Half-life extending moieties and methods of use thereof

Abstract

The present disclosure relates generally to biology and medicine, and more specifically to compounds that act as half-life (t1 / 2) extending moieties for use with therapeutic agents, particularly to improve the t1 / 2 of biological therapeutic agents (i.e., biotherapeutics or biologics). The present disclosure further relates to fusions and conjugates comprising one or more of the compounds that act as t1 / 2 extending moieties, as well as pharmaceutical compositions comprising same, and their use in treating various conditions, diseases, or disorders.

Description

[Technical Field]

[0001] The present disclosure relates generally to biology and medicine, and more specifically to single domain antibodies known as variable domains of heavy chain-only antibodies (VHHs) that have been engineered / modified to act as half-life (t) extending moieties for use with therapeutic agents, particularly to improve the t of biological therapeutic agents (i.e., biotherapeutics or biologics). The disclosure further relates to fusions and conjugates comprising one or more of the VHH-based t extending moieties and therapeutic agents, as well as pharmaceutical compositions comprising same, and their uses in treating various conditions, diseases, or disorders. [Background technology]

[0002] Biotherapeutics are natural or modified components of physiological pathways and tend to be highly selective, effective, and safe. However, they have several limitations. One limitation is that, with some exceptions, biotherapeutics cannot be administered orally. Another limitation is that many biotherapeutics have a relatively short t1 / 2 when used in clinical settings.

[0003] Several strategies exist for extending the t of biotherapeutics, which can improve their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles. Such strategies typically use bulking moieties or recycling via neonatal Fc receptors (FcRn). In this manner, antibodies (Ab) or fragments thereof (e.g., Fab, Fc, etc.), polymers such as polyethylene glycol (PEG), polysialic acid (PSA), hyaluronic acid (HA), and hydroxyethyl starch (HES), fatty acids and other lipids, N- or O-glycosylation, and serum albumin or other plasma proteins (e.g., transferrin) can be covalently and / or noncovalently attached to certain biotherapeutics to extend their t. See, e.g., Hamers-Casterman et al. (1993) Nature 363:446-448, Harmsen & Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22, Kontermann (2016) Expert Opin. Biol. Ther. 16:903-915, Muller et al. (2012) MAbs 4:673-685, Podust et al. (2013) Protein Eng. Des. Sel. 26:743-753, Strohl (2015) BioDrugs 29:215-239, and Werle U Bernkop-Schnurch (2006) Amino Acids 30:351-367.

[0004] Despite the vast number of t1 / 2 extension strategies, additional mechanisms are needed to extend or improve the PK properties, such as t1 / 2, of biotherapeutics. Summary of the Invention

[0005] To address this need, the present disclosure first describes compounds that can be used as t1 / 2 extending moieties for biotherapeutics. In one example, a compound is provided that includes an amino acid sequence selected from any one of SEQ ID NOS: 1-37 and 124-126.

[0006] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 1). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 1.

[0007] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 2). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 2.

[0008] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 3). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 3.

[0009] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 4). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 4.

[0010] Another example is the amino acid sequence: Provided is a compound comprising EVQLVESGGGLVQAGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFTAGIGGSVDITYYLDSVKGRFTISKDNTKNTVYLQMNSLKPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTQVTVSS (SEQ ID NO: 5). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 5.

[0011] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS (SEQ ID NO: 6). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 6.

[0012] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGTLVTVSS (SEQ ID NO: 7). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 7.

[0013] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 8). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 8.

[0014] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 9). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 9.

[0015] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 10). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 10.

[0016] Another example is the amino acid sequence: A compound is provided comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 11). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 11.

[0017] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 12). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 12.

[0018] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 13). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 13.

[0019] Another example is the amino acid sequence: A compound is provided comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 14). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 14.

[0020] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 15). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 15.

[0021] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 16). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 16.

[0022] Another example is the amino acid sequence: A compound is provided comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 17). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 17.

[0023] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 18). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 18.

[0024] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 19). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 19.

[0025] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 20). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 20.

[0026] Another example is the amino acid sequence: A compound is provided comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 21). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 21.

[0027] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 22). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 22.

[0028] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 23). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 23.

[0029] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 24). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 24.

[0030] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 25). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 25.

[0031] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 26). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 26.

[0032] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGTLVTVSS (SEQ ID NO: 27). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 27.

[0033] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRPGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 28). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 28.

[0034] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARQGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 29). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 29.

[0035] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRQGRPLITSKVADLYPYWGQGTLVTVSS (SEQ ID NO: 30). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 30.

[0036] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSQVADLYPYWGQGTLVTVSS (SEQ ID NO: 31). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 31.

[0037] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQADLYPYWGQGTLVTVSS (SEQ ID NO: 32). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 32.

[0038] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAELYPYWGQGTLVTVSS (SEQ ID NO: 33). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 33.

[0039] Another example is the amino acid sequence: Provided is a compound comprising EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVASLYPYWGQGTLVTVSS (SEQ ID NO: 34). Alternatively, the compound may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 34.

[0040] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQAELYPYWGQGTLVTVSS (SEQ ID NO: 35). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 35.

[0041] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQASLYPYWGQGTLVTVSS (SEQ ID NO: 36). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 36.

[0042] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC (SEQ ID NO: 37). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 37.

[0043] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSC (SEQ ID NO: 124). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 124.

[0044] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 125). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 125.

[0045] Another example is the amino acid sequence: Compounds are provided that include EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGTRPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 126). Alternatively, the compounds may have an amino acid sequence having at least about 90% to about 99% sequence similarity to SEQ ID NO: 126.

[0046] Second, the present disclosure describes compounds comprising at least one of the VHH-based t1 / 2 extension moieties described herein and a biotherapeutic. In some examples, the compounds can comprise the following structure from the amino terminus (N-terminus) to the carboxy terminus (C-terminus): M-X1, X1-M, M-X2, X2-M, M-L1-X1, M-L2-X1, M-L1-X2, M-L2-X2, X1-L1-M, X1-L2-M, X2-L1-M, X2-L2-M, X1-M-X2, X2-M-X1, X1-L1-M-X2, X2-L1-M-X1, X1-M-L1-X2, X2-M-L1-X1, X1-L1-M-L2-X2, X2-L1-M-L2-X1, M-L1-X1-L2-X2, M-L1-X2-L2-X1, X1-L2-X2-L1-M, X2-L2-X1-L1-M, or M-L1-X1 combined (i.e., non-covalently associated) with M-L2-X2 wherein M acts as a t1 / 2 extender and is a compound having an amino acid sequence selected from SEQ ID NOs: 1-37 and 124-126, or an amino acid sequence having at least about 90% to about 99% sequence similarity thereto; L1 (if present) is a first linker; L2 (if present) is a second linker; X1 is a biotherapeutic protein, peptide, or oligomer; and X2 is a biotherapeutic protein, peptide, or oligomer that can be the same as or different from X1 (e.g., if the biotherapeutic is a homodimer or if the biotherapeutic is a heterodimer, X1 can be one chain (the α chain) and X2 can be the other chain (the β chain)). X1 and X2 can also be completely different from each other. In some examples, L1 is (GGGGQ) n (SEQ ID NO: 38), (GGGQ) n (SEQ ID NO: 39), (GGGGS) n (SEQ ID NO: 40), (PGPQ) n (SEQ ID NO: 41), (PGPA) n (SEQ ID NO: 42), GGGG(AP) n GGGG (SEQ ID NO: 43), (GGE) n (SEQ ID NO: 44), (GGGGE) n (SEQ ID NO: 45), (GGK) n (SEQ ID NO: 46), (GGGGK) n (SEQ ID NO: 47), GGGG(EP) n GGGG (SEQ ID NO: 48), GGGG (KP) n GGGG, (SEQ ID NO: 49), (PGPE) n (SEQ ID NO: 50), or (PGPK) n (SEQ ID NO: 51), where n can be 1 to 15, particularly about 5 to about 10. In another example, L1 can have an amino acid sequence selected from SEQ ID NOs: 52 to 63. In yet another example, L1 can have one or more additions, deletions, insertions, or substitutions such that L1 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 52 to 63.

[0047] In some examples, L2 can have an amino acid sequence selected from SEQ ID NOs: 64-65. In still other examples, L2 can have one or more additions, deletions, insertions, or substitutions such that L2 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 64-65.

[0048] In yet another example, L1 or L2 is polyethylene glycol (PEG), particularly (PEG) n and n can be 1-20.

[0049] In some examples, X1, X2, or X1 / X2 is a peptide or protein (and even an oligomer, e.g., a homodimer or heterodimer, which may or may not be covalently linked). Examples of such peptides or proteins include, but are not limited to, an antibody (Ab), an antibody fragment (e.g., Fab, scFv, Fab-Fab, VH, VL, or VHH of different specificities), ciliary neurotrophic factor (CNTF), growth / differentiation factor 15 (GDF15), an incretin (INC), an interleukin (IL), a neuregulin (NRG), or a hormone. In certain examples, the INC can be insulin (INS), glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), GIP / GLP-1, or even an INC with triple receptor activity (i.e., glucagon-GIP-GLP-1 receptor activity). In certain examples, the IL is interleukin-2 (IL-2). In certain instances, the NRG is neuregulin 1 (NRG1) or neuregulin 4 (NRG4). In certain instances, the hormone is adrenocorticotropic hormone (ACTH) or relaxin-2 (RLN-2). In certain instances, the Fab binds to GITR, and in some instances, the Fab binds to GITR and becomes a GITR antagonist.

[0050] In certain examples, the compound may have the amino acid sequence of any one of SEQ ID NOs: 100 to 118. Alternatively, the compound may have at least about 90% to about 99% sequence similarity to the amino acid sequence of any one of SEQ ID NOs: 100 to 118.

[0051] Third, the present disclosure describes pharmaceutical compositions comprising at least one compound herein and a pharmaceutically acceptable carrier.

[0052] Fourth, the disclosure describes methods of using the compounds and pharmaceutical compositions for medicines and for extending the t1 / 2 of biotherapeutics.

[0053] Fifth, the disclosure describes the use of the compounds herein in the manufacture of medicaments and in extending the t1 / 2 of biotherapeutics.

[0054] An advantage of t1 / 2 extensions and compounds containing them is that they can be synthesized chemically or recombinantly as single-chain polypeptides (i.e., monomers) and therefore do not require intracellular proteolytic processing for biological activity. However, in some instances, it is contemplated that compounds acting as t1 / 2 extensions may be fused not only to single-chain peptides and proteins, but also to peptides with two or more chains, e.g., double-chain peptides, multi-chain peptides, and proteins. Compounds acting as t1 / 2 extensions may be chemically conjugated not only to the N-terminus and C-terminus, but also to any surface-exposed amino acid of the t1 / 2 extension (although such conjugation does not completely abolish albumin binding).

[0055] The advantage of compounds that act as t1 / 2 extending moieties and compounds containing such moieties is that the t1 / 2 extending moiety extends the duration of action in mammals, such as humans, to t1 / 2 of about 20 to about 30 days, allowing administration at least weekly or biweekly compared to natural peptides and proteins, which can improve compliance and quality of life, particularly in the case of chronic diseases requiring lifelong treatment.

[0056] An advantage of the compounds herein that act as t1 / 2 extending moieties is that they have tunable pharmacokinetics, which is achieved by altering the albumin affinity of the t1 / 2 extending moiety.

[0057] An advantage of compounds acting as t1 / 2 extension moieties herein is that they may be amenable to recombinant expression in standard production organisms such as yeast, mammalian, or prokaryotes.

[0058] Furthermore, an advantage of the compounds that act as t1 / 2 extending moieties herein is that they have similar binding to serum albumin not only from humans but also from monkeys, mice, rats, dogs, and pigs, allowing pharmacodynamic, pharmacokinetic, and toxicological studies to be more easily transferred from these species to humans. Thus, the t1 / 2 extending moieties herein can be used in animal therapy as well as human therapy. DETAILED DESCRIPTION OF THE INVENTION

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0060] Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements are present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0061] definition As used herein, "about" means within a statistically significant range of a value or values, such as, for example, a specified concentration, length, molecular weight, pH, sequence identity, time frame, temperature, volume, etc. Such values ​​or ranges may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0062] As used herein, with respect to one or more receptors, "activity," "activate," "activating," and the like, refer to the ability of a compound, such as a fusion herein, to bind to a receptor and induce a response thereat, as measured using assays known in the art, such as the in vitro assays described below.

[0063] As used herein, "adrenocorticotropic hormone" or "ACTH" refers to ACTH obtained or derived from any species, including mammalian species, particularly humans. ACTH includes both native ACTH (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native ACTH). One sequence of ACTH is set forth in SEQ ID NO: 95 (UniProt / SwissProt database accession number P01189). In humans, ACTH binds to the ACTH receptor (ACTHR, also known as melanocortin receptor 2 or MC2R), one sequence of which is set forth in SEQ ID NO: 96 (UniProt / SwissProt database accession number Q01718). ACTHR is a G protein-coupled receptor located in the outer cell plasma membrane that is coupled to Gα and upregulates cAMP levels by activating adenylyl cyclase.

[0064] As used herein, "amino acid" refers to a molecule that is characterized, from a chemical standpoint, by the presence of one or more amine groups and one or more carboxylic acid groups, and may contain other functional groups. As is known in the art, there is a set of 20 amino acids that are designated as standard amino acids and can be used as building blocks for the majority of peptides / proteins produced by all living organisms. The amino acid sequences of the present disclosure include the standard one-letter or three-letter codes for the 20 naturally occurring amino acids.

[0065] As used herein, "analog" means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and induces at least one in vivo or in vitro effect induced by the receptor's natural agonist.

[0066] As used herein, "biotherapeutic agent" and the like refers to amino acid or nucleic acid based compounds such as antibodies, coagulation factors, clotting factors, cytokines, enzymes, growth factors, hormones, and fragments thereof, as well as therapeutic DNA and / or RNA molecules, which have at least one therapeutic activity / application.

[0067] As used herein, "CNTF" or "ciliary neurotrophic factor" refers to CNTF obtained or derived from any species, including mammalian species, particularly humans. CNTF includes both native CNTF (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native CNTF). One sequence of CNTF is set forth in SEQ ID NO: 97 (UniProt / SwissProt database accession number P26441). In humans, CNTF binds to the CNTFα receptor (CNTFRα), one sequence of which is set forth in SEQ ID NO: 98 (UniProt / SwissProt database accession number P26992). CNTFRα also utilizes two signaling transmembrane subunits, LIFRβ and gp130, which together activate the Jak-STAT signaling pathway. See Stahl et al. (1994) Science 263:92-95 and Stahl & Yancopoulos (1994) J. Neurobiol. 25:1454-1466.

