Therapeutic variable domain of heavy chain (VHH) antibodies neutralizing interleukin 13 (il-13)

Abstract

The present invention provides VHH antibodies that neutralize the pro-inflammatory cytokine interleukin IL-13 and their uses for treatment of inflammatory, fibrotic and other diseases and disorders.

Description

[0001] THERAPEUTIC VARIABLE DOMAIN OF HEAVY CHAIN (VHH) ANTIBODIES NEUTRALIZING INTERLEUKIN 13 (IL-13)

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains in the fields of antibody technology, medicine, pharmacology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that neutralize the pleiotropic cytokine interleukin 13 (IL- 13).

[0004] BACKGROUND OF THE INVENTION

[0005] Cytokines mediate communication between immune cells and coordinate the immune response. While essential for fighting pathogens, they are also key to the pathogenesis of autoimmune conditions. As such, they represent drug targets for therapeutic intervention.

[0006] The proinflammatory cytokines IL- 13, IL-4, and TSLP (thymic stromal lymphopoietin) are here of particular relevance (Ebina- Shibuya and Leonard, 2023). IL-4 and IL- 13 are mainly produced by T helper 2 (Th2) cells but also by mast cells and basophils. TSLP is primarily secreted by epithelial cells, e. g. by epithelial cells in the skin, lungs, and intestines in response to damage or inflammation.

[0007] IL-4 and IL- 13 stimulate B cell proliferation, differentiation, and antibody production, particularly of IgE that is a hallmark of allergic reactions. They induce mucus production by epithelial cells and airway hyperresponsiveness in the lungs, contributing, for example, to asthma symptoms.

[0008] IL-4 and IL-13 utilize a shared receptor system, triggering overlapping effects, though there are subtle difference in their receptor usage and signalling. IL-4 binds the type I IL-4 receptor (LaPorte et aL, 2008), which comprises IL-4Ra (UniProt ID: P24394) and the common cytokine receptor y-chain (yc) subunit (UniProt ID: P31785). This receptor type is primarily expressed on hematopoietic cells (e.g., T cells, B cells, and macrophages). Alternatively, IL-4 can bind to the type II IL-4 receptor (LaPorte et al., 2008), which consists (again) of the IL-4Ra subunit and the IL-13Ral subunit (UniProt ID: Q13437). This receptor type is expressed in both hematopoietic and non-hematopoietic cells (e.g. epithelial cells).IL-13 also signals through the Type II IL-4 receptor, comprising IL-4Ra and IL-13Ral. Binding of IL-4 and / or IL-13 to their respective receptor complexes leads to activation of protein kinases, JAK1, JAK3 or Tyk2, and the subsequent phosphorylation of the transcription factor STAT6. Phosphorylated STAT6 dimerizes and translocates to the nucleus to activate transcription. IL- 13 also binds IL-13Ra2 (UniProt ID: Q14627), which is considered a decoy receptor that does not initiate signaling but might attenuate IL-13 effects (Lupardus et al., 2010).

[0009] The cytokines IL-4 and IL- 13 represent the key proximal cytokines that activate the Th2 pathway, i.e. the activation of T helper 2 response involving the transcription factor GATA3. Antibodies to these cytokines proved clinically useful against a subtype of asthma called the “allergic” phenotype, but they are now also used more broadly to treat severe asthma. They are also useful to treat diseases that are often co-morbid with asthma — atopic dermatitis and chronic sinusitis with nasal polyps.

[0010] Type 2 immunity is characterized by T helper 2 (Th2) CD4+T cells and B cells producing antibodies of the immunoglobulin E (IgE) class. In addition, the innate cellular response includes group 2 innate lymphoid cells, eosinophils, basophils, mast cells as well as IL-4- and / or IL- 13 -activated macrophages. Epithelial -derived cytokines, TSLP, IL-25 and IL-33 also propagate or initiate type 2 responses, but their functions are not limited to type 2 immune responses.

[0011] TSLP activates dendritic cells and drives the differentiation of naive T cells into Th2 cells, which produce cytokines like IL-4, IL-5, and IL-13. This may amplify an allergic response (Nakajima etal., 2020). It also induces mast cell activation, degranulation, and thus release of inflammatory mediators.

[0012] To date, several biologies targeting the IL-4 / IL-13 pathway have been evaluated in clinical trials (Bernstein etal., 2023): an antibody targeting IL-4 (pascolizumab), three antibodies targeting IL-13, lebrikizumab, anrukinzumab, and tralokinumab (Wollenberg et al., 2021), and one antibody targeting IL-4Ra, dupilumab (Simpson et al., 2016). tralokinumab (Adbry) and dupilomab (Dupixent) have been approved and are in clinical use. Pitrakinra is a dominant negative mutant of IL-4 (Tony et al., 1994). In addition, IL-5-specific blockers work against asthma of the eosinophilic subtype. Also, a blocker of IgE, omalizumab (Dantzer and Wood, 2021), is effective against allergic asthma.

[0013] TSLP also signals through a heterodimeric receptor species, namely a receptor comprising the TSLP receptor (CRLF2, UniProt ID: Q9HC73) and IL-7Ra (Verstraete et al., 2017). Signalinginvolves activation of the JAK1 and JAK2 kinases, phosphorylation, subsequent dimerization, and nuclear import of STAT5, which then activates downstream genes (Ebina-Shibuya and Leonard, 2023). TSLP is particularly relevant target for treating asthma (Nakajima et al., 2020). An antibody to it, tezepelumab, is in clinical use (Corren et al., 2017). Besides TSLP, epithelial cells produce the “alarmins” IL-25 and IL-33 with overlapping functions.

[0014] Given the synergy of TSLP, IL-4 and IL- 13 in promoting allergic and autoimmune conditions, it is a reasonable therapeutic strategy to block these cytokines at the same time. Indeed, dupilumab, directed against IL-4Ra, interferes with IL-4 and IL- 13 signalling and has proven a highly effective drug. Likewise, abispecific antibody format against TSLP and IL-13 has been reported (Venkataramani et al., 2018), though it appears not to have proceeded through clinical studies, perhaps because it involved a rather complex engineering with covalent fusions between light and heavy antibody chains and combining them into a heterodimer. In any case, this design strategy appears impossible to extend to a three-specific format.

[0015] Nanobodies (VHH antibodies) are the best-known single domain antibodies. They represent the isolated antigen-binding domain of camelid heavy-chain-only antibodies (Hamers-Casterman et al., 1993; Arbabi Ghahroudi et al., 1997). In contrast to monoclonal IgGs, which comprise a heavy and a light chain, and which need to be manufactured in mammalian cells, nanobodies can also be produced in bacteria or yeast. Apart from their low molecular weight, certain nanobodies displayed a particularly high affinity and proved to be hyper-thermostable (Guttler etal., 2021). Gevenois P., et al., (2021) disclose neutralizing multimeric nanobody constructs directed against IL-13. The monomers had a good affinity profile but were not able to strongly inhibit IL- 13 biological activity in vitro.

