EA048693B1 - HALF-LIFE EXTENDING FRAGMENTS AND METHODS OF THEIR APPLICATION - Google Patents

Description

The present invention relates generally to biology and medicine and, more particularly, it relates to single-domain antibodies, known as variable domains of heavy-chain-only antibodies (VHH), which are designed/modified to act as half-life (t1/ ₂ ) enhancing moieties for use with therapeutic agents, in particular for improving the t1/ ₂ of biologically based therapeutic agents (i.e., biotherapeutics or biologics). The invention also relates to fusion molecules and conjugates that include one or more VHH-based t1/ ₂ enhancing moieties and a therapeutic agent, as well as pharmaceutical compositions comprising them and their use in the treatment of various conditions, diseases or disorders.

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

There are several strategies for increasing the t1/ ₂ of biotherapeutics that may improve their pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles. Such strategies typically utilize volume-expanding moieties or neonatal Fc receptor (FcRn)-mediated recycling. Thus, antibodies (Abs) 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 (such as transferrin) can be covalently and/or non-covalently linked to a single biotherapeutic to increase its t1/ ₂ . 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.

Despite the vast number of strategies to increase t ¹ /2, there is a need for additional structures to increase or improve PK properties, such as t ¹ /2, of biotherapeutics.

To address this need, the disclosure first describes compounds that can be used as t ¹ / ₂ enhancing moieties for biotherapeutics. In one instance, a compound is provided that comprises an amino acid sequence selected from any of SEQ ID NOs: 1-37 and 124-126. In another instance, a compound is provided that comprises the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ Ш NO:1).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 1.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ Ш NO:2).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 2.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ Ш NO:3).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 3.

In another case, a compound is proposed that includes the following amino acid sequence:

- 1 048693

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ Ш NO:4).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 4.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLVESGGGLVQAGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFTAGIGGSVDITY

YLDSVKGRFTISKDNTKNTVYLQMNSLKPEDTAVYYCAVRPGRPLITSRDANLYDYWG QGTQVTVSS (SEQ ID NO:5).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 5.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGG

SVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPPLITSRDANL

YDYWGQGTLVTVSS (SEQ ID NO:6).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 6.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLTSRVANLYPYWG QGTLVTVSS (SEQ Ш NO:7).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 7.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ Ш NO:8).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 8.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:9).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 9.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDET YYADSVI<GRFTISRDNSI<NTLYLQMNSLRPEDTAVYYCAARPGRPLITSI<VADLYPYW GQGTLVTVSS (SEQ ID NO: 10).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 10.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDQT YYADSVI<GRFTISRDNSI<NTLYLQMNSLRPEDTAVYYCAARPGRPLITSI<VADLYPYW GQGTLVTVSS (SEQ ID NO: AND).

Alternatively, the compound may have an amino acid sequence comprising

- 2 048693 providing at least about 90% to about 99% similarity to SEQ ID NO: 11.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITA

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 12).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 12.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITE

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 13).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 13.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITQ

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 14).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 14.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITS

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 15).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 15.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITT

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 16).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 16.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 17).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 17.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO: 18).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 18.

In another case, a compound is proposed that includes the following amino acid sequence:

- 3 048693

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO: 19).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 19.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:20).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 20.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:21).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 21.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:22).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 22.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:23).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 23.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:24).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 24.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:25).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 25.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWG QGTLVTVSS (SEQ ID NO:26).

Alternatively, the compound may have an amino acid sequence comprising

- 4 048693 providing at least about 90% to about 99% similarity to SEQ ID NO: 26.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWG

QGTLVTVSS (SEQ ID NO:27).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 27.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRPGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:28).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 28.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARQGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:29).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 29.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRQGRPLITSKVADLYPYWG

QGTLVTVSS (SEQ ID NO:30).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 30.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSQVADLYPYWG QGTLVTVSS (SEQ ID NO:31).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 31.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQADLYPYWG

QGTLVTVSS (SEQ ID NO:32).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 32.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAELYPYWG

QGTLVTVSS (SEQ ID NO:33).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 33.

In another case, a compound is proposed that includes the following amino acid sequence:

- 5 048693

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVASLYPYWGQ GTLVTVSS (SEQ Ш NO:34).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 34.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQAELYPYWG

QGTLVTVSS (SEQ ID NO:3 5).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 35.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQASLYPYWGQ GTLVTVSS (SEQ ID NO:36).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 36.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC (SEQ ID NO:37).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 37.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWG QGTLVTVSSC (SEQ ID NO: 124).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 124.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ Ш NO: 125).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 125.

In another case, a compound is proposed that includes the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGTRPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ ID NO: 126).

Alternatively, the compound may have an amino acid sequence that is at least about 90% to about 99% similar to SEQ ID NO: 126.

Secondly, the present invention describes compounds that include at least one of the VHH-based t1/ ₂ enhancing fragments as described herein and a biotherapeutic agent. In some cases, the compounds may include the following structure, from amino-terminus (N-terminus) to carboxy-terminus (C-terminus):

M-X1, X1-M,

M-X2,

X2-M,

- 6 048693

M-L1-X1,

ML ₂ -Xi,

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 in combination with M-L2-X2 (i.e., non-covalently linked), wherein M is a compound that acts as a t1/ ₂ enhancing moiety and has an amino acid sequence selected from SEQ ID NOs: 1-37 and 124-126, or has an amino acid sequence that is at least about 90% to about 99% similar thereto, wherein L1 (if present) is a first linker, wherein L2 (if present) is a second linker, wherein X ₁ is a biotherapeutic protein, peptide, or oligomer, and wherein X ₂ is a biotherapeutic protein, peptide, or oligomer that may be the same as or different from X1 (e.g., when the biotherapeutic agent is a homodimer or when the biotherapeutic agent is a heterodimer, X1 may be one chain thereof (an α-chain), and X2 may be another chain thereof (β-chain)). X1 and _X2 may also be completely different from each other. In some cases, L1 may have the amino acid sequence (GGGGQ)n (SEQ ID NO:38), (GGGQ) _n (SEQ ID NO:38)

(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), wherein n may be from 1 to 15, in particular from about 5 to about 10. In other cases, L1 may have an amino acid sequence, selected from SEQ ID NOs: 52-63. In other cases, L ₁ may contain one or more additions, deletions, insertions, or substitutions such that L ₁ has an amino acid sequence that is at least about 90% to about 99% similar to any of SEQ ID NOs: 52-63.

In some cases, L ₂ may have an amino acid sequence selected from SEQ ID NOs: 64-65. In other cases, L ₂ may contain one or more additions, deletions, insertions, or substitutions such that L ₂ has an amino acid sequence that is at least about 90% to about 99% similar to any of SEQ ID NOs: 64-65.

In other cases, L1 or L2 may be a polymer such as polyethyleneglycol (PEG), especially (PEG) _n , where n may be from 1 to 20. In some cases, X1, X2, or X1/X2 is a peptide or protein (and even an oligomer, such as 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 other specificity), ciliary neurotrophic factor (CNTF), growth/differentiation factor 15 (GDF15), incretin (INC), interleukin (IL), neuregulin (NRG), or a hormone. In some cases, INC may be insulin (INS), glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), GIP/GLP-1, or even INC with triple receptor activity (i.e., glucagon-GIP-GLP-1 receptor activity). In some cases, IL is interleukin-2 (IL-2). In some cases, NRG is neuregulin-1 (NRG1) or neuregulin-4 (NRG4). In some cases, the hormone is adrenocorticotropic hormone (ACTH) or relaxin-2 (RLN-2). In some cases, Fab binds to GITR; and in some cases, Fab binds to GITR and is a GITR antagonist.

- 7 048693

In particular cases, the compounds may have any amino acid sequence of SEQ ID NOs: 100-118. Alternatively, the compounds may have from about 90% to about 99% sequence similarity to any amino acid sequence of SEQ ID NOs: 100-118. Third, the present invention provides pharmaceutical compositions that include at least one compound provided herein and a pharmaceutically acceptable carrier.

Fourth, the present invention describes methods of using the compounds and pharmaceutical compositions as drugs and for increasing the t ^1/2 of biotherapeutic agents.

Fifth, the present invention describes the use of the compounds provided herein for the production of medicaments and for increasing the t1/ ₂ of biotherapeutic agents.

An advantage of t1/ ₂ enhancing fragments and compounds comprising them is that they can be chemically or recombinantly synthesized as a single-chain polypeptide (i.e., monomeric) and thus do not require endoproteolytic processing to provide biological activity. However, it is envisaged that in some cases, compounds acting as t1/ ₂ enhancing fragments can be fused not only to single-chain peptides and proteins, but also to peptides with more than one chain, such as double-chain peptides, multichain peptides and proteins. Compounds acting as t1/ ₂ enhancing fragments can be chemically conjugated not only to the N- and C-terminus, but also to any surface-located amino acid of the t1/ ₂ enhancing fragments (provided that such conjugation does not completely neutralize albumin binding).

An advantage of the compounds acting as t1/ _2- increasing fragments and compounds that include them is that the t1/ ₂ -increasing fragments provide an extended duration of action in mammals such as humans and can have a t1/ ₂ of about 20 days to about 30 days, thereby allowing for at least weekly or biweekly administration compared to native peptides and proteins, which can improve compliance and can improve quality of life, especially in cases of chronic diseases requiring lifelong therapy. An advantage of the compounds acting as t1/ ₂ -increasing fragments provided herein is that they have tunable pharmacokinetics achieved by altering the albumin affinity of the t1/ ₂ -increasing fragments.

An advantage of the compounds acting as t1/ ₂ enhancing fragments proposed herein is that they can be recombinantly expressed in standard producer organisms such as yeast, mammals or prokaryotes.

Moreover, the advantage of the compounds acting as t1/ ₂ -increasing fragments provided herein is that they have similar binding not only to human serum albumin but also to monkey, mouse, rat, dog and pig serum albumin, which allows pharmacodynamic, pharmacokinetic and toxicological studies to be carried out for easier transfer from these species to humans. Accordingly, the t1/ ₂ -increasing fragments provided herein can be used not only for the treatment of humans but also for the treatment of animals.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the analogs, pharmaceutical compositions and methods, the preferred methods and materials are described herein.

Furthermore, a reference to an element in the singular does not exclude the possibility of more than one element being present unless the context clearly requires one and only one element. Accordingly, singular terms generally mean at least one.

Definitions.

As used herein, the term "approximately" means within a statistically significant range of a value or values, such as, for example, a specified concentration, length, molecular weight, pH, sequence similarity, time interval, temperature, volume, etc. Such a value or range may be within one order of magnitude, typically within 20%, more often within 10%, and even more often within 5% of the given value or range. The tolerance covered by the term "approximately" will depend on the particular system being studied and can be readily estimated by one skilled in the art.

As used herein, and with respect to one or more receptors, activity, activate, activating, and the like means the ability of a compound, such as a fusion molecule, as provided herein, to bind to and induce a response from a receptor(s), as measured by assays known in the art, such as in

- 8 048693 vitro, described below.

As used herein, adrenocorticotropic hormone or ACTH means ACTH obtained from or originating in any species, such as mammalian species, especially humans. ACTH includes both native ACTH (i.e., full-length) and variants (i.e., additions, deletions, insertions, and/or substitutions of native ACTH). One sequence for ACTH is provided in SEQ ID NO: 95 (UniProt/SwissProt Accession Number P01189). In humans, ACTH binds to the ACTH receptor (ACTHR, also known as the melanocortin receptor type 2 or MC2R), and one sequence for ACTHR is provided in SEQ ID NO: 96 (UniProt/SwissProt Accession Number Q01718). ACTHR is a G protein-coupled receptor located on the outer plasma membrane of cells; it is associated with Gas and increases cAMP levels by activating adenylate cyclase. As used herein, an amino acid is a molecule that is chemically characterized by having one or more amino 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 twenty amino acids, called standard amino acids, that can be used as building blocks for most peptides/proteins produced by any living being. The amino acid sequences in this specification contain standard one-letter or three-letter codes for the twenty natural amino acids.

As used herein, an analog means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and produces at least one effect in vivo or in vitro produced by a native agonist of that receptor. As used herein, a biotherapeutic agent, etc. means amino acid or nucleic acid-based compounds such as antibodies, coagulation factors, blood clotting factors, cytokines, enzymes, growth factors, hormones and fragments thereof that have at least one therapeutic activity/use, as well as therapeutic DNA and/or RNA molecules. As used herein, CNTF or ciliary neurotrophic factor means CNTF obtained from or derived from any species, such as mammalian species, especially 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 for CNTF is provided by SEQ ID NO: 97 (UniProt/SwissProt Accession Number P26441). In humans, CNTF binds to the CNTFa receptor (CNTFRa), and one sequence for CNTFRa is provided by SEQ ID NO: 98 (UniProt/SwissProt Accession Number P26992). CNTFRa also contains two transmembrane signal transduction subunits, LIFRP 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. As used herein, a conservative substitution refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for the presence of one or more conservative amino acid substitutions in its amino acid sequence. Typically, a variant obtained by conservative modification comprises an amino acid sequence that is 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 replacement of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, conformation and rigidity of the main chain, etc.) and has a minimal effect on the biological activity of the peptide or polypeptide obtained by substitution. Conservative substitutions of functionally similar amino acids are well known in the art and therefore need not be described exhaustively herein.

As used herein, an effective amount means an amount or dose of one or more compounds provided herein, or a pharmaceutically acceptable salt thereof, that upon single or multiple dose administration to an individual in need thereof provides the desired effect in such individual being diagnosed or treated (i.e., can cause a clinically measurable difference in the individual's condition, such as, for example, increased angiogenesis, increased vascular elasticity, increased cardiac blood flow, increased hepatic blood flow, increased pulmonary blood flow, increased renal blood flow, increased glomerular filtration rate, decreased blood pressure, decreased (or prevented) inflammation, and/or decreased (or prevented) fibrosis of the heart, kidney, liver, or lungs). An effective amount can be readily determined by one of skill in the art by the use of known methods and by analyzing results obtained under similar circumstances. In determining an effective amount for an individual, a number of factors are taken into account, including, but not limited to, the mammalian species, size, age, and general health, the particular disease or disorder being considered, the extent or lesion or severity of the disease or disorder, the subject's response, the particular compound being administered, the route of administration, the bioavailability characteristics of the administered drug, the selected dosing regimen, the use of concomitant therapy, and other relevant circumstances.

- 9 048693

As used herein, an extended duration of action means that the binding affinity and activity for a fusion molecule comprising at least one compound provided herein and a biotherapeutic agent provided herein is maintained for a period of time exceeding that of the native biotherapeutic agent, allowing for a less frequent dosing, such as once daily or even three times per week, twice per week, or once per week. The action profile can be measured using known pharmacokinetic assays, such as the methods used in the examples below.

As used herein, glucocorticoid-inducible TNFR-related protein or GITR, also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18), means GITR obtained from or originating from any species, such as mammalian species, especially 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 for full-length human GITR (but without the signal peptide) is provided in SEQ ID NO: 122 (see also UniProt/SwissProt Database Accession Number Q9Y5U5). One sequence for the ECD of human GITR (but without the signal peptide) is provided in SEQ ID NO: 123.

As used herein, glucose-dependent insulinotropic peptide(s), gastric inhibitory peptide(s), or GIP (GIP) means GIP obtained from or originating from any species, such as mammalian species, in particular humans. GIP includes both native GIP (i.e., full-length) and variants (i.e., additions, deletions, insertions, and/or substitutions of native GIP). GIP is formed by processing from a precursor, proGIP. One sequence for proGIP is shown in SEQ ID NO: 67 (see also UniProt/SwissProt Accession Number P09681), and one sequence for GIP is shown in SEQ ID NO: 68. An alternative GIP is GIPi _-3 o (see Hansen et al. (2016) Br. J. Pharmacol. 173:826–838). Humans have one GIP receptor (GIPR; SEQ ID NO: 69; see also UniProt/SwissProt Database Accession Number P48546), which acts as a G protein-coupled receptor. See Yaqub et al. (2010) Mol. Pharmacol. 77:547–558. As used herein, glucagon-like peptide-1 or GLP-1 means GLP-1 obtained from or derived from any species, such as mammalian species, especially humans. GLP-1 includes both native GLP-1 (i.e., full-length) and variants (i.e., additions, deletions, insertions, and/or substitutions of native GLP-1). GLP-1 is formed by processing from a precursor, proglucagon (proGCG). One sequence for proGIP is given as SEQ ID NO: 70 (see also UniProt/SwissProt Accession Number P01275) and one sequence for GLP-1 is given as SEQ ID NO: 71. Two active physiological variants are known, which are given as SEQ ID NOs: 72 and 73. In humans, there is one GLP-1 receptor (GLP-1R; SEQ ID NO: 74; see also UniProt/SwissProt 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 42:16781682.

As used herein, growth/differentiation factor 15 or GDF15 means GDF15 obtained from or derived from any species, such as mammalian species, in particular 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 that is formed by processing from a precursor, proGDF15. One sequence for the precursor is provided in SEQ ID NO: 75 and one sequence for GDF15 is provided in SEQ ID NO: 76 (see also UniProt/SwissProt Accession Number Q99988). In humans, there is one receptor, GDF15 (GFRAL; SEQ ID NO: 77; see also UniProt/SwissProt database accession number Q6UXV0), which 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.

As used herein, half-life or t1/ ₂ means the time required for half of an amount of a compound, such as a fusion molecule described herein, to be removed from a fluid or other physiological space, such as serum or plasma of an individual, by biological processes. Alternatively, t1/ ₂ may also mean the time it takes for a certain amount of such a fusion molecule to lose half of its pharmacological, physiological or radiological activity. In this document, the half-maximal effective concentration or EC ₅₀ means the concentration of a 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 PLCy; GDF15: PI3K/AKT and MAPK/ERK and Smad; 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).

In this document, in combination with means the administration of at least one of the fusion molecules provided herein, simultaneously, sequentially, or as a single combination.