[0068] As used herein, "conservative substitution" refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for one or more conservative amino acid substitutions in its amino acid sequence. Generally, conservatively modified variants include amino acid sequences that are at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference amino acid sequence. More specifically, a conservative substitution refers to the substitution of an amino acid with an amino acid that has similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, and rigidity) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution of functionally similar amino acids is well known in the art and need not be described exhaustively herein.

[0069] As used herein, "effective amount" means an amount or dose of one or more compounds herein, or pharmaceutically acceptable salts thereof, that, upon single or multiple administration to an individual in need thereof, provides a desired effect (i.e., that can produce a clinically measurable difference in the individual's condition, such as, for example, increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, lowered blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) cardiac, renal, liver, or pulmonary fibrosis) in such individual during diagnosis or treatment. An effective amount can be readily determined by one of ordinary skill in the art by the use of known techniques and by observing results obtained under similar circumstances. In determining the effective amount for an individual, numerous factors are taken into account, including, but not limited to, the species of mammal, its size, age, and general health, the particular disease or disorder involved, the extent or involvement or severity of the disease or disorder, the individual's response, the particular compound administered, the mode of administration, the bioavailability characteristics of the administered preparation, the selected dosage, the use of concomitant medications, and other relevant circumstances.

[0070] As used herein, "long-lasting action" means that the binding affinity and activity of a fusion comprising at least one compound herein and a biotherapeutic herein continues for a longer period of time than the native biotherapeutic, allowing for dosing at least as frequently as once daily, or even three times a week, twice a week, or even once a week. Time-action profiles can be measured using known pharmacokinetic testing methods, such as those utilized in the Examples below.

[0071] As used herein, "glucocorticoid-inducible TNFR-related protein" or "GITR," also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18), refers to GITR obtained or derived from any species, including mammalian species, particularly humans. GITR includes both native GITR (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native GITR). One sequence of full-length human GITR (but without the signal peptide) is set forth in SEQ ID NO: 122 (see also UniProt / SwissProt database accession number Q9Y5U5). One sequence of human GITR ECD (but without the signal peptide) is set forth in SEQ ID NO: 123.

[0072] As used herein, "glucose-dependent insulinotropic peptide," "gastric inhibitory peptide," or "GIP" refers to GIP obtained or derived from any species, such as mammalian species, particularly humans. GIP includes both native GIP (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native GIP). GIP is processed from a precursor, proGIP. One sequence for proGIP is set forth in SEQ ID NO: 67 (see also UniProt / SwissProt database accession number P09681), and one sequence for GIP is set forth in SEQ ID NO: 68. Alternative GIPs include GIP 1-30(See Hansen et al. (2016) Br. J. Pharmacol. 173:826-838). In humans, there is one GIP receptor (GIPR; SEQ ID NO: 69; also see UniProt / SwissProt database accession number P48546) that acts as a G protein-coupled receptor. See Yaqub et al. (2010) Mol. Pharmacol. 77:547-558.

[0073] As used herein, "glucagon-like peptide-1" or "GLP-1" refers to GLP-1 obtained or derived from any species, including mammalian species, particularly humans. GLP-1 includes both native GLP-1 (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native GLP-1). GLP-1 is processed from a precursor, proglucagon (proGCG). One sequence of proGCG is set forth in SEQ ID NO: 70 (see also UniProt / SwissProt database accession number P01275), and one sequence of GLP-1 is set forth in SEQ ID NO: 71. Two active physiological variants are known, which are set forth in SEQ ID NOs: 72 and 73. In humans, there is one GLP-1 receptor (GLP-1R; SEQ ID NO: 74; see also UniProt / SwissProt database accession number P43220), which acts as a G protein-coupled receptor. See Dillon et al. (1993) Endocrinol. 133:1907-1910, Graziano et al. (1993) Biochem. Biophys. Res. Commun. 196:141-146, and Thorens et al. (1993) Diabetes 42:1678-1682.

[0074] As used herein, "growth / differentiation factor 15" or "GDF15" refers to GDF15 obtained or derived from any species, including mammalian species, particularly humans. GDF15 includes both native GDF15 (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native GDF15). GDF15 is a homodimeric peptide processed from the precursor proGDF15. One sequence of the precursor is set forth in SEQ ID NO: 75, and one sequence of GDF15 is set forth in SEQ ID NO: 76 (see also UniProt / SwissProt database accession number Q99988). In humans, there is one GDF15 receptor (GFRAL; SEQ ID NO: 77; see also UniProt / SwissProt database accession number Q6UXV0) that acts as a RET receptor tyrosine kinase. See Emmerson et al. (2017) Nat Med. 23:1215-1219, Yang et al. (2017) Nat. Med. 23:1158-1166, and Baek & Eling (2019) Pharmacol. Ther. 198:46-58.

[0075] As used herein, "half-life" or "t1 / 2" refers to the time it takes for half of the amount of a compound, such as a fusion described herein, to be removed by biological processes from a fluid, such as serum or plasma, or other physiological space of an individual. Alternatively, t1 / 2 can refer to the time it takes for an amount of such a fusion to lose half of its pharmacological, physiological, or radiological activity.

[0076] As used herein, "50% effective concentration" or "EC 50" refers to the concentration of compound that results in 50% activation / stimulation of an assay endpoint such as a dose-response curve (e.g., CNTF: Jak, STAT, Ras, PI3K / Akt, and MAPK / ERK; NRG: PI3K / Akt, Jak, STAT, Ras, and PLCγ; GDF15: PI3K / AKT, MAPK / ERK, and Smads; IL-2: JAK-STAT, PI3K / Akt, and MAPK / ERK; GLP1: cAMP, PI3K, MAPK / ERK, PKCδ; TNF: TRAF, MKK, IKK, and NFkB; ACTH: cAMP and PKA).

[0077] As used herein, "in combination with" means that at least one of the fusions herein is administered simultaneously, sequentially, or in a single combined formulation with one or more additional therapeutic agents.

[0078] As used herein, "an individual in need thereof" means a mammal, such as a human, having a condition, disease, disorder, or symptom that requires treatment or therapy, including, for example, those listed herein. In particular, preferred individuals to be treated are humans.

[0079] As used herein, "incretin" or "INC" refers to a peptide secreted by enteroendocrine cells that can increase insulin secretion after a meal. INCs can be incretins obtained or derived from any species, including mammalian species, particularly humans. In humans, INCs include INS, GIP, and GLP-1, as discussed above.

[0080] As used herein, "insulin" or "INS" refers to insulin obtained or derived from any species, e.g., mammalian species, particularly humans, and the native form is a heterodimeric peptide having two peptide chains (e.g., A and B chains) connected by two disulfide bonds, with the A chain further having a single intramolecular disulfide bond. In humans, INS processing begins with preproinsulin (see UniProt / SwissProt database accession number P01308), which is processed into proinsulin (comprising an A chain, a B chain, and a C peptide; native INS has the structure BCA; see SEQ ID NO: 78; also see UniProt / SwissProt database accession number P01308). Proinsulin undergoes further processing, in which the C-peptide is cleaved to reach INS (see SEQ ID NO: 79 for the A chain of native human INS, SEQ ID NO: 80 for the B chain of native human INS, and also see UniProt / SwissProt database accession number P01308).

[0081] As used herein, "interleukin" or "IL" refers to an interleukin obtained or derived from any species, including mammalian species, particularly humans. IL includes both naturally occurring ILs (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of naturally occurring ILs). In humans, a number of naturally occurring IL isoforms exist, but of interest herein is IL-2. IL-2 is a cytokine that can signal through three different signaling pathways, including the JAK-STAT, PI3K / Akt / mTOR, and MAPK / ERK pathways. One sequence for human IL-2 is set forth in SEQ ID NO: 83 (see also UniProt / SwissProt database accession number P60568). In humans, there is one IL-2 receptor containing α, β, and γ subunits (IL-2R; SEQ ID NOs: 84-86; see also UniProt / SwissProt database accession numbers P01589, P14784, and P31785). See Liao et al. (2011) Curr. Opin. Immunol. 23:598-604 and Malek & Castro (2010) Immunity 33:153-165.

[0082] As used herein, "long-acting" means that the binding affinity and activity of the compositions herein persist for a longer period of time than the native peptide or protein, allowing for dosing at least as frequently as once daily, or even three times a week, twice a week, once a week, or even once a month. The time-action profiles of the compounds herein can be measured using known pharmacokinetic testing methods, such as those described in the Examples below.

[0083] As used herein, "neuregulin" or "NRG" refers to neuregulin obtained or derived from any species, including mammalian species, particularly humans. NRG includes both native NRGs (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and / or substitutions of native NRGs). In humans, there are several native NRG family members, but the focus here is on NRG1. Like all NRGs, NRG1 is processed from a larger precursor. The sequence of one precursor is set forth in SEQ ID NO: 87, and the sequence of one NRG1 is set forth in SEQ ID NO: 88 (see also UniProt / SwissProt database accession number Q02297). In humans, there are two NRG1 receptors: ErbB3 (SEQ ID NO: 89; see also UniProt / SwissProt database accession number P21860) and ErbB4 (SEQ ID NO: 90; see also UniProt / SwissProt database accession number Q15303). See Mei & Xiong (2008).

[0084] As used herein, "non-standard amino acid" refers to an amino acid that may occur naturally in cells but is not involved in peptide synthesis. Non-standard amino acids can be building blocks of peptides and are often produced by modification of standard amino acids in peptides (i.e., via post-translational modification). Non-standard amino acids can include D-amino acids that have the opposite absolute chirality to the standard amino acids listed above.

[0085] As used herein, "oligomer" refers to a molecule having several similar or identical repeating units, monomers thereof, which may be derived from copies of smaller molecules. These monomers may be joined by bonds that are either strong or weak, covalent or non-covalent (e.g., intramolecular) bonds.

[0086] As used herein, "patient," "subject," and "individual" are used interchangeably herein and refer to animals, particularly humans. In certain instances, the individual is further characterized by a condition, disease, disorder, or symptom that would benefit from the administration of a compound or composition herein.

[0087] As used herein, "pharmaceutically acceptable buffer" means any of the standard pharmaceutical buffers known to those of skill in the art.

[0088] As used herein, "relaxin-2" or "RLN-2" refers to relaxin obtained or derived from any species, including mammalian species, particularly humans, where the native form is a heterodimeric peptide consisting of two peptide chains (e.g., A and B chains) connected via two disulfide bonds, with the A chain also having a single intramolecular disulfide bond. In humans, RLN-2 processing begins with preprorelaxin (see UniProt / SwissProt database accession number P04090), which is processed into prorelaxin (comprising A chain, B chain, and C peptide; native RLN has the structure BCA; see SEQ ID NO: 91; also see UniProt / SwissProt database accession number P04090). Prorelaxin undergoes further processing, where the C-peptide is cleaved to reach RLN-2 (see SEQ ID NO: 92 for RLN-2 A chain and SEQ ID NO: 93 for RNL-2 B chain, also see UniProt / SwissProt database accession number P04090).

[0089] As used herein, "sequence similarity" refers to a quantitative property of two or more nucleic acid or amino acid sequences of biological compounds, such as the match over the entire length or comparison window of two or more sequences. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of identical residues between two biological compounds divided by the length of the shortest sequence, while percent similarity measures identity and also includes sequence gaps and residue similarity in the assessment. Methods and algorithms for determining sequence similarity are well known in the art and need not be described exhaustively herein. A particular percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0090] As used herein, "treating" or "to treat" means managing or caring for an individual having a condition, disease, disorder, or condition for which administration of a compound herein is indicated for the purpose of alleviating, inhibiting, reversing, slowing, or halting the progression of the condition, disease, disorder, or condition or its severity. Treating includes administering a compound herein or a composition comprising a compound herein to an individual to prevent the onset of the condition or complications, alleviate the symptoms or complications, or eliminate the condition, disease, disorder, or condition. Treating includes administering a compound herein or a composition comprising a compound herein to an individual to produce, for example, increased angiogenesis, increased vascular compliance, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, lowered blood pressure, reduced (or prevented) inflammation, and / or reduced (or prevented) fibrosis in the heart, kidney, liver, or lung. The individual to be treated is a mammal, particularly a human.

[0091] As used herein, "VHH" or "VHH moiety" refers to an antibody fragment comprising a single monomeric variable region of a heavy-chain-only antibody (HcAb), which may be approximately 15 kDa in size, in the form of a single-domain antibody. It has now been discovered that engineered / modified VHH-based compounds can be used as pharmacokinetic enhancers to extend the duration of action and / or improve the t1 / 2 of biotherapeutics. While the present VHH-based compounds bind to serum albumin, they can also be used to bind to IgG (including the Fc domain), neonatal Fc receptor (FcRn), or other long-lasting serum proteins. Thus, the present VHH-based compounds can be used to improve the t1 / 2 of compounds such as peptides or proteins, or even other molecules, such as small molecules.

[0092] Certain abbreviations are defined as follows: "ACR" refers to urinary albumin / urinary creatinine ratio, "AUC" refers to area under the curve, "cAMP" refers to cyclic adenosine monophosphate, "CMV" refers to cytomegalovirus, "DNA" refers to deoxyribonucleic acid, "ECD" refers to ectodomain, "EDC" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, "ETA" refers to ethanolamine, "GS" refers to glutamine synthetase, "HC" refers to heavy chain, "HIC" refers to hydrophobic interaction chromatography, "hr" refers to hour(s), "IV" refers to intravenous, "kDa" refers to kilodaltons, "LC" refers to light chain, "LC / MS" refers to liquid chromatography mass spectrometry, and "min" refers to the total number of hours. refers to minute(s), "MS" refers to mass spectrometry, "MSX" refers to methionine sulfoximine, "NHS" refers to N-hydroxysuccinimide, "OtBu" refers to O-tert-butyl ether, "PEI" refers to polyethyleneimine, "RP-HPLC" refers to reverse-phase high performance liquid chromatography, "sec" refers to second(s), "NaOAc" refers to sodium acetate, "rcf" means relative centrifugal force, "RT" means room temperature, "RU" means resonance units, "SQ" refers to subcutaneous, "SEC" refers to size exclusion chromatography, "SEM" refers to standard error of the mean, "SPR" means surface plasmon resonance, "TFA" refers to trifluoroacetic acid, and "Trt" refers to trityl.