[0016] WO2010060486 discloses specific single variable domain antibodies that bind IL-13. The single domain antibodies have a binding affinity to human IL-13 of 70-260 pM and thermal stability of maximum 57°C.

[0017] WO2021116182 discloses specific single domain antibodies targeting IL-13 or TSLP. The single domain antibodies show thermal stability of up to 70°C and KD in the nM range to human IL-13. Because of the high number of its plausible effects in inflammatory and fibrotic functions, many preclinical studies involving the inhibition of IL- 13 have been conducted. These studies have shown promising results for many pathologic conditions in addition to asthma and atopicdermatitis, such as idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, ulcerative colitis, eosinophilic esophagitis, and different types of cancers.

[0018] Most of these applications will benefit from an antibody with high affinity, stability, and versatility regarding application routes and fusion to additional entities such as inhibitors of IL-4 and / or TSLP signaling, and stabilizing entities. This illustrates the immense spectrum of benefits expected from VHH antibodies targeting IL-13.

[0019] There is a clear unmet need for novel stable and non-immunogenic therapeutic agents that will target IL- 13 and that will be safely used to treat inflammatory and fibrotic pathologies. Such agents may advantageously be used to treat upper respiratory disorders by local (e.g., inhalation) or systemic administration and to treat fibrotic skin disorders by local administration.

[0020] SUMMARY OF THE INVENTION

[0021] The present invention provides novel classes of single-domain VHH antibodies that inhibit or neutralize the human cytokine IL- 13 and compositions and methods for the amelioration of symptoms associated with this proinflammatory cytokine, and for treating patients with inflammatory, fibrotic and other disorders caused by and / or associated with the dysregulation of this cytokine.

[0022] The novel VHH antibodies of the present invention are herein shown to bind to IL- 13 with sub-nanomolar affinities and inhibit its binding to its receptor, therefore preventing or inhibiting receptor activation. Some of the VHH antibodies of the present invention are also hyperthermostable and aggregation-resistance making them ideal candidates for use as therapeutic entities. The VHH antibodies of the present invention are highly efficacious, specific, yet safer and more convenient for the treatment of inflammatory, fibrotic and other diseases and disorders associated with IL-13. Depending on the condition to be treated and on the structural and functional properties of the VHH antibodies they may be administered systematically, locally (e.g., by respiratory delivery), or topically.

[0023] For some of the VHH antibodies, the binding to the human IL- 13 has been confirmed and further elucidated using crystal and modelled structures of complexes of the novel VHH antibodies and the protein. These crystal and modelled structures are used to gain structural and functional information about the binding and to design improved VHH antibodies.In certain embodiments, the VHH antibodies having very high affinity (KD of nanomolar to picomolar) to human IL-13, are in monovalent form, e.g., as a single VHH domain or as a fusion to a heterologous protein such as Fc fragment or serum albumin. In certain embodiments, The VHH antibodies are in multivalent form, e.g., as bivalent polypeptide optionally comprising a linking polypeptide, such as an antibody Fc domain, or as multimers comprising two or more, identical or different VHH antibodies to IL- 13 and optionally to other human cytokines.

[0024] The present invention provides, according to one aspect, a VHH antibody recognizing the human IL- 13 polypeptide with high affinity and prevents or inhibits activation of a human IL- 13 receptor. The VHH antibody belongs to a class of structurally-related antibodies, wherein the class is selected from:

[0025] i. a class A VHH antibody comprising a CDR3 sequence selected from:

[0026] 1) wherein X7is selected from L, V,

[0027]

[0028] and X10is Y or F, and

[0029] ii. a class C VHH antibody comprising a CDR3 comprising the sequence STTICSVVAGLISNKYDSR (SEQ ID NO: 2),

[0030] wherein the sequence identity between different VHH antibodies within a specific class is 80% or more.

[0031] According to some embodiments, class A VHH antibodies comprising CDR3 sequence defined in SEQ ID NO: 1 and CDR2 and 3 sequences exemplified below can be generated by allowing substitutions that are present in any of the class members disclosed below. These substitutions are within at least one framework (FW) and / or CDR sequences, as long as the intra-class homology of the entire VHH antibody sequence is 80% or more.

[0032] According to some embodiments, the sequence identity between different VHH antibodies within a specific class is at least 90% or at least 95%.

[0033] According to some embodiments, the VHH antibody comprises a CDR3 sequence selected from: LADLDFTITTIQGDEYW (SEQ ID NO: 3), CADLDFTIKTIEGDEFW (SEQ ID NO: 4), VADLDFTIKTIEGDEFW (SEQ ID NO: 48), and STTICSVVAGLISNKYDSR (SEQ ID NO: 2).

[0034] According to some embodiments, the negatively charged sidechains of the CDR3 sequence of a class A VHH antibody form coordinative bonds to a cation, which in turn constrains the conformation of CDR3 and makes it shape-complementary to its IL- 13 target. According to someembodiments, the cation is selected from Na+, K+, Ca2+and Mg2+. According to some embodiments, the CDR3 sequence comprises 4 or 5 negatively-charged residues. According to specific embodiments, amino acid residues D99, T103, T106, E108 and Elll of class A VHH antibody Bm41A08, or D99, T103, T106, Q108 and Elll of class A VHH Bm46A10 form coordinative bonds with the said cation. Electron donors of these coordinative bonds are the backbone oxygens of T 103 and T106 as well as the sidechain carboxylate or carboxamide oxygens of D99, El 08 / QI 08 and Elll.

[0035] According to some embodiments, the VHH antibody is a class A antibody comprising a CDR1 comprising the sequence X1X2SGRTFNSX3GM (SEQ ID NO: 5), wherein X1is Thr (T) or Vai (V), X2is Asn (N) or Ala (A) and X3is Tyr (Y) or Phe (F); a CDR2 comprising the sequence AISWX4SGX5TX6(SEQ ID NO: 6) wherein X4is Asp (D) or Ser (S), X5is Thr (T) or Glu (E), and X6is Tyr (Y) or Ser (S); and a CDR3 comprising the sequence X7ADLDFTIX8TIX9GDX11X10W (SEQ ID NO: 1) wherein X7is selected from Leu (L), Vai (V) and Cys (C), X8is Thr (T) or Lys (K), X9is Q or E, X11is D or E, and X10is Tyr (Y) or Phe (F). According to some embodiments, the invention provides a VHH antibody that binds to IL- 13 and inhibits or neutralizes its activity wherein the VHH antibody is selected from: a class A antibody comprising a CDR set selected from:

[0036] i. a CDR1 comprising TNSGRTFNSYGM (SEQ ID NO: 7), a CDR2 comprising AISWDSGTTY (SEQ ID NO: 8), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);

[0037] ii. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising CADLDFTIKTIEGDEFW (SEQ ID NO: 4);

[0038] iii. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);

[0039] iv. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising VADLDFTIKTIEGDEFW (SEQ ID NO: 48);

[0040] v. a CDR1 comprising ANSGRTFNSYAM (SEQ ID NO: 11), a CDR2 comprising SISWSEGRVT (SEQ ID NO: 12), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3); andvi. a CDR1 comprising ANSGRTFNSYAM (SEQ ID NO: 11), CDR2 comprising AISWSGGRTT (SEQ ID NO: 19), and CDR3 comprising VADLDFTITTIEDDDYW (SEQ ID NO: 20); and

[0041] a class C VHH antibody comprising the CDR sequences TASGSGFTLNNEDI (SEQ ID NO: 13) as CDR1, CLSFKNNKTYTY (SEQ ID NO: 15) as CDR2, and STTICSVVAGLISNKYDSR (SEQ ID NO: 2) as CDR3.