- 10 048693 a non-uniform preparation with one or more additional therapeutic agents.

As used herein, an individual in need thereof means a mammal, such as a human, having a condition, disease, disorder, or symptom requiring treatment or therapy, including, for example, those listed herein. In particular, a preferred individual to be treated is a human.

As used herein, incretin(s) or INC refers to a peptide secreted by enteroendocrine cells that can increase postprandial insulin secretion. INC may be an incretin derived from or originating in any species, such as mammalian species, especially humans. In humans, INCs include INS, GIP, and GLP-1, which were discussed above. As used herein, insulin or INS refers to insulin derived from or originating in any species, such as mammalian species, especially humans, wherein the native form is a heterodimeric peptide having two peptide chains (e.g., chain A and chain B) joined by two disulfide bonds, wherein chain A additionally has one intramolecular disulfide bond. In humans, INS processing begins with preproinsulin (see UniProt/SwissProt Accession No. P01308), which is processed to proinsulin (comprising the A chain, B chain, and C-peptide; native INS has the structure B-C-A; see SEQ ID NO: 78; see also UniProt/SwissProt Accession No. P01308). Proinsulin undergoes further processing, in which the C-peptide is cleaved to form INS (for the A chain of native human INS, see SEQ ID NO: 79, and for the B chain of native human INS, see SEQ ID NO: 80; see also UniProt/SwissProt Accession No. P01308).

As used herein, interleukin(s) or IL refers to an interleukin obtained from or derived from any species, such as mammalian species, especially humans. IL includes both native IL (i.e., full-length) and variants thereof (i.e., additions, deletions, insertions, and/or substitutions of native IL). There are a number of native IL isoforms in humans; however, 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 provided in SEQ ID NO: 83 (see also UniProt/SwissProt Accession Number P60568). In humans, there is one IL-2 receptor, which comprises α-, β-, and γ-subunits (IL-2R; SEQ ID NOs: 84–86; see also UniProt/SwissProt 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.

In this document, long-acting means that the binding affinity and activity of the composition provided herein is maintained for a period of time longer than that of the native peptide or protein, allowing for less frequent dosing, such as once daily or even three times a week, twice a week, once a week or monthly. The profile of action of the compounds provided herein can be measured using known pharmacokinetic assays, such as the methods described in the examples below.

As used herein, neuregulin(s) or NRG refers to neuregulin obtained from or derived from any species, such as mammalian species, in particular humans. NRG includes both native NRG (i.e., full-length) and variants (i.e., additions, deletions, insertions, and/or substitutions of native NRG). There are a number of native members of the NRG family in humans; however, of interest herein is NRG1. Like all NRGs, NRG1 is formed by processing from a larger precursor. One sequence for the precursor is provided in SEQ ID NO: 87, and one sequence for NRG1 is provided in SEQ ID NO: 88 (see also UniProt/SwissProt Accession Number Q02297). In humans, there are two NRG1 receptors, ErbB3 (SEQ ID NO: 89; see also UniProt/SwissProt Accession Number P21860) and ErbB4 (SEQ ID NO: 90; see also UniProt/SwissProt Accession Number Q15303). See Mei & Xiong (2008).

As used herein, a non-standard amino acid means an amino acid that may naturally occur in cells but is not involved in peptide synthesis. Non-standard amino acids may be constituents of a peptide and are often formed by modifying standard amino acids in a peptide (i.e., by post-translational modification). Non-standard amino acids may include D-amino acids that have an absolute chirality opposite to the standard amino acids listed above.

As used herein, an oligomer is a molecule having several similar or identical repeating units, which may be derived from copies of a smaller molecule, its monomer. These monomers may be linked by bonds, which may be strong or weak, covalent or non-covalent (e.g., intramolecular).

In this document, patient, subject, and individual are used interchangeably to mean a mammal, especially a human. In some cases, individual is additionally referred to as

- 11 048693 is characterized by the presence of a condition, disease, disorder, or symptom that can be improved by administration of the compound or composition provided herein.

As used herein, a pharmaceutically acceptable buffer means any of the standard pharmaceutical buffers known to those skilled in the art.

As used herein, relaxin-2 or RLN-2 refers to relaxin-2 obtained from or originating from any species, such as mammalian species, in particular humans, wherein the native form is a heterodimeric peptide having two peptide chains (e.g., chain A and chain B) linked by two disulfide bonds, wherein chain A additionally has one intramolecular disulfide bond. In humans, RLN-2 processing begins with preprorelaxin (see UniProt/SwissProt Accession Number P04090), which is processed to prorelaxin (comprising chain A, chain B, and C-peptide; native RLN has the structure B-C-A; see SEQ ID NO: 91; see also UniProt/SwissProt Accession Number P04090). Prorelaxin undergoes further processing, in which the C-peptide is cleaved to form RLN-2 (for chain A of RLN-2, see SEQ ID NO: 92, and for chain B of RNL-2, see SEQ ID NO: 93; see also UniProt/SwissProt accession number P04090).

In this document, sequence similarity means a quantitative property of two or more nucleic acid sequences or amino acid sequences of biological compounds, such as, for example, agreement over the entire length or within a comparison window of said two or more sequences. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures the percentage of residues that are identical for two biological compounds divided by the length of the shortest sequence; while percent similarity measures identity and, in addition, includes gaps in sequences and residue similarity in the assessment. Methods and algorithms for determining sequence similarity are well known in the art and therefore do not need to be described exhaustively herein. The stated 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 higher.

As used herein, treating or treating means the management and care of an individual having a condition, disease, disorder, or symptom for which administration of a compound as provided herein is indicated, in order to alleviate, inhibit, reverse, slow, or halt the progression of, or reduce the severity of, said condition, disease, disorder, and/or symptom. Treating includes administering a compound as provided herein or a composition comprising a compound as provided herein to an individual to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate the condition, disease, disorder, or symptom. Treating includes administering a compound as provided herein or a composition comprising a compound as provided herein to an individual to, for example, enhance angiogenesis, increase vascular compliance, increase cardiac blood flow, increase hepatic blood flow, increase pulmonary blood flow, increase renal blood flow, increase glomerular filtration rate, lower blood pressure, reduce (or prevent) inflammation, and/or reduce (or prevent) fibrosis of the heart, kidney, liver, or lung. The individual to be treated is a mammal, especially a human.

As used herein, a VHH or VHH fragment refers to a form of single-domain antibody, in particular an antibody fragment comprising a single monomeric variable region of a heavy chain-only antibody (HcAb), which may be approximately 15 kDa in size. It has been found herein that engineered/modified VHH-based compounds can be used as a pharmacokinetic enhancer to increase the duration of action and/or improve the t ¹ /2 of biotherapeutics. VHH-based compounds bind serum albumin; however, VHH-based compounds can be used to bind IgG (including the Fc domain), neonatal Fc receptor (FcRn), or other long-acting serum proteins. Thus, a VHH-based compound can be used to improve the t ¹ /2 of a compound such as a peptide or protein, or even other molecules such as, for example, small molecules. Some abbreviations are defined as follows: ACR refers to urine albumin-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 extracellular domain; EDC refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ETA refers to ethanolamine; GS refers to glutamine synthetase; HC refers to heavy chain; CHB refers to hydrophobic interaction chromatography; h refers to hour or hours; IV refers to intravenous administration; kDa refers to kilodaltons; LC refers to light chain; LC-MS refers to liquid chromatography with tandem mass spectrometry; min refers to minute or minutes; MS refers to mass spectrometry; MSX refers to methionine sulfoximine; NHS refers to N

- 12 048693 hydroxysuccinimide; OtBu refers to O-tert-butyl; PEI refers to polyethyleneimine; RP-HPLC refers to reversed-phase high-performance liquid chromatography; s refers to second or seconds; NaOAc refers to sodium acetate; rcf means relative centrifugal force; rt means room temperature; RU means resonance units; sc refers to subcutaneous injection; 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 Tit refers to trityl.

VHH-based compounds acting as half-life extenders and including fusion or conjugate compounds thereof.

Briefly, the compounds provided herein may 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,

X ₁ -L ₂ -M,

X ₂ -L ₁ -M,

X ₂ -L ₂ -M,

X1-M-X2,

X ₂ -MX ₁ ,

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

ML ₁ -X ₁ in combination with M-L2-X2 (i.e., non-covalently linked), wherein M is a VHH-based compound that serves as a t1/ ₂ enhancing moiety and has an amino acid sequence selected from SEQ ID NOs: 1-37 and 124126, or has an amino acid sequence that is at least about 90% to about 99% similar thereto, wherein L1 (if present) is a first linker, wherein L ₂ (if present) is a second linker, wherein X ₁ (if present) is a biotherapeutic protein, peptide, or oligomer, and wherein X ₂ (if present) is also a biotherapeutic protein, peptide, or oligomer, which may be the same as or different from X ₁ (e.g., when the biotherapeutic agent is a homodimer or when the biotherapeutic agent is a heterodimer, X ₁ may be one chain thereof (α-chain), and X ₂ may represent its other chain (β-chain)). X1 and X ₂ may also be completely different from each other. In some cases, L ₁ may have the amino acid sequence (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)nGGGG (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), wherein n may be from 1 to 15, in particular from about 5 to about 10. In other cases, L ₁ may have an amino acid sequence selected from any of SEQ ID NOs:52-63. In other cases, L ₁ may comprise one or more additions, deletions, insertions or substitutions such that L ₁ has an amino acid sequence having at least about 90% to about 99% similarity to any of SEQ ID NOs:52-63. In some cases, L2 may have an amino acid sequence selected from any of SEQ ID NOs:64-65. In other cases, L ₂ may comprise one or more additions, deletions, insertions or substitutions such that L ₂ has an amino acid sequence having at least about 90% to about 99% similarity to any of SEQ ID NOs:64-65.

In other cases, L1 or L2 may be a polymer such as polyethylene glycol (PEG), especially maleimide-(PEG) ₁₂ .

Collectively, the illustrative fusion molecules are:

Compound 1, a VHH-CNTF fusion molecule that includes CNTF, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as a t ^1/2 enhancing _moiety (underlined), has the following amino acid sequence:

MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVA STDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAA FAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKKLWGLKVLQELSQWTVRSIHDLR FISSHOTGGGGGOGGGGOGGGGOGGGGOGGGGQEVOLLESGGLVOPGGSLRLSCAA SGRYIDETAVAWFROAPGI<EREFVAGIGGGVDITYYADSVI<GRFTISRDNSI<NTLYLQM

NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ Ш NO: 100).

Compound 2, a VHH-CNTF fusion molecule that includes a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), a (G ₄ Q) ₅ linker (in italics), and CNTF, has the following amino acid sequence:

EVQLLESGGGLVQPGGSLRESCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGOGGGGOGGGGOGGGGOGGGGQMAFTEHSPLTPHRRDLASRSIWL ARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYR TFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADG MPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTG (SEQ ID ΝΟΤΟΙ).

Compound 3, a VHH-NRG1 fusion molecule that includes NRG1, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), has the following amino acid sequence:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHL GIEFMEAEELYQGGGGOGGGGOGGGGOGGGGOGGGGQEVQLLESGGGLVQPGGSLRL SCAASGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTL YLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 102).

Compound 4, a VHH-NRG1 fusion molecule that includes a VHH-based compound acting as a t1/ ₂ enhancing fragment (underlined), a (G ₄ Q) ₅ linker (in italics), and NRG1, has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGOGGGGOGGGGOGGGGOGGGGQSHLVKCAEKEKTFCVNGGECFM VKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQ (SEQ Ш NO: 103).

Compound 5, a VHH-GDF15 fusion molecule that includes a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), a (G ₄ Q) ₅ linker (in italics), and GDF15, has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGOGGGGOGGGGOGGGGOGGGGQGDHCPLGPGRCCRLHTVRASLE DLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ Ш NO: 104).

Compound 6, a VHH-IL-2 fusion molecule that includes IL-2, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), has the following amino acid sequence:

- 14 048693

APQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAOSIISTLTGGGGOGGGGOGGGGOGGGGOGGGGQEVOLLESGGGLVOPGGSLRL SCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTL

YLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 105).

Compound 7, a VHH-IL-2 fusion molecule that includes a VHH-based compound acting as a t1/ ₂ enhancing fragment (underlined), a (G4Q)5 linker (in italics), and IL-2, has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVT VS SGGGGQGGGGOGGGGOGGGGOGGGGQAPO SS STQOTOLOLEHLLLDLQ MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 106).

Compound 8, a VHH-GLP-1 fusion molecule that includes GLP-1, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), has the following amino acid sequence:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGG2GGGG2GGGG2GGGG2GGGG2 EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ Ш NO: 107).

Compound 9, a VHH-Fab fusion molecule that includes HC, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as _a t ^1/2 enhancing fragment (underlined), has the following amino acid sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGG2G GGGQGGGGOGGGGQGGGGOEVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFR OAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCA ARPGRPLITSKVADLYPYWGOGTLVTVSSPP (SEQ ID NO: 108).

Compound 9 further comprises LC having the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO: 109).

Compound 10, a VHH-Fab fusion molecule that includes LC, a (G ₄ Q) ₅ linker (in italics), and a VHH-based compound acting as _a t ^1/2 enhancing fragment (underlined), has the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGG2GGGG2GGGG2GGGG2G

- 15 048693

GGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGG VDITYYADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTL VT VSSPP (SEQ ID NO: 111).

Compound 10 further comprises HC having the following amino acid sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ Ш NO: 110).

Compound 11, a VHH-Fab fusion molecule that includes a VHH-based compound acting as a t ¹ /2 enhancing fragment (underlined), a (G4Q)5 linker (in italics), and HC, has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQEVOLVESGGGLVQPGRSLRLSC AASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCD (SEQ Ш NO: 112).

Compound 11 further comprises LC having the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: FROM).

Compound 12, a VHH-Fab fusion molecule that includes a VHH-based compound serving as _a t ^1/2 enhancing fragment (underlined), a (G4Q)5 linker (in italics), and an LC, has the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OGTLVTVSSGGGGOGGGGOGGGGOGGGGOGGGGODIOMTQ SPS SLS AS VGDRVTTITC RASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDV ATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 115).

Compound 12 further comprises HC having the following amino acid sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 114).

Compound 13, a VHH-GLP-1 fusion molecule that includes GLP-1, a (G ₄ Q) ₅ linker (in italics), a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), and a C-terminal Cys, has the following amino acid sequence:

- 16 048693

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGG2GGGG2GGGG2GGGG2GGGG2 EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC (SEQ Ш NO: 116).

Compound 14, a VHH-GLP-1/ACTH fusion conjugate that comprises GLP-1, a (G4Q)5 linker (in italics), a VHH-based compound acting as a t1/ ₂ enhancing moiety (underlined), a C-terminal Cys, and ACTH linked in a C-terminal to N-terminal direction by a maleimide(PEG) ₁₂ linker, has the following amino acid sequence:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGG£>GGGG£>GGGG£>GGGG£>GGGG£>

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLУТVS 5C-maleimide-(PEO) j ?-ACTH (in the direction from C-terminus to N-terminus) (SEQ ID NO

NO: 117).

Compound 15, a VHH-ACTH fusion conjugate that comprises a VHH-based compound acting as a t ¹ / ₂ enhancing moiety (underlined), a C-terminal Cys, and ACTH linked in a C-terminal to N-terminal direction with a maleimide(PEG) ₁₂ linker, has the following amino acid sequence:

EVOLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFROAPGKGREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLOMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG

QGTLVTVSSC- _mal imid-(PEG) ₁₂ -(ACTH in C-terminal to N-terminal direction) (SEQ ID NO: 118). Pharmaceutical compositions and kits.

The compounds (i.e., VHH-based fusion molecules or VHH-based conjugates, such as, for example, compounds 1-15 above) provided herein can be formulated into pharmaceutical compositions that can be administered by parenteral routes (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally). Such pharmaceutical compositions and methods for preparing them are well known in the art. See, for example, Remington, The Science and Practice of Pharmacy (DB Troy ed., ^21st Ed., Lippincott, Williams & Wilkins, 2006). In specific instances, the compositions are administered subcutaneously or intravenously. Alternatively, however, the compositions can be formulated into other pharmaceutically acceptable routes, such as, for example, tablets or other solid dosage forms for oral administration; modified release capsules; and any other forms currently in use, including creams, lotions, inhalation formulations, etc.

As noted previously, and to improve their compatibility and efficacy in vivo, the VHH-based fusion molecules or VHH-based conjugates provided herein can be reacted with any number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts. Pharmaceutically acceptable salts and conventional techniques for their preparation are well known in the art (see, e.g., Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use (2 ^nd Revised Ed. Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use provided herein include the sodium, trifluoroacetate, hydrochloride, and acetate salts.

The compounds proposed in this document can be administered by a physician or self-administered by injection. It is understood that the gauge of the needle and the amount of injection volume can be easily determined by a person skilled in the art. However, the injection volume can be < about 2 ml or even < about 1 ml, and the gauge of the needle can be > about 27 G or even > about 29 G.

The invention also provides and therefore encompasses novel intermediates and processes useful for synthesizing the compounds provided herein or a pharmaceutically acceptable salt thereof. The intermediates and compounds can be prepared by various techniques that are well known in the art. For example, a method using recombinant synthesis is illustrated in the examples below. The specific steps for each of the described techniques can be combined in various ways to produce the compounds. The reagents and starting materials are readily available to one skilled in the art.

The compounds provided herein are generally effective over a wide dosage range. Illustrative dosages of the compounds or pharmaceutical compositions comprising them may be in milligram (mg) or microgram (mcg), nanogram (ng) or picogram (pg) amounts per kilogram (kg) of an individual's body weight. Thus, a daily dosage may range from

- 17 048693 approximately 1 mcg to approximately 100 mg.

In this case, the effective amount of the compound in the pharmaceutical composition may be a dose of about 0.25 mg to about 5.0 mg. However, one skilled in the art will appreciate that in some cases the effective amount (i.e., dose/dosage) may be below the lower limit of the above range and be more than sufficient, while in other cases the effective amount may be a higher dose and may be used with acceptable side effects.