[0093] VHH-based compounds and fusion or conjugate compounds containing same that act as half-life extenders Briefly, the compounds herein can comprise an amino acid sequence from N-terminus to C-terminus having one of the following structures: M-X1, X1-M, M-X2, X2-M, M-L1-X1, M-L2-X1, M-L1-X2, M-L2-X2, X1-L1-M, X1-L2-M, X2-L1-M, X2-L2-M, X1-M-X2, X2-M-X1, X1-L1-M-X2, X2-L1-M-X1, X1-M-L1-X2, X2-M-L1-X1, X1-L1-M-L2-X2, X2-L1-M-L2-X1, M-L1-X1-L2-X2, M-L1-X2-L2-X1, X1-L2-X2-L1-M, X2-L2-X1-L1-M, or M-L1-X1 combined (i.e., non-covalently associated) with M-L2-X2 wherein M acts as a t1 / 2 extension and is a VHH-based compound having an amino acid sequence selected from SEQ ID NOs: 1-37 and 124-126, or an amino acid sequence having at least about 90% to about 99% sequence similarity thereto; L1 (if present) is a first linker; L2 (if present) is a second linker; X1 (if present) is a biotherapeutic protein, peptide, or oligomer; and X2 (if present) is a biotherapeutic protein, peptide, or oligomer, which may be the same as or different from X1 (e.g., if the biotherapeutic is a homodimer or if the biotherapeutic is a heterodimer, X1 can be one chain (the α chain) and X2 can be the other chain (the β chain)). X1 and X2 can also be completely different from each other.

[0094] In some instances, L1 is (GGGGQ) n (SEQ ID NO: 38), (GGGQ) n (SEQ ID NO: 39), (GGGGS) n (SEQ ID NO: 40), (PGPQ) n (SEQ ID NO: 40), (PGPA) n(SEQ ID NO: 42), GGGG(AP) n GGGG (SEQ ID NO: 43), (GGE) n (SEQ ID NO: 44), (GGGGE) n (SEQ ID NO: 45), (GGK) n (SEQ ID NO: 46), (GGGGK) n (SEQ ID NO: 47), GGGG(EP) n GGGG (SEQ ID NO: 48), GGGG (KP) n GGGG (SEQ ID NO: 49), (PGPE) n (SEQ ID NO: 50), or (PGPK) n (SEQ ID NO: 51), where n can be 1 to 15, particularly about 5 to about 10. In another example, L1 can have an amino acid sequence selected from any one of SEQ ID NOs: 52 to 63. In yet another example, L1 can have one or more additions, deletions, insertions, or substitutions such that L1 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 52 to 63.

[0095] In some examples, L2 can have an amino acid sequence selected from any one of SEQ ID NOs: 64-65. In still other examples, L2 can have one or more additions, deletions, insertions, or substitutions such that L2 has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOs: 64-65.

[0096] In yet another embodiment, L1 or L2 is polyethylene glycol (PEG), particularly maleimide-(PEG). 12 The polymer may be, for example, a methyl acrylate or a methyl acrylate.

[0097] In summary, exemplary fusions are as follows:

[0098] Compound 1, a VHH-CNTF fusion comprising CNTF, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0099] Compound 2, a VHH-CNTF fusion comprising a VHH-based compound acting as a t1 / 2 extender (underlined), a (G4Q)5 linker (italicized), and CNTF, has the following amino acid sequence: [ka]

[0100] Compound 3, a VHH-NRG1 fusion comprising NRG1, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0101] Compound 4, a VHH-NRG1 fusion comprising a VHH-based compound acting as a t1 / 2 extender (underlined), a (G4Q)5 linker (italicized), and NRG1, has the following amino acid sequence: [ka]

[0102] Compound 5, a VHH-GDF15 fusion comprising a VHH-based compound acting as a t1 / 2 extender (underlined), a (G4Q)5 linker (italicized), and GDF15, has the following amino acid sequence: [ka]

[0103] Compound 6, a VHH-IL-2 fusion comprising IL-2, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0104] Compound 7, a VHH-IL-2 fusion comprising a VHH-based compound acting as a t1 / 2 extension (underlined), a (G4Q)5 linker (italicized), and IL-2, has the following amino acid sequence: [ka]

[0105] Compound 8, a VHH-GLP-1 fusion comprising GLP-1, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0106] Compound 9, a VHH-Fab fusion comprising HC, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0107] Compound 9 further comprises an LC having the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109)

[0108] Compound 10, a VHH-Fab fusion comprising an LC, a (G4Q)5 linker (italicized), and a VHH-based compound acting as the t1 / 2 extension (underlined), has the following amino acid sequence: [ka]

[0109] Compound 10 further comprises an HC having the following amino acid sequence: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 110)

[0110] Compound 11, a VHH-Fab fusion comprising a VHH-based compound acting as a t1 / 2 extension (underlined), a (G4Q)5 linker (italicized), and a HC, has the following amino acid sequence: [ka]

[0111] Compound 11 further comprises an LC having the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

[0112] Compound 12, a VHH-Fab fusion comprising a VHH-based compound acting as a t1 / 2 extension (underlined), a (G4Q)5 linker (italicized), and an LC, has the following amino acid sequence: [ka]

[0113] Compound 12 further comprises an HC having the following amino acid sequence: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 114)

[0114] Compound 13, a VHH-GLP-1 fusion containing GLP-1, a (G4Q)5 linker (italicized), a VHH-based compound acting as a t1 / 2 extension (underlined), and a C-terminal Cys, has the following amino acid sequence: [ka]

[0115] GLP-1, a (G4Q)5 linker (italicized), a VHH-based compound acting as a t1 / 2 extender (underlined), a C-terminal Cys, and maleimide (PEG) in a C-terminal to N-terminal orientation. 12 Compound 14, a VHH-GLP-1 / ACTH fusion conjugate containing ACTH connected via a linker, has the following amino acid sequence: [ka]

[0116] VHH-based compounds acting as t1 / 2 extenders (underlined), C-terminal Cys, and maleimide (PEG) in a C-terminal to N-terminal orientation 12 Compound 15, a VHH-ACTH fusion conjugate containing ACTH connected via a linker, has the following amino acid sequence: [ka]

[0117] Pharmaceutical compositions and kits The compounds herein (i.e., VHH-based fusions or VHH-based conjugates, such as compounds 1-15 above) can be formulated as pharmaceutical compositions that can be administered parenterally (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally). Such pharmaceutical compositions and techniques for their preparation are well known in the art. See, for example, Remington, "The Science and Practice of Pharmacy" (D.B. Troy ed., 21 st See, e.g., Lippincott, Williams & Wilkins, Ed., 2006). In certain cases, the compositions are administered SQ or IV. Alternatively, however, the compositions may be formulated in forms for other pharmaceutically acceptable routes, such as, for example, tablets or other solid forms for oral administration; sustained-release capsules; and any other form currently in use, including creams, lotions, inhalants, etc.

[0118] As mentioned above, to improve their in vivo compatibility and efficacy, the VHH-based fusions or VHH-based conjugates herein may be reacted with any number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and general techniques for their preparation are well known in the art (see, for example, Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use" (2002). ndRevised Ed. Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts.

[0119] The compound herein can be administered by a doctor or self-administered by injection.It is understood that the gauge size and injection volume can be easily determined by those skilled in the art.However, injection volume can be about 2mL or less, or even about 1mL or less, and needle gauge can be about 27G or more, or even about 29G or more.

[0120] The present disclosure also provides and thus encompasses novel intermediates and methods useful for synthesizing the compounds herein, or pharmaceutically acceptable salts thereof. The intermediates and compounds can be prepared by a variety of techniques well known in the art. For example, methods using recombinant synthesis are illustrated in the examples below. The specific steps for each of the techniques described can be combined in various ways to prepare the compounds. The reagents and starting materials are readily available to those skilled in the art.

[0121] The compounds of the present invention are generally effective over a wide range of dosages. Exemplary dosages of the compounds or pharmaceutical compositions containing them may be milligrams (mg), micrograms (μg), nanograms (ng), or picograms (pg) per kilogram (kg) of an individual. Thus, the daily dosage may be about 1 μg to about 100 mg.

[0122] Here, the effective amount of the compound in the pharmaceutical composition can be about 0.25 mg to about 5.0 mg. However, those skilled in the art will understand that in some cases, the effective amount (i.e., dosage / dosage) may be more than sufficient even if it is below the lower limit of the aforementioned range, while in other cases, the effective amount may be a higher dosage and may be used with acceptable side effects.

[0123] In addition to the compounds herein, the pharmaceutical compositions can also include at least one additional therapeutic agent, such as, for example, a therapeutic agent typically used as a standard of care for a particular condition, disease, or disorder (e.g., a cardiovascular, neurological, immune, metabolic, oncology, psychiatric, pulmonary, and / or renal condition, disease, or disorder).

[0124] Thus, a pharmaceutical composition can include an effective amount of one or more compounds herein, a pharmaceutically acceptable carrier, and optionally at least one additional therapeutic agent. For example, the pharmaceutical composition can include an effective amount of SEQ ID NO: 100 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 101 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 102 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 103 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 104 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 105 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 106 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 107 and a pharmaceutically acceptable carrier, or an effective amount of the compound of SEQ ID NO: 108 and a pharmaceutically acceptable carrier. carrier, an effective amount of the compounds of SEQ ID NOs: 108 and 109 and a pharmaceutically acceptable carrier, an effective amount of the compounds of SEQ ID NOs: 110 and 111 and a pharmaceutically acceptable carrier, an effective amount of the compounds of SEQ ID NOs: 112 and 113 and a pharmaceutically acceptable carrier, an effective amount of the compounds of SEQ ID NOs: 114 and 115 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 116 and a pharmaceutically acceptable carrier, an effective amount of the compound of SEQ ID NO: 117 and a pharmaceutically acceptable carrier, and an effective amount of the compound of SEQ ID NO: 118 and a pharmaceutically acceptable carrier.

[0125] Alternatively, the compounds herein may be provided as part of a kit. In some examples, the kit includes a device for administering at least one compound (and optionally at least one additional therapeutic agent) to an individual. In certain examples, the kit includes a syringe and needle for administering at least one compound (and optionally at least one additional therapeutic agent) herein. In certain cases, the compound (and optionally at least one additional therapeutic agent) is pre-formulated in an aqueous solution in the syringe.

[0126] Methods of making and using VHH-based compounds or fusions and conjugates thereof that act as half-life extenders The compounds herein can be made via any number of standard recombinant DNA methods known in the art or standard chemical peptide synthesis methods. With regard to recombinant DNA methods, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence encoding the amino acid sequence of the compound (i.e., a fusion peptide or fusion conjugate), incorporate the polynucleotide into a recombinant expression vector, and introduce the vector into host cells, such as bacteria, yeast, and mammalian cells, to produce the compound. See, for example, Green & Sambrook, "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 4 th ed. 2012).

[0127] With respect to recombinant DNA methods, the compounds herein can be prepared by producing protein or precursor protein molecules using recombinant DNA technology. DNA, including cDNA and synthetic DNA, can be double-stranded or single-stranded, and the coding sequence therein encoding the compounds herein can vary as a result of redundancy or degeneracy in the genetic code. Briefly, a DNA sequence encoding the compounds herein is introduced into a host cell to produce the compounds or their precursors. The host cell can be a bacterial cell, such as Escherichia coli K12 or B strain, a fungal cell, such as a yeast cell, or a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0128] To produce the compound of the present invention or its precursor, an appropriate host cell is transiently or stably transfected or transformed with an expression system such as an expression vector. The expression vector is typically replicable in the host organism, either as an episome or as an integrated part of the host chromosomal DNA. Generally, the expression vector contains a selection marker, such as tetracycline, neomycin, G418, and dihydrofolate reductase, to allow detection of cells transformed with the desired DNA sequence.

[0129] The specific biosynthetic or synthetic steps for each of the steps described herein may be used, not used, or combined in different ways to prepare the compounds of the present invention.

[0130] For chemical peptide synthesis, standard manual or automated solid-phase synthesis procedures can be used. For example, automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial suppliers. Solid-phase synthesizers can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during the reaction, coupling, deprotecting, and capping unreacted amino acids.

[0131] One use of the compounds herein is in treating cardiovascular, neurological, immune, metabolic, oncology, psychiatric, pulmonary, and / or renal conditions, diseases, or disorders.

[0132] The method may include the steps described herein, which may be, but are not necessarily, performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. Furthermore, the method may include additional, unspecified steps.

[0133] Thus, such methods may include, for example, selecting an individual having or susceptible to a neurological condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a metabolic condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a cardiovascular condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a neoplastic condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a psychiatric condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a pulmonary condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to a renal condition, disease, or disorder. Alternatively, the method may include selecting an individual having or susceptible to an autoimmune condition, disease, or disorder.

[0134] The method may also include administering to the individual an effective amount of at least one compound herein, which may be in the form of a pharmaceutical composition as described herein. In some examples, the compound / pharmaceutical composition may include an additional therapeutic agent.

[0135] The concentrations / dosages / administration of the compound and optional additional therapeutic agents are discussed elsewhere herein.

[0136] With regard to the route of administration, the compounds or pharmaceutical compositions comprising same may be administered according to known methods, for example, orally, by injection (i.e., intra-arterial, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraportal, or intralesional), by sustained release system, or by implanted device, etc. In certain cases, the compounds or pharmaceutical compositions comprising same may be administered SQ by bolus injection or continuously.

[0137] With regard to dosing frequency, the compound or pharmaceutical composition comprising it can be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every other week), or monthly. In certain cases, the compound or pharmaceutical composition comprising it is administered SQ every other day, SQ three times a week, SQ twice a week, SQ once a week, SQ every other week, or SQ monthly. In certain cases, the compound or pharmaceutical composition comprising it is administered SQ once a week (QW).

[0138] Alternatively, when administered IV, the compound or pharmaceutical composition comprising same can be administered IV every other day, IV three times a week, IV twice a week, IV once a week, IV every other week, or IV monthly. In certain cases, the compound or pharmaceutical composition comprising same is administered IV once a week (IW).

[0139] When the present compound or a pharmaceutical composition comprising it is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent may be administered simultaneously with the present compound or a pharmaceutical composition comprising it, separately, or sequentially.

[0140] Moreover, the additional therapeutic agent may be administered at the same frequency as the compound or pharmaceutical composition comprising same (i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent may be administered at a different frequency than the compound or pharmaceutical composition comprising same. In other cases, the additional therapeutic agent may be administered SQ. In other cases, the additional therapeutic agent may be administered IV. In still other cases, the additional therapeutic agent may be administered orally.

[0141] It is further contemplated that these methods may be combined with diet and exercise, and / or may be combined with additional therapeutic agents other than those discussed above. [Example]

[0142] The following non-limiting examples are offered by way of illustration and not limitation.