[0042] According to some embodiments, the class A VHH antibody comprises a CDR1 selected from TNSGRTFNSYGM (SEQ ID NO: 7), VASGRTFNSFGM (SEQ ID NO: 9) and ANSGRTFNSYAM (SEQ ID NO: 11), a CDR2 selected from AISWDSGTTY (SEQ ID NO: 8), AISWSSGETS (SEQ ID NO: 10) and SISWSEGRVT (SEQ ID NO: 12), and a CDR3 selected from LADLDFTITTIQGDEYW (SEQ ID NO: 3), VADLDFTIKTIEGDEFW (SEQ ID NO: 48) and CADLDFTIKTIEGDEFW (SEQ ID NO: 4).

[0043] According to some embodiments, the VHH antibody is a class A antibody comprising a set of 3 CDR sequences, wherein the set is selected from:

[0044] i. a CDR1 comprising TNSGRTFNSYGM (SEQ ID NO: 7), a CDR2 comprising AISWDSGTTY (SEQ ID NO: 8), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);

[0045] ii. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising CADLDFTIKTIEGDEFW (SEQ ID NO: 4);

[0046] iii. a CDR1 comprising TNSGRTFNSYGM (SEQ ID NO: 7), a CDR2 comprising AISWDSGTTY (SEQ ID NO: 10), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);

[0047] iv. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising VADLDFTIKTIEGDEFW (SEQ ID NO: 48);

[0048] v. a CDR1 comprising ANSGRTFNSYAM (SEQ ID NO: 11), a CDR2 comprising SISWSEGRVT (SEQ ID NO: 12), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3); and

[0049] vi. a CDR1 comprising ANSGRTFNSYAM (SEQ ID NO: 11), CDR2 comprising AISWSGGRTT (SEQ ID NO: 19), and CDR3 comprising VADLDFTITTIEDDDYW (SEQ ID NO: 20).According to other embodiments, the VHH antibody is a class C antibody comprising a set of 3 CDR sequences wherein CDR1 comprises the sequence TASGSGFTLNNEDI (SEQ ID NO: 13), CDR2 comprises the sequence CLSFKNNKTYTY (SEQ ID NO: 15), and CDR3 comprises the sequence: STTICSVVAGLISNKYDSR (SEQ ID NO: 2).

[0050] According to some embodiments, the VHH antibody that binds to IL- 13 and inhibits or neutralizes its activity, comprises an amino acid sequence selected from SEQ ID NOs: 21-30 (class A); and SEQ ID NOs. 32-33 (class C), or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any of these sequences.

[0051] According to some embodiments, the VHH antibody is a class A VHH antibody comprising a sequence selected from SEQ ID NOs: 21-24 and 29-30, or a variant thereof comprising at least 89% sequence identity. According to particular embodiments, the class A VHH antibody comprises a sequence selected from SEQ ID NOs: 21-24 and 29-30. According to some specific embodiments, the class A VHH antibody consists of a sequence selected from SEQ ID NOs: 21-24 and 29-30.

[0052] According to some embodiments, the class A VHH antibody comprises a variant of a sequence selected from SEQ ID NOs: 21-24 and 29-30 in which one or more of the following framework (FW) positions are (counting from N-terminus): position 6 is Q or E, position 10 is G or S, position 11 is S or L, position 14 is A or P, position 16 is E or G, position 61 is A or G, position 62 is D or E, position 72 is R or S, position 75 is T or A, position 76 is N or K, position 77 is T or N, position 78 is A or T, and position 85 is N or S.

[0053] According to some embodiments, the class A VHH antibody comprises a sequence selected from SEQ ID NOs: 21-24 and 29-30, wherein 1-13 FW residues are substituted. According to some embodiments, the 1-13 substitutions are in FW1 and / or FW3 residues. According to some specific embodiments the substitutions are at positions 6, 10, 11, 14, 16, 61, 62, 72, 75, 76, 77, 78, and / or 85.

[0054] According to some specific embodiments, the class A VHH antibody comprises a variant sequence of any of SEQ ID NOs: 23, 24, 29, and 30 in which FW position 6 is Q or E, FW position 16 is E or G, and FW position 61 is A or G.

[0055] A VHH antibody according to the present invention comprises the structure FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. According to some embodiments, a class A VHH antibody comprises a FW1 having the sequence QVQLVXSGGXXVQXGXSLRLSC (SEQ ID NO: 52) wherein Xat position 6 is Q or E, X at position 10 is G or S, X at position 11 is S or L, X at position 14 is A or P, and X at position 16 is E or G; a FW2 having the sequence GWFRQAPGKEREFVA (SEQ ID NO: 53); a FW3 having the sequence YXXSVKGRFTISXDNXXXXVYLQMNXLKPEDTAVYYC (SEQ ID NO: 54), wherein X at position 2 (position 61 of the entire VHH antibody) is A or G, X at position 3 (position 62 of the entire VHH antibody) is D or E, X at position 13 (position 72 of the entire VHH antibody) is R or S, X at position 16 (position 75 of the entire VHH antibody) is T or A, X at position 17 (position 76 of the entire VHH antibody) is N or K, X at position 18 (position 77 of the entire VHH antibody) is T or N, X at position 19 (position 78 of the entire VHH antibody) is A or T, and X at position 26 (position 85 of the entire VHH antibody) is N or S; and a FW4 having the sequence GQGTQVTVSS (SEQ ID NO: 55).

[0056] The class A VHH antibodies of the present invention comprises, according to some embodiments, a combination of the FWs of SEQ ID NOs: 52, 53, 54 and 55 and a set of CDR1, CDR2 and CDR3 sequences selected from: SEQ ID NOs: 7, 8 and 3; SEQ ID NOs: 9, 10, and 4; SEQ ID NOs: 9, 10, and 3; SEQ ID NOs: 9, 10, and 48; SEQ ID NOs: 11, 12, and 3; and SEQ ID NOs: 11, 19, and 20.