In addition to the compound provided herein, the pharmaceutical composition may also include at least one additional therapeutic agent, such as, for example, a therapeutic agent commonly used as a standard of treatment for a particular condition, disease, or disorder (e.g., cardiovascular, neurological, immunological, metabolic, oncological, psychological, pulmonary, and/or renal condition, disease, or disorder).

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

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

Methods for the preparation and use of VHH-based compounds acting as half-life extenders or fusion molecules and conjugates containing them.

The compounds provided herein can be prepared using any number of standard recombinant DNA techniques or standard chemical peptide synthesis techniques known in the art. With respect to recombinant DNA techniques, standard recombinant techniques can be used to construct a polynucleotide having a nucleic acid sequence that encodes an amino acid sequence for the compound (i.e., a fusion peptide or fusion protein or fusion conjugate), incorporate the polynucleotide into recombinant expression vectors, and introduce the vectors into host cells, such as bacterial cells, yeast cells, and mammalian cells, to produce the compound. See, for example, Green & Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, ^4th ed. 2012). [00134] With respect to recombinant DNA techniques, the compounds provided herein can be produced by producing a protein molecule or a precursor protein using recombinant DNA techniques. DNA, including kDNA and synthetic DNA, can be double-stranded or single-stranded, and the coding sequences contained therein that encode a compound provided herein can vary as a result of redundancy or degeneracy of the genetic code. Briefly, DNA sequences encoding the compounds provided herein are introduced into a host cell to produce the compound or a precursor thereof. The host cell can be bacterial cells, such as K12 or B strains of Escherichia coli, fungal cells, such as yeast cells, or mammalian cells, such as Chinese hamster ovary (CHO) cells.

A suitable host cell is transiently or stably transfected or transformed with an expression system, such as expression vectors, to produce a compound provided herein.

- 18 048693 this document, or its predecessor. Expression vectors can typically replicate in host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors will typically contain selection markers, such as for tetracycline, neomycin, G418, and dihydrofolate reductase, to allow selection of those cells transformed with the desired DNA sequences.

To prepare the compounds provided herein, the specific biosynthetic or synthetic steps for each of the steps described herein may be used, not used, or combined in various combinations. With respect to the methods of chemical peptide synthesis, standard solid-phase synthesis procedures may be used, either manually or automatically. 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 sources. Solid-phase synthesizers may be used according to the manufacturer's instructions to block interfering groups, protect amino acids during the reaction, couple, deprotect, and cap unreacted amino acids.

One of the uses of the compounds proposed herein is the treatment of cardiovascular, neurological, immunological, metabolic, oncological, psychological, pulmonary and/or renal conditions, diseases or disorders.

The methods may include the steps described herein, and they may be performed, but are not necessarily performed, in the sequence described. However, other sequences of execution are also possible. Moreover, individual steps or several steps may be performed in parallel, and/or overlapping in time, and/or separately, or in multiple repeated steps. In addition, the methods may include additional unspecified steps.

Thus, such methods may include selecting an individual having, for example, a neurological condition, disease, or disorder, or being predisposed thereto. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, a metabolic condition, disease, or disorder. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, a cardiovascular condition, disease, or disorder. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, an oncological condition, disease, or disorder. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, a psychological condition, disease, or disorder. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, a pulmonary condition, disease, or disorder. Alternatively, the methods may include selecting an individual having, or being predisposed thereto, a renal condition, disease, or disorder. Alternatively, the methods may include selecting an individual who has or is predisposed to an autoimmune condition, disease, or disorder.

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

The concentration/dose/dosage of the compound and optional additional therapeutic agent are discussed elsewhere in this document.

With regard to the route of administration, the compound or a pharmaceutical composition comprising it can be administered by known methods, such as, for example, orally; by injection (i.e., intraarterially, intravenously, intraperitoneally, intracerebrally, intracerebroventricularly, intramuscularly, intraocularly, intraportally or intralesionally); by means of sustained release systems or by means of implantable devices. In some cases, the compound or a pharmaceutical composition comprising it can be administered subcutaneously by bolus injection or continuously.

With respect to dosing frequency, the compound or a pharmaceutical composition comprising it may be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), once every two weeks (i.e., every two weeks), or monthly. In some cases, the compound or a pharmaceutical composition comprising it is administered subcutaneously every other day, subcutaneously three times a week, subcutaneously twice a week, subcutaneously once a week, subcutaneously every two weeks, or subcutaneously monthly. In certain cases, the compound or a pharmaceutical composition comprising it is administered subcutaneously once a week (QW).

Alternatively, and when administered intravenously, the compound or a pharmaceutical composition comprising it is administered intravenously every other day, intravenously three times a week, intravenously twice a week, intravenously once a week, intravenously once every two weeks, or intravenously monthly. In certain cases, the compound or a pharmaceutical composition comprising it is administered intravenously once a week (once a week).

As regards those cases where the compound or a pharmaceutical composition including it

- 19 048693 is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent can be administered simultaneously, separately or sequentially with the compound or a pharmaceutical composition including it.

Moreover, the additional therapeutic agent may be administered at the same frequency as the compound or pharmaceutical composition comprising it (i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent may be administered at a frequency different from the frequency of administration of the compound or pharmaceutical composition comprising it. In other cases, the additional therapeutic agent may be administered subcutaneously. In other cases, the additional therapeutic agent may be administered intravenously. In other cases, the additional therapeutic agent may be administered orally.

It is also contemplated that the methods may be combined with diet and exercise and/or may be combined with additional therapeutic agents other than those discussed above.

Examples

The following non-limiting examples are provided for purposes of illustration and not limitation.

Expression, purification and conjugation of polypeptides.

Example 1. Recombinant expression and purification of VHH-based fusion molecule 1.

Example 1 is a CNTF-VHH fusion molecule having the following amino acid sequence:

MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVA STDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAA FAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKKLWGLKVLQELSQWTVRSIHDLR FISSHQTGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQM NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ Ш NO: 100).

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

To produce the VHH-based fusion molecule by transient transfection, CHOK1 cells were transfected with the recombinant expression plasmid by a PEI-based method. Briefly, an appropriate volume of CHOK1 cell suspension at a density of 4 x ¹⁰⁶ cells/mL was transferred to shake flasks, and both PEI and recombinant plasmid DNA were added to the cells. The cells were incubated in suspension culture at 32°C for 6 days. At the end of the incubation period, the cells were removed by low-speed centrifugation, and the VHH-based fusion molecule was purified from the conditioned medium.

Alternatively, and to generate a VHH-based fusion molecule via stable transfections, CHOK1 cells were stably transfected by electroporation with an appropriate amount of recombinant expression plasmid, and the transfected cells were maintained in suspension culture at an appropriate cell density. Selection of transfected cells was performed by growing in serum-free medium containing 25 μM MSX and incubating at approximately 37°C and approximately 6% CO2.

The VHH-based fusion molecule secreted into the medium from CHO cells was purified by protein A affinity chromatography followed by ion exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography. Specifically, the VHH-based fusion molecule from the pooled medium was captured on Mab Select Protein A resin (GE Healthcare). The resin was then quickly washed with a running buffer such as sodium phosphate buffer (PBS; pH 7.4) or Tris-containing running buffer to remove non-specifically bound material. The VHH-based fusion molecule was eluted from the resin with a low pH solution such as 10 mM citric acid at pH 3. The fractions containing the VHH-based fusion molecule were pooled and allowed to stand at low pH to inactivate potential viruses. The pH could be neutralized by adding a base such as 1 M Tris pH 8.0. The VHH-based fusion molecule could be further purified by ion exchange chromatography using resins such as Poros 50 HS (ThermoFisher). The VHH-based fusion molecule was eluted from the ion exchange column using a gradient of 0 to 500 mM NaCl in 20 mM NaOAc, pH 5.0, in 15 column volumes.

The fused VHH-based molecule can be further purified by chromatography.

- 20 048693 hydrophobic interactions using a Capto Phenyl ImpRes CHB column (GE Healthcare). Purification was accomplished by adjusting the column loading solution to approximately 0.5 M sodium sulfate and eluting with a 10 column volume (CV) gradient going from 0.5 M to 0 M sodium sulfate in 20 mM Tris pH 8. Following CHB, the VHH-based fusion molecule can be further purified by SEC by loading a concentrated pool of Capto Phenyl ImpRes onto Superdex200 or Superdex75 (GE Healthcare) with isocratic elution in PBS pH 7.4 or in 20 mM histidine, 50 mM NaCl pH 6.0.

The purified VHH-based fusion molecule can be passed through a virus-retaining filter such as Planova 20N (Asahi Kasei Medical), followed by concentration/diafiltration in 20 mM histidine, 20 mM NaCl pH 6, using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore).

Thus, a fusion molecule based on VHH is obtained by this or a similar method, which can be easily determined by one skilled in the art.

Example 2: Recombinant expression and purification of VHH-based fusion molecule 2.

Example 2 is a VHH-CNTF fusion molecule having the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQMAFTEHSPLTPHRRDLASRSIWL ARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYR TFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADG MPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTG (SEQ Ш NO: 101).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 101 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 101 was used in the expression plasmid.

Example 3. Recombinant expression and purification of VHH-based fusion molecule 3.

Example 3 is a NRG1-VHH fusion molecule having the following amino acid sequence:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHL

GIEFMEAEELYQGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRL SCAASGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID

NO: 102).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 102 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 102 was used in the expression plasmid.

Example 4. Recombinant expression and purification of VHH-based fusion molecule 4.

Example 4 is a VHH-NRG1 fusion molecule having the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSHLVKCAEKEKTFCVNGGECFM VKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQ (SEQ ID

NO: 103).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 103 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 103 was used in the expression plasmid.

Example 5. Recombinant expression and purification of VHH-based fusion molecule 5.

Example 5 is a VHH-GDF15 fusion molecule having the following amino acid sequence:

- 21 048693

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGDHCPLGPGRCCRLHTVRASLE DLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ Ш NO: 104).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 104 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 104 was used in the expression plasmid.

Example 6. Recombinant expression and purification of VHH-based fusion molecule 6.

Example 6 is an IL-2-VHH fusion molecule having the following amino acid sequence:

APQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRL SCAASGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 105).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 105 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 105 was used in the expression plasmid.

Example 7. Recombinant expression and purification of VHH-based fusion molecule 7.

Example 7 is a VHH-IL-2 fusion molecule analogue having the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVT VS SGGGGQGGGGQGGGGQGGGGQGGGGQAPQS S STQQTQLQLEHLLLDLQ MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 106).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 106 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 106 was used in the expression plasmid.

Example 8. Recombinant expression and purification of VHH-based fusion molecule 8.

Example 8 is a GLP-1-VHH fusion molecule having the following amino acid sequence:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ GO NO: 107).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 107 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 107 was used in the expression plasmid.

Example 9. Recombinant expression and purification of VHH-based fusion molecule 9.

Example 9 is a Fab-VHH fusion molecule containing HC-VHH and LC, where HCVHH has the following amino acid sequence:

- 22 048693

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQG GGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFR QAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCA ARPGRPLITSKVADLYPYWGQGTLVTVSSPP (SEQ ID NO: 108).

and LC has the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 109).

In this case, a fusion molecule based on VHH with the sequences of SEQ ID NOs: 108 and 109 was prepared essentially as described in Example 1, except that the cDNA sequences encoding SEQ ID NOs: 108 and 109 were cloned into two expression plasmids, and then a 1:1 mixture of both plasmids was used for transfection.

Example 10. Recombinant expression and purification of VHH-based fusion molecule 10.

Example 10 is a Fab-VHH fusion molecule comprising HC and LC-VHH, where HC has the following amino acid sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ Ш NO: 110).

and LC-VHH has the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL PYWGQGTLVTVSSPP (SEQ ID NO: 111).

In this case, a VHH-based fusion molecule analog with the sequences of SEQ ID NOs: 110 and 111 was prepared essentially as described in Example 1, except that the cDNA sequences encoding SEQ ID NOs: 110 and 111 were cloned into two expression plasmids, and then a 1:1 mixture of both plasmids was used for transfection.

Example 11. Recombinant expression and purification of VHH-based fusion molecule 11.

Example 11 is a Fab-VHH fusion molecule comprising VHH-HC and LC, where VHHHC has the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQEVQLVESGGGLVQPGRSLRLSC AASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCD (SEQ ID NO: 112).

and LC has the following amino acid sequence:

- 23 048693

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO: 113).

In this case, a fusion molecule based on VHH with the sequences of SEQ ID NOs: 112 and 113 was prepared essentially as described in Example 1, except that the cDNA sequences encoding SEQ ID NOs: 112 and 113 were cloned into two expression plasmids, and then a 1:1 mixture of both plasmids was used for transfection.

Example 12. Recombinant expression and purification of VHH-based fusion molecule 12.

Example 12 is a VHH-Fab comprising HC and VHH-LC, where HC has the following amino acid sequence:

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (SEQ Ш NO: 114).

and VHH-LC has the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDIQMTQSPSSLSASVGDRVTITC RASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDV ATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ GO NO: 115).

In this case, a VHH-based fusion molecule analog with the sequences of SEQ ID NOs: 114 and 115 was prepared essentially as described in Example 1, except that the cDNA sequences encoding SEQ ID NOs: 114 and 115 were cloned into two expression plasmids, and then a 1:1 mixture of both plasmids was used for transfection.

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ GO NO: 121).

In this case, Fabs with the sequences of SEQ ID NOs: 120 and 121 were prepared essentially as described in Example 1, except that the cDNA sequences encoding SEQ ID NOs: 120 and 121 were cloned into two expression plasmids, and then a 1:1 mixture of both plasmids was used for transfection.

Example 14. Recombinant expression and purification of VHH-based fusion molecule 13.

Example 14 is a GLP-1-VHH fusion molecule having the following amino acid sequence:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC (SEQ GO NO: 116).

In this case, a fusion molecule based on VHH with the sequence of SEQ ID NO: 116 was prepared essentially as described in Example 1, except that the cDNA sequence encoding SEQ ID NO: 116 was used in the expression plasmid.

Example 15. Preparation of conjugate 1 based on VHH.

Example 15 is a GLP-1/AKTT-VHH conjugate having the following chemical structure:

- 24 048693

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGGQGGGGGQGGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC-ManeHMiig-(PEG)i2-AKTF (from C-terminus to N-terminus; SEQ ID NO: 117). The VHH conjugate of SEQ ID NO: 117 was prepared as follows. 1 mg of the VHH fusion molecule from Example 14 (SEQ ID NO: 116) was prepared at a concentration of 1 mg/mL (~50 μM) by diluting a 10-fold stock solution of 10 mg/mg with PBS, pH 7.2. Four equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in PBS, pH 7.2, were added and incubated for 4 h at room temperature. Successful reduction to homogeneous monomers was confirmed by mass spectrometry.

The reaction mixture was desalted using a 2 mL ZEBA desalting column with a MW cutoff of 7K. The column storage buffer was removed by centrifugation at 1000 rcf for 1 min. The column was washed by adding 2 mL PBS, pH 7.2, and centrifuging at 1000 rcf for 1 min. The reaction mixture was loaded onto the ZEBA column and desalted by centrifugation for 1 min at 1000 rcf into a clean 15 mL conical vial. The desalted sample was concentrated to ~700 µM (~100 µL) using an Amicon Ultra spin column with a MW cutoff of 3K. ACTH(PEG) ₁₂ -maleimide-NH2 (SEQ ID NO: 119) was prepared at a concentration of 20 mg/mL (5.2 mM) in PBS, pH 7.2. 20 μL of ACTH-(PEG) ₁₂ -maleimide-NH2, corresponding to 2 stoichiometric equivalents, were added to the desalted and concentrated solution of the VHH-based fusion molecule from Example 14 (SEQ ID NO: 116). A white precipitate formed immediately. The reaction was allowed to proceed for 10 min and the solution was solubilized by lowering the pH to 5.5 by slowly adding 0.1 M HCl until the solution became clear. Successful conjugation was confirmed by mass spectrometry.

The desired product was purified in an ACTH purification system using a 5 ml MabSelect Protein A resin column (GE Healthcare) with a loading buffer of PBS, pH 7.2, and an elution buffer of 20 mM citrate, pH 3. The sample was loaded onto the column and the loading buffer was continued until the unreacted ACTH-(PEG) ₁ 2-maleimide-NH2 peak passed through the column. When the signal returned to baseline, the buffer was changed to 20 mM citrate, pH 3, to elute the desired product from the column. As elution progressed, 1 ml fractions were collected, pooled, and the pH was adjusted to 7 with Tris-HCl, pH 8.

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

Example 16. Preparation of conjugate 2 based on VHH.

Example 16 is an ACTH-VHH conjugate having the following chemical structure:

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGG

GVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADL

YPYWGQGTLVTVSSC-A^eHMiig-(PEG)i2-(AKTr) (in the direction from C-terminus to N-terminus;

SEQ ID NO:118).

The VHH conjugate of SEQ ID NO: 118 was prepared as follows. 1 mg of control VHH (SEQ ID NO: 37) was prepared at a concentration of 1 mg/mL (approximately 70 μM) by diluting a 10-fold stock solution of 10 mg/mg with PBS, pH 7.2. Four equivalents of TCEP in PBS, pH 7.2, were added and incubated for 4 h at room temperature. Successful reduction to homogeneous monomers was confirmed by mass spectrometry. The reaction mixture was desalted using a 2 mL ZEBA desalting column with a MW cutoff of 7K. The column storage buffer was removed by centrifugation at 1000 rcf for 1 min. The column was washed by adding 2 mL PBS pH 7.2 and centrifuging at 1000 rcf for 1 min. The reaction mixture was loaded onto the ZEBA column and desalted by centrifugation for 1 min at 1000 rcf into a clean 15 mL conical vial. The desalted sample was concentrated to approximately 700 µM (~100 µL) using an Amicon Ultra spin column with a MW cutoff of 3K.