[0143] Polypeptide Expression, Purification, and Conjugation Example 1: Recombinant expression and purification of VHH-based fusion 1 Example 1 is a CNTF-VHH fusion having the following amino acid sequence: MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 100)

[0144] Here, a VHH fusion of SEQ ID NO: 100 is produced in a mammalian cell expression system using a CHOK1 cell derivative. The cDNA sequence encoding SEQ ID NO: 100 is subcloned into an expression plasmid backbone (pEE12.4-based plasmid) containing GS. The cDNA sequence is fused in frame with the coding sequence for the signal peptide sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 66) to enhance secretion of the VHH fusion into tissue culture medium. Expression is driven by a viral CMV promoter.

[0145] To generate VHH fusions via transient transfection, CHOK1 cells are transfected with recombinant expression plasmids using a PEI-based method. Briefly, at a density of 4 x 10 6 An appropriate volume of CHOK1 suspension cells in cells / mL is transferred to a shake flask and both PEI and recombinant plasmid DNA are added to the cells. The cells are incubated in suspension culture at 32°C for 6 days. At the end of the incubation period, the cells are removed by low-speed centrifugation and the VHH fusions are purified from the conditioned medium.

[0146] Alternatively, to generate VHH fusions via stable transfection, CHOK1 cells are stably transfected using electroporation and an appropriate amount of recombinant expression plasmid, and the transfected cells are maintained in suspension culture at the appropriate cell density. Selection of transfected cells is achieved by growing them in serum-free medium containing 25 μM MSX and incubating at approximately 37° C. and approximately 6% CO2.

[0147] VHH fusions secreted into the medium from CHO cells are purified by Protein A affinity chromatography, followed by ion exchange, hydrophobic interaction, or size exclusion chromatography. Specifically, VHH fusions from harvested medium are captured on Mab Select Protein A resin (GE Healthcare). The resin is then briefly washed with a running buffer such as phosphate-buffered saline (PBS; pH 7.4) or a running buffer containing Tris to remove nonspecifically bound material. VHH fusions are eluted from the resin with a low pH solution, such as 10 mM citric acid at pH 3. Fractions containing the VHH fusions can be pooled at low pH to inactivate potential viruses. The pH can also be neutralized by adding a base such as 1 M Tris at pH 8.0. VHH fusions can also be further purified by ion exchange chromatography using a resin such as Poros 50 HS (ThermoFisher). The VHH fusions are eluted from the column using a NaCl gradient from 0 to 500 mM in 20 mM NaOAc at pH 5.0 over 15 column volumes.

[0148] VHH fusions may be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes HIC Column (GE Healthcare). Purification is performed by adjusting the column loading solution to approximately 0.5 M sodium sulfate and eluting with a 10 column volume (CV) gradient from 0.5 M to 0 M sodium sulfate in 20 mM Tris, pH 8. After HIC, VHH fusions may be further purified by SEC by loading the concentrated Capto Phenyl ImpRes pool onto a Superdex200 or Superdex75 (GE Healthcare) column using an isocratic elution in PBS, pH 7.4, or in 20 mM histidine, 50 mM NaCl, pH 6.0.

[0149] The purified VHH fusion is passed through a virus-retaining filter such as Planova 20N (Asahi Kasei Medical) and then concentrated / diafiltered into 20 mM histidine, 20 mM NaCl, pH 6 using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

[0150] Thus, VHH fusions are prepared in this manner, or in a similar manner readily determined by one skilled in the art.

[0151] Example 2: Recombinant expression and purification of VHH-based fusion 2 Example 2 is a VHH-CNTF fusion with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQMAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTG (SEQ ID NO: 101)

[0152] Here, a VHH fusion of SEQ ID NO: 101 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 101 is used in the expression plasmid.

[0153] Example 3: Recombinant expression and purification of VHH-based fusion 3 Example 3 is an NRG1-VHH fusion having the following amino acid sequence: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 102)

[0154] Here, a VHH fusion of SEQ ID NO: 102 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 102 is used in the expression plasmid.

[0155] Example 4: Recombinant expression and purification of VHH-based fusion 4 Example 4 is a VHH-NRG1 fusion with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQ (SEQ ID NO: 103)

[0156] Here, a VHH fusion of SEQ ID NO: 103 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 103 is used in the expression plasmid.

[0157] Example 5: Recombinant expression and purification of VHH-based fusion 5 Example 5 is a VHH-GDF15 fusion with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 104)

[0158] Here, a VHH fusion of SEQ ID NO: 104 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 104 is used in the expression plasmid.

[0159] Example 6: Recombinant expression and purification of VHH-based fusion 6 Example 6 is an IL-2-VHH fusion having the following amino acid sequence: APQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 105)

[0160] Here, a VHH fusion of SEQ ID NO: 105 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 105 is used in the expression plasmid.

[0161] Example 7: Recombinant expression and purification of VHH-based fusion 7 Example 7 is a VHH-IL-2 fusion with the following amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQAPQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT SEQ ID NO: 106)

[0162] Here, a VHH fusion of SEQ ID NO: 106 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 106 is used in the expression plasmid.

[0163] Example 8: Recombinant expression and purification of VHH-based fusion 8 Example 8 is a GLP-1-VHH fusion having the following amino acid sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 107)

[0164] Here, a VHH fusion of SEQ ID NO: 107 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 107 is used in the expression plasmid.

[0165] Example 9: Recombinant expression and purification of VHH-based fusion 9 Example 9 is a Fab-VHH fusion comprising an HC-VHH and an LC, and the HC-VHH amino acid sequence is as follows: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 108)

[0166] The LC amino acid sequence is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109).

[0167] Here, VHH fusions of SEQ ID NOs: 108 and 109 are produced essentially as described for Example 1, except that the cDNA sequences encoding SEQ ID NOs: 108 and 109 are cloned into two expression plasmids, and then a 1:1 mix of both plasmids is used for transfection.

[0168] Example 10: Recombinant expression and purification of VHH-based fusion 10 Example 10 is a Fab-VHH fusion comprising an HC and an LC-VHH, where the HC amino acid sequence is: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (sequence 110)

[0169] The LC-VHH amino acid sequence is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 111).

[0170] Here, VHH fusion analogs of SEQ ID NOs: 110 and 111 are produced essentially as described for Example 1, except that the cDNA sequences encoding SEQ ID NOs: 110 and 111 are cloned into two expression plasmids, and then a 1:1 mix of both plasmids is used for transfection.

[0171] Example 11: Recombinant expression and purification of VHH-based fusion 11 Example 11 is a Fab-VHH fusion comprising a VHH-HC and a LC, the VHH-HC amino acid sequence being: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 112)

[0172] The LC amino acid sequence is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113).

[0173] Here, VHH fusions of SEQ ID NOs: 112 and 113 are produced essentially as described for Example 1, except that the cDNA sequences encoding SEQ ID NOs: 112 and 113 are cloned into two expression plasmids, and then a 1:1 mix of both plasmids is used for transfection.

[0174] Example 12: Recombinant expression and purification of VHH-based fusion 12 Example 12 is a VHH-Fab comprising an HC and a VHH-LC, the HC amino acid sequence of which is as follows: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 114)

[0175] The VHH-LC amino acid sequence is as follows: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 115).

[0176] Here, VHH fusion analogs of SEQ ID NOs: 114 and 115 are produced essentially as described for Example 1, except that the cDNA sequences encoding SEQ ID NOs: 114 and 115 are cloned into two expression plasmids, and then a 1:1 mix of both plasmids is used for transfection.

[0177] Example 13: Recombinant expression and purification of control Fab Example 13 is a Fab containing an HC and an LC, the HC amino acid sequence being: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 120)

[0178] The LC amino acid sequence is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 121).

[0179] Here, the Fabs of SEQ ID NOs: 120 and 121 are produced essentially as described for Example 1, except that the cDNA sequences encoding SEQ ID NOs: 120 and 121 are cloned into two expression plasmids, and then a 1:1 mix of both plasmids is used for transfection.

[0180] Example 14: Recombinant expression and purification of VHH-based fusion 13 Example 14 is a GLP-1-VHH fusion having the following amino acid sequence: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC (SEQ ID NO: 116)

[0181] Here, a VHH fusion of SEQ ID NO: 116 is produced essentially as described for Example 1, except that a cDNA sequence encoding SEQ ID NO: 116 is used in the expression plasmid.

[0182] Example 15: Production of VHH-based conjugate 1 Example 15 is a GLP-1 / ACTH-VHH conjugate having the following chemical structure: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC-Maleimide-(PEG) 12 -ACTH (C-terminal to N-terminal orientation; SEQ ID NO: 117). The VHH conjugate of SEQ ID NO: 117 is produced as follows: 1 mg of the VHH fusion of Example 14 (SEQ ID NO: 116) is prepared at 1 mg / mL (approximately 50 μM) by diluting 10 times the 10 mg / mg stock solution with PBS, pH 7.2. Four equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in PBS, pH 7.2 is added and incubated for 4 hours at room temperature. Successful reduction to a homogeneous monomer is confirmed via mass spectrometry.

[0183] The reaction mixture is desalted using a 2 mL 7K MW Cutoff ZEBA desalting column. The column storage buffer is removed by spinning at 1000 rcf for 1 minute. 2 mL of PBS, pH 7.2, is added and the column is washed by spinning at 1000 rcf for 1 minute. The reaction mixture is loaded onto a ZEBA column and desalted by spinning at 1000 rcf for 1 minute into a clean 15 mL conical vial. The desalted sample is concentrated to approximately 700 μM (approximately 100 μL) using a 3K MW cutoff Amicon Ultra spin column.

[0184] ACTH-(PEG) 12 -Maleimide-NH2 (SEQ ID NO: 119) is prepared at 20 mg / mL (5.2 mM) in PBS, pH 7.2. 20 μL of ACTH-(PEG) corresponding to 2 stoichiometric equivalents is added. 12 -Maleimide-NH2 is added to a desalted, concentrated solution of the VHH fusion of Example 14 (SEQ ID NO: 116). A white precipitate forms immediately. The reaction is allowed to continue for 10 minutes and the solution is solubilized by slowly adding 0.1 M HCl to lower the pH to 5.5 until the solution is clear. Successful conjugation is confirmed via mass spectrometry.

[0185] The desired product is purified on an AKTA purification system using a 5 mL MabSelect Protein A resin (GE Healthcare) column with a loading buffer of PBS, pH 7.2, and an elution buffer of 20 mM citrate, pH 3. The sample is loaded onto the column and the unreacted ACTH-(PEG) 12 Hold the loading buffer phase until the -maleimide-NH2 peak has passed through the column. Once the signal returns to baseline, switch the buffer to 20 mM citrate, pH 3, to elute the desired product from the column. Collect and pool 1 mL fractions during elution and adjust the pH to 7 using Tris-HCl, pH 8.

[0186] To open the maleimide ring, the pH is adjusted to 8 by adding 0.1 M NaOH and left overnight at room temperature. To complete the partial ring opening, the pH is adjusted to 8.5 and incubated for an additional 24 hours at room temperature. Completion of the ring opening is confirmed by mass spectrometry, and a final pH adjustment to approximately 7.2 is made by adding 0.1 N HCl. The VHH conjugate of Example 15 is then stored at 4°C.

[0187] Example 16: Production of VHH-based conjugate 2 Example 16 is an ACTH-VHH conjugate with the following chemical structure: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC-Maleimide-(PEG) 12 -(ACTH) (C-terminal to N-terminal orientation; SEQ ID NO: 118).

[0188] The VHH conjugate of SEQ ID NO: 118 is produced as follows: 1 mg of control VHH (SEQ ID NO: 37) is prepared at 1 mg / mL (approximately 70 μM) by diluting 10-fold from a 10 mg / mg stock solution with PBS, pH 7.2. Four equivalents of TCEP in PBS, pH 7.2, is added and incubated for 4 hours at room temperature. Successful reduction to a homogeneous monomer is confirmed via mass spectrometry.

[0189] The reaction mixture is desalted using a 2 mL 7K MW Cutoff ZEBA desalting column. The column storage buffer is removed by spinning at 1000 rcf for 1 minute. 2 mL of PBS, pH 7.2, is added and the column is washed by spinning at 1000 rcf for 1 minute. The reaction mixture is loaded onto a ZEBA column and desalted by spinning at 1000 rcf for 1 minute into a clean 15 mL conical vial. The desalted sample is concentrated to approximately 700 μM (approximately 100 μL) using a 3K MW cutoff Amicon Ultra spin column.

[0190] ACTH-(PEG) 12 -maleimide-NH2 (SEQ ID NO: 119) was prepared and conjugated to VHH as described in Example 15.

[0191] In vitro function – albumin binding Example 17: Albumin binding studies via SPR of the VHH moiety The in vitro binding of various VHH moieties to human, cynomolgus monkey, mouse, rat, pig, dog, and bovine serum albumin (SA) is determined by SPR. In particular, the binding of the VHH moieties herein to SA of these species is summarized below in Table 1. The binding of the VHH moieties of SEQ ID NOs: 3 and 8-28 to various SA is performed on a Biacore 8K instrument.

[0192] Immobilization of SA onto the surface of a Series S Sensor Chip CM5 was performed according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, the carboxyl groups on the sensor chip surface (flow cells 1 and 2) were activated by injecting 70 μL of a mixture containing 75 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 11.5 mg / mL N-hydroxysuccinimide (NHS) at 10 μL / min. SA from human, cynomolgus monkey, mouse, rat, pig, dog, and bovine species was diluted to 1, 1, 3, 1, 1, 1, and 1 μg / mL in 10 mM sodium acetate (BR-1003-49) at pH 4.0 and then injected over the activated chip surface (flow cell 2, channels 1–8) at 10 μL / min for 90 s. Human SA is obtained from Sigma Aldrich (St. Louis, MO, Catalog No. A8763). Cynomolgus SA is obtained from Athens R&T (Athens, GA, Catalog No. 16-16-011202-CM). Mouse SA is obtained from Sigma Aldrich (Cat. No. A3559). Rat SA is obtained from Sigma Aldrich (Cat. No. A4538). Pig SA is obtained from Sigma Aldrich (Cat. No. A4414). Canine SA is obtained from Molecular Innovations (Novi, MI, Catalog No. DSA-1213 NC0739153). Bovine SA is obtained from Sigma Aldrich (Cat. No. A7030). Various SAs were covalently immobilized via free amines onto a carboxymethyldextran-coated sensor chip CM5, targeting an average surface density of approximately 77 (range, 58-98) RU. Excess reactive groups on the surface (flow cells 1 and 2) were deactivated by injecting 70 μL of 1 M ethanolamine hydrochloride-NaOH, pH 8.5, at 10 μL / min.