[0057] According to some embodiments, the class A antibody comprises the sequence of QVQLVESGGX12LVQX13GGSLRLSCANSGRTFNSYAMGWFRQAPGKEREFVAAISWSG GRTTYAESVKGRFIISSDNAKNTVYLQMNSLKPEDTAVYYCVADLDFTITTIEDDDYW GQGTQVTVSS, wherein X12is G or S and X13is A or P (SEQ ID NO: 51), or a sequence having at least 90%, at least 95%, or at least 98% identity. According to some embodiments, the invention provides a class A VHH antibody variant comprising 1-10 substitutions to SEQ ID NO: 51. According to some embodiments, the substitutions are in FW residues.

[0058] According to some specific embodiments, the VHH antibody is selected from Bm60Gl 1 (class A, SEQ ID NO: 30), Bm41Hl 1 (class C, SEQ ID NO: 33), Bm46A10 (class A, SEQ ID NO: 24), Bm46H07 (class A, SEQ ID NO: 29), Bm41F06 (class C, SEQ ID NO: 32), and Bm41 A08 (class A, SEQ ID NO: 23). According to some embodiments, the variant has at least 91%, 92%, 93%, 94% or 95% sequence identity to the VHH antibody. According to more particular embodiments, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity to the VHH antibody. Variants comprising substitutions in 1-10 amino acid residues are also included in the scope of the present invention. The substitutions may be selected from conservative substitutions, nonconservative substitutions, and combinations thereof. According to some embodiments, thesubstitutions are in the framework and / or in the CDR sequences. According to other embodiments, the substitutions are in the framework sequences.

[0059] According to some embodiments, a variant of a VHH antibody described above, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are substituted, deleted, or added is provided. According to particular embodiments, 1-5 amino acids in a VHH antibody are substituted, deleted, or added. According to some specific embodiments, the substitution, deletion, or addition retains or improves at least one physical property of the VHH antibody. According to some embodiments a substitution or a combination of 2-5 substitutions, additions or deletions improve the affinity, stability, aggregation-resistance and / or producibility of a specific VHH antibody according to the present invention.

[0060] Variants comprising 1-3 amino acid substitutions in one, two or even three CDR sequences are also included in the scope of the present invention. According to some embodiments, the substitution is a conservative substitution. According to other embodiments, the substitution is a non-conservative substitution.

[0061] According to particular embodiments, the present invention provides the VHH antibody Bm60Gl 1 comprising a VHH sequence as shown in SEQ ID NO: 30, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0062] According to particular embodiments, the present invention provides the VHH antibody Bm41Hl 1 comprising a VHH sequence as shown in SEQ ID NO: 33, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0063] According to particular embodiments, the present invention provides the VHH antibody Bm46A10 comprising a VHH sequence as shown in SEQ ID NO: 24, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0064] According to particular embodiments, the present invention provides the VHH antibody Bm46H07 comprising a VHH sequence as shown in SEQ ID NO: 29, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0065] According to particular embodiments, the present invention provides the VHH antibody Bm41F06 comprising a VHH sequence as shown in SEQ ID NO: 32, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.According to particular embodiments, the present invention provides the VHH antibody Bm41 A08 comprising a VHH sequence as shown in SEQ ID NO: 23, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0066] According to particular embodiments, the present invention provides the VHH antibody Bml9G08 comprising a VHH sequence as shown in SEQ ID NO: 21, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0067] According to particular embodiments, the present invention provides the VHH antibody Bm20D05 comprising a VHH sequence as shown in SEQ ID NO: 22, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0068] According to particular embodiments, the present invention provides the VHH antibody Bm46A10 comprising a VHH sequence as shown in SEQ ID NO: 24, or a VHH antibody, which is a variant thereof having at least 90%, at least 95%, or at least 98% identity.

[0069] According to some embodiments, class A VHH antibodies comprise the following residues that interact with the human IL-13 molecule: F29, N30, S31, D99, L100, F102, T103, T106, 1107, E108, G109, DUO, El 11, Fl 12. A representative VHH antibody of this embodiment is Bm41A08 comprising SEQ ID NO: 23.

[0070] According to some embodiments, a class A VHH antibody binds to residues R44, 147, E48, V51, Q55, L133, L134, K137, F140, R141, and G143 of the human IL-13 molecule (SEQ ID NO. 47 corresponding to IL-13 HUMAN without the signal peptide, according to UniProt numbering the first residue is then number 25).

[0071] The present invention also provides, according to yet another aspect, a humanized VHH antibody of a VHH antibody described above. According to some embodiments, the humanized VHH antibody comprises the three CDR sequences of any VHH antibody described above. According to other embodiments, the humanized VHH antibody comprises at least one CDR sequence that differs in 1 to 3 amino acid residues from the CDR sequence of a VHH antibody described above.

[0072] In certain embodiments, the VHH antibody is a humanized VHH variant which differs from the original VHH sequence by at least one framework mutation, e.g., by 1 to 15, 1 to 10 or 1 to 5 framework mutations, wherein the sequence identity to a human immunoglobulin heavy chain variable region, e.g., a human germline immunoglobulin variable region is increased. In particularembodiments, a humanized VHH variant comprises humanized framework regions wherein at least one amino acid, e.g.,1 to 10 amino acids in the framework regions, i.e., the regions outside the hypervariable CDR1, CDR2 and CDR3 regions are replaced by other amino acids found in a human framework region.

[0073] According to some embodiments, the binding affinity of a monomeric VHH antibody, expressed as a dissociation constant KD, to an immobilized human IL-13 is about 1 nM, 700 pM, 500 pM, 300 pM, 100 pM, 50 pM or less.

[0074] According to some embodiments, the VHH antibody prevents binding of human IL- 13 to human IL- 13 receptor at a concentration of about 10 nM or less, of about 3 nM or less, of about 1 nM or less, of about 0.3 nM or less, when tested in an appropriate cell-based assay.

[0075] The VHH antibody according to some embodiments of the present invention, neutralizes human IL- 13 and thus signaling through the IL-13Ra and IL-4Ra receptors, at a concentration of about 10 nM or less, of about 3 nM or less, of about 1 nM or less, of about 0.3 nM or less, of about 0.1 nM or less, when tested in an appropriate cell-based assay under affinity-limited test conditions. According to some embodiments, the VHH antibody is stable, particularly thermostable or hyperthermostable. According to some specific embodiments, the VHH antibody has a melting temperature of at least about 65°C, of at least about 80°C, of at least 90°C or of at least about 95°C when the structural disulfide bond of the VHH antibody has been formed and the measurement has been performed under non-reducing conditions.

[0076] According to other embodiments, the structural disulfide bond (class A), or the disulfide bonds (class C), of a VHH according to the present invention is formed by expression in a secretory system, e.g. by periplasmic expression in E. coli or by protein secretion from a yeast or an animal cell. Alternatively, the disulfide bond can be formed post-production, e.g. by using oxidized glutathione as an oxidant and protein disulfide isomerases as catalysts, as disclosed in WO 2024 / 256467.