ACTH-(PEG) ₁ 2-maleimide-NH2 (SEQ ID NO: 119) was prepared and conjugated to VHH as described in Example 15.

In vitro function - albumin binding.

Example 17. SPR studies of albumin binding to VHH fragments.

In vitro binding of different VHH fragments to human serum albumin (SA), Java

- 25 048693 macaque, mouse, rat, pig, dog and cow SAs were determined using SPR. In particular, the binding of the VHH fragments proposed in this document to the SAs of these species is summarized below in Table 1. The binding of the VHH fragments with the sequences SEQ ID NOs: 3 and 8-28 to different SAs was performed on a Biacore 8K instrument.

The immobilization of CA on the surface of the S-series CM5 sensor chip was performed according to the manufacturer's instructions (BR-1000-50 Amino Group Immobilization Kit). Briefly, the carboxyl groups on the sensor chip surfaces (flow cell 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 a flow rate of 10 μL/min. Human, cynomolgus monkey, mouse, rat, pig, dog, and bovine SA were diluted in 10 mM sodium acetate, pH 4.0 (BR-1003-49) at concentrations of 1, 1.3, 1, 1, 1, and 1 μg/mL and then flowed over the surfaces of the activated chip (flow cell 2, channels 1 through 8) at 10 μL/min for 90 s. Human SA was purchased from Sigma Aldrich (St. Louis, MO; Cat. #A8763). Cynomolgus monkey SA was purchased from Athens R&T (Athens, GA; Cat. #16-16-011202-CM). Mouse SA was purchased from Sigma Aldrich (Cat. #A3559). Rat SA was obtained from Sigma Aldrich (Cat. No. A4538). Porcine SA was obtained from Sigma Aldrich (Cat. No. A4414). Canine SA was obtained from Molecular Innovations (Novi, MI; Cat. No. DSA-1213 NC0739153). Bovine SA was obtained from Sigma Aldrich (Cat. No. A7030). The various SAs were covalently immobilized via free amine groups onto a carboxymethyldextran-coated CM5 sensor chip to achieve a surface density of approximately 77 (58-98) RU. Excess reactive groups on the surfaces (flow cell 1 and 2) were inactivated by adding 70 μL of 1 M ethanolamine hydrochloride NaOH, pH 8.5, at a flow rate of 10 μL/min.

VHH fragments were diluted in HBS-EP+ buffer (10 mM HEPES, pH 7.6, 150 mM NaCl, 3 mM EDTA, and 0.05% polysorbate 20) at concentrations of 300 nM. 150 μL of sample were passed sequentially over the surface with immobilized SA and dissociated for 600 s at a flow rate of 50 μL/min at 25 °C. The surface was regenerated by adding 10 mM glycine-HCl, pH 1.5 (BR1003-54) at 50 μL/min for 100 s. The resulting sensorgrams were analyzed using Biacore 8K Insight Evaluation Software (version 2.0.15.12933) to calculate the dissociation rate constant (kd).

Table 1 Binding of illustrative VHH fragments to SA from human, cynomolgus monkey, mouse, rat, pig, dog and cow at 25°C

VHH fragment Human SA SA cynomolgus macaque SA mice SA rats SA pigs SA dogs CA cows SEQID NO: kd (1/s) kd (1/s) kd (1/s) kd (1/s) kd (1/s) kd (1/s) kd (1/s) 3 8,0E-04 2,0E-03 4,ZE-OZ 3,5E-03 5,2E-03 2,2E-03 1,5E-02 3 7,8E-04 1,9E-03 4,ZE-OZ 3,4E-03 5,2E-03 2,ZE-OZ 1,4E-02 8 1,0E-03 3,0E-03 3,2E-03 7,5E-03 9,2E-03 2,2E-03 1,5E-02 9 1,2E-03 3,2E-03 1,ZE-02 Ι,ΙΕ-02 1,2E-02 Ι,ΙΕ-02 very weak 10 weak Very Very Very Very Very binding

- 26 048693

weak weak weak weak weak absent 11 7DE-03 very weak linking absent linking absent linking absent linking absent linking absent 12 1DE-02 very weak linking absent linking absent linking absent linking absent linking absent 13 very weak linking absent linking absent linking absent linking absent linking absent linking absent 14 1,ZE-02 linking absent linking absent linking absent linking absent linking absent linking absent 15 5,8E-03 very weak linking absent linking absent linking absent linking absent linking absent 16 3,9E-03 1,5E-02 linking absent linking absent linking absent very weak linking absent 17 1,5E-03 2,2E-03 1,0E-02 8DE-OZ 1,ZE-02 8,2E-03 1,5E-02 3 7,7E-04 1,7E-03 4,2E-03 ZDE-OZ 5 DE-OZ 2,0E-03 1,5E-02 18 7,6E-03 7,2E-03 very weak 1,4E-02 very weak 1,5E-02 linking absent 19 4DE-03 7, ZE-OZ 1,5E-02 1,5E-02 1DE-02 1,4E-02 linking absent 20 9,2E-03 linking absent linking absent linking absent linking absent very weak linking absent 21 2DE-03 1,4E-02 1,ZE-02 1DE-02 1,0E-02 8DE-03 very weak 22 2,8E-03 1,4E-02 1,0E-02 1,0E-02 1DE-02 7,4E-03 very weak 23 linking absent linking absent linking absent linking absent linking absent linking absent linking absent 24 linking absent linking absent linking absent linking absent linking absent linking absent linking absent 25 very weak linking absent very weak very weak very weak very weak linking absent 26 very weak linking absent linking absent linking absent linking absent linking absent linking absent 27 1DE-02 linking absent linking absent linking absent linking absent linking absent linking absent 3 8,6E-04 1,5E-03 4,2E-03 3,OE-O3 5,0E-03 1,9E-03 1,5E-02 4 2DE-04 5,ZE-04 4,ZE-OZ ZDE-OZ 5,7E-03 1,7E-03 n/a 125 9DE-05 6,0E-04 5,6E-03 3.8E-O3 6,5E-03 2DE-03 n/a 126 1DE-04 7,2E-04 5,4E-03 5,ZE-OZ 5,8E-03 2DE-03 n/a

Example 18. SPR studies of albumin binding of VHH-based fusion molecules.

In vitro binding of the VHH-based fusion molecules to the SAs of human, cynomolgus monkey, mouse, rat, pig, cow, and rabbit was determined by SPR at 25°C. In particular, the affinity of the VHH-based fusion molecules of Examples 1-9 to the SAs of these species is summarized below in Tables 2-10.

The coupling of the VHH-based fusion molecules from Examples 1-9 to different CAs was performed on a Biacore 8K instrument. Immobilization of the different CA orthologs on the surface of the S-series CM5 sensor chip (BR-1006-68) was performed according to the manufacturer's instructions (BR-1000-50 Amino Group Immobilization Kit). Briefly, the carboxyl groups on the sensor chip surfaces (flow cell 1 and 2) were activated by injecting 70 μl of a mixture containing 75 mg/ml hydrochloride

- 27 048693 yes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 11.5 mg/ml N-hydroxysuccinimide (NHS) at a rate of 10 μl/min. Human, cynomolgus macaque, rat, mouse, dog, pig, cow, and rabbit SA were diluted in 10 mM sodium acetate, pH 4.0 (BR-1003-49), at concentrations of 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 passed over the surfaces of the activated chip (flow cell 2, channels 1 through 8) at 10 μL/min for 100 s. Human SA was purchased from Sigma Aldrich (Cat. No. A8763). Cynomolgus monkey SA was obtained from Athens R&T (cat. #16-16-011202-CM). Mouse SA was obtained from Sigma Aldrich (cat. #A3139). Rat SA was obtained from Sigma Aldrich (cat. #A4538). Porcine SA was obtained from Sigma Aldrich (cat. #A4414). Canine SA was obtained from Molecular Innovations (Novi, MI; cat. #DSA-1213 NC0739153). Bovine SA was obtained from Sigma Aldrich (cat. #A7030). Rabbit SA was obtained from Fitzgerald Industries International (Acton, MA; cat. #30R-3303). SA were covalently immobilized via free amines onto a carboxymethyldextran-coated CM5 sensor chip at a surface density of 2578 resonance units (RU) for human, cynomolgus monkey, rat, mouse, dog, pig, and cow SA and 118-372 resonance units (RU) for rabbit SA. Excess reactive groups on the surfaces (flow cell 1 and 2) were deactivated by adding 70 μl of 1 M ethanolamine hydrochloride-NaOH, pH 8.5. The VHH-based fusion molecules were diluted in HBS-EP+ buffer (10 mM HEPES, pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20) at 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 were individually sequentially passed over the surface of the chip with immobilized SA and dissociated for 600 sec at a flow rate of 60 μl/min at 25°C. The surface was regenerated by adding 10 mM glycineHCl, pH 1.5 (BR-1003-54) at 60 μl/min for 100 sec. The resulting sensorgrams were analyzed using a 1:1 binding kinetics model fit in Biacore 8K Insight Evaluation Software (version 3.0.11.15423) to calculate the binding kinetics parameters: association rate constant ( _ka ), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

Table 2

Kinetics of binding of the VHH-based fusion molecule from Example 1 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 7.6E+04 2,4E-04 3,2E-09 SA cynomolgus macaque 6.8E+04 8,6E-04 1,ZE-08 SA mice 8.1E+04 8,2E-03 1,0E-07 SA rats 7.5E+04 6, ZE-OZ 8,ZE-08 SA pigs 5.8E+04 Ι,ΙΕ-02 1,9E-07 SA dogs 7.9E+04 3,2E-03 4.0E-08 CA cows 9.1E+04 7,4E-02 8.2E-07 SA rabbit No linking

The obtained KD values were 3.2, 13, 100, 83, 190, 40 and 820 nM for binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule of Example 1.

- 28 048693

Table 3

Kinetics of binding of the VHH-based fusion molecule from Example 2 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 4,ZE+05 2,5E-04 5.8E-10 SA cynomolgus macaque 5.0E+05 6,0E-04 1,2E-09 SA mice 4.8E+05 4,2E-03 8,8E-09 SA rats 4.9E+05 3,0E-03 6.1E-09 SA pigs 3.0E+05 5,7E-03 1,9E-08 SA dogs 4.6E+05 1,7E-03 3.7E-09 CA cows 4.6E+05 5,2E-02 Ι,ΙΕ-07 SA rabbit No linking

The obtained KD values were 0.58, 1.2, 8.8, 6.1, 19, 3.7, and 110 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog, and bovine SA, respectively, to the VHH-based fusion molecule of Example 2.

Table 4

Kinetics of binding of the VHH-based fusion molecule from Example 3 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 1,2E+05 2,ZE-04 1,9E-09 SA cynomolgus macaque 1.4E+05 8,7E-04 6.1E-09 SA mice 1.4E+05 8,8E-03 6.2E-08 SA rats 1,2E+05 6,0E-03 5,2E-08 SA pigs 9,ZE+04 1,2E-02 1,ZE-07 SA dogs 1.7E+05 3,2E-03 1,9E-08 CA cows 1.6E+05 7,2E-02 4.7E-07 SA rabbit No linking

The obtained KD values were 1.9, 6.1, 62, 52, 130, 19 and 470 nM for binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule from Example 3.

Table 5

Kinetics of binding of the VHH-based fusion molecule from Example 4 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 1.0E+06 1,9E-04 1,9E-10 SA cynomolgus macaque 6.8E+05 6,ZE-04 9.4E-10 SA mice 6.6E+05 4,2E-03 6.4E-09 SA rats 9.7E+05 3,0E-03 3.1E-09 SA pigs 4.0E+05 6,0E-03 1,5E-08 SA dogs 7.8E+05 1,7E-03 2,2E-09 CA cows 5.7E+05 5,8E-02 1,0E-07 SA rabbit No linking

The obtained KD values were 0.19, 0.94, 6.4, 3.1, 15, 2.2 and 100 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog and cow SA, respectively, to the fusion molecule on

- 29 048693 based on VHH from example 4.

Table 6

Kinetics of binding of the VHH-based fusion molecule from Example 5 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 6.7E+05 1,4E-04 2,0E-10 SA cynomolgus macaque 7.4E+05 3,4E-04 4.5E-10 SA mice 1.4E+06 1,2E-03 8.6E-10 SA rats 1.0E+06 7,7E-04 7.4E-10 SA pigs 8.5E+05 2,8E-03 3,ZE-09 SA dogs Ι,ΙΕ+06 6,8E-04 6.0E-10 CA cows *not applicable *not applicable 2,8E-08 SA rabbit No linking

The obtained KD values were 0.2, 0.45, 0.86, 0.74, 3.3, 0.6 and 28 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule of Example 5.

Table 7

Kinetics of binding of the VHH-based fusion molecule from Example 6 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 1.8E+05 2,6E-04 1,5E-09 SA cynomolgus macaque 1.8E+05 8,8E-04 5,0E-09 SA mice 1.7E+05 8,7E-03 5,0E-08 SA rats 1.9E+05 6,6E-03 3,4E-08 SA pigs 1,2E+05 1,2E-02 1,0E-07 SA dogs 1.7E+05 3,4E-03 1,9E-08 CA cows 1.9E+05 7DE-02 3.8E-07 SA rabbit No linking

The obtained KD values were 1.5, 5.0, 50, 34, 100, 19 and 380 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule of Example 6.

- 30 048693

Table 8

Kinetics of binding of the VHH-based fusion molecule from Example 7 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 9.1E+05 2DE-04 2,ZE-10 SA cynomolgus macaque 1.4E+06 5,6E-04 3.9E-10 SA mice 9.7E+05 3,8E-03 3.9E-09 SA rats 9.7E+05 2,9E-03 3.0E-09 SA pigs 4.5E+05 5,8E-03 1,ZE-08 SA dogs 9.2E+05 1,6E-03 1,8E-09 CA cows 6.7E+05 5,2E-02 7,8E-08 SA rabbit No linking

The obtained KD values were 0.23, 0.39, 3.9, 3.0, 13, 1.8 and 78 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule of Example 7.

Table 9

Kinetics of binding of the VHH-based fusion molecule from Example 8 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 7.2E+05 1,1E-04 1,5E-10 SA cynomolgus macaque 9.1E+05 8,7E-04 9.6E-10 SA mice 5.6E+05 1,0E-02 1,8E-08 SA rats 6.6E+05 7DE-03 Ι,ΙΕ-08 SA pigs 4.9E+05 1,ZE-02 2,7E-08 SA dogs 7.1E+05 3,8E-03 5.4E-09 CA cows 8.0E+05 9,ZE-02 1,2E-07 SA rabbit No linking

The obtained KD values were 0.15, 0.96, 18, 11, 27, 5.4 and 120 nM for the binding of human, cynomolgus monkey, mouse, rat, pig, dog and bovine SA, respectively, to the VHH-based fusion molecule of Example 8.

Table 10

Kinetics of binding of the VHH-based fusion molecule from Example 9 to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit SA at 25°C

Binding to immobilized SA ka (1/Ms) kd (1/s) K _D (M) Human SA 2.1E+05 2,8E-04 1,ZE-09 SA cynomolgus macaque 2,2E+05 9,4E-04 4,ZE-09 SA mice 2,ZE+05 1,0E-02 4,ZE-08 SA rats 2.1E+05 7DE-03 3,ZE-08 SA pigs 1.5E+05 1,ZE-02 8,ZE-08 SA dogs 2.4E+05 3,7E-03 1,5E-08 CA cows 2.5E+05 8,5E-02 3,4E-07 SA rabbit No linking

The obtained KD values were 1.3, 4.3, 43, 33, 83, 15 and 340 nM for binding to human CA,

- 31 048693 cynomolgus monkey, mouse, rat, pig, dog and cow, respectively, with a fusion molecule based on the VHH of Example 9.

In vitro function - specific activity of payloads.

Example 19. In vitro activity of the VHH-based fusion molecules of Examples 1 and 2.

Human pSTAT3 CNTFR assay: IMR-32 cells (ATCC Cat# CCL-127) endogenously expressing human CNTFR, LIFR, and gp130 were cultured at 37°C, 5% _CO2 , 90% humidity in RPMI-1640 medium (ATCC Cat# 30-2001) supplemented with a final 10% FBS, 1 mM sodium pyruvate, and 1x antibiotic-antimycotic (Thermo Fisher Scientific, 15240062). On day -1 (the day before pSTAT3 assay), cells were washed once with 1x PBS, lifted from flasks with Cell Dissociation Buffer (Gibco, 13151), and resuspended in the above-mentioned culture medium. Cells were seeded in 96-well plates coated with poly-O-lysine (Corning; Cat. #354640) at 150,000 cells/0.1 ml/well. Cells were cultured at 37°C, 5% _CO2 , 90% humidity overnight. On day 1 (the day of pSTAT3 assay), the medium was aspirated and replaced with 100 μl of EMEM (ATCC, Cat. #30-2003). Cells were incubated in serum-free medium at 37°C, 5% _CO2 , 90% humidity for 4 h, then 50 μl of 3-fold serially diluted human CNTF (R&D Systems; Cat. #257-NT-010), Example 1 or Example 2, in 0.3% IgG-free BSA (Jackson ImmunoResearch Laboratories, Inc.; Cat. #001-000-162) were added. EMEM was added to a final concentration of 1x. Cells were incubated with these serially diluted proteins for an additional 10 min at 37°C, 5% _CO2 , 90% humidity. After completion of the incubation period, the contents of the plate were removed. Cells were lysed and pSTAT3 was determined using the AlphaLISA Surefire Ultra p-STAT3 (Tyr705) Assay Kit (Perkin Elmer; ALSU-PST3) and its dual-plate assay protocol for adherent cells. Plates were read on an Envision 2102 instrument (Perkin Elmer).