[0193] The VHH moiety is diluted to a concentration of 300 nM in HBS-EP+ buffer (10 mM HEPES, pH 7.6, 150 mM NaCl, 3 mM EDTA, and 0.05% polysorbate 20). 150 μL of sample is injected individually and sequentially across the immobilized SA surface and allowed to dissociate for 600 seconds at a flow rate of 50 μL / min at 25°C. The surface is regenerated by injecting 10 mM glycine-HCl (BR-1003-54), pH 1.5, at 50 μL / min for 100 seconds. The resulting sensorgrams are analyzed using Biacore 8K Insight Evaluation Software (version 2.0.15.12933) and the dissociation rate (kd) is calculated. [Table 1]

[0194] Example 18: VHH-based fusion albumin binding studies via SPR In vitro binding of VHH-based fusions to human, cynomolgus monkey, mouse, rat, pig, dog, bovine, and rabbit SA is determined by SPR at 25° C. In particular, the affinities of the VHH fusions of Examples 1-9 for SA of these species are summarized below in Tables 2-10.

[0195] Binding of the VHH-based fusions of Examples 1-9 to various SAs was performed on a Biacore 8K instrument. Immobilization of various SAs onto the surface of a Series S Sensor Chip CM5 (BR-1006-68) was performed according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, the carboxyl groups on the sensor chip surface (flow cells 1 and 2) were activated by injecting 70 μL of a mixture containing 75 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 11.5 mg / mL N-hydroxysuccinimide (NHS) at 10 μL / min. Human, cynomolgus monkey, rat, mouse, dog, pig, cow, and rabbit SA were diluted in 10 mM sodium acetate (BR-1003-49) at pH 4.0 to 1 and 0.8 μg / ml, 1 and 0.8 μg / ml, 1.5 and 0.8 μg / ml, 4 and 2.5 μg / ml, 1 and 0.8 μg / ml, 1 and 1 μg / ml, 1 and 1 μg / ml, and 1 and 1.5 μg / ml and then injected over the activated chip surface (flow cell 2, channels 1-8) at 10 μL / min for 100 seconds. Human SA was obtained from Sigma-Aldrich (catalog no. A8763). Cynomolgus monkey SA was obtained from Athens R&T (catalog no. 16-16-011202-CM). Mouse SA was obtained from Sigma-Aldrich (catalog no. A3139). Rat SA is obtained from Sigma Aldrich (catalog number A4538). Porcine SA is obtained from Sigma Aldrich (catalog number A4414). Canine SA is obtained from Molecular Innovations (Novi, MI, catalog number DSA-1213 NC0739153). Bovine SA is obtained from Sigma Aldrich (catalog number A7030). Rabbit SA is obtained from Fitzgerald Industries International (Acton, MA, catalog number 30R-3303).SA is covalently immobilized via free amines onto a carboxymethyldextran-coated sensor chip CM5 at a surface density of 25-78 resonance units (RU) for human, cynomolgus monkey, rat, mouse, dog, pig, and bovine SA, and 118-372 resonance units (RU) for rabbit SA. Excess reactive groups on the surface (flow cells 1 and 2) are deactivated by injecting 70 μl of 1 M ethanolamine hydrochloride-NaOH, pH 8.5.

[0196] VHH fusions are diluted in HBS-EP+ buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) to concentrations of 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37, 0.457, 0.152, 0.051, and 0.017 nM. 180 μl of sample is injected individually and sequentially across the immobilized SA on the surface of the chip and allowed to dissociate for 600 seconds at a flow rate of 60 μL / min at 25°C. The surface is regenerated by injecting 10 mM glycine-HCl (BR-1003-54), pH 1.5, at 60 μL / min for 100 seconds. The resulting sensorgrams were analyzed using a 1:1 binding kinetics model fitting in Biacore 8K Insight Evaluation Software (version 3.0.11.15423) to obtain the binding kinetic parameters association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K D ) is calculated. [Table 2]

[0197] K for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 1 D are determined to be 3.2, 13, 100, 83, 190, 40, and 820 nM, respectively. [Table 3]

[0198] The KDs for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 2 are determined to be 0.58, 1.2, 8.8, 6.1, 19, 3.7, and 110 nM, respectively. [Table 4]

[0199] The KDs for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 3 are determined to be 1.9, 6.1, 62, 52, 130, 19, and 470 nM, respectively. [Table 5]

[0200] The KDs for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 4 are determined to be 0.19, 0.94, 6.4, 3.1, 15, 2.2, and 100 nM, respectively. [Table 6]

[0201] The KD for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 5 are determined to be 0.2, 0.45, 0.86, 0.74, 3.3, 0.6, and 28 nM, respectively. [Table 7]

[0202] The KD for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 6 are determined to be 1.5, 5.0, 50, 34, 100, 19, and 380 nM, respectively. [Table 8]

[0203] The KD for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 7 are determined to be 0.23, 0.39, 3.9, 3.0, 13, 1.8, and 78 nM, respectively. [Table 9]

[0204] The KD for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 8 are determined to be 0.15, 0.96, 18, 11, 27, 5.4, and 120 nM, respectively. [Table 10]

[0205] The KD for SA binding in human, cynomolgus monkey, mouse, rat, pig, dog, and cow using the VHH-based fusions of Example 9 are determined to be 1.3, 4.3, 43, 33, 83, 15, and 340 nM, respectively.

[0206] In vitro function - payload specific activity Example 19: In vitro potency of VHH-based fusions of Examples 1 and 2 Human CNTFR pSTAT3 assay: IMR-32 cells (ATCC, catalog no. CCL-127), which endogenously express human CNTFR, LIFR, and gp130, were cultured in RPMI-1640 (ATCC, catalog no. 30-2001) medium supplemented with a final 10% FBS, 1 mM sodium pyruvate, and 1x Antibiotic-Antimycotic (Thermo Fisher Scientific, 15240062) at 37°C, 5% CO2, and 90% humidity. On day -1 (the day before the pSTAT3 assay), cells were washed once with 1x PBS, removed from the flask using cell dissociation buffer (Gibco, 13151), and resuspended in the above-mentioned culture medium. Cells were plated at 150,000 cells / 0.1 mL / well in 96-well poly-D-lysine-coated plates (Corning, catalog no. 354640). The cells are cultured overnight at 37°C, 5% CO2, and 90% humidity. On day 1 (the day of the pSTAT3 assay), the medium is aspirated and replaced with 100 μL of EMEM (ATCC, catalog number 30-2003). The cells are serum-starved for 4 hours at 37°C, 5% CO2, and 90% humidity, after which 50 μL of human CNTF (R&D Systems, catalog number 257-NT-010), Example 1, or Example 2, serially diluted 3-fold in 0.3% IgG-free BSA (Jackson ImmunoResearch Laboratories, Inc., catalog number 001-000-162), is added. EMEM is added to a final concentration of 1x. The cells are incubated with these serially diluted proteins for an additional 10 minutes at 37°C, 5% CO2, and 90% humidity. After the incubation period is complete, the contents are removed from the plate. Cells are lysed and pSTAT3 is detected using the AlphaLISA Surefire Ultra p-STAT3 (Tyr705) Assay Kit (Perkin Elmer, ALSU-PST3) and its two-plate assay protocol for adherent cells. Plates are read on an Envision 2102 instrument (Perkin Elmer).

[0207] Statistical analysis of data: Data are imported from the Envision 2102 instrument reader into Excel® (Microsoft). The % of maximum (1 nM) human CNTF stimulated pSTAT3 signal is calculated for each concentration of protein tested. EC 50 Values ​​are generated from these calculations by variable slope-four parameter dose-response curve fitting using GraphPad Prism® software (GraphPad Software, LLC, La Jolla, CA, version 8.4.3). The results of the assay are presented below in Table 11. [Table 11]

[0208] Example 20: In vitro potency of VHH-based fusions of Examples 3 and 4 A DiscoverX / eurofins cell-based dimerization bioassay (PathHunter eXpress ErbB4 / ErbB4 Dimerization Assay, Catalog No. 93-0961E3) is performed to test the potency of the VHH fusions of Examples 3 and 4 on the human ErbB4 receptor (NCBI Reference Sequence No. NP_001036064.1). This assay detects ligand-induced dimerization of the two subunits of a receptor-dimer pair and is designed to assess potency. Tests are performed according to the protocol provided by the manufacturer. Briefly, cells are thawed and plated at 100 μL per well into the provided 96-well plate. After 24 hours of incubation at 37°C in 5% CO2, 10 μL of 11-fold dilutions of Examples 3, 4, and the control agonist rhNRG-1 (eurofins, Catalog No. 92-1091) are added in duplicate to the appropriate wells in an 11-point serial dilution curve (1:3). Plates are incubated at 37°C for 6 hours. After the 6-hour incubation, 110 μL of detection reagent is added to each well. Plates are incubated for an additional hour in the dark at room temperature. Chemiluminescent signals are read on a plate reader (Molecular Devices SpectraMax i3x plate reader) using a 0.5-second integration time. Statistical analysis of raw data is performed using GraphPad PRISM version 9.0. The average background reading is subtracted from all values, and raw data are transformed and nonlinear regression analysis (log (agonist) vs. response - variable slope (4 parameters)) is performed to determine EC 50 Obtain values ​​and determine goodness of fit (n=2 for all data). [Table 12]

[0209] Example 21: In vitro potency of VHH-based fusions of Example 5 Generation of HEK293 human GFRAL and human RET-expressing cell lines: HEK293 cells (ATCC) were cultured in DMEM containing 10% FBS, 25 mM HEPES, and 1x antibiotics and split 1:16 every 3–4 days using TrypLE™ Express (Gibco). Cells were transfected with plasmid DNA for human GFRAL (GDNF receptor alpha-like, NCBI reference sequence NP_997293.2), human RET (proto-oncogene tyrosine-protein kinase receptor RET, NCBI reference sequence NP_066124.1), and Fugene 6 (Promega) according to the manufacturer's instructions. Transfected cells were selected for 3–4 weeks using Geneticin (Gibco, 1 mg / ml) and Puromycin (Gibco, 0.1 mg / ml). Clonal lines were obtained by limiting dilution cloning into 96-well plates and confirmed for GDF15 response using the AlphaLISA SureFire Ultra p-ERK1 / 2 (Thr202 / Tyr204) Assay Kit (PerkinElmer). Clones were expanded, harvested, resuspended in freezing medium, aliquoted into cryovials, and stored in liquid nitrogen for long-term storage. Clonal line #7, the top responder with the best GDF15 response (signal-to-background ratio), was selected.

[0210] Human GFRAL and RET Receptor AlphaLISA SureFire Ultra p-ERK1 / 2 (Thr202 / Tyr204) Assay: HEK293 cell lines expressing human GFRAL and human RET are cultured in selection medium (DMEM containing 10% FBS, 25 mM HEPES, 1x antibiotics, 1 μg / mL puromycin, 1 mg / mL geneticin). On day 3 (the day of cell plating), cells are washed once with PBS, removed from flasks using TrypLE™ Express, and resuspended in plating medium (DMEM containing 25 mM HEPES, 1x antibiotics, 10% FBS). Cells are plated at 20,000 cells / 0.1 mL / well in 96-well plates (Corning Cat. No. 356461). Cells are cultured at 37°C, 5% CO2 for 72 hours. On day 1 (the day of the assay), the medium is removed and replaced with 50 μL of serum-free medium (DMEM with 25 mM HEPES and 1x antibiotics). The plate is incubated at 37°C for 4 hours, after which 50 μL of 2x ligand is added (GDF15, 1x final). The plate is incubated for an additional 10 minutes at 37°C. After the incubation period is complete, the medium is removed from the plate by decanting and blotting onto a white utility wipe. Next, 50 μL of 1x AlphaLISA SureFire Ultra Lysis Buffer is added to each well, and the plate is incubated on a plate shaker at 350 rpm for 10 minutes at room temperature. Subsequently, 10 μL cell lysate, 5 μL Acceptor Mix, and 5 μL Donor Mix are added to an OptiPlate™-384 plate (PerkinElmer catalog number 6007290) according to the manufacturer's instructions for a 2-plate / 1 incubation protocol for adherent cells (AlphaLISA SureFire Ultra p-ERK (Thr202 / Tyr204) Assay Kit, PerkinElmer catalog number ALSU-pERK-A10K).Plates are sealed, wrapped in foil, and incubated at room temperature on a plate shaker at 250 rpm for 1 minute, then incubated in the dark at room temperature for 8 hours before being read on an EnVision 2102 Multilabel Reader with EnVision Manager software (PerkinElmer).

[0211] Statistical analysis of data: Data are imported from the EnVision 2102 Multilabel Reader into GraphPad Prism® software (GraphPad Software, LLC; La Jolla, CA; version 8). 50 Values ​​are generated by variable slope-four parameter dose-response curves. [Table 13]

[0212] Example 22: In vitro potency of VHH-based fusions of Examples 6 and 7 The potency of the VHH-based fusions of Examples 6 and 7 is evaluated using the PathHunter IL-2 BioAssay Kit (eurofins / DiscoverX part number 93-1003Y3-00091), a cell-based dimerization bioassay from DiscoverX. This assay detects IL-2-induced dimerization of the two subunits of an IL-2 receptor-dimer pair and is designed to assess potency. The assay is performed according to the manufacturer's protocol. Briefly, cells are thawed and plated at 80 μL per well into the provided 96-well plate. After 20 hours of incubation at 37°C in 5% CO2, 20 μL of the 5x stock concentrate of Examples 6 and 7 and the provided IL-2 reference standard are added in an 11-point serial dilution curve (1:3) to appropriate wells in duplicate. The plate is incubated at 37°C for 6 hours. After 6 hours of incubation with Examples 6 and 7 and the reference standard, 10 μL of Detection Reagent 1 is added to each well. Mixing is achieved for 1 minute on a plate shaker at 350 rpm, and the plate is incubated for 15 minutes in the dark at room temperature. 40 μL of Detection Reagent 2 is then added to each well and incubated for an additional hour in the dark at room temperature. Chemiluminescent signals are read on a plate reader (Molecular Devices SpectraMax i3x plate reader) using a 1 second integration time. Statistical analysis of the raw data is performed using GraphPad PRISM version 8.4.3. The raw data are transformed and nonlinear regression analysis (log (agonist) vs. response - variable slope (4 parameters)) is performed to determine EC 50 Obtain the values ​​and determine the goodness of fit (n=2 for all data). [Table 14]

[0213] Example 23: In vitro potency of VHH-based fusions of Examples 9 and 13 An L929 cell-based cytotoxicity assay is used to assess the in vitro neutralization capacity of Examples 9 and 13 of soluble human TNFα, soluble cynomolgus TNFα, or membrane-bound human TNFα.