[0077] According to some embodiments, the VHH antibody has an aggregation temperature of at least about 60°C, of at least 70°C, of at least about 80°C, of at least about 90°C, or of at least about 95°C, when measured under non-reducing conditions.

[0078] According to some embodiments, the VHH antibody is resistant to at least 10 freeze-thaw cycles.The VHH antibody according to any one of the previous embodiments may be non-glycosylated, or glycosylated.

[0079] A further aspect of the present invention relates to a set of two or more different VHH antibodies, wherein at least one VHH antibody is as described above. The set may include two or more different VHH antibodies that are mixed together or that are fused or are covalently or non-covalently conjugated together, directly or through a linker or a carrier protein in a monovalent, bivalent or multivalent format.

[0080] According to some embodiments, the two or more VHH antibodies bind IL-13, at the same epitope or at different epitopes. According to other embodiments, the set comprises at least one VHH antibody that recognizes and neutralizes IL-13, and at least one VHH antibody that recognizes a different human protein. According to some embodiments, the different human protein is a cytokine. According to some embodiments, the cytokine is selected from the group consisting of TSLP, IL-4, and IL-17. According to yet other embodiments, the different human protein comprises a sequence or a domain of a constant region of an antibody, a human serum albumin or a fragment thereof, or a collagen or a fragment thereof.

[0081] In certain embodiments, the VHH antibody as described above is covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a carrier protein, a labeling group, a capture group or an effector group.

[0082] In certain embodiments, the VHH antibody as described above is fused to a heterologous polypeptide moiety, e.g., to an IgG Fc fragment, to serum albumin or to an albumin-binding moiety, e.g., a VHH antibody against human serum albumin. According to some embodiments, the IgG Fc fragment does not have an effector function. According to specific embodiments, the Fc fragment is of a human IgG4. In certain embodiments, the VHH antibody is conjugated or fused to one or several polymer moieties, particularly hydrophilic polymer moieties, such as polyethylene glycol (PEG), to increase the molecular weight of the antibody conjugate and, thus, delay renal clearance. The molecular weight of a polymer moiety may vary over a broad range, for example in the range of about 5 kDa to about 80 kDa. Such coupling or fusion may be performed through e.g., amino or carboxyl groups already present in the VHHs (e.g., amino and carboxy terminals) and / or through the side chains of lysine, aspartic acid, glutamic acid or cysteine residues, or through engineered backbone or side chains of other amino acids, and involve known chemistries for forming amide bonds, secondary amine bonds, urea bonds or thioether bonds.According to some embodiments, the VHH antibody or variant thereof is in a monovalent format. According to some embodiments, the VHH antibody or variant thereof is in a dimeric or multimeric format, wherein at least two identical or different VHH antibodies are attached to each other, directly or through a linker or a heterologous sequence or moiety, in a tandem format, or in a branched multimeric format.

[0083] According to some embodiments, a homodimeric bivalent, or homo-multimeric molecule is provided comprising two or more identical VHH antibodies that recognizes human IL-13, wherein the two or more identical VHH molecules are fused or conjugated directly to each other or through a linker or a heterologous polypeptide. According to some embodiments, the heterologous polypeptide is capable of increasing the half-life of the VHH antibody in the circulation. According to some embodiments, the heterologous polypeptide is an immunoglobulin fragment, e.g., an antibody Fc region. According to other embodiments, the heterologous polypeptide is human serum albumin or a portion thereof.

[0084] According to other embodiments, a heterodimeric or a hetero-multimeric molecule is provided comprising one or more, identical or different, VHH antibodies that recognizes human IL-13, and one or more VHH antibodies that recognizes at least one other human protein, in a bivalent or a multivalent branched or tandem format. According to some embodiments, the other human protein is selected from TSLP, IL-4, serum albumin, and IL-17. The VHH molecules in the heterodimeric or hetero-multimeric molecule are expressed as a single protein or are fused or conjugated directly to each other or through a linker or a heterologous polypeptide sequence. According to some embodiments, the heterologous polypeptide is capable of prolonging the halflife of the VHH antibody in the circulation. According to some embodiments, the heterologous polypeptide is an immunoglobulin fragment, e.g., an antibody Fc region. According to other embodiments, the heterologous polypeptide is human serum albumin or a portion thereof, or human collagen or a portion thereof.

[0085] According to some embodiments, the invention provides a single polypeptide of two or three tandem-fused single domain antibodies, one that targets IL-13 as disclosed above and the others that target IL-4, and / or TSLP.

[0086] A VHH antibody described above or a set of VHH antibodies are suitable for use in medicine, e.g., human medicine, particularly for use in therapy, e.g., in the prevention or treatment of a disorder caused by and / or associated with an activity or overactivity of human IL-13, or indiagnostics, e.g., for detecting IL-13 in a patient sample, e.g., in a body fluid or tissue sample, or in research.

[0087] The present invention also provides, according to another aspect, a nucleic acid molecule encoding a VHH antibody or a subunit of a VHH antibody, or a dimeric or multimeric molecule described above, preferably in operative linkage with a heterologous expression control sequence, or included in a vector. According to some embodiments, the nucleic acid molecule is a DNA molecule. According to other embodiments, the nucleic acid molecule is an RNA molecule. Non-limiting DNA sequences encoding the VHH antibodies of the present invention are disclosed in SEQ ID NOs: 34-43, but it should be noted that as the genetic code is degenerate, each VHH antibody may be encoded by additional DNA sequences that are all within the scope of the present invention.

[0088] According to some embodiments, the invention provides a recombinant construct of a nucleic acid sequence encoding a thermostable VHH antibody that binds to and neutralizes human IL-13 or a conjugate or an active fragment thereof, wherein the nucleic acid sequence is operatively linked to at least one transcription control sequence.

[0089] According to some embodiments, the recombinant nucleic acid molecule or construct encodes a VHH antibody comprising a sequence selected from SEQ ID Nos. 21-30 and 32-33 or a sequence having at least 90%, 95%, or 98% identity with any of these sequences.

[0090] According to some embodiments, the nucleic acid sequence encoding a VHH antibody comprises a sequence encoding a signal peptide that may be cleaved or not from the mature polypeptide sequence. According to some embodiments, the nucleic acid sequence encoding a VHH antibody further comprises a translation start codon at the N-terminus of the polypeptide sequence.

[0091] According to some embodiments, the construct comprises two different nucleic acid sequences encoding two different VHH antibodies wherein at least one is a VHH antibody that binds to and neutralizes human IL-13.