Statistical data analysis: Data were imported from the Envision 2102 reader into Excel® (Microsoft). The % maximal (1 nM) pSTAT3 signal stimulated by human CNTF was calculated for each protein concentration tested. _EC50 values were obtained from these calculations by four-parameter variable-slope dose-response curve analysis using GraphPad Prism® software (GraphPad Software, LLC; La Jolla, CA; version 8.4.3). The results of the analysis are presented below in Table 11.

Table 11

In vitro activity of VHH-based fusion molecules towards the human CNTF receptor

Connection EC ₅ about hCNTFR, pM, geometric mean SEM N CNTF of a person 3.50 0.45 2 Example 1 7.09 0.35 2 Example 2 8.39 0.65 2

Example 20. In vitro activity of the VHH-based fusion molecules of Examples 3 and 4.

A cell-based dimerization bioassay from DiscoverX/eurofins (PathHunter eXpress ErbB4/ErbB4 Dimerization Assay, Cat# 93-0961E3) was used to test the activity of the VHH-based fusions from Examples 3 and 4 against the human Eb4 receptor (NCBI reference sequence no. NP_001036064.1). The assay detects ligand-induced dimerization of the two subunits of a receptor-dimer pair and is designed to assess activity. Testing was performed according to the protocol provided by the manufacturer. Briefly, cells were thawed and seeded into the provided 96-well plate at 100 µl per well. After 24 h incubation at 37°C in 5% CO2, 10 µl of 11x Example 3, Example 4 and control agonist, rhNRG-1 (eurofins, Cat# 92-1091) were added in an 11-point serial dilution curve (1:3) to the appropriate wells in duplicate. The plate was incubated at 37°C for 6 h. After 6 h incubation, 110 µl of detection reagents were added to each well. The plate was incubated at room temperature in the dark for another hour. The chemiluminescent signal was read on a plate reader using an integration time of 0.5 s (SpectraMax i3x plate reader from Molecular Devices). Statistical analysis of raw data was performed using GraphPad PRISM version 9.0. The mean background value was subtracted from all values, raw data were transformed and nonlinear regression analysis (NLA) was performed against the response-variable slope (four parameters) to obtain EC ₅₀ values and determine the goodness of fit (n=2 for all data).

- 32 048693

Table 12

Activity of VHH-based fusions from Examples 3 and 4 in the DiscoverX PathHunter ErbB4 Bioassay

rhNRG-1 Example 3 Example 4 ECso (ng/ml) 16.51 33.55 112.2 R ² 0.9884 0.9863 0.9986

Example 21. In vitro activity of the VHH-based fusion molecule from Example 5.

Construction of HEK293 cell line expressing human GFRAL and RET: HEK293 cells (ATCC) were cultured in DMEM with 10% FBS and 25 mM HEPES, 1x antibiotics, and split 1:16 every 3-4 days with TrypLE™ Express (Gibco). Cells were transfected with plasmid DNA of human GFRAL (GDNF receptor alpha-like; NCBI Reference Sequence No. NP_997293.2), human RET (proto-oncogenic receptor tyrosine kinase RET; NCBI Reference Sequence No. NP_066124.1), and Fugene. 6 (Promega) according to the manufacturer's instructions. Transfected cells were selected with geneticin (Gibco, 1 mg/ml) and puromycin (Gibco, 0.1 mg/ml) for 3-4 weeks. Clonal lines were generated by limiting dilution cloning into 96-well plates and response to GDF15 was confirmed using the AlphaLISA SureFire Ultra p-ERK1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer). Clones were expanded, pooled, resuspended in cell freezing medium, aliquoted into cryovials and stored in liquid nitrogen for long-term storage. The sample with the best response to GDF15 (signal to background ratio), clonal line #7, was selected.

AlphaLISA SureFire Ultra p-ERK1/2 (Thr202/Tyr204) Human GFRAL and RET Receptor Assay: HEK293 cell lines expressing human GFRAL and human RET were cultured in selection medium (DMEM with 10% FBS with 25 mM HEPES, 1x antibiotics, 1 μg/ml puromycin, 1 mg/ml geneticin). On day -3 (the day of cell seeding), cells were washed once with PBS, lifted from flasks using TrypLE™ Express, and resuspended in seeding medium (DMEM with 25 mM HEPES, 1x antibiotics, 10% FBS). Cells were seeded in 96-well plates (Falcon, Cat. #356461) at 20,000 cells/0.1 ml/well. Cells were cultured at 37°C and 5% CO2 for 72 h. On day 1 (the day of assay), the medium was removed and replaced with 50 μl serum-free medium (DMEM with 25 mM HEPES, 1x antibiotics). Plates were incubated at 37°C for 4 h, then 50 μl of 2x ligand (GDF15, final 1x) was added. Plates were incubated for an additional 10 min at 37°C. After completion of the incubation period, the medium was removed from the plates by pouring off and blotting with white all-purpose wipes. Then, 50 μl of 1x AlphaLISA SureFire Ultra Lysis Buffer was added to each well and the plates were incubated on a plate shaker at 350 rpm at room temperature for 10 min. Then, according to the manufacturer's instructions (AlphaLISA SureFire Ultra p-ERK (Thr202/Tyr204) Assay Kit, PerkinElmer, Cat# ALSU-pERK-A10K) for the 2-plate/1-incubation protocol for adherent cells, 10 µl of cell lysate, 5 µl of acceptor mix, and 5 µl of donor mix were added to an OptiPlate™-384 plate (PerkinElmer, Cat# 6007290). The plate was sealed, wrapped in foil, incubated for 1 min on a plate shaker at 250 rpm at room temperature, incubated at room temperature in the dark for 8 h, and then read on an En Vision 2102 Multi-Purpose Reader with EnVision Manager Software (PerkinElmer).

Statistical data analysis: Data were imported from the EnVision 2102 Multi-Purpose Reader into GraphPad Prism® software (GraphPad Software, LLC; La Jolla, CA; version 8). EC ₅₀ values were obtained using a four-parameter variable slope dose-response curve analysis.

Table 13

Stimulation of ERK1/2 phosphorylation in HEK293 cells expressing human GFRAL and RET51 using the VHH-based fusion molecule from Example 5

Connection EC50 hGFRAL/RET (pM) SEM N Human GDF15 2.85 1.08 2 Example 5 101 19,5 2

Example 22. In vitro activity of the VHH-based fusion molecules of Examples 6 and 7.

To assess the activity of the VHH-based fusion molecules from Examples 6 and 7, a cellular dimerization bioassay from DiscoverX, PathHunter IL-2 BioAssay Kit (eurofins/DiscoverX, art. no. 931003Y3-00091), was used. The assay detects IL-2-induced dimerization of the two subunits of the IL-2 receptor-dimer pair and is designed to assess IL-2 activity. The assay was performed according to the manufacturer's protocol. Briefly, cells were thawed and seeded in the provided 96-well plate at 80 µl per well. After 20 h of incubation at 37°C in 5% CO2 _, 20 µl of 5x the initial concentration were added to the plate.

- 33 048693 of Examples 6 and 7 and the provided IL-2 reference standard were added in an 11-point serial dilution curve (1:3) to the appropriate wells in duplicate. The plate was incubated at 37°C for 6 h. After 6 h of incubation with Examples 6 and 7 and the reference standard, 10 μl of detection reagent 1 were added to each well. Mixing was performed on a plate shaker at 350 rpm for 1 min, and the plate was incubated at room temperature in the dark for 15 min. Then 40 μl of detection reagent 2 were added to each well and incubated at room temperature in the dark for an additional 1 h. The chemiluminescent signal was read on a plate reader using an integration time of 1 s (SpectraMax i3x plate reader from Molecular Devices). Statistical analysis of raw data was performed using GraphPad PRISM version 8.4.3. Raw data were transformed and nonlinear regression analysis (log(agonist) versus response - variable slope (four parameters)) was performed to obtain EC ₅₀ values and determine the goodness of fit (n=2 for all data).

Table 14

Activity of VHH-based fusion molecules from Examples 6 and 7 in the DiscoverX PathHunter IL-2 Bioassay

EC ₅₀ (nM) R ² N Human IL-2 0,13 0.9983 2 Example 6 6,7 0.9978 2 Example 7 0.49 0.9980 2

Example 23. In vitro activity of the VHH-based fusion molecules of Examples 9 and 13.

An L929 cell-based cytotoxicity assay was used to evaluate the in vitro neutralization potency of Examples 9 and 13 against soluble human TNFa, soluble cynomolgus monkey TNFa, or membrane-bound human TNFa. L929 cells (ATCC) endogenously expressing the human TNFa receptor were cultured in high-glucose DMEM with 10% heat-inactivated FBS, 2 mM L-glutamine, 1x nonessential amino acids, 1x sodium pyruvate, 1x antibiotics, and split 1:20 every 3-4 days with TrypLE™ Express (Gibco).

Soluble human TNFa protein was prepared by custom synthesis from Syngene (Bangalore, India). Soluble cynomolgus monkey TNFa protein was obtained from R&D Systems (Minneapolis, MN; Cat# 1070-RM/CF).

A stable cell line, MT104 H2 CHO, expressing membrane-bound human TNFa was obtained from Eli Lilly and Company (Indianapolis, IN). Cells were cultured in Lilly LM7300 medium with 8 mM L-glutamine and G418 selection agent (500 μg/ml) and split 1:10 every 3-4 days.

L929 cytotoxicity assay: On day 1, L929 cells were trypsinized with 5 ml TrypLE™ (Gibco #12605036), complete medium (1:3 volume) was added, and the cells were centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was carefully aspirated and the cells were resuspended in 15 ml complete medium (high glucose DMEM with 10% heat-inactivated FBS, 2 mM L-glutamine, 1x nonessential amino acids, 1x sodium pyruvate, 1x antibiotics), and an aliquot of the cells was counted using a Viacell counter. Cells were seeded in 96-well plates coated with poly-O-lysine (Corning #354496) at 10,000 cells/0.1 ml/well and cultured at 37°C with 5% CO2 overnight. On day 2, samples 9 and 13 were exposed to fixed amounts of antigens: soluble human TNFa (200 μg/ml), soluble cynomolgus monkey TNFa (750 pg/ml), or cells expressing membrane human TNFa (5,000 cells/ml) in DMEM+FBS with actinomycin D (6.25 μl). The initial concentrations were 1.5 μg/ml, 3 μg/ml, and 10 μg/ml to neutralize soluble human TNF, soluble cynomolgus TNF, and human membrane TNF, respectively, and were reduced by 3-fold serial dilution to obtain 8 points. The dosed compound-antigen complex was added to 96-well plates containing L929 cells after removing the medium, and the plates were incubated at 37°C with 5% _CO2 overnight. Stimulated negative control wells contained L929 cells+actinomycin D+TNFa antigen, while unstimulated control wells contained L929 cells+actinomycin D only.

On day 3, the medium was removed from the 96-well plates and 120 μl of Cell Titer AQueous ONE substrate solution (1:6 dilution in DMEM+FBS) was added to each well on all plates. Plates were read at OD 490 nm after 2 h on a Spectra max plate reader using Softmax 4.7 software. Data analysis: OD readings versus concentration were plotted in Excel® (Microsoft; Redmond, WA); EC ₅₀ calculation and statistical analysis were performed using Eli Lilly statistical software tools (Global Stats Discovery Team, Lilly).

- 34 048693

Table 15

In vitro activity of VHH-based fusion molecules from Examples 9 and 13 to neutralize soluble human TNFa

Connection EC ₅ o, geometric mean (mcg/ml) Error N Logarithmic mean Sd Example 9 0,0562 0,0115 3 -1.2506 0,0153 Example 13 0,0333 0,0004 3 -1.4781 0,0089 Adalimumab 0,0392 0,0009 3 -1,407 0,01678

Table 16

In vitro activity of VHH-based fusion molecules from Examples 9 and 13 to neutralize soluble cynomolgus macaque TNFa

Connection es ₅₀ , geometric mean (mcg/ml) Error N Mean logarithmic Sd Example 9 0,1748 0,0040 3 -0.7574 0,0170 Example 13 0,1025 0,0045 3 -0.9893 0,0333 Adalimumab 0,0944 0,0021 3 -0.01633 0,0163

Table 17

In vitro activity of VHH-based fusion molecules from Examples 9 and 13 to neutralize human membrane TNFa

Connection EC ₅ o, geometric mean (mcg/ml) Error N Mean logarithmic Sd Example 9 0.2943 0,0040 3 -0.5312 0,0102 Example 13 0,2005 0,0196 3 -0.6979 0,0736 Adalimumab 0,2165 0,0068 3 -0.6645 0,0238

Table 18

In vitro activity of VHH-based fusion molecules from Examples 9 and 13 to neutralize various forms of human and cynomolgus monkey TNFa

ECso (nM) - geometric mean Connection Inhibition of soluble human TNFa Inhibition of soluble TNFa in cynomolgus monkey Inhibition of human membrane TNFa Example 9 0,886 2,7565 4,641 Example 13 0.703 2,1668 4,238 Adalimumab 0.2613 0,6292 1,443

Example 24. In vitro activity of a multispecific fusion molecule and a VHH-based conjugate

- 35 048693 from examples 15 and 16.

Cell-based cyclic AMP assay kits purchased from DiscoverX (eurofins) were used to assess the activity of the VHH-based conjugates from Examples 15 and 16 on GLP1R and MC3R. For cAMP, the Hunter eXpress GLP1R CHO-Κι GPCR assay (Cat# 95-0062E2) was used and the internal GLP-1 standard (SEQ ID NO: 99) was used as a positive control. For cAMP, the Hunter eXpress MC3R CHO-Κ1 GPCR assay (Cat# 95-0045E2) was used and α-MSH was used as a positive control. The assays were performed according to the GPCR assay protocol provided by the manufacturer. Briefly, cells were thawed and seeded in a 96-well plate at 100 μl per well. After 24 h incubation at 37°C in 5% CO2 _, 15 μl of 3x agonist (VHH conjugate/fusion and control) and cAMP standard were added to the appropriate wells in duplicate. The plate was incubated at 37°C for 30 min. The cAMP working solution was prepared and stored at room temperature protected from light. After 30 min incubation with the VHH fusion or conjugate, 15 μl of cAMP antibody reagent were added to each well. Immediately after the addition of the antibody reagent, 60 μl of the cAMP working solution, which had been prepared during the incubation period, were added. The plate was incubated at room temperature in the dark for 1 h, 60 μl of cAMP solution A were added to each well and the plate was incubated at room temperature in the dark for 3-18 h. The chemiluminescent signal was read on a plate reader (SpectraMax i3x plate reader from Molecular Devices). Statistical analysis of raw data was performed using GraphPad PRISM version 8. Raw data were transformed and nonlinear regression analysis (log(agonist) vs. response - variable slope (4 parameters)) was performed to obtain EC50 values and determine the goodness of fit (n=2 for all data).

Table 19

In vitro activity of VHH-based conjugates from Examples 15 and 16 against MC3R

Example 15 Example 16 cAMP standard yes ₅₀ 54.2 46.17 36.25 R-square 0.9941 0.9888 0.9932

Table 20

In vitro activity of the VHH-based conjugate of Example 15 against GLP1R

Example 15 Positive control GLP-1 cAMP standard yes ₅₀ 1,807 0,5044 39.89 R-square 0.9925 0.9832 0.9971

In vivo function - specific long-acting PD for each payload.

Example 25. In vivo activity (weight loss in healthy mice) of the VHH-based fusion compounds from Examples 1 and 2.

The study was performed on 48 male DIO C57BL6 mice obtained from Taconic. Mice were 15 weeks old at the start of the study. Animals were housed individually in microisolator cages with unlimited access to an automated source of tap water (2-5 ^ppm chlorine) and high-fat chow (TD95217). Mice were randomly assigned to 8 groups (n=6) based on body weight using a block random assignment tool developed by Lilly statisticians for random assignment to groups in vivo. To reduce potential stress-related effects of the study, all animals were acclimated to daily handling for at least 3 days prior to the start of the study. Body weight and food weight were recorded 2 days prior to the start of the study and then daily throughout the study. Drugs were administered subcutaneously in the interscapular space throughout the study (once daily dosing schedule) using a 28G 0.5 mL insulin syringe (BD Cat. #329461). Native CNTF (R&D systems, recombinant human CNTF, Cat. #257-NT/CF, Lot #GL402101A), Example 1 and Example 2 were diluted to the appropriate concentrations in sterile PBS, pH 7.2, no later than 30 min prior to administration. The dose of native CNTF was 0.25 mg/kg. The dose of Example 1 and Example 2 was either 0.25 μg/kg or 0.1 μg/kg. Dilutions were calculated for each drug daily based on the average body weight of the group on the previous day. 200 μL of PBS was administered to animals in the vehicle control group. Changes in body weight were analyzed using an in-house statistical tool and a rigorous model-based approach developed by

- 36 048693 by Lilly statisticians for in vivo PD studies. Animals were excluded from the study regardless of dosing date when 20% body weight loss was achieved.