[0214] L929 cells (ATCC), which endogenously express human TNFα receptors, are cultured in DMEM high glucose medium containing 10% heat-inactivated FBS, 2 mM L-glutamine, 1x non-essential amino acids, 1x sodium pyruvate, and 1x antibiotics, and split 1:20 every 3–4 days using TrypLE™ Express (Gibco).

[0215] Soluble human TNFα protein is obtained from custom synthesis by Syngene (Bangalore, India). Soluble cynomolgus monkey TNFα protein is obtained from R&D Systems (Minneapolis, MN, catalog number 1070-RM / CF).

[0216] The stable MT104 H2 CHO membrane human TNFα-expressing cell line was generated at Eli Lilly and Company (Indianapolis, IN). Cells were cultured in Lilly medium LM7300 containing 8 mM L-glutamine and the selection agent G418 (500 μg / mL) with a 1:10 split every 3–4 days.

[0217] L929 cytotoxicity assay: On day 1, L929 cells were trypsinized with 5 mL TrypLE™ (Gibco #12605036), complete medium was added (1:3 volume), and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was gently aspirated, and the cells were resuspended in 15 mL of complete medium (DMEM high glucose medium containing 10% heat-inactivated FBS, 2 mM L-glutamine, 1x non-essential amino acids, 1x sodium pyruvate, and 1x antibiotics). An aliquot of cells was counted using a Viacell counter. Cells were plated at 10,000 cells / 0.1 mL / well into 96-well poly-D-lysine-coated plates (Corning #354496) and cultured overnight at 37°C and 5% CO2. On day 2, Examples 9 and 13 were titrated with a fixed amount of antigen: soluble human TNFα (200 pg / mL) or soluble cynomolgus monkey TNFα (750 pg / mL) or membrane human TNFα-expressing cells (5000 cells / mL) in DMEM+FBS containing actinomycin D (6.25 μg / mL). The starting concentrations were 1.5 μg / mL, 3 μg / mL, and 10 μg / mL for soluble human TNF, soluble cynomolgus monkey TNF, and membrane human TNF neutralization, respectively, and titrated by 8-point 3-fold serial dilutions. After removing the medium, the titrated compound-antigen complexes were added to a 96-well plate containing L929 cells, and the plate was incubated overnight at 37°C and 5% CO2. Stimulated negative control wells contain L929 cells + actinomycin D + TNFα antigen, while unstimulated control wells contain L929 cells + actinomycin D only.

[0218] On day 3, remove the medium from the 96-well plates and add 120 μL of Cell Titer AQueous ONE substrate solution (diluted 1:6 in DMEM medium + FBS) to each well of all plates. After 2 hours, read the plates at OD 490 nm on a Spectra max plate reader using Softmax 4.7 software.

[0219] Data analysis: OD readings versus concentration are graphed in Excel® (Microsoft, Redmond, WA). 50 Calculations and statistical analyses are performed using Eli Lilly statistical software tools (Global Stats Discovery Team, Lilly). [Table 15] [Table 16] [Table 17] [Table 18]

[0220] Example 24: In vitro activity of multispecific VHH-based fusions and conjugates of Examples 15 and 16 The activity of the VHH-based conjugates of Examples 15 and 16 at GLP1R and MC3R is assessed using a cell-based cyclic AMP assay kit purchased from DiscoverX (eurofins). For cAMP, the Hunter eXpress GLP1R CHO-K1 GPCR Assay (Cat. No. 95-0062E2) is used, with an internal GLP-1 standard (SEQ ID NO: 99) as a positive control. For cAMP, the Hunter eXpress MC3R CHO-K1 GPCR Assay (Cat. No. 95-0045E2) is used, with α-MSH as a positive control. The assay is performed according to the GPCR assay protocol provided by the manufacturer. Briefly, cells are thawed and plated at 100 μL per well in a 96-well plate. After 24 hours of incubation at 37°C in 5% CO2, 15 μL of 3x agonists (VHH conjugates / fusions and controls) and cAMP standards are added to appropriate wells in duplicate. Plates are incubated for 30 minutes at 37°C. cAMP agonist detection solution is prepared and kept at room temperature, protected from light. After the 30-minute incubation with VHH fusions or conjugates, 15 μL of cAMP antibody reagent is added to each well. Immediately after adding the antibody reagent, 60 μL of cAMP agonist detection solution is added, which is prepared during the incubation period. After incubating the plate for 1 hour in the dark at room temperature, 60 μL of cAMP solution A is added to each well, and the plate is incubated for 3 to 18 hours in the dark at room temperature. Chemiluminescent signals are read using a plate reader (Molecular Devices SpectraMax i3x plate reader). Statistical analysis of the raw data is performed using GraphPad PRISM version 8. The raw data are transformed and nonlinear regression analysis is performed (log (agonist) vs. response - variable slope (4 parameters)) to determine EC 50 Obtain values ​​and determine goodness of fit (n=2 for all data). [Table 19] [Table 20]

[0221] In vivo function - long-acting specific PD for each payload Example 25: In vivo activity of VHH-based fusions of Examples 1 and 2 (weight loss in healthy mice) This study will be conducted using 48 male DIO C57BL6 mice supplied by Taconic. Mice will be 15 weeks old at the start of the study. Animals will be housed singly in microisolator cages with automated household water (2-5 ppm chlorine) and high-fat diet (TD95217) available ad libitum. Mice will be randomized into eight groups (n=6) based on body weight using a block randomization allocation tool developed by Lilly statisticians for in vivo group randomization. To reduce potential stress-related effects on the study, all animals will be habituated to daily handling for at least three days prior to the start of the study. Body weight and food weight will be recorded for two days prior to the start of the study and daily thereafter throughout the study. Treatments will be administered subcutaneously in the interscapular region using a 0.5 mL 28G insulin syringe (BD catalog number 329461) throughout the course of the study (QD dosing schedule). Native CNTF (R&D systems, recombinant human CNTF catalog number 257-NT / CF, lot number GL402101A), Example 1, and Example 2 are diluted to the appropriate concentration in sterile PBS, pH 7.2, within 30 minutes of administration. Native CNTF is dosed at 0.25 mpk. Examples 1 and 2 are dosed at either 0.25 mpk or 0.1 mpk. Dilution rates are calculated for each treatment each day based on the group's mean weight from the previous day. 200 μL of PBS is administered to animals in the vehicle control group. Body weight changes are analyzed using an internal statistical tool that employs a rigorous model-based approach developed by Lilly statisticians for in vivo PD studies. Animals are removed from the study once they reach 20% weight loss, regardless of dosing day. [Table 21] [Table 22]

[0222] As shown in Tables 21 and 22, all treatment groups show significant weight loss compared to the vehicle group by day 3 of the study. Example 1 and Example 2, both dosed at 0.1 mpk, show weight loss comparable to rhCNTF dosed at 0.25 mpk. Animals given Example 1 at 0.25 mpk show significantly greater weight loss by study day 2 when compared to rhCNTF at 0.25 mpk. Animals given Example 1 at 0.25 mpk show significantly greater weight loss by study day 3 when compared to rhCNTF at 0.25 mpk. Animals in the 0.25 mpk dose groups of both Example 1 and 2 are not dosed on days 5 or 6 due to clinical signs of dehydration and / or achievement of 20% BWL.

[0223] Example 26: In vivo activity of VHH-based fusions of Examples 3 and 4 (blood chemistry analysis of healthy mice) This study will be conducted using 48 female C57BL6 mice, 6 weeks of age, supplied by Envigo. Animals will be pair-housed in microisolator cages with automated water supply (2-5 ppm chlorine) and standard diet (Teklad 2014 Rodent Maintenance Diet) available ad libitum. Mice will be randomized into six groups (n=8) based on body weight using a block randomization allocation tool developed by Lilly statisticians for in vivo group randomization. To reduce potential stress-related effects on the study, all animals will be habituated to daily handling for at least three days prior to the start of the study. Body weight and food weight will be recorded for two days prior to the start of the study and then monitored and recorded daily throughout the study. Animals will be 8 weeks of age at the time of the first dose. Treatment will be administered subcutaneously in the interscapular region using a 0.5 mL 28G insulin syringe (BD catalog number 329461) every other day for seven days (Q2D dosing schedule) for a total of three doses. Native NRG-1 (R&D systems, recombinant human NRG1-beta1 EGF domain, CF, catalog number 396-HB / CF, batch number ACD182101A), Example 3, and Example 4 are diluted to the appropriate concentration in sterile PBS at pH 7.2 within 30 minutes of administration. Native NRG-1 is dosed at 1 mpk. Examples 3 and 4 are dosed at 1 and 0.3 mpk. The dilution rate for each treatment is calculated daily based on the previous day's group average body weight. Animals in the vehicle control group receive 200 μL of PBS SC. Treatments are administered in the morning on study days 1, 3, and 5. On study day 7, all animals are anesthetized with isofluorane (4%) on the surgical surface and exsanguinated via retroorbital bleeding according to the Animal Care and Use Protocol. Blood is collected in serum separator tubes (BD catalog number 365967) and kept at room temperature for a maximum of 90 minutes. The blood is centrifuged at 10,000 RPM for 10 minutes. Serum is collected from each tube and aliquoted for Chem 18 analysis. Serum is analyzed on a Roche Cobas 8000 Modular chemistry analyzer. All reagents for Chem 18 analysis are supplied by Roche, except for the triglyceride test, which uses Fujifilm WAKO reagents. Statistical analysis of blood chemistry values ​​uses JMP software one-way CI analysis by group using Dunnett's method. [Table 23]

[0224] As shown in Table 23, the VHH fusions of Examples 3 and 4 demonstrate a significant effect on blood chemistry compared to the vehicle control. A decrease in serum creatinine was observed across all treatment groups, with statistically significant decreases occurring at both dose levels for Example 4 and the native rhNRG1 control. A significant decrease in ALP from baseline occurs in animals dosed with the native rhNRG1 control and the 1 mpk dose group for both Examples 3 and 4. Cholesterol levels increase for both dose levels of Example 3, at the 1 mpk dose of Example 4, and in the native rhNRG1 control group. The native rhNRG1 control and the 0.3 mpk dose group of Example 4 also significantly decrease BUN.

[0225] Example 27: In vivo activity of VHH-based fusions of Example 5 (weight loss in healthy mice) Twenty-four male DIO C57BL6 mice were procured from Taconic at 12 weeks of age. Animals were housed singly in microisolator cages with automated water (2-5 ppm chlorine) and high-fat diet (TD95217) available ad libitum. Mice were randomized into four groups (n = 6) based on body weight using a block randomization allocation tool developed by Lilly statisticians for in vivo group randomization. To reduce potential stress-related effects on the study, all animals were habituated to daily handling for at least three days prior to the start of the study. Body weight and food weight were recorded for two days prior to the start of the study and then monitored and recorded daily throughout the study. Five doses of each treatment were administered subcutaneously in the interscapular region using 0.5 mL 28G insulin syringes (BD catalog number 329461) over the course of the study (Q3D dosing schedule). Native GDF-15 (R&D systems, recombinant, catalog number 957-GD / CF, batch EHF232101A) and the VHH fusion protein of Example 5 are diluted to the appropriate concentrations in sterile PBS, pH 7.2, within 30 minutes of administration. Native GDF-15 is dosed at 0.1 mpk, and the VHH fusion protein of Example 5 is dosed at either 1 mpk or 0.1 mpk based on the group mean weight from the previous day. Animals in the vehicle control group receive 200 μL of PBS. Body weight changes are analyzed using an internal statistical tool that employs a rigorous model-based approach developed by Lilly statisticians for in vivo PD studies. [Table 24] [Table 25]

[0226] As shown in Tables 24 and 25, significant weight loss occurs in both groups treated with the VHH fusion protein of Example 5 at either 0.1 mpk or 1 mpk by day 3 compared to vehicle controls. Weight loss in the native GDF-15 treated group reaches significance by day 9. There is no difference in weight loss at any time point between the 1 mpk and 0.1 mpk dose groups of the VHH fusion protein of Example 5. Animals receiving 1 mpk or 0.1 mpk VHH fusion protein of Example 5 lost significantly more weight by day 6 of the study compared to animals treated with native GDF15. This significance persists for the remainder of the study.

[0227] Example 28: In vivo activity of the VHH-based conjugates of Example 15 (corticosterone induction and weight loss in healthy mice) Healthy 6-week-old female C57BL6 mice were purchased from Envigo and group-housed in microisolator cages (3 mice per cage), bottle-fed with household filtered water, and fed a standard chow diet (2014 Teklad Global Rodent Maintenance Diet). Mice were allowed to acclimate for at least 72 hours after receipt. Mice were weighed one day prior to dosing. A single subcutaneous dose of 0.1, 0.3, 1, or 3 nmol / kg of the VHH fusion conjugate of Example 15 or vehicle (DPBS) was administered on day 1. n=3 per group. Tail sticks to collect dried blood spot (DBS) samples for corticosterone level analysis were performed immediately prior to dosing (TO) and 2, 6, 24, 30, 48, and 72 hours post-dosing. Approximately 20-30 μL of blood was collected from the tail of each animal onto a Whatman DMPK card (WB129243). DBS cards were provided to in-house LC-MS specialists for corticosterone level analysis. Animal weights were also recorded daily throughout the study. All in vivo experimental procedures were conducted in accordance with IACUC standards and in accordance with approved animal use protocol (19-033). Data are reported in Tables 26 and 27, respectively, as mean corticosterone levels (ng / mL) or mean percent change from body weight recorded on day 0. [Table 26]

[0228] As shown in Table 26, treatment with the VHH-based conjugate of Example 15 results in a significant induction of corticosterone levels at 2 and 6 hours post-dosing compared to vehicle controls at all dose levels. For animals in the 3mpk dose group, the increase in corticosterone levels remains significantly higher than the vehicle control group up to 30 hours post-dose. [Table 27]

[0229] As shown in Table 27, treatment with the VHH-based conjugate of Example 15 results in prevention of weight gain in the 1 nmol / kg and 3 nmol / kg treatment groups for the duration of the study. The difference in percent weight gain between these two treatment groups by day 3 is significant when compared to vehicle control. p-values ​​are calculated using an unpaired t-test of treatment group vs. vehicle in GraphPad PRISM software.