[0092] According to some embodiments, the two different nucleic acid sequences encoding two different VHH antibodies are in two expression cassettes within said recombinant construct. According to some embodiments, the construct comprises two different nucleic acid sequences that encode two different VHH antibodies the bind to and neutralize human IL-13. According to other embodiments, the construct comprises two different nucleic acid sequences, one encodes a VHH antibody that binds to and neutralizes human IL-13, and one encodes a VHH antibody that bindsto a different target, e.g., a human polypeptide other than IL- 13. According to some embodiments, the different target is selected from HSA, human antibody Fc region and a human cytokine, other than IL-13. According to some embodiments, the human cytokine is selected from TSLP, IL-4, and IL-17. According to some embodiments, the different target is human serum albumin (HSA) and the construct encodes a VHH antibody that binds to and neutralizes human IL-13, and a VHH antibody that binds HSA. According to some embodiments, the different target is human immunoglobulin (IgG) constant region or a fragment thereof and the construct encodes a VHH antibody that binds to and neutralizes human IL- 13, and a VHH antibody that binds human Fc. The present invention also provides a recombinant cell or a non-human organism transformed or transfected with a nucleic acid molecule or a vector.

[0093] According to some embodiments, the cell or organism is selected from a bacterium such as E. coli, a Bacillus sp., a unicellular eukaryotic organism, e.g., yeast such as Pichia pastoris, or I.eishmauia, an insect cell, a mammalian cell, and a plant cell.

[0094] According to some embodiments, the VHH antibody or variant thereof is produced in a bacterium, e.g., E. coli, or in a yeast, e.g., Pichia pastoris.

[0095] The invention also provides a method for recombinant production of a VHH antibody, fragment, dimer, multimer, conjugate or fusion polypeptide described above, comprising cultivating a cell or an organism in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium.

[0096] According to some embodiments, the method comprises cultivating cells from a bacterium, a yeast, an insect, a mammalian, or a plant. According to some embodiments, the cells are mammalian cells. According to some embodiments, the mammalian cells are Chinese Hamster Ovary (CHO) cells.

[0097] According to some embodiments, the method comprises cultivating yeast cells such as Pichia pastoris cells and obtaining the VHH antibody from the medium.

[0098] The invention further provides, according to another aspect, a pharmaceutical composition comprising at least one VHH antibody defined above, and a pharmaceutically acceptable excipient, or diluent.According to some embodiments, the pharmaceutical composition comprises one specific VHH antibody described above. According to other embodiments, the pharmaceutical composition comprises a plurality of VHH antibodies described above, e.g., a set of different VHH antibodies. According to some embodiments, the pharmaceutical composition comprises a single polypeptide comprising two or three tandem-fused single domain antibodies, one that targets IL-13 as disclosed above and the other / s that target IL-4, and / or TSLP.

[0099] Pharmaceutical compositions provided according to the present invention may be formulated as liquid, semi-liquid, suspension, spray, aerosol, mist, solid or semi-solid states. According to some embodiments, the pharmaceutical composition is a liquid formulation. According to other embodiments, the pharmaceutical composition is formulated as a mist, spray, aerosol, cream, paste, gel, hydrogel, ointment, lotion, and emulsion.

[0100] The pharmaceutical composition may be formulated according to the intended administration mode. According to some embodiments, the pharmaceutical composition is formulated for systemic administration, including but not limited to intravenous administration by injection or infusion, subcutaneous administration, or intramuscular administration. According to other embodiments, the formulation is for local administration. According to some embodiments, the formulation is for administration to the respiratory tract, for example by pulmonary or nasal administration, by inhalation, e.g., using a mist inhaler, or nasal spray. According to other embodiments, the pharmaceutical composition is formulated for topical administration, e.g., on skin, such as an injured skin or wound.

[0101] The present invention also provides a pharmaceutical composition comprising at least one VHH antibody described above for use in medicine, particularly for use in therapy or a diagnostic composition for us in research or diagnosis.

[0102] According to some embodiments, the pharmaceutical composition is for use in preventing, treating, alleviating, delaying the progression, preventing worsening, or relieving the symptoms of a disorder caused by and / or associated with an activity or overactivity of IL-13.

[0103] Several disorders associated with IL-13 and IL-4 might become amenable to treatment with the IL-13 antagonists of the present invention. These include but are not limited to Th2 diseases such as asthma, and eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, and different types of cancers, including but not limited to Hodgkin’s lymphoma. Inaddition, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-IL-4Ra, may be amendable with the anti-IL-13 VHH antibodies of the present invention, optionally together with anti TSLP, and / or anti-IL-4.

[0104] According to some embodiments, the pharmaceutical composition is for use in the prevention or treatment of an inflammatory and / or fibrotic disorder.

[0105] According to some embodiments, the pharmaceutical composition is for prevention or treatment of a skin fibrosis or disorder. According to some embodiments, the skin fibrosis or disorder is selected from atopic dermatitis skin eczema and aberrant wound healing process, e.g., scarring or keloids.

[0106] According to some embodiments, the pharmaceutical composition is for use in combination with anti-IL-4, anti-TSLP and / or anti-IL-17.

[0107] According to some embodiments, the pharmaceutical composition is for use in preventing or treating allergic asthma, moderate asthma or severe asthma.

[0108] According to some embodiments, the pharmaceutical composition is for prevention or treatment of pulmonary or liver fibrosis in response to chronic inflammation, or for prevention or treatment of liver fibrosis in the context of schistosomiasis. According to some embodiments, the pharmaceutical composition is for prevention or treatment of idiopathic pulmonary fibrosis. The invention further provides, according to another aspect, a diagnostic composition comprising at least one VHH antibody defined above, and an acceptable excipient or diluent.

[0109] Pharmaceutical and diagnostic kits comprising at least one VHH antibody and instructions for use are also provided. According to some embodiments, the diagnostic composition comprises a standard or reference sample of a known amount of human IL-13, for calculating the amount or concentration of the protein in the tested sample.

[0110] The present invention also provides a method of detection or quantitation of human IL- 13 using a VHH antibody or a conjugate or fusion thereof, described above.

[0111] According to some embodiments, the diagnostic composition is for use in detecting the amount or concentration of human IL-13 in a biological sample, for example in a body sample, e.g., a body fluid or tissue sample. The diagnostic composition may be used for research or for clinical diagnosis or prognosis of a disease or disorder associated with IL-13 expression, activity oroveractivity. For detecting IL- 13 in a sample obtained from a patient or from a subject suspected to have a certain disease or disorder, or in research or manufacturing.

[0112] Still a further aspect of the invention relates to a method for the preventing, treating, alleviating, delaying the progression, preventing worsening, or relieving the symptoms of a disorder caused by and / or associated with an activity or overactivity of IL-13, comprising administering an effective dose of the VHH antibody or conjugate or fusion polypeptide as described above or the set of at least two different VHH antibodies as described above or a pharmaceutical composition described above, to a subject in need thereof.

[0113] According to some embodiments, the subject is a human subject suffering from a disorder caused by and / or associated with IL-13 expression, overexpression, activity or overactivity.