Table 21

Effect of different doses of VHH-based fusion molecules from Examples 1 and 2 on body weight in healthy mice at different time points

Connection Dose N % change in body weight on different study days 0 1 2 3 4 5 6 Carrier n/a 6 102.47 102.11 101,01 99.98 99.50 98.61 99.32 Example 1 0.1 mg/kg 6 101.09 100.51 97.61 95.38 94.24 92.73 91.44 Example 1 0.25 mg/kg 6 100.81 97.18 91.21 85.31 81,18 79.19 82.31 Example 2 0.1 mg/kg 6 102.13 100,70 98,04 95.39 94.87 93,02 91,91 Example 2 0.25 mg/kg 6 102.77 99.81 95.46 90.42 85.29 82.23 82.91 rhCNTF 0.25 mg/kg 6 100.51 98.95 96,96 94.76 94.03 91.74 90.62

Table 22

Statistical analysis of the effect of different doses of VHH-based fusion molecules from Examples 1 and 2 on body weight in healthy mice at different time points

Connection Dose Day of the study 0 1 2 3 4 5 6 Example 1 0.1 mg/kg magnitude of influence pvalue 0.580 1.566 0.650 0.626 0.208 0.989 0.823 0.997 0.751 0.995 0.996 1.000 0.958 0.983 Example 1 0.25 mg/kg magnitude of influence pvalue 0.292 -1.764 -5.750 -9.442 -12.853 -12.552 -8.309 1.000 0.650 0.00026 <0.0001 <0.0001 <0.0001 <0.0001 Example 2 0.1 mg/kg magnitude of influence pvalue 1.617 1.754 1.077 0.638 0.837 1.281 1.289 0.726 0.655 0.939 0.995 0.981 0.876 0.873 Example 2 0.25 mg/kg magnitude of influence pvalue 2.252 0.867 -1.501 -4.332 -8.743 -9.516 -7.715 0.403 0.977 0.783 0.012 <0.0001 <0.0001 <0.0001

P-values are shown relative to the group receiving 0.25 mg/kg native CNTF. As shown in Tables 21 and 22, all treatment groups exhibited significant body weight loss compared to the vehicle group by Day 3 of the study. Example 1 and Example 2 at 0.1 mg/kg both exhibited body weight loss equivalent to rhCNTF at 0.25 mg/kg. Animals receiving Example 1 at 0.25 μg/kg exhibited significantly greater body weight loss by Day 2 of the study compared to rhCNTF at 0.25 μg/kg. Animals receiving Example 1 at 0.25 μg/kg exhibited significantly greater body weight loss by Day 3 of the study compared to rhCNTF at 0.25 μg/kg. Animals in the 0.25 mg/kg dose groups of both Examples 1 and 2 were not dosed on Day 5 or 6 due to clinical signs of dehydration and/or reaching 20% body weight loss.

Example 26. In vivo activity (biochemical analysis of blood of healthy mice) of fusion molecules based on VHH from Examples 3 and 4.

- 37 048693

The study was conducted on 48 female C57BL6 mice obtained from Envigo, 6 weeks old. Animals were housed pairwise in microisolator cages with unlimited access to an automated tap water source (2-5 ^ppm chlorine) and standard chow (Teklad 2014 rodent diet). Mice were randomly assigned to 6 groups (n=8) based on body weight using a block random assignment tool developed by Lilly statisticians for in vivo random assignment. To reduce potential stress-related effects of the study, all animals were acclimated to daily handling for at least 3 days prior to study start. Body weight and food weight were recorded 2 days prior to study start and then monitored and recorded daily throughout the study. Animals were 8 weeks old at the time of first dose. The drugs were administered subcutaneously in the interscapular space every other day for 7 days for a total of 3 doses (once every other day dosing schedule) using a 28G 0.5 mL insulin syringe (BD Cat. #329461). Native NRG-1 (R&D systems, recombinant human EGF domain NRG1-beta 1, CF, Cat. #396HB/CF, Lot #ACD182101A), Example 3 and Example 4 were diluted to the appropriate concentrations in sterile PBS, pH 7.2, no later than 30 min prior to administration. The dose of NRG-1 was 1 mg/kg. The dose of Example 3 and Example 4 was 1 and 0.3 μg/kg. Dilutions for each drug were calculated daily based on the average body weight of the group on the previous day. Animals in the vehicle control group received 200 μl of PBS s.c. Administration was performed in the morning on study days 1, 3, and 5. All animals were anesthetized with isoflurane (4%) on the surgical table and exsanguinated by retro-orbital hemorrhage according to animal care and use protocols on study day 7. Blood was collected into serum separator tubes (BD, cat# 365967) and maintained at room temperature for up to 90 min. Blood was centrifuged for 10 min at 10,000 rpm. Serum was collected from each tube and aliquoted for the Chem 18 assay. Serum was analyzed on a Roche Cobas 8000 modular chemistry analyzer. All reagents for the Chem 18 assay were supplied by Roche except for the triglyceride assay, which used the Fujifilm WAKO reagent. JMP software for one-way confidence interval analysis by groups using Dunnett's method was used for statistical analysis of blood biochemical parameters.

- 38 048693

Table 23

Statistical analysis of the effect of different doses of VHH-based fusion molecules from examples 3 and 4 on blood biochemical parameters in healthy mice

Blood urea nitrogen creatinine cholesterol alkaline phosphatase

Compound Dose N mg/dl mg/dl mg/dl IU/l

Carrier n/a 8 mean value p- value 17.51 0.1025 79.125 173 11 11 Example 3 1 mg/kg 8 mean value p- value 16.63 0.08 98.375 128.75 0.99 0.0462 0.0004* 0.0001* Example 3 0.3 mg/kg 8 mean value p- value 17.22 0.08 85 156.625 0.8 0.0462 0.5397 0.1347 Example 4 1 mg/kg 8 mean value p- value 17.39 0.0675 173 81.25 1 0.0008* <0.0001* ^< 0.0001* Example 4 0.3 mg/kg 8 mean value p- value 14.7 0.0787 99.75 138.25 0.015* 0.0324* 0.0001* 0.0002* rhNRG-1 (native control) 1 mg/kg 8 mean value p- value 14.55 0.0775 79.375 157.5 0.01* 0.0224* 1 0.1695

*Indicates statistically significant difference from vehicle group.

As shown in Table 23, the VHH-based fusion molecules of Examples 3 and 4 exhibit significant effects on blood chemistry parameters compared to the vehicle control. Decreases in serum creatinine levels are observed in all drug-treated groups, with both dose levels of Example 4 and the native rhNRG1 control providing statistically significant decreases. Significant decreases in ALP from baseline are observed in animals receiving the native rhNRG1 control and the 1 mg/kg dose group of both Examples 3 and 4. Cholesterol levels are elevated for both dose levels of Example 3, the 1 mg/kg dose of Example 4, and the native rhNRG1 control group. The native rhNRG1 control group and the 0.3 mg/kg dose group of Example 4 also exhibited significant decreases in blood urea nitrogen.

Example 27. In vivo activity (weight loss in healthy mice) of the VHH-based fusion compound from Example 5.

Male DIO C57BL6 mice were obtained from Taconic at 12 weeks of age. Animals were housed individually in microisolator cages with ad libitum access to an automated tap water source (2-5 ^ppm chlorine) and high-fat diet (TD95217). Mice were randomly assigned to four groups (n=6) based on body weight using a block random assignment tool developed by Lilly statisticians for random assignment to groups in vivo. To reduce potential study-related confounding effects,

- 39 048693 stress, all animals were allowed to acclimate to daily handling for at least 3 days prior to study initiation. Body weight and food intake were recorded 2 days prior to study initiation and then monitored and recorded daily throughout the study. Five doses of each treatment were administered subcutaneously into the interscapular space throughout the study (once every 3 days dosing schedule) using a 28G, 0.5 ml insulin syringe (BD Cat. #329461). Native GDF15 (R&D systems, recombinant, Cat. #957-GD/CF; Lot #EHF2321O1A) and the VHH-based fusion protein from Example 5 were diluted to appropriate concentrations in sterile PBS, pH 7.2, no later than 30 min prior to administration. Native GDF-15 was administered at 0.1 μg/kg and the VHH-based fusion protein from Example 5 was administered at either 1 μg/kg or 0.1 μg/kg depending on the average body weight of the group the previous day. Animals in the vehicle control group received 200 μl of PBS. Body weight changes were analyzed using an in-house statistical tool and a rigorous model-based approach developed by Lilly statisticians for in vivo PD studies.

Table 24

Effect of different doses of the VHH-based fusion molecule from Example 5 on body weight in healthy mice at different time points

Connection Dose N % change in body weight on different study days 0 3 6 9 12 Carrier n/a 6 100.3 101.9 100.2 102.3 104.4 Example 5 0,1 6 100.9 95.8 90.7 89.2 86.2 Example 5 1 6 101.9 95.6 91.3 87.7 85.5 Native GDF-15 0,1 6 99.7 99.2 97.1 95.7 94.8

Table 25

Statistical analysis of the effect of different doses of the VHH-based fusion molecule from Example 5 on body weight in healthy mice at different time points______________

Connection Dose Day of the study 0 3 6 9 12 Example 5 0,1 Magnitude of influence P-value 0.583 -6.02 -9.48 -13.08 -18.15 0.971 6.88E-04 <0.001 <0.001 <0.001 Example 5 1 Magnitude of influence P-value 1.657 -6.247 -8.862 -14.644 -18.825 0.616 4.53E-04 <0.001 <0.001 <0.001 Native GDF-15 0,1 Magnitude of influence P-value -0.631 -2.673 -3.030 -6.611 -9.589 0.963 0.242 0.158 1.85E-04 <0.001

P-values are compared to vehicle-treated controls.

As shown in Tables 24 and 25, significant body weight loss occurs in both groups receiving either 0.1 μg/kg or 1 μg/kg of the VHH fusion protein of Example 5 on day 3 compared to the vehicle control. The body weight loss in the native GDF-15 group reaches significance by day 9. There is no difference in body weight loss at any time point between the 1 and 0.1 mg/kg of the VHH fusion protein of Example 5 groups. Animals receiving either 1 mg/kg or 0.1 mg/kg of the VHH fusion protein of Example 5 lost significantly more body weight by day 6 of the study compared to animals receiving native GDF15. This significance persists throughout the remainder of the study.

Example 28. In vivo activity (corticosterone induction and body weight loss in healthy mice) of the VHH-based conjugate from Example 15.

Healthy 6-week-old female C57BL6 mice were purchased from Envigo and housed in groups (3 mice per cage) in micro-isolator cages with filtered bottled tap water and fed a standard chow diet (2014 Teklad global rodent maintenance diet). Mice were allowed to acclimate after receipt for at least 72 h. Mice were weighed 1 day before

- 40 048693 dosing. A single subcutaneous dose of the VHH fusion conjugate from Example 15 at 0.1, 0.3, 1, or 3 nmol/kg or vehicle (DPBS) was administered on day 1; n=3 per group. A tail nick was used to collect dried spot samples (DBS) for corticosterone analysis immediately before dosing ( _T0 ) and at 2, 6, 24, 30, 48, and 72 h post-dose. Approximately 20-30 μL of tail blood from each animal was collected onto Whatman DMPK cards (WB129243). DBS cards were provided to staff LC-MS technicians for corticosterone analysis. Animal body weights were also recorded daily throughout the study. All in vivo experimental procedures were performed according to IACUC standards and in accordance with an approved animal use protocol (19-033). Data are presented in Tables 26 and 27 as mean corticosterone levels (ng/mL) or mean percent change from day 0 body weight, respectively.

Table 26

Effect of different doses of the VHH-based conjugate from Example 15 on corticosterone levels in healthy mice at different time points

Hours after dose administration: Dose N 0 2 6 24 30 48 72 0.1 nmol/kg 3 Average value 8.53 331,44 542,62 52,60 32.77 16.36 7.54 SEM 0.65 48.23 43.88 33.65 1.43 4.00 2.73 Meaning 0.17 0,018 0,007 0.27 0,032 OD 0,002 0.3 nmol/kg 3 Average value 44.71 788,87 1278,7 132.09 53.99 5.86 18.96 SEM 11.96 125.20 120,20 96.47 11.99 0.18 1.83 Meaning 0.49 0,006 0,0013 0.974 0,053 0,078 0,015 1 nmol/kg 3 Average value 25,16 1027.8 1315.4 1003.8 275.68 8.02 8.28 SEM 15.44 52,55 109.56 297.4 151,60 3.08 0.16 Meaning o,z <,0001 0,0008 0,045 0.63 0,098 0,0006 3 nmol/kg 3 Average value 52.85 1071.9 1315,5 1746.4 1748,0 260.69 10.24 SEM 19.44 63,01 15,20 82.73 92.80 187.38 2.95 Meaning 0.62 0,0001 < ,0001 <,0001 0,0001 0.302 0,005 Carrier 3 Average value 77.11 126.99 321,41 135.83 192.73 38,37 30.81 SEM 41,11 21.57 2.44 55.38 49.68 13.82 2.30

P-values were calculated to determine significance using unpaired t-tests of drug versus vehicle groups at individual time points in GraphPad PRISM software.

As shown in Table 26, administration of the VHH-based conjugate of Example 15 results in a significant induction of corticosterone levels compared to vehicle control at 2 and 6 h post-dose for all dose levels. In animals in the 3 μg/kg groups, the increase in corticosterone levels remains significantly greater than in the vehicle control group for 30 h post-dose.

- 41 048693

Table 27

Effect of different doses of the VHH-based conjugate from Example 15 on body weight in healthy mice 72 h after injection

Group receiving the drug Dose (nmol/kg) N Average %MT SEM Meaning Carrier n/a 3 106.7 1,125 1 Example 15 OD 3 105.1 0.563 0.2846 Example 15 OD 3 102.1 2,183 0,1346 Example 15 1 3 97.9 0,671 0,0026 Example 15 3 3 100,1 1,385 0,0211

As shown in Table 27, administration of the VHH-based conjugate of Example 15 prevented body weight gain in both the 1 nmol/kg and 3 nmol/kg drug groups over the course of the study. The difference in percent body weight gain was significant for the two drug groups at day 3 compared to the vehicle control. P-values were calculated using an unpaired t-test of drug versus vehicle groups in GraphPad PRISM software.

In vivo function - FC.

Example 29. PK of fusion molecules based on VHH from Example 4 in male Sprague-Dawley rats.

Male Sprague-Dawley rats were administered a single intravenous (i.v.) or subcutaneous (s.c.) dose of 106.8 nmol/kg of Example 4 incorporated in PBS buffer (pH 7.4) at a dose volume of 2.6 mL/kg. Blood for PK characterization was collected at 1.6, 12, 24, 48, 96, 144, 168, and 240 h post-dose for i.v. administration; and at 6, 12, 24, 48, 96, 144, 168, and 240 h post-dose for s.c. administration.

Plasma concentrations of the VHH fusion molecules from Example 4 were determined by qualified LC/MS analysis at Altascience (Laval, Quebec, Canada) using a Q/Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The VHH fusion molecules from Example 4 and the internal standard were isolated from rat plasma in K3EDTA by immunoprecipitation with an anti-VHH antibody-biotin conjugate and streptavidin-coated magnetic beads. Following wash steps to remove interfering endogenous proteins, the isolated VHH fusion molecules from Example 4 were reduced, alkylated, and digested with trypsin, and the tryptic peptides were analyzed by LC/MS as a surrogate for intact fusion molecules. Plasma concentrations of the VHH-based fusion molecules from Example 4 were used to calculate the PK parameters given in Table 28.

Table 28

Mean plasma pharmacokinetic parameters for the VHH-based fusion molecules from Example 4 following single i.v. or s.c. dose administration to male Sprague-Dawley rats

Connection Route of administration Dose (nmol/kg) Tshchah (h) Co (i.v.) Stach (s.c.) (nmol/l) AUCo-inf (h*nmol/l) CL (IV) CL/F (SC) (ml/h/kg) 1½ (h) Example 4 in/in 106.8 nd 2310 (461) 4.40E4 (5.27E3) 2.45 (0.274) 30 (1,3) p/k 106.8 24 (0) 127 (27) 6.83EZ (1.51EZ) 16.2 (3.50) 38 (3.2)

Note: Abbreviations: Δ/ ₂ = half-life, T _max = time to reach maximum concentration, C max = maximum observed plasma concentration, C ₀ = plasma concentration extrapolated to time 0, AUC _0-H11 = area under the curve from time 0 h to infinity, CL/B = clearance/bioavailability. Standard deviation values are included in the table below the mean values of PK parameters in brackets. (^=3/group).

As shown in Table 28, the VHH-based fusion molecule from Example 4 exhibits an enhanced PK profile in Sprague-Dawley rats compared to non-fusion proteins (data not shown).

Example 30. PK of the VHH-based fusion molecules from Examples 5, 6 and 7 in male Sprague-Dawley rats.

Male Sprague-Dawley rats were given a single intravenous (i.v.) or subcutaneous (s.c.) dose of 50 nmol/kg for Example 5, 25 nmol/kg for Example 6 or 7, included in buffer

- 42 048693

PBS (pH 7.4), at a dose volume of 4 ml/kg. Blood for PK characterization was collected at 1.6, 12, 24, 48, 96, 144, 168, and 240 h post-dose for i.v. administration; and at 6, 12, 24, 48, 96, 144, 168, and 240 h post-dose for s.c. administration.

Plasma concentrations of the VHH fusion molecules of Examples 5-7 were determined by qualified LC/MS analysis at Covance Laboratories (Greenfield, IN) using a Q/Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The VHH fusion molecules of Examples 5-7 and the internal standard were isolated from rat plasma in EDTA by immunoprecipitation with an anti-VHH antibody-biotin conjugate and streptavidin-coated magnetic beads. Following wash steps to remove interfering endogenous proteins, the isolated VHH fusion molecules of Examples 5-7 were reduced, alkylated, and digested with trypsin, and the tryptic peptides were analyzed by LC/MS as a surrogate for intact fusion molecules. Plasma concentrations of the VHH-based fusion molecules from Examples 5–7 were used to calculate the PK parameters presented in Table 29.

Table 29

Mean plasma pharmacokinetic parameters for the VHH-based fusion molecules of Examples 5, 6 ______and 7 following single IV or SC dose administration to male Sprague-Dawley rats______

Connection Route of administration Dose (nmol/kg) Tshchah (h) Co (i.v.) Stach (s.c.) (nmol/l) AUCo-inf (h*nmol/l) CL (IV) CL/F (SC) (ml/h/kg) 1½ (h) Example 5 in/in 50 nd 1480 (231) 1.48E5* (7.08E3) 0.438* (0.0290) 106* (12.9) p/k 50 64 (28) 269 (56) 8.77E4* (1.89E4) 1.14* (0.160) 206* (102) Example 6 in/in 25 nd 383 (42.4) 8390 (892) 3.00 (0.317) 15.7 (2.35) p/k 25 32 (14) 57.8 (12.0) 3240 (746) 7.96 (1.62) 12.6 (0.968) Example 7 in/in 25 ND 309 (29.2) 5830 (180) 4.30 (0.134) 13.9 (4.78) p/k 25 24 (0) 31.6 (16.9) 1520 (755) 20.2 (P.6) 13.2 (3.19)

Comments: The study duration is too short to fully characterize the elimination phase of the PK profile. Data represent mean values (standard deviation in parentheses): for Example 5, the values are N=3 (IV and SC, 1 to 240 h); for Example 6, the IV values are N=3 (1 to 168 h) and N=1 (240 h), and the SC values are N=3 (6 to 144 h); for Example 7, the IV values are N=3 (1 to 96 h), N=2 (144 h), and N=1 (168, 240 h); and the SC values are N=3 (6 to 96 h), N=2 (144 h), and N=1 (168 h).