[0230] In vivo function - PK Example 29: PK of the VHH-based fusion of Example 4 in male Sprague Dawley rats Male Sprague Dawley rats are administered a single intravenous (IV) or subcutaneous (SC) dose of 106.8 nmol / kg of Example 4 formulated in PBS buffer (pH 7.4) at a dose volume of 2.6 mL / kg. Blood is collected for PK characterization at 1, 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the IV route and at 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the SC route.

[0231] The plasma concentrations of the VHH-based fusions of Example 4 are determined by a regular LC / MS assay from Altascience Company (Laval, Quebec, Canada) using a Q / Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The VHH fusions of Example 4 and internal standards are isolated from K3EDTA rat plasma via immunoprecipitation using an anti-VHH-antibody-biotin conjugate and streptavidin-coated magnetic beads. After a washing step to remove interfering endogenous proteins, the isolated VHH fusions of Example 4 are reduced, alkylated, and digested with trypsin, and the tryptic peptides are analyzed by LC / MS as a surrogate measure of the intact fusion. The plasma concentrations of the VHH fusions of Example 4 are used to calculate the PK parameters shown in Table 28. [Table 28]

[0232] As shown in Table 28, the VHH fusions of Example 4 exhibit an enhanced PK profile in Sprague Dawley rats compared to the non-fusion protein (data not shown).

[0233] Example 30: PK of VHH-based fusions of Examples 5, 6, and 7 in male Sprague Dawley rats Male Sprague Dawley rats are administered a single intravenous (IV) or subcutaneous (SQ) dose of 50 nmol / kg for Example 5 or 25 nmol / kg for Examples 6 or 7 formulated in PBS buffer (pH 7.4) at a dose volume of 4 mL / kg. Blood is collected for PK characterization at 1, 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the IV route and at 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the SQ route.

[0234] The plasma concentrations of the VHH-based fusions of Examples 5-7 are determined by a formal LC / MS assay from Covance Laboratories (Greenfield, IN) using a Q / Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The VHH fusions of Examples 5-7 and internal standards are isolated from K3EDTA rat plasma via immunoprecipitation using an anti-VHH-antibody-biotin conjugate and streptavidin-coated magnetic beads. After a washing step to remove interfering endogenous proteins, the isolated VHH fusions of Examples 5-7 are reduced, alkylated, and digested with trypsin, and the tryptic peptides are analyzed by LC / MS as a surrogate measure of the intact fusion. The plasma concentrations of the VHH fusions of Examples 5-7 are used to calculate the PK parameters shown in Table 29. [Table 29]

[0235] As shown in Table 29, the VHH fusions of Examples 5, 6, and 7 exhibit enhanced PK profiles in Sprague Dawley rats compared to the non-fusion protein (data not shown).

[0236] Example 31: PK of the Fab-VHH fusion of Example 9 and the corresponding unfused Fab (Example 13) in male Sprague Dawley rats Male Sprague Dawley rats are administered a single intravenous (IV) or subcutaneous (SC) dose of 63.4 nmol / kg for Example 9 and 78.9 nmol / kg for Example 13 formulated in PBS buffer (pH 7.4) at a dose volume of 4 mL / kg. Blood is collected for PK characterization at 1, 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the IV route and at 6, 12, 24, 48, 96, 144, 168, and 240 hours post-dose for the SC route.

[0237] The plasma concentrations of the Fab-VHH fusions of Example 9 and the corresponding unfused Fabs of Example 13 are determined by a standard LC / MS assay from Altascience Company (Laval, Quebec, Canada) using a Q / Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The Fab-VHH fusions of Example 9 and the Fabs of Example 13, as well as the internal standard, are isolated from K3EDTA rat plasma via immunoprecipitation using an anti-human kappa light chain-biotin conjugate and streptavidin-coated magnetic beads, or an anti-VHH-antibody-biotin conjugate and streptavidin-coated magnetic beads. After a washing step to remove interfering endogenous proteins, the isolated proteins of Examples 9 and 13 are reduced, alkylated, and digested with trypsin, and the tryptic peptides are analyzed by LC / MS as a surrogate measure of the intact fusion. The plasma concentrations of the Fab-VHHs of Example 9 (Table 30) are used to calculate the PK parameters shown in Table 31. The plasma concentrations of the unfused Fab of Example 13 are so low that the PK parameters cannot be calculated. [Table 30]

[0238] As shown in Table 30, the Fab-VHH fusion of Example 9 exhibits significantly higher mean plasma concentrations in Sprague Dawley rats compared to the corresponding unfused Fab (Example 13). [Table 31]

[0239] As shown in Table 31, the Fab-VHH fusion of Example 9 exhibits an enhanced PK profile in Sprague Dawley rats, whereas the mean plasma concentrations of the corresponding unfused Fab (Example 13) are so low that PK parameters cannot even be calculated.

[0240] array The following nucleic acid and / or amino acid sequences are referred to in this disclosure and are provided below for reference.

[0241] SEQ ID NO:1 - VHH portion 1 (MC6.1C22) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0242] SEQ ID NO:2 - VHH portion 2 (MC6.1C80) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0243] SEQ ID NO:3 - VHH portion 3 (MC6.1C22.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0244] SEQ ID NO:4 - VHH portion 4 (MC6.1C80.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0245] SEQ ID NO:5 - VHH portion 5 (MC6) EVQLVESGGGLVQAGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFTAGIGGSVDITYYLDSVKGRFTISKDNTKNTVYLQMNSLKPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTQVTVSS

[0246] SEQ ID NO:6 - VHH portion 6 (MC6.1) EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWGQGTLVTVSS

[0247] SEQ ID NO: 7-VHH portion 7 (MC6.1C6) EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWGQGTLVTVSS

[0248] SEQ ID NO:8 - VHH portion 8 (MC6.1C22-G26Y) EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0249] SEQ ID NO: 9-VHH portion 9 (MC6.1C22-R27A) EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0250] SEQ ID NO:10 - VHH portion 10 (MC6.1C22-I57E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDETYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0251] SEQ ID NO:11 - VHH portion 11 (MC6.1C22-I57Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDQTYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0252] SEQ ID NO:12 - VHH portion 12 (MC6.1C22-Y59A) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITAYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0253] SEQ ID NO:13 - VHH portion 13 (MC6.1C22-Y59E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITEYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0254] SEQ ID NO:14 - VHH portion 14 (MC6.1C22-Y59Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITQYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0255] SEQ ID NO: 15 - VHH portion 15 (MC6.1C22-Y59S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0256] SEQ ID NO: 16 - VHH portion 16 (MC6.1C22-Y59T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITTYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSS

[0257] SEQ ID NO: 17 - VHH portion 17 (MC6.1C22-R102K) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWGQGTLVTVSS

[0258] SEQ ID NO: 18 - VHH portion 18 (MC6.1C22-R102Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWGQGTLVTVSS

[0259] SEQ ID NO: 19 - VHH portion 19 (MC6.1C22-R102S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWGQGTLVTVSS

[0260] SEQ ID NO:20 - VHH portion 20 (MC6.1C22-P103) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWGQGTLVTVSS

[0261] SEQ ID NO:21 - VHH portion 21 (MC6.1C22-P103Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWGQGTLVTVSS

[0262] SEQ ID NO: 22 - VHH portion 22 (MC6.1C22-P103S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWGQGTLVTVSS

[0263] SEQ ID NO:23 - VHH portion 23 (MC6.1C22-L104E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWGQGTLVTVSS

[0264] SEQ ID NO:24 - VHH portion 24 (MC6.1C22-L104G) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWGQGTLVTVSS

[0265] SEQ ID NO: 25 - VHH portion 25 (MC6.1C22-L104Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWGQGTLVTVSS

[0266] SEQ ID NO:26 - VHH portion 26 (MC6.1C22-L104T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWGQGTLVTVSS

[0267] SEQ ID NO: 27 - VHH portion 27 (MC6.1C22-S107E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWGQGTLVTVSS

[0268] SEQ ID NO: 28 - VHH portion 28 (MC6.1C80-A98T) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRPGRPLITSKVADLYPYWGQGTLVTVSS

[0269] SEQ ID NO:29 - VHH portion 29 (MC6.1C80-P100Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARQGRPLITSKVADLYPYWGQGTLVTVSS

[0270] SEQ ID NO:30 - VHH portion 30 (MC6.1C80-A98T, P100Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRQGRPLITSKVADLYPYWGQGTLVTVSS

[0271] SEQ ID NO:31 - VHH portion 31 (MC6.1C80-K108Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSQVADLYPYWGQGTLVTVSS

[0272] SEQ ID NO: 32 - VHH portion 32 (MC6.1C80-V109Q) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQADLYPYWGQGTLVTVSS

[0273] SEQ ID NO: 33 - VHH portion 33 (MC6.1C80-D111E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAELYPYWGQGTLVTVSS

[0274] SEQ ID NO: 34 - VHH portion 34 (MC6.1C80-D111S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVASLYPYWGQGTLVTVSS

[0275] SEQ ID NO: 35 - VHH portion 35 (MC6.1C80-V109Q, D111E) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQAELYPYWGQGTLVTVSS

[0276] SEQ ID NO: 36 - VHH portion 36 (MC6.1C80-V109Q, D111S) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQASLYPYWGQGTLVTVSS

[0277] SEQ ID NO: 37 - VHH portion 37 (MC6.1C80Cys) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC

[0278] SEQ ID NO: 38-L1((GGGGQ) n (nucleotide sequence of GGGGQ

[0279] SEQ ID NO: 39-L1((GGGQ) n (nucleotide sequence of GGGQ

[0280] SEQ ID NO: 40-L1((GGGGS) n (nucleotide sequence of GGGGS

[0281] SEQ ID NO: 41-L1((PGPQ) n (nucleotide sequence of PGPQ

[0282] SEQ ID NO: 42-L1((PGPA) n (nucleotide sequence of PGPA

[0283] SEQ ID NO: 43-L1(GGGG(AP) n GGGG base sequence) GGGG-AP-GGGG

[0284] SEQ ID NO: 44-L1((GGE) n (nucleotide sequence of GGE

[0285] SEQ ID NO: 45-L1((GGGGE) n (nucleotide sequence of GGGGE

[0286] SEQ ID NO: 46-L1((GGK) n (nucleotide sequence of GGK

[0287] SEQ ID NO: 47-L1((GGGGK) n (nucleotide sequence of GGGGK

[0288] SEQ ID NO: 48-L1(GGGG(EP) n GGGG base sequence) GGGG-EP-GGGG

[0289] SEQ ID NO: 49-L1(GGGG(KP) n GGGG base sequence) GGGG-KP-GGGG

[0290] SEQ ID NO: 50-L1((PGPE) n (nucleotide sequence of PGPE

[0291] SEQ ID NO: 51-L1((PGPK) n (nucleotide sequence of PGPK

[0292] SEQ ID NO:52-L11(GGGGQ)5 GGGGQGGGGQGGGGQGGGGQGGGGQ

[0293] SEQ ID NO:53-L12(PGPQ)8 PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQ

[0294] SEQ ID NO:54-L13(PGPA)8 PGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPA

[0295] SEQ ID NO: 55-L14((GGE)8) GGEGGEGGEGGEGGEGGEGGEGGE

[0296] SEQ ID NO: 56-L15((GGGGE)5) GGGGEGGGGEGGGGEGGGGEGGGGE

[0297] SEQ ID NO: 57-L16((GGK)8) GGKGGKGGKGGKGGKGGKGGKGGK

[0298] SEQ ID NO: 58-L17((GGGGK)5) GGGGKGGGGKGGGGKGGGGKGGGGK

[0299] SEQ ID NO: 59-L18((GGGG(AP) 10 GGGG)) GGGGAPAPAPAPAPAPAPAPAPAPAPGGGG

[0300] SEQ ID NO: 60-L19((GGGG(EP) 10 GGGG)) GGGGEPEPEPEPEPEPEPEPEPEPEPGGGG

[0301] SEQ ID NO: 61-L110((GGGG(KP) 10 GGGG)) GGGGKPKPKPKPKPKPKPKPKPKPKPGGGG

[0302] SEQ ID NO: 62-L111((PGPE)8) PGPEPGPEPGPEPGPEPGPEPGPEPGPEPGPE

[0303] SEQ ID NO: 63-L112((PGPK)8) PGPKPGPKPGPKPGPKPGPKPGPKPGPKPGPK

[0304] SEQ ID NO:64-L21 GGGSGGSGGG

[0305] SEQ ID NO:65-L22 GGGSGGSGGSGGG

[0306] SEQ ID NO: 66 - Signal peptide METDTLLLWVLLLWVPGSTG

[0307] SEQ ID NO: 67 - Human proGIP MVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSSPA KNPSDEDLLLR DLLIQELLACLLDQTNLCRLRSR

[0308] SEQ ID NO: 68 - Human GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

[0309] SEQ ID NO: 69 - Human GIP receptor RAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHHHVAAGFVLRQCGSDGQWGLWRDHTQCENPEKNEAFLDQRL ILERLQVMYTVGYSLSLATLLLALLILSLFRRLHCTRNYIHINLFTSFMLRAAAILSRDRLLLPRPGPYLGDQALALWNQALAACRTAQIVTQYCVGANYTWLLVEGVYLHS LLVLVGGSEEGHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLGVHE VVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSGSGPGEVPTSRGLSSGTLPGPGNEASRELESYC

[0310] SEQ ID NO: 70 - Human proglucagon MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK

[0311] SEQ ID NO: 71 - Human GLP-1 HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0312] SEQ ID NO: 72 - Human GLP-1 7-37 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0313] SEQ ID NO: 73 - Human GLP-1 7-36 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0314] SEQ ID NO: 74 - Human GLP-1 receptor RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGE RSSPEEQLLFLYIIYTVGYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANYYW LLVEGVYLYTLLAFSVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKST LTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGSSMYTATCQASCS

[0315] SEQ ID NO: 75 - Human proGDF15 MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPA LHLRLSPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0316] SEQ ID NO:76 - Human GDF15 ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0317] SEQ ID NO:77 - Human GDF15 (GFRAL) receptor MIVFIFLAMGLSLENEYTSQTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIP CQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL

[0318] SEQ ID NO:78 - Human proINS FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0319] SEQ ID NO: 79 - Human INS A chain GIVEQCCTSICSLYQLENYCN

[0320] SEQ ID NO:80 - Human INS B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0321] SEQ ID NO:81 - Human INS receptor alpha-subunit HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERC WTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEIS GYLKIRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNH PGWLMRGLGKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRKTSSGTGAEDPRPS

[0322] SEQ ID NO:82 - Human INS receptor β-subunit SLGDVGNVTVAVPTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLS PGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAKIIIGPLIFVFLFSVVIGSIYLFLRKRQPDGPLGPLYASSNPEYLSASDVFPCSVYVPDEWEVSREKITLLRELGQGSFGMVYEGNARDIIKGEAETRVAVKTVNESASLRERIEFL NEASVMKGFTCHHVVRLLGVVSKGQPTLVVMELMAHGDLKSYLRSLRPEAENNPGRPPPTLQEMIQMAAEIADGMAYLNAKKFVHRDLAARNCMVAHDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMAPESLKDGVFTTSSDMWSFGVVLW EITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCPERVTDLMRMCWQFNPKMRPTFLEIVNLLKDDLHPSFPEVSFFHSEENKAPESEELEMEFEDMENVPLDRSSHCQREEAGGRDGGSSLGFKRSYEEHIPYTHMNGGKKNGRILTLPRSNPS

[0323] SEQ ID NO:83 - Human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0324] SEQ ID NO:84 - Human IL-2 receptor alpha-subunit ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQ MVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0325] SEQ ID NO:85—Human IL-2 receptor beta-subunit AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQA SHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKF FSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0326] SEQ ID NO:86—Human IL-2 receptor gamma subunit LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDH SWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0327] SEQ ID NO:87 - Human proNRG1 MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPRLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVST EGANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKVQNQEKAEELYQKRVLTITGICIALVVGIMCVVAYCKTKKQRKKLHDRLRQSLRSERNNMMNIANGPHHPNPPPENVQLVNQYVSKNVISS EHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSNGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEERPLLL VTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

[0328] SEQ ID NO:88 - Human NRG1 SGKKPESAAGSQSPALPPRLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIV ESNEIITGMPASTEGAYVSSESPIRISVSTEGANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKVQNQEKAEELYQK

[0329] SEQ ID NO: 89 - Human NRG1 receptor ErbB3

[0330] SEQ ID NO: 90 - Human NRG1 receptor ErbB4

[0331] SEQ ID NO: 91 - Human proRLN2 MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC

[0332] SEQ ID NO: 92 - Human RLN2 A chain QLYSALANKCCHVGCTKRSLARFC

[0333] SEQ ID NO: 93 - Human RLN2 B chain DSWMEEVIKLCGRELVRAQIAICGMSTWS

[0334] SEQ ID NO: 94 - Human RXFP1 receptor MTSGSVFFYILIFGKYFSHGGGQDVKCSLGYFPCGNITKCLPQLLHCNGVDDCGNQADEDNCGDNNGWSLQFDKYFASYYKMTSQYPFEAETPECLVGSVPVQCLCQGLELDCDETNLRAVPSVSSNVTAMSLQWNLIRKLPPDCFKNYHDLQKLYLQNNKITSISIYAFRGLNSLTKLYLSHNRITFL KPGVFEDLHRLEWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPDKPLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRKNKINHLNENTFAPLQKLDELDLGSNKIENLPPLIFKDLKELSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQQRMFRPLMNLSHIYFKKFQ YCGYAPHVRSCKPNTDGISSLENLLASIIQRVFVWVVSAVTCFGNIFVICMRPYIRSENKLYAMSIISLCCADCLMGIYLFVIGGFDLKFRGEYNKHAQLWMESTHCQLVGSLAILSTEVSVLLLTFLTLEKYICIVYPFRCVRPGKCRTITVLILIWITGFIVAFIPLSNKEFFKNYYGTNGVCFPLH SEDTESIGAQIYSVAIFLGINLAAFIIIVFSYGSMFYSVHQSAITATEIRNQVKKEMILAKRFFFIVFTDALCWIPIFVVKFLSLLQVEIPGTITSWVVIFILPINSALNPILYTLTTRPFKEMIHRFWYNYRQRKSMDSKGQKTYAPSFIWVEMWPLQEMPPELMKPDLFTYPCEMSLISQSTRLNSYS

[0335] SEQ ID NO: 95 - Human ACTH SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF

[0336] SEQ ID NO:96 - Human ACTH receptor (ACTHR, MC2R) MKHIINSYENINNTARNNSDCPRVVLPEEIFFTISIVGVLENLIVLLAVFKNKNLQAPMYFFICSLAISDMLGSLYKILENILIILRNMGYLKPRGSFETTADDIIDSLFVLSLLGSIFSLSVIAADRYITIFHALRYHSIVTMRRTV VVLTVIWTFCTGTGITMVIFSHHVPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRKISTLPRANMKGAITLTILLGVFIFCWAPFVLHVLLMTFCPSNPYCACYMSLFQVNGMLIMCNAVIDPFIYAFRSPELRDAFKKMIFCSRYW

[0337] SEQ ID NO:97 - Human CNTF MAFTEHSPLTPHRRDLCSRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDQWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTGIPARGSHYIANNKKM

[0338] SEQ ID NO: 98 - Human CNTF receptor alpha QRHSPQEAPHVQYERLGSDVTLPCGTANWDAAVTWRVNGTDLAPDLLNGSQLVLHGLELGHSGLYACFHRDSWHLRHQVLLHVGLPPREPVLSCRSNTYPKGFYCSWHLPTPTYIPNTFNVTVLHGSKIMVCEKDPALKNRCHIRYMHLFSTIKYKVSIS VSNALGHNATAITFDEFTIVKPDPPENVVARPVPSNPRRLEVTWQTPSTWPDPESFPLKFFLRYRPLILDQWQHVELSDGTAHTITDAYAGKEYIIQVAAKDNEIGTWSDWSVAAHATPWTEEPRHLTTEAQAAETTTSTTSSLAPPPTTKICDPGELGS

[0339] SEQ ID NO: 99-GLP1-standard HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0340] SEQ ID NO: 100 - VHH fusion 1 (CNTF-(G4Q)5-MC6.1C22.43) MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWT VRSIHDLRFISSHQTGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0341] SEQ ID NO: 101 - VHH fusion 2 (MC6.1C22-(G4Q)5-CNTF) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQMAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTG

[0342] Sequence number 102 - VHH fusion 3 (NRG1-(G4Q)5-MC6.1C22.43) SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0343] Sequence number 103 - VHH fusion 4 (MC6.1C22-(G4Q)5-NRG1) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQ

[0344] SEQ ID NO: 104 - VHH fusion 5 (MC6.1C22-(G4Q)5-GDF15) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGG QGGGGQGGGGQGGGGQGGGGQGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0345] SEQ ID NO: 105 - VHH fusion 6 (IL2-(G4Q)5-MC6.1C22.43) APQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLTGGGGQGGGGQ GGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0346] SEQ ID NO: 106 - VHH fusion 7 (MC6.1C22-(G4Q)5-IL2) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGGGGQAPQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0347] Sequence number 107 - VHH fusion 8 (GLP1-(G4Q)5-MC6.1C22.43) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0348] Sequence number 108 - HC - VHH (AdaFabVHCH1-(G4Q)5-MC6.1C22.43) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0349] SEQ ID NO: 109-LC (AdaFabVLCL) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0350] SEQ ID NO: 110-HC (AdaFabVHCH1) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

[0351] SEQ ID NO:111-LC-VHH(AdaFabVLCL-(G4Q)5-MC6.1C22.43) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYICQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESSGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0352] SEQ ID NO:112-VHH-HC(MC6.1C22-(G4Q)5-AdaFabVHCH1) EVQLLESSGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWV RQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

[0353] SEQ ID NO:113-LC(AdaFabVLCL) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0354] SEQ ID NO:114-HC(AdaFabVHCH1) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSCD

[0355] SEQ ID NO:115-VHH-LC(MC6.1C22-(G4Q)5-AdaFabVLCL) EVQLLESSGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYCAARPGRPLITSKVADLYPYWGQGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDIQMTQSPSSLSASVGDRVTITCRASQGIRN YLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0356] SEQ ID NO: 116 - VHH fusion 13 (GLP1-MC6.1C80Cys) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC

[0357] SEQ ID NO: 117 - VHH fusion conjugate 1 (GLP1-C80Cys-ACTH conjugate) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC-Maleimide-(PEG) 12 -ACTH (C-terminal to N-terminal orientation) [ka]

[0358] SEQ ID NO: 118 - VHH fusion conjugate 2 (MC6.1C80Cys-ACTH conjugate) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSC-Maleimide-(PEG) 12 -ACTH (C-terminal to N-terminal orientation) [ka]

[0359] SEQ ID NO: 119 - Intermediate 1 (ACTH-(PEG) 12 -maleimide conjugate) SYSMEHFRWGKPVGKKRRPVKVYPK-(PEG) 12 -Maleimide-NH2 [ka]

[0360] SEQ ID NO: 120 - Fab HC (AdaFabVHCH1) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

[0361] SEQ ID NO: 121 - Fab LC (AdaFabVLCL) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0362] SEQ ID NO: 122 - Human GITR full length (without signal peptide) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTD CTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0363] SEQ ID NO: 123 - Human GITR ECD (without signal peptide) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAE

[0364] SEQ ID NO: 124 - VHH portion 38 (MC6.1C90Cys) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSC

[0365] SEQ ID NO: 125 - VHH portion 39 (MC6.1C90.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0366] SEQ ID NO: 126 - VHH portion 40 (MC6.1C95.43) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGTRPGRPLITSKVADLYPYWGQGTLVTVSSPP

Claims

1. A compound comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37 and 124-126, or a sequence having at least 90% sequence similarity thereto.

2. The structure: M-X １ 、 X １ -M、 M-L １ -X １ 、 X １ -L １ -M、 X １ -M-X ２ 、 X ２ -M-X １ 、 X １ -L １ -M-X ２ 、 X ２ -L １ -M-X １ 、 X １ -M-L １ -X ２ 、 X ２ -M-L １ -X １ 、 X １ -L １ -M-L ２ -X ２ 、 X ２ -L １ -M-L ２ -X １、 M-L １ -X １ -L ２ -X ２ 、 M-L １ -X ２ -L ２ -X １ 、 X １ -L ２ -X ２ -L １ -M, or X ２ -L ２ -X １ -L １ -M [In the formula, M has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 124, 125, and 126, or has at least about 90% sequence similarity thereto; X １ is the first biotherapeutic agent, X ２ (if present) is a second biological therapeutic agent; L １ (if present) is (GGGGQ) ｎ (SEQ ID NO: 38), (GGGQ) ｎ (SEQ ID NO: 39), (GGGGS) ｎ (SEQ ID NO: 40), (PGPQ) ｎ (SEQ ID NO: 41), (PGPA) ｎ (SEQ ID NO: 42), (GGGG(AP) ｎ GGGG) (SEQ ID NO: 43), (GGE) ｎ (SEQ ID NO: 44), (GGGGGE) ｎ (SEQ ID NO: 45), (GGK) ｎ (SEQ ID NO: 46), (GGGGK) ｎ (SEQ ID NO: 47), (GGGG(EP) ｎ GGGG) (SEQ ID NO: 48), (GGGG(KP) ｎ GGGG) (SEQ ID NO: 49), (PGPE) ｎ (SEQ ID NO:50), and (PGPK) ｎ (SEQ ID NO:51), wherein n can be 1 to 10; L ２ (if present) is an amino acid sequence selected from the group consisting of SEQ ID NOs: 64 and 65, or a second linker having at least 90% sequence similarity thereto. A compound comprising:

3. The compound of claim 2 , wherein the first biotherapeutic agent is a peptide, protein, or oligomer.

4. 4. The compound of claim 2 or 3, wherein the second biotherapeutic agent is a peptide, protein, or oligomer.

5. The compound of any one of claims 2 to 4, wherein the first biotherapeutic agent and the second biotherapeutic agent are different from each other.

6. L １ is an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-63, or has at least about 90% sequence similarity thereto.

7. 7. The compound of any one of claims 2 to 6, wherein the first biotherapeutic agent and / or the second biotherapeutic agent is a peptide or protein, such as an antibody (Ab) or fragment thereof, adrenocorticotropic hormone (ACTH), growth / differentiation factor 15 (GDF15), incretin (INC), insulin (INS), interleukin (IL), ciliary neurotrophic factor (CNTF), neuregulin (NRG), or relaxin (RLN).

8. The compound of claim 7, wherein the peptide or protein is INC.

9. 9. The compound of claim 8, wherein the INC is selected from the group consisting of glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and GIP / GLP-1.

10. The compound of claim 7, wherein the peptide or protein is an IL, and the IL is IL-2.

11. The compound of claim 7, wherein the peptide or protein is an NRG, and the NRG is neuregulin 1 (NRG1) or neuregulin 4 (NRG4).

12. 8. The compound of claim 7, wherein the peptide or protein is RLN, and the RLN is relaxin 2 (RLN2).

13. A pharmaceutical composition comprising a compound according to any one of claims 2 to 12 and a pharma- ceutically acceptable buffer.

14. 14. The pharmaceutical composition of claim 13, further comprising an additional therapeutic agent.

15. A medicament for treating an individual comprising a compound according to any one of claims 2 to 12 or a pharmaceutical composition according to claim 13 or 14, comprising the step of administering said compound or said pharmaceutical composition to said individual.

16. 13. A pharmaceutical composition comprising a compound of claim 1 for use in extending the t1 / 2 of a biotherapeutic agent.

17. A pharmaceutical composition comprising a compound according to any one of claims 2 to 12, or a pharmaceutical composition according to claim 13 or 14, for use in therapy.

18. A pharmaceutical composition comprising a compound according to any one of claims 2 to 12 or a pharmaceutical composition according to claim 13 or 14 for use in treating cardiovascular, neurological, immune, metabolic, oncology, psychiatric, pulmonary and / or renal conditions, diseases and / or disorders.

19. Use of a compound according to any one of claims 2 to 12 for the manufacture of a medicament.

20. A compound comprising any one of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118, or a sequence having at least about 90% sequence similarity thereto.

21. 21. A pharmaceutical composition comprising a compound of claim 20 and a pharma- ceutically acceptable buffer.

22. 22. The pharmaceutical composition of claim 21 further comprising an additional therapeutic agent.

23. 23. A medicament for treating an individual comprising a compound according to claim 20 or a pharmaceutical composition according to claim 21 or 22, comprising the step of administering said compound or said pharmaceutical composition to said individual.

24. 23. A pharmaceutical composition comprising a compound according to claim 20, or a pharmaceutical composition according to claim 21 or 22, for use in therapy.

25. 23. A pharmaceutical composition comprising a compound of claim 20, or a pharmaceutical composition of claim 21 or 22, for use in treating cardiovascular, neurological, immune, metabolic, oncology, psychiatric, pulmonary and / or renal conditions, diseases and / or disorders.

26. 21. Use of a compound according to claim 20 for the manufacture of a medicament.