold serial dilutions to achieve molar excesses (VHH:canine albumin) ranging from 40:1 to 0.31:1, and incubated for 2 h. To detect VHH binding, mouse THE™ c-Myc Tag Antibody at 0.2 pg / ml (Genscript, cat nr. A00704) was added, followed by an anti- mouse-HRP secondary antibody at 0.08 pg / ml (JI R, cat nr. 715-035-150), both in 0.25% casein. To detect the FcRn binding to albumin, streptavidin-HRP at 0.5 pg / ml (JI R, cat nr. 016-030-084) in 0.25% casein was added to the plate and incubated for 1 hour. Of course, detection of VHH binding and detection of FcRn binding were each done in separate, parallel, wells of the plate.

[0653] Rabb...

Claims

CLAIMS1. A construct comprising:(a) at least one VHH antibody which binds to canine albumin but does not bind significantly to human albumin; and(b) at least one agent which binds to a mammalian protein other than albumin.

2. The construct of claim 1 , wherein said VHH antibody comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises(i) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially homologous thereto;(ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto;(iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:33 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:34 or a sequence substantially homologous thereto;(iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:41 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:42 or a sequence substantially homologous thereto;(v) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and aCDR3 that has the amino acid sequence of SEQ ID NO:50 or SEQ ID NO:91 or a sequence substantially homologous thereto; wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

3. The construct of claim 1 or claim 2, wherein said VHH antibody comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises(i) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:10 or a sequence substantially homologous thereto;(ii) a CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:57 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:10 or a sequence substantially homologous thereto;(iii) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or a sequence substantially homologous thereto; or(iv) a CDR1 that has the amino acid sequence of SEQ ID NO:48 or a sequence substantially homologous thereto, a CDR2 that has the amino acid sequence of SEQ ID NO:49 or a sequence substantially homologous thereto and a CDR3 that has the amino acid sequence of SEQ ID NO:91, or a sequence substantially homologous thereto wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.

4. The construct of any one of claims 1 to 3, wherein said VHH antibody comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises(i) a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:57, and a CDR3 that has the amino acid sequence of SEQ ID NO: 10;(ii) a CDR1 that has the amino acid sequence of SEQ ID NO:24, a CDR2 that has the amino acid sequence of SEQ ID NO:25, and a CDR3 that has the amino acid sequence of SEQ ID NO:26;(iii) a CDR1 that has the amino acid sequence of SEQ ID NO:32, a CDR2 that has the amino acid sequence of SEQ ID NO:33, and a CDR3 that has the amino acid sequence of SEQ ID NO:34;(iv) a CDR1 that has the amino acid sequence of SEQ ID NQ:40, a CDR2 that has the amino acid sequence of SEQ ID NO:41, and a CDR3 that has the amino acid sequence of SEQ ID NO:42; or(v) a CDR1 that has the amino acid sequence of SEQ ID NO:48, a CDR2 that has the amino acid sequence of SEQ ID NO:49, and a CDR3 that has the amino acid sequence of SEQ ID NQ:50 or SEQ ID NO:91.

5. The construct of any one of claims 1 to 4, wherein said VHH antibody comprises a VHH domain comprising three CDRs, wherein said VHH domain comprises(i) a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:9 and a CDR3 that has the amino acid sequence of SEQ ID NO: 10;(ii) a CDR1 that has the amino acid sequence of SEQ ID NO:8, a CDR2 that has the amino acid sequence of SEQ ID NO:57 and a CDR3 that has the amino acid sequence of SEQ ID NO: 10;(iii) a CDR1 that has the amino acid sequence of SEQ ID NO:48, a CDR2 that has the amino acid sequence of SEQ ID NO:49 and a CDR3 that has the amino acid sequence of SEQ ID NQ:50; or(iv) a CDR1 that has the amino acid sequence of SEQ ID NO:, a CDR2 that has the amino acid sequence of SEQ ID NO:49 and a CDR3 that has the amino acid sequence of SEQ ID NO:91.

6. The construct of any one of claims 1 to 5, wherein said VHH antibody comprises a VHH domain having (i) an amino acid sequence selected from the group consisting of SEQ ID NQ:107, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71 ,SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83,SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NQ:104,SEQ ID NQ:105 and SEQ ID NQ:106, or (ii) an amino acid sequence having at least80% sequence identity to an amino acid sequence of (i).

7. The construct of any one of claims 1 to 6, wherein said VHH antibody comprises a VHH domain having an amino acid sequence selected from the group consisting of SEQ ID NQ:107, SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:81 , SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NQ:104, SEQ ID NQ:105 and SEQ ID NQ:106.

8. The construct of any one of claims 1 to 7, wherein said VHH antibody binds to canine albumin at pH7.4 and at pH5.5.

9. The construct of claim 1 , wherein said VHH antibody binds to the same epitope on canine albumin as a VHH antibody as defined in any one of claims 2 to 8.

10. The construct of any one of claims 1 to 9, wherein said construct is a polypeptide.

11. The construct of any one of claims 1 to 10, wherein said agent of (b) is a VHH antibody.

12. The construct of claim 11 , wherein said agent of (b) is a VHH antibody which binds to canine IL-31 , or a VHH antibody which binds to canine TNFa, or a VHH antibody which binds to canine GnRH.

13. The construct of any one of claims 1 to 10, wherein said agent of (b) is selected from the group consisting of (i) Exendin-4, (ii) GLP-1 , (iii) insulin, (iv) p75NTR, (v) a fragment of any one of (i) to (iv), and (vi) an analogue of any one of (i) to (v).

14. A VHH antibody which binds to canine albumin, wherein said VHH antibody comprises a VHH domain as defined in any one of claims 2 to 7.

15. The VHH antibody of claim 14, wherein(i) said VHH antibody binds to canine albumin at pH7.4 and at pH5.5; and / or(ii) said VHH antibody does not bind significantly to human albumin.

16. A construct comprising a VHH antibody as defined in claim 14 or claim 15.

17. The construct of claim 16, wherein said construct is a polypeptide.

18. A composition comprising a construct of any one of claims 1 to 13, 16 or 17, or a VHH antibody of claim 14 or claim 15, wherein said composition comprises a diluent, carrier or excipient, preferably a pharmaceutically acceptable diluent, carrier or excipient.

19. A nucleic acid molecule comprising a nucleotide sequence that encodes a construct of any one of claims 1 to 13, 16 or 17, or a VHH antibody of claim 14 or claim 15.

20. An expression vector comprising a nucleic acid molecule of claim 19.

21. A host cell comprising the nucleic acid molecule of claim 19, or the expression vector of claim 20, or expressing a construct of any one of claims 1 to 13, 16 or 17, or a VHH antibody of claim 14 or claim 15.

22. A method of producing a construct of any one of claims 1 to 13, 16 or 17, or a VHH antibody of claim 14 or claim 15, comprising the steps of:(i) culturing a host cell comprising (a) one or more nucleic acid molecules encoding a construct of any one of claims 1 to 13, 16 or 17, or a VHH antibody of claim 14 or claim 15 or (b) one or more expression vectors comprising one or more of said nucleic acid molecules, under conditions suitable for the expression of the encoded VHH antibody or construct; and(ii) isolating or obtaining the construct or the VHH antibody from the host cell or from the growth medium / supernatant.

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