Note: Abbreviations: C/ ₂ = half-life, ^^time to peak concentration, Cmax = maximum observed plasma concentration, _C0 = plasma concentration extrapolated to time 0, AUC0 _-inf = area under the curve from time 0 h to infinity, OU/P^clearance/bioavailability. As shown in Table 29, the VHH-based fusion molecules of Examples 5, 6, and 7 exhibit an enhanced PK profile in Sprague-Dawley rats compared to the unfused proteins (data not shown).

Example 31. PK of the Fab-VHH fusion molecule from Example 9 and the corresponding unfused Fab (Example 13) in male Sprague-Dawley rats.

Male Sprague-Dawley rats were administered a single intravenous (i.v.) or subcutaneous (s.c.) dose of 63.4 nmol/kg for Example 9, 78.9 nmol/kg for Example 13, incorporated in PBS buffer (pH 7.4), at a dose volume of 4 ml/kg. Blood for PK characterization was collected at 1.6, 12, 24, 48, 96, 144, 168, and 240 h after dosing for i.v. administration; and at 6, 12, 24, 48, 96, 144, 168, and 240 h after dosing for s.c. administration. Plasma concentrations of the Fab-VHH fusion molecule from Example 9 and the corresponding unfused Fab from Example 13 were determined by qualified LC/MS analysis at Altascience (Laval, Quebec, Canada) using a Q/Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA). The Fab-VHH fusion molecule from Example 9 and the Fab from Example 13 and the internal standard were isolated from rat plasma in EDTA by immunoprecipitation with anti-kappa light chain conjugate.

- 43 048693 human-biotin and streptavidin-coated magnetic beads or an anti-VHH antibody-biotin conjugate and streptavidin-coated magnetic beads. After wash steps to remove interfering endogenous proteins, the isolated proteins from Examples 9 and 13 were reduced, alkylated, and digested with trypsin, and the tryptic peptides were analyzed by LC/MS as a surrogate for intact fusion molecules. The plasma concentrations of Fab-VHH from Example 9 (Table 30) were used to calculate the PK parameters shown in Table 31. The plasma concentrations of the unfused Fabs from Example 13 were too low to calculate the PK parameters.

Table 30

Mean plasma concentration (expressed in nmol/L) of the Fab-VHH fusion molecule from Example 9 and the corresponding unfused Fab (Example 13) after a single i.v. or s.c. dose in male Sprague-Dawley rats

Connection Route of administration Time (hours) 1 6 12 24 48 96 144 168 240 Example 9 in/in 1909.8 (35.4) 1378.2 (93.2) 999.7 (19.7) 797.8 (40.3) 515.3 (44.0) 257.4 (10.6) 133.1 (17.4) 96.9 (14.2) 34.6 (5.0) p/k nd 28.1 (7.1) 79.2 (26.5) 159.6 (30.0) 229.8 (16.2) 183.1 (16.4) 103.0 (13.4) 85.0 (8.3) 46.0 (20.9) Example 13 in/in 75.7 (7.1) 18.8 (ID) 6.9 (4.2) 2.7 (N/R) Below PKO Below PKO Below PKO Below PKO Below PKO p/k ND 12.1 (1.2) 5.6 (1.2) Below PKO Below PKO Below PKO Below PKO Below PKO Below PKO

Standard deviation values are included in the table below the mean concentration values in brackets. ^=3/group.

As shown in Table 30, the fused Fab-VHH molecule from Example 9 exhibited significantly higher mean plasma concentrations in Sprague-Dawley rats compared to the corresponding unfused Fab (Example 13).

Table 31

Mean pharmacokinetic parameters of the Fab-VHH fusion molecule from Example 9 after a single i.v. or s.c. dose in male Sprague-Dawley rats

Connection Route of administration Dose (nmol/kg) Tshchah (h) Co (i.v.) Stach (s.c.) (nmol/l) AUCo-mf (h*nmol/l) CL (IV) CL/F (SC) (ml/h/kg) 1½ (h) Example 9 in/in 78.9 ND 2039 (23) 8D7E4 (3.16E3) 0.966 (0.037) 49.4 (1.7) p/k 78.9 48 (0) 230 (16) 3.56E4 (6.24E3) 2,259 (0,361) 73.8 (20.9)

Note: Abbreviations: ^/2=half-life, ^^time to reach maximum concentration, Cmax maximum observed plasma concentration, C ₀ =plasma concentration extrapolated to time 0, AUC _0-in f=area under the curve from time 0 h to infinity, CL/F=clearance/bioavailability.

Standard deviation values are included in the table below the mean values of the PK parameters in brackets. ^=3/group.

As shown in Table 31, the Fab-VHH fusion molecule from 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 calculation of PK parameters is not possible.

- 44 048693

Sequences

The description of the present invention contains references to the following nucleotide and/or amino acid sequences, which are provided below for information.

SEQ ID NO: 1 - VHH fragment 1 (MC6.1C22)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY

YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:2 - fragment VHH 2 (MC6.1C80)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:3 - fragment VHH 3 (MC6.1C22.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ ID NO:4 - VHH fragment 4 (MC6.1C80.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ ID NO:5 - VHH fragment 5 (MC6)

- 45 048693

EVQLVESGGGLVQAGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFTAGIGGSVDITY YLDSVKGRFTISKDNTKNTVYLQMNSLKPEDTAVYYCAVRPGRPLITSRDANLYDYWG QGTQVTVSS

SEQ ID NO:6 - fragment VHH 6 (MC6.1)

EVQLLESGGGLVQPGGSLRLSCAASGRTVSSTAVAWFRQAPGKEREFVAGIGGSVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVRPGRPLITSRDANLYDYWG QGTLVTVSS

SEQ ID NO:7 - fragment VHH 7 (MC6.1C6)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDSTAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSRVANLYPYWG QGTLVTVSS

SEQ ID NO:8 - VHH fragment 8 (MC6.1C22-G26Y)

EVQLLESGGGLVQPGGSLRLSCAASYRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:9 - fragment VHH 9 (MC6.1C22-R27A)

EVQLLESGGGLVQPGGSLRLSCAASGAYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 10 - VHH fragment 10 (MC6.1C22-I57E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDET YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSS

SEQ ID NO: I - fragment VHH 11 (MC6.1C22-I57Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDQT YYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYW GQGTLVTVSS

- 46 048693

SEQ ID NO: 12 - fragment VHH 12 (MC6.1C22-Y59A)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITA YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 13 - fragment VHH 13 (MC6.1C22-Y59E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITE YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 14 - VHH fragment 14 (MC6.1C22-Y59Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITQ YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ IDNO:15- fragment VHH 15 (MC6.1C22-Y59S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITS YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 16 - fragment VHH 16 (MC6.1C22-Y59T)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITT YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 17 - fragment VHH 17 (MC6.1C22-R102K)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGKPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO: 18 - fragment VHH 18 (MC6.1C22-R102Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGQPLITSKVADLYPYWG QGTLVTVSS

- 47 048693

SEQ ID NO: 19 - fragment VHH 19 (MC6.1C22-R102S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGSPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:20 - fragment VHH 20 (MC6.1C22-P103)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRELITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:21 - fragment VHH 21 (MC6.1C22-P103Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRQLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:22 - fragment VHH 22 (MC6.1C22-P103S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRSLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:23 - fragment VHH 23 (MC6.1C22-L104E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPEITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:24 - fragment VHH 24 (MC6.1C22-L104G)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPGITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:25 - fragment VHH 25 (MC6.1C22-L104Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPQITSKVADLYPYWG QGTLVTVSS

- 48 048693

SEQ ID NO:26 - fragment VHH 26 (MC6.1C22-L104T)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPTITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:27 - fragment VHH 27 (MC6.1C22-S107E)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITEKVADLYPYWG QGTLVTVSS

SEQ Ш NO:28 - fragment VHH 28 (MC6.1C80-A98T)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRPGRPLITSKVADLYPYWG QGTLVTVSS

SEQ ID NO:29 - fragment VHH 29 (MC6.1C80-P100Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARQGRPLITSKVADLYPYWG QGTLVTVSS

SEQ Ш NO:30 - fragment VHH 30 (MC6.1C80-A98T, P100Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCATRQGRPLITSKVADLYPYWG QGTLVTVSS

SEQ IDNO:31 - fragment VHH 31 (MC6.1C80-K108Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSQVADLYPYWG QGTLVTVSS

SEQ ID NO:32 - fragment VHH 32 (MC6.1C80-V109Q)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQADLYPYWG QGTLVTVSS

- 49 048693

SEQ Ш NO:33 - fragment VHH 33 (MC6.1C80-D11 IE)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVAELYPYWG QGTLVTVSS

SEQ Ш NO:34 - fragment VHH 34 (MC6.1C80-D111S)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVASLYPYWGQ GTLVTVSS

SEQ ID NO:35 - fragment VHH 35 (MC6.1C80-V109Q, DI HE)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQAELYPYWG QGTLVTVSS

SEQ ID NO:36 - fragment VHH 36 (MC6.1C80-V109Q, Dll IS)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKQASLYPYWGQ GTLVTVSS

SEQ ID NO:37 - fragment VHH 37 (MC6.1C80Cys)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC

SEQ ID NO:38 - Li (base sequence (GGGGQ) _n ) GGGGQ

SEQ ID NO:39 - Li (base sequence (GGGQ) _n ) GGGQ

SEQ ID NO:40 - Li (base sequence (GGGGS) _n ) GGGGS

SEQ ID NO:41 - Li (main sequence (PGPQ) _n )

- 50 048693

PGPQ

SEQ ID NO:42 - Li (main sequence (PGPA) _n )

PGPA

SEQ ID NO:43 - Li (base sequence GGGG(AP)nGGGG) GGGG-AP-GGGG

SEQ ID NO:44 - Li (main sequence (GGE) _n ) GGE

SEQ ID NO:45 - Li (main sequence (GGGGE) _n ) GGGGE

SEQ ID NO:46 - Li (main sequence (GGK) _n ) GGK

SEQ ID NO:47 - Li (base sequence (GGGGK) _n ) GGGGK

SEQ ID NO:48 - Li (base sequence GGGG(EP) _n GGGG) GGGG-EP-GGGG

SEQ ID NO:49 - Li (base sequence GGGG(KP) _n GGGG) GGGG-KP-GGGG

SEQ ID NO:50 - Li (primary sequence (PGPE) _n )

PGPE

SEQ ID NO:51 - Li (primary sequence (PGPK) _n )

PGPK

SEQ ID NO:52 - Li 1 (GGGGQ)s

GGGGQGGGGGQGGGGGQGGGGQGGGGQ

- 51 048693

SEQ ID NO:53 - Li 2 (PGPQ)s

PGPQPGPQPGPQPGPQPGPQPGPQPGPQPGPQ

SEQ ID NO:54 - Li 3 (PGPA) ₈

PGPAPGPAPGPAPGPAPGPAPGPAPGPAPGPA

SEQ Ш NO:55 - Li 4 ((GGE) ₈ )

GGEGGEGGEGGEGGEGGEGGEGGE

SEQ Ш NO:56 - Li 5 ((GGGGE)s)

GGGGEGGGGGGGGGGGGGGGGGE

SEQ Ш NO:57 - Li 6 ((GGK) ₈ )

GGKGGKGGKGGKGGKGGKGGKGGK

SEQ ID NO:58 - Li 7 ((GGGGK)s)

GGGGKGGGGGKGGGGKGGGGKGGGGK

SEQ ID NO:59 - Li 8 ((GGGG(AP)ioGGGG))

GGGGAPAPAPAPAPAPAPAPAPAPAPGGGG

SEQ ID NO:60 - Li 9 ((GGGG(EP)ioGGGG))

GGGGEPEPEPEPEPEPEPEPEPEPEPGGGG

SEQ Ш N0:61 - Li 10 ((GGGG(KP)ioGGGG))

GGGGKPKPKPKPKPKPKPKPKPKPKPGGGG

SEQ ID NO:62 - Li 11 ((PGPE) ₈ )

PGPEPGPEPGPEPGPEPGPEPGPEPGPEPGPE

SEQ ID NO:63 - Li 12 ((PGPK) ₈ )

PGPKPGPKPGPKPGPKPGPKPGPKPGPKPGPK

SEQ ID NO:64- _L21

GGGSGGSGGG

- 52 048693

SEQ NO:65 - L ₂ 2

GGGSGGSGGSGGG

SEQ ID NO:66 - signal peptide

METDTLLLWVLLLLWVPGSTG

SEQ ID NO:67 - human proGIP

MVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISD YSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSS PA KNPSDEDLLR DLLIQELLACLLDQTNLCRLRSR

SEQ ID NO:68 - Human GIP

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ

SEQ ID NO:69 - Human GIP receptor

RAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDMYVCWDYAAPNA TARASCPWYLPWHHHVAAGFVLRQCGSDGQWGLWRDHTQCENPEKNEAFLDQRLILE RLQVM YTVGYSLSLATLLL ALLIL SLFRRLHCTRNYIHINLFTSFMLRAAAILSRDRLLPR PGPYLGDQALALWNQALAACRTAQIVTQYCVGANYTWLLVEGVYLHSLLVLVGGSEE GHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILMTILINFLIF IRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLGVHEWFAPVTEEQARGALRFAKL GFEIFLSSFQGFLVSVLYCFINKEVQSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSGS GPGEVPTSRGLSSGTLPGPGNEASRELESYC

SEQ ID NO:70 - human proglucagon

MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTF TSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAA KEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKI TDRK

SEQ ID NO:71 - Human GLP-1

HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO:72 - Human GLP-17-37

- 53 048693

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

SEQ ID NO:73 - Human GLP-I7-36 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

SEQ ID NO:74 - Human GLP-1 receptor

RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFV NVSCPWYLPWAS S VPQGHVYRFCT AEGLWLQKDNS SLPWRDLSECEESKRGERS SPEE QLLFLYIIYTVGYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAAL KWMYSTAAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAF SVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAI GVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGT LRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKC PTS SLS SGATAGS SMYTATCQ ASC S

SEQ ID NO:75 - human proGDF15

MPGQELRTVNGSQMLLVLLVLSWLPHGGALLSLAEASRASFPGPSELHSEDSRFRELRKR YEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASR LHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQL ELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQV TMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

SEQ ID NO:76 - Human GDF15

ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

SEQ ID NO:77 - Human GDF15 (GFRAL) receptor

MIVFIFLAMGLSLENEYTSQTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPC KMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWN LTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRF FYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELC RRHYRTFQSI<CWQRVTRI<CHEDENCISTLSI<QDLTCSGSDDCI<AAYIDILGTVLQVQCT CRTITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEV IYAAMCMT VTCGILLLVMVKLRTSRIS SKARDPS SIQIPGEL

- 54 048693

SEQ ID NO:78 - human proINS

FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQKRGIVEQCCTSIC SLYQLENYCN

SEQ ID NO:79 - human INS A chain GIVEQCCTSICSLYQLENYCN

SEQ ID NO:80 - human INS chain B FVNQHLCGSHLVEALYLVCGERGFFYTPKT

SEQ ID NO:81 - Human INS receptor α-subunit

HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYL LLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEK NNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVE RCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCV ETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSS NLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANL GLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNL TITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIR TSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPL RSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSV PLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTW SPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRKT SSGTGAEDPRPS

SEQ ID NO:82 - Human INS receptor β-subunit

SLGDVGNVTVAVPTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQ ACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLI VLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPT YFYVTDYLDVPSNIAKIIIGPLIFVFLFSVVIGSIYLFLRKRQPDGPLGPLYASSNPEYLSAS DVFPCSVYVPDEWEVSREKITLLRELGQGSFGMVYEGNARDIIKGEAETRVAVKTVNES ASLRERIEFLNEASVMKGFTCHHVVRLLGVVSKGQPTLVVMELMAHGDLKSYLRSLRP EAENNPGRPPPTLQEMIQMAAEIADGMAYLNAKKFVHRDLAARNCMVAHDFTVKIGD FGMTRDIYETDYYRKGGKGLLPVRWMAPESLKDGVFTTSSDMWSFGVVLWEITSLAEQ

- 55 048693

PYQGLSNEQVLKFVMDGGYLDQPDNCPERVTDLMRMCWQFNPKMRPTFLEIVNLLKD DLHPSFPEVSFFHSEENKAPESEELEMEFEDMENVPLDRSSHCQREEAGGRDGGSSLGFK RSYEEHIPYTHMNGGKKNGRILTLPRSNPS

SEQ ID NO:83 - Human IL-2

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT

SEQ ID NO:84 - Human IL-2 receptor alpha subunit

ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSL YMLCTGNS SHS SWDNQ CQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATER IYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLL SGLTWQRRQRKSRRTI

SEQ Ш NO:85 - β-subunit of the human IL-2 receptor AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQAS WACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVH VETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDT QYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILV YLLINCRNTGPWLKK VLKCNTPDP SKFF SQL S SEHGGD VQKWLS SPFP S S SF SPGGLAPEI SPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYF TYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPST APGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPD AGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

SEQ ID NO:86 - Human IL-2 receptor γ-subunit

LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQP TNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQ ATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWT EQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFL FALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGL AESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