[0114] According to some embodiments, the disease or disorder is an inflammatory and / or fibrotic disease or disorder.

[0115] According to some embodiments, the disease is a Th2 disease.

[0116] According to some embodiments, the disorder is selected from asthma, eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, cancer, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-IL-4Ra antibody.

[0117] According to some embodiments, the disorder is a skin fibrosis or disorder. According to some embodiments, the skin fibrosis or disorder is selected from atopic dermatitis skin eczema and aberrant wound healing process, e.g., scarring or keloids.

[0118] According to some embodiments, disease is allergic asthma, moderate asthma or severe asthma. According to some embodiments, the disease is a pulmonary or liver fibrosis caused in response to chronic inflammation, or for liver fibrosis in the context of schistosomiasis. According to some embodiments, the disease is idiopathic pulmonary fibrosis.

[0119] According to some embodiments, the cancer is Hodgkin’s lymphoma.

[0120] According to some embodiments, the method comprises systemic administration, including but not limited to intravenous administration by injection or infusion, subcutaneous injection, or intramuscular inj ection, of the VHH antibody, conjugate, or fusion thereof, or the pharmaceutical composition. According to other embodiments, the method comprises local administration.According to other embodiments, the method comprises topical administration, e.g., on skin, injured skin or wound.

[0121] According to some embodiments, the method of treating diseases residing in the airway system, e.g., pulmonary fibrosis, asthma, eosinophilic COPD, chronic sinusitis, nasal polyps, and rhinitis conjunctivitis, comprises direct administration of the anti-IL-13 VHH antibodies or pharmaceutical compositions comprising them, through respiratory delivery, by inhalation (e.g., by mist inhaler) or nasal spraying.

[0122] According to some embodiments, the method comprises administration of at least two different anti IL- 13 VHH antibodies or conjugates or fusions thereof, in one, two or more compositions. The methods of preventing, treating, alleviating, delaying the progression, preventing worsening, or relieving the symptoms of a disease or disorder using the VHH antibodies of the present invention may be stand-alone methods or as part of a treatment regimen with any other treatment for the disease or disorder.

[0123] According to some embodiments, the method of administering anti-IL-13 VHH antibodies of the present invention, comprises also administration of anti-TSLP, anti-IL-4, and anti-IL-17, or an agent directed against another pro-inflammatory cytokine.

[0124] According to some embodiments, the method comprises administration of a polypeptide comprising two or three tandem-fused single domain antibodies, one that targets IL-13 as disclosed above and the other / s that target IL-4, and / or TSLP

[0125] In some embodiments, the method comprises administration of a single dose of the composition or multiple doses of the composition. In other embodiments, the composition is administered at regular intervals. In yet other embodiment, the composition is administered at day, week, month, bi-annual or annual intervals.

[0126] According to some embodiments, the subject is tested, using methods known in the art, before treatment. The blood levels and content of IL-13 proteins and the timing and amounts of the compositions described above to be administered are decided based on the levels of the IL- 13 proteins. According to other embodiments, the timing and amount of administration is based on the severity of the disease and / or on other treatments of said subject.

[0127] According to some embodiments, the circulation level of the anti-human IL-13 VHH antibody or antibodies administered is monitored at least once following the treatment.The present invention is explained in more detail by the following Figures and Examples.

[0128] BRIEF DESCRIPTION OF THE FIGURES

[0129] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0130] Figure 1: Sequences of selected anti-IL-13 VHH antibodies.

[0131] The figure shows an alignment of sequences from selected VHH antibodies. Variable residues (in framework and CDRs) are highlighted by a grey background. The class assignments and three CDR regions are indicated.

[0132] Figures 2A-2i: Affinities of VHH antibodies for human IL-13 measured by biolayer interferometry (BLI). IL-13 (biotinylated Fc-Avi-tag fusion, produced in mammalian cells) was allowed to bind to High Precision Streptavidin biosensors of an Octet RED96e instrument (ForteBio / Sartorius), as detailed in Example 2. After thorough washing, class A (Bm41A08, Bm46A10, Bm46H07, Bm46G09, Bm46D05, Bm46E09, and Bm46H02, Figures 2A-2G, respectively) and class C (Bm41F06 and Bm41Hll, Figures 2H and 2i, respectively) VHH antibodies at indicated concentrations were allowed to bind and to dissociate as indicated. Binding signals were recorded as wavelength shifts (in nm). Baselines were recorded in parallel as ‘minus VHH’ controls. On-rates (kon), off-rates (kog), and apparent dissociation constant values (KDS) were calculated by the Octet Data Analysis HT 12.0 software, using a mass transport model to fit the data. Graphs show the fits as well as the raw data after baseline subtraction.

[0133] Figures 3A-3i: Thermal stability of selected anti-IL-13 VHH antibodies.

[0134] VHH antibodies (same as in Figures 2A-2i), were subjected to Differential Scanning Fluorescence (DSF) as detailed in Example 3. Thermal unfolding is measured as an enhanced fluorescence of added SYBR Orange following a stepwise increase in temperature with 532 nmexcitation and a 555 nm long pass filter. The melting temperature (Tm) is defined as the inflection point of the first melting peak.

[0135] Figures 4A-4D: Dose-response of IL-13 reporter cells to recombinant IL-13 cytokine. Figures 4A and 4B - IL-4 / IL-13 bioassay cells (Promega CS2018H) were used according to the manufacturer’s instructions. They were stimulated overnight with increasing concentrations of human IL-13. Stimulation results in the expression of a Luciferase reporter, whose activity was assessed using Bio-Glo-NL™ as substrate and measuring the resulting bioluminescence with a microplate luminometer. The relative light units (RLU) are plotted in Figure 4A. The plot in Figure 4B depicts the range of induction as calculated by dividing the RLU readings at the respective IL- 13 dilution by the RLU value of the minus IL- 13 control. The results are the mean ± SD of one experiment measured in triplicate. The dashed vertical line indicates the IL- 13 concentration that was used in the subsequent VHH neutralization experiments (Figure 5). Figures 4C and 4D - Cells of the HEK-Blue™ IL-4 / IL-13 reporter line were stimulated with increasing concentrations of human IL-13. Stimulation results in the secretion of an embryonic alkaline phosphatase (SEAP) reporter, whose activity was determined colorimetrically using QUANTI-Blue™ as a substrate and measuring the resulting absorbance at 620 nm. The OD620 values are plotted in Figure 4C. The plot in Figure 4D depicts the range of induction as calculated by dividing the OD620 readings at the respective IL- 13 dilution by the OD620 value of the minus IL- 13 control. The results are the mean ± SD of one experiment measured in triplicate. The dashed vertical line indicates the IL-13 concentration that was used in the subsequent VHH neutralization experiments (Figure 6).