- 56 048693

SEQ ID NO:87 - human proNRGl

MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPRLKEMKSQESAAGSKLVLRCE TSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKL GNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVSTEGANTSSSTSTSTTGTSHLVK WS NGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYR DSPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEER PLLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEP VKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAA SLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV

SEQ ID NO:88 - Human NRG1

SGKKPESAAGSQSPALPPRLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELN RKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGM PASTEGAYVSSESPIRISVSTEGANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVK DLSNPSRYLCKCQPGFTGARCTENVPMKVQNQEKAEELYQK

SEQ ID NO:89 - Human NRG1 receptor EbBS

MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCE WMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKF AIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEI WKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECA GGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASC PHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNI DGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHN FSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLN WTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGG VCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHC VSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLT MALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLA RIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHML AIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAK GMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKW

- 57 048693

MALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQI CTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHG LTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGY MPMNQGNLGESCQES AVSGS SERCPRP VSLHPMPRGCL ASES SEGHVTGSEAELQEKVS MCSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSR EGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSA SLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEE MRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT

SEQ ID NO:90 - Human ErbB4 NRG1 receptor

MKPATGLWVWVSLLVAAGTVQPSDSQSVCAGTENKLSSLSDLEQYRALRKYYENCE WMGNLEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYLPLENLRIIRGTKLYEDRYAL AIFLNYRKDGNFGLQELGLKNLTEILNGGVYVDQNKFLCYADTIHWQDIVRNPWPSNLT LVSTNGSSGCGRCHKSCTGRCWGPTENHCQTLTRTVCAEQCDGRCYGPYVSDCCHREC AGGCSGPKDTDCFACMNFNDSGACVTQCPQTFVYNPTTFQLEHNFNAKYTYGAFCVK KCPHNFVVDSSSCVRACPSSKMEVEENGIKMCKPCTDICPKACDGIGTGSLMSAQTVDS SNIDKFINCTKINGNLIFLVTGIHGDPYNAIEAIDPEKLNVFRTVREITGFLNIQSWPPNMT DFSVFSNLVTIGGRVLYSGLSLLILKQQGITSLQFQSLKEISAGNIYITDNSNLCYYHTINW TTLFSTINQRIVIRDNRKAENCTAEGMVCNHLCSSDGCWGPGPDQCLSCRRFSRGRICIES CNLYDGEFREFENGSICVECDPQCEKMEDGLLTCHGPGPDNCTKCSHFKDGPNCVEKCP DGLQGANSFIFKYADPDRECHPCHPNCTQGCNGPTSHDCIYYPWTGHSTLPQHARTPLI AAGVIGGLFILVIVGLTFAVYVRRKSIKKKRALRRFLETELVEPLTPSGTAPNQAQLRILK ETELKRVKVLGSGAFGTVYKGIWVPEGETVKIPVAIKILNETTGPKANVEFMDEALIMAS MDHPHLVRLLGVCLSPTIQLVTQLMPHGCLLEYVHEHKDNIGSQLLLNWCVQIAKGMM YLEERRLVHRDLAARNVLVKSPNHVKITDFGLARLLEGDEKEYNADGGKMPIKWMAL ECIHYRI<FTHQSDVWSYGVTIWELMTFGGI<PYDGIPTREIPDLLEI<GERLPQPPICTIDVY MVMVI<CWMIDADSRPI<FI<ELAAEFSRMARDPQRYLVIQGDDRMI<LPSPNDSI<FFQNL LDEEDLEDMMDAEEYLVPQAFNIPPPIYTSRARIDSNRSEIGHSPPPAYTPMSGNQFVYR DGGFAAEQGVSVPYRAPTSTIPEAPVAQGATAEIFDDSCCNGTLRKPVAPHVQEDSSTQ RYSADPTVFAPERSPRGELDEEGYMTPMRDKPKQEYLNPVEENPFVSRRKNGDLQALD NPEYHNASNGPPKAEDEYVNEPLYLNTFANTLGKAEYLKNNILSMPEKAKKAFDNPDY WNHSLPPRSTLQHPDYLQEYSTKYFYKQNGRIRPIVAENPEYLSEFSLKPGTVLPPPPYR HRNTVV

SEQ ID NO:91 - human proRLN2

- 58 048693

MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQ EDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVL KDSSLLFEEFKKLIRNRQSEAADSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTK RSLARFC

SEQ ID NO:92 - human RLN2 A chain QLYSALANKCCHVGCTKRSLARFC

SEQ ID NO:93 - human RLN2 chain B

DSWMEEVIKLCGRELVRAQIAICGMSTWS

SEQ ID NO:94 - Human RXFP1 receptor

MTSGSVFFYILIFGKYFSHGGGQDVKCSLGYFPCGNITKCLPQLLHCNGVDDCGNQADE DNCGDNNGWSLQFDKYFASYYKMTSQYPFEAETPECLVGSVPVQCLCQGLELDCDETN LRAVPSVSSNVTAMSLQWNLIRKLPPDCFKNYHDLQKLYLQNNKITSISIYAFRGLNSLT KLYLSHNRITFLKPGVFEDLHRLEWLIIEDNHLSRISPPTFYGLNSLILLVLMNNVLTRLPD I<PLCQHMPRLHWLDLEGNHIHNLRNLTFISCSNLTVLVMRI<NI<INHLNENTFAPLQI<LD ELDLGSNKTENLPPLIFKDLKELSQLNLSYNPIQKIQANQFDYLVKLKSLSLEGIEISNIQQ RMFRPLMNLSHIYFKKFQYCGYAPHVRSCKPNTDGISSLENLLASIIQRVFVWVVSAVTC FGNIFVICMRPYIRSENKLYAMSIISLCCADCLMGIYLFVIGGFDLKFRGEYNKHAQLWM ESTHCQLVGSLAILSTEVSVLLLTFLTLEKYICIVYPFRCVRPGKCRTITVLILIWITGFIVA FIPLSNKEFFKNYYGTNGVCFPLHSEDTESIGAQIYSVAIFLGINLAAFIIIVFSYGSMFYSV HQSAITATEIRNQVKKEMILAKRFFFIVFTDALCWIPIFVVKFLSLLQVEIPGTITSWVVIFI LPINSALNPILYTLTTRPFI<EMIHRFWYNYRQRI<SMDSI<GQI<TYAPSFIWVEMWPLQEM PPELMKPDLFTYPCEMSLISQSTRLNSYS

SEQ ID NO:95 - Human ACTH

SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF

SEQ ID NO:96 - human ACTH receptor (ACTHR, MC2R)

MKHIINSYENINNTARNNNSDCPRVVLPEEIFFTISIVGVLENLIVLLAVFKNKNLQAPMYF FICSLAISDMLGSLYKILENILILRNMGYLKPRGSFETTADDIIDSLFVLSLLGSIFSLSVIA ADRYITIFHALRYHSIVTMRRTVVVLTVIWTFCTGTGITMVIFSHHVPTVITFTSLFPLML VFILCLYVHMFLLARSHTRKISTLPRANMKGAITLTILLGVFIFCWAPFVLHVLLMTFCPS NPYCACYMSLFQVNGMLIMCNAVIDPFIYAFRSPELRDAFKKMIFCSRYW

- 59 048693

SEQ ID NO:97 - Human CNTF

MAFTEHSPLTPHRRDLCSRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVA STDQWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAA FAYOIEELMILLEYI<IPRNEADGMPINVGDGGLFEI<I<LWGLI<VLOELSOWTVRSIHDLR FIS SHQTGIP ARGSHYIANNKKM

SEQ ID NO:98 - Human CNTF receptor alpha

QRHSPQEAPHVQYERLGSDVTLPCGTANWDAAVTWRVNGTDLAPDLLNGSQLVLHGL ELGHSGLYACFHRDSWHLRHQVLLHVGLPPREPVLSCRSNTYPKGFYCSWHLPTPTYIP NTFNVTVLHGSKIMVCEKDPALKNRCHIRYMHLFSTIKYKVSISVSNALGHNATAITFDE FTIVKPDPPENVVARPVPSNPRRLEVTWQTPSTWPDPESFPLKFFLRYRPLILDQWQHVE LSDGTAHTITDAYAGKEYIIQVAAKDNEIGTWSDWSVAAHATPWTEEPRHLTTEAQAA ETTTSTTSSLAPPPTTKICDPGELGS

SEQ ID NO:99 - GLP1 standard

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVF SCS VMHEALHNHYTQKSLSL SLG

SEQ ID NO: 100 - VHH Fusion Molecule 1 (CNTF-(G ₄ Q) ₅ -MC6.1C22.43) MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVA STDRWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAA FAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLR FISSHQTGGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAA SGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTLYLQM NSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

SEQ ID NO: 101 - VHH Fusion Molecule 2 (MC6.1C22-(G ₄ Q)s-CNTF) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQMAFTEHSPLTPHRRDLASRSIWL ARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDRWSELTEAERLQENLQAYR

- 60 048693

TFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADG MPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTG

SEQ Ш NO: 102 - VHH-based fusion molecule 3 (NRG1-(G4Q)s-MC6.1C22.43) SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHL GIEFMEAEELYQGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRL SCAASGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

SEQ ID NO: 103 - VHH-based fusion molecule 4 (MC6.1C22-(G4Q)s-NRG1) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQSHLVKCAEKEKTFCVNGGECFM VKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKHLGIEFMEAEELYQ

SEQ ID NO:104 - VHH Fusion Molecule 5 (MC6.1C22-(G4Q)s-GDF15) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQGDHCPLGPGRCCRLHTVRASLE DLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASY NPMVLIQKTDTGVSLQTYDDLLAKDCHCI

SEQ ID NO: 105 - VHH Fusion Molecule 6 (IL2-(G4Q)5-MC6.1C22.43) APQSSSTQQTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRL SCAASGRYIDETAVAWFRQAPGI<EREFVAGIGGVDITYYADSVI<GRFTISRDNSI<NTL YLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

SEQ ID NO: 106 - VHH fusion molecule 7 (MC6.1C22-(G4Q)5-IL2) EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVT VS SGGGGQGGGGQGGGGQGGGGQGGGGQAPQS S STQQTQLQLEHLLLDLQ MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

- 61 048693

SEQ Ш NO: 107 - VHH-based fusion molecule 8 (GLP1-(G ₄ Q)5-MC6.1C22.43) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGQGGGGQGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ Ш NO: 108 - HC-VHH (AdaFabVHCHi-(G ₄ Q) ₅ -MC6.1C22.43)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQG GGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFR QAPGKEREFVAGIGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCA ARPGRPLITSKVADL YPYWGQGTLVT VS SPP

SEQ Ш NO: 109 - LC (AdaFabVLCL)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 110 - HC (AdaFabVHCHi)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

SEQ ID NO: 111 - LC-VHH (AdaFabVLCL-(G ₄ Q) ₅ -MC6.1C22.43)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQG GGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGG

- 62 048693

VDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTL VT VS SPP

SEQ Ш NO: 112 - VHH-HC (MC6.lC22-(G ₄ Q) ₅ -AdaFabVHCHi)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQEVQLVESGGGLVQPGRSLRLSC AASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCD

SEQ Ш NO: 113 - LC (AdaFabVLCL)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 114 - HC (AdaFabVHCHi)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

SEQ Ш NO: 115 - VHH-LC (MC6.1C22-(G ₄ Q) ₅ -AdaFabVLCL)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKEREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSGGGGQGGGGQGGGGQGGGGQGGGGQDIQMTQSPSSLSASVGDRVTITC RASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDV ATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

SEQ ID NO:116 - VHH-based fusion molecule 13 (GLP1-MC6. lC80Cys)

- 63 048693

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC

SEQ ID NO: 117 - VHH-based fusion conjugate 1 (GLPl-C80CysACTH conjugate)

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGQGGGGGQGGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSSC-MAJIEHMRI[-(PEG)i2-AKTr (in the direction from C-terminus to N-terminus)

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGQGGGGQGGGGQGGGGQGGGGQ EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG QGTLVTVSS. _n

SEQ ID NO: 118 - VHH-based fusion conjugate 2 (MC6.1C80CysACTH conjugate)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWG OOTEUTU88C-maleimide-(PEO)12-ACTH (in the direction from C-terminus to N-terminus)

- 64 048693

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVD ITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLY PYWGQGTLVTVSS

SEQ ID NO: 119 - intermediate 1 (ACTH-(PEG) 12-maleimide conjugate) 8¥8MENEK№OKRUOKKKRUKU¥RK-(PEO)12-maleimide-НН2

SEQ ID NO: 120 - Fab HC (AdaFabVHCHi)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

SEQ Ш NO: 121 -Fab LC (AdaFabVLCL)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 122 - Full-length human GITR (without signal peptide)

- 65 048693

QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGD PCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTV FPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPR ETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

SEQ ID NO: 123 - Human GITR ECD (without signal peptide)

QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGD PCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTV FPGNKTHNAVCVPGSPPAE

SEQ ID NO: 124 - fragment VHH 38 (MC6.1C90Cys)

EVQLLESGGGLVQPGGSLRLSCAASGRYroETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWG QGTLVTVSSC

SEQ ID NO: 125 - fragment VHH 39 (MC6.1C90.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYroETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWG QGTLVTVSSPP

SEQ ID NO: 126 - fragment VHH 40 (MC6.1C95.43)

EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGTRPGRPLITSKVADLYPYWG QGTLVTVSSPP

Claims (28)

1. A compound for increasing the half-life of a therapeutic moiety, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37 and 124-126, or having at least about 90% similarity thereto. 2. The compound according to claim 1, containing the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ ID NO: 125) or a sequence having at least about 90% similarity thereto. 3. The compound according to claim 1, comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITY YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGTRPGRPLITSKVADLYPYWG QGTLVTVSSPP (SEQ ID NO: 126) or a sequence having at least about 90% similarity thereto. 4. A compound for increasing the half-life of a therapeutic moiety, comprising the structure: M-X1, X1-M, M-L1-X1, X1-L1-M, - 66 048693 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 or x ₂ -l ₂ -x ₁ -l ₁ -m, wherein M is an amino acid sequence selected from the group consisting of SEQ ID NO: 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 having at least about 90% similarity thereto, wherein X1 is a first biotherapeutic agent, wherein X2 (if present) is is a second biotherapeutic agent, wherein L1 (if present) is a first linker having an amino acid sequence selected from the group consisting of (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)nGGGG) (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 from 1 to 10, and wherein L2 (if present) is a second linker having an amino acid sequence selected from the group consisting of SEQ ID NOs:64 and 65, or having at least about 90% similarity thereto. 5. The compound of claim 4, wherein the first biotherapeutic agent is a peptide, protein or oligomer. 6. The compound of claim 4 or 5, wherein the second biotherapeutic agent is a peptide, protein or oligomer. 7. A compound according to any one of claims 4 to 6, wherein the first biotherapeutic agent and the second biotherapeutic agent are different from each other. 8. The compound of any one of claims 4 to 7, wherein L1 has an amino acid sequence selected from the group consisting of SEQ ID NO: 52-63, or having at least about 90% similarity thereto. 9. The compound of any one of claims 4 to 8, wherein the first biotherapeutic agent and/or the second biotherapeutic agent is a peptide or protein, such as an antibody (Ab) or a fragment thereof, adrenocorticotropic hormone (ACTE), growth/differentiation factor 15 (GDF15), incretin (INC), insulin (INS), interleukin (IL), ciliary neurotrophic factor (CNTF), neuregulin (NRG) or relaxin (RLN). 10. The compound of claim 9, wherein the peptide or protein is INC. 11. The compound of claim 10, wherein INC is selected from the group consisting of glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and GIP/GLP-1. 12. The compound of claim 9, wherein the peptide or protein is IL, and wherein the IL is IL2. 13. The compound of claim 9, wherein the peptide or protein is NRG, and wherein NRG is neuregulin-1 (NRG1) or neuregulin-4 (NRG4). 14. The compound of claim 9, wherein the peptide or protein is RLN, and wherein RLN is relaxin-2 (RLN2). 15. A pharmaceutical composition comprising a compound according to any one of claims 4 to 14 and a pharmaceutically acceptable buffer. 16. The pharmaceutical composition according to claim 15, additionally containing an additional therapeutic agent. 17. A method for treating an individual, comprising the step of administering to said individual an effective amount of a compound according to any one of claims 4-14 or a pharmaceutical composition according to any one of claims 15, 16. - 67 048693 18. Use of a compound according to claim 1 to increase the t ^1/2 _of a biotherapeutic agent. 19. Use of a compound according to any one of claims 4 to 14 for the treatment of cardiovascular, neurological, immunological, metabolic, oncological, psychological, pulmonary and/or renal conditions, diseases and/or disorders. 20. Use of a compound according to any of claims 4 to 14 for the production of a medicinal product. 21. A compound for increasing the half-life of a therapeutic moiety comprising any of the sequences of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 111, 112, 115, 116, 117 or 118, or a sequence having at least about 90% similarity thereto. 22. A compound for increasing the half-life of a therapeutic moiety comprising any of the sequences of SEQ ID NO: 109, 113, or a sequence having at least about 90% similarity thereto. 23. A compound for increasing the half-life of a therapeutic moiety comprising any of the sequences of SEQ ID NO: 110, 114, or a sequence having at least about 90% similarity thereto. 24. A pharmaceutical composition comprising a compound according to any one of claims 21-23 and a pharmaceutically acceptable buffer. 25. The pharmaceutical composition according to claim 24, additionally containing an additional therapeutic agent. 26. A method of treating an individual, comprising the step of administering to said individual an effective amount of a compound according to any one of claims 21-23 or a pharmaceutical composition according to any one of claims 24, 25. 27. Use of a compound according to any one of claims 21 to 23 for the treatment of cardiovascular, neurological, immunological, metabolic, oncological, psychological, pulmonary and/or renal conditions, diseases and/or disorders. 28. Use of a compound according to any one of claims 21-23 for the production of a medicinal product.