[0136] Figures 5A-5F: Neutralization of IL-13 by VHH antibodies assessed by the IL-4 / IL-13 Bioassay reporter cell line

[0137] Plots show the expression of a luciferase reporter by IL-4 / IL-13 Bioassay cells induced with 1.6 nM IL- 13, which had been pre-incubated with indicated concentrations of anti-IL-13 class A and class B VHH antibodies in culture medium. A number of 1.0 indicates (full) induction in the absence of a VHH, and a number of 0 indicates no induction above the background of the minus VHH controls. Figure 5A shows neutralization by first-generation (Bml9 and Bm20 series) class A representatives, compared to a class B member. Figure 5B shows class A member Bm41 A08in comparison to Bml9G08. Figures 5C-5F show class A members of the second and third generation. The results are the mean ± SD of three experiments, each measured in triplicate. For clarity, each graph lists the VHHs in an order of increasing neutralizing potency.

[0138] Figures 6A-6D: IL-13 neutralization potency of VHH antibodies measured by HEK-Blue IL-4 / IL-13 cells

[0139] The graphs show the expression of a phosphatase reporter by HEK-Blue™ IL-4 / IL-13 cells after induction with 160 pM IL- 13, which had been pre-incubated for two hours with indicated concentrations of anti-IL-13 VHH antibodies in culture medium. A number of 1.0 indicates (full) induction in the absence of a VHH, while 0 indicates no induction above the background of the minus VHH controls. Figures 6A and 6B show neutralization by class A members. Figures 6C and 6D show neutralization by class C members and Bm41A08 (class A) is shown for comparison. The clinically used anti-IL-13 monoclonal antibody tralokinumab was included as benchmark in Figures 6B and 6D. Because of its higher molecular weight and the presence of two IL- 13 binding sites in tralokinumab, identical mass concentrations (ng / mL) were compared. The results are the mean ± SD of three experiments, each measured in triplicate. For clarity, each graph lists the VHHs in an order of increasing neutralizing potency.

[0140] Figures 7A-7D: Structural basis for IL-13-neutralisation by the class A VHH antibodies.

[0141] The VHH antibody Bm46H07 was co-expressed with IL- 13 in E. coh. the complex was purified to homogeneity, crystallised, and an x-ray diffraction dataset was collected at a synchrotron source. The corresponding structure was solved by molecular replacement to a resolution of 2.9 A with good crystallographic statistics.

[0142] Figure 7A Previously reported structure of IL- 13 in complex with the IL- 13 and IL-4 receptors (pdb 3BPO; LaPorte et al., 2008). The color code for the individual proteins is indicated.

[0143] Figure 7B Shows the structure of the Bm46H07-IL-13 complex with the IL-13 in cartoon (right side) and the Nanobody in surface presentation (left side). The structure is shown in the same orientation with respect to IL- 13 as Figure 7 A.

[0144] Figure 7C Superposition of the structures shown in Figures 7A and 7B. Note the frontal clash between Bm46H07 and the IL-13Ra receptor, which indicates that Bm46H07-neutralized IL-13 molecules cannot bind their receptor. A minor clash would also prevent binding of the IL-4receptor a. The structure of IL-13-Bm46A10 (not shown) complex is virtually identical, except for an additional hydrogen-bond between CDR3 and IL- 13.

[0145] Figure 7D Panel shows the structure of the Bm46H07-IL-13 complex again, but in a different orientation, with a cartoon representation of the VHH antibody and showing the cation, which is tightly bound by CDR3 and which restrains the conformation of CDR3 to perfect shapecomplementarity with its IL- 13 target.

[0146] Figures 8A-8B: Bm60Gll - a hyperthermostable, picomolar IL-13 binder

[0147] Figure 8A - The thermostabilities of two class A members, Bm60Gll and Bm46A10, were compared by DSF as in Figures 3A-3i.

[0148] Figure 8B - Affinity of Bm60Gll for IL-13, measured by biolayer interferometry as in Figures 2A-2i.

[0149] Figures 9A-9F: Extreme thermostability of Bm60Gll as assessed by ligand binding The BLI assay of Figures 2A-2i and 8B was used to measure the concentration of active VHH that remains after a heat treatment. This approach takes advantage of the fact that the rate, by which the binding signal increases, is directly proportional to the concentration of active binder. lOOpM of the class A VHH antibody Bm46A10 (Figure 9B), Bm46H02 (Figure 9D), and Bm60Gll (Figure 9F) in 50mM Tris / HCl pH 8.0, 300mM NaCl, was heated to 95°C for 10 min, placed on ice, aggregates were removed by centrifugation at 21,000g for 10 min. The supernatant was diluted 1 :5000 in assay buffer (20mM Tris / HCl pH 7.5, 150mM NaCl, 0.02% Tween 20 and 0.1% BSA) to a nominal concentration of 20 nM. The untreated samples were handled the same but without the heating step. Association to IL- 13 (biotinylated and pre-bound to the sensor chips) was allowed for 1200 seconds, followed by a dissociation step of 1800 seconds. Results for untreated samples Bm46A10, Bm46H02, and Bm60Gll are shown in Figure 9A, Figure 9C, and Figure 9E, respectively. Note that the heat treatment caused a severe loss of activity of VHH antibodies Bm46A10 (by 85%, Figure 9B) and Bm46H02 (by 97%, Figure 9D), while Bm60Gll (Figure 9F) remained fully active.

[0150] Figure 10A-10B: Freeze-tolerance of Bm60GllClass A VHH antibodies Bm46A10 (Figure 10A) and Bm60Gll (Figure 10B) were either left untreated or they were repeatedly frozen in liquid nitrogen and thawed in a room temperature water bath. BLI assay was then used to compare their residual binding activity. This revealed that Bm46A10 (Figure 10A) lost 97% of its activity after 10 freeze-thaw cycles, while Bm60Gll remained fully active even after 20 freeze-thaw cycles (Figure 10B). Freezing was at a VHH concentration of 150 pM in storage buffer (50mM Tris / HCl pH 8.0, 300mM NaCl, 250mM sucrose). The BLI assay was performed after a 1:7500 dilution to a nominal VHH concentration of 20 nM in assay buffer (20mM Tris / HCl pH 7.5, 150mM NaCl, 0.02% Tween 20 and 0.1% BSA). 600 s each were allowed for binding and dissociation.

[0151] DETAILED DESCRIPTION OF THE INVENTION

[0152] The present invention relates to stable VHH antibodies recognizing the human IL- 13 polypeptide with high affinity and inhibiting or neutralizing its activity, for use in prevention, treatment and diagnosis of variety of inflammatory, fibrotic and other conditions.

[0153] The VHH antibodies of the present invention were identified in a long process of immunization, testing and selection, overcoming binding, stability and activity issues. The present invention provides thermostable neutralizing VHH antibodies with picomolar affinity to human IL-13. It is remarkable to notice that when compared to tralokinumab, a clinically approved antibody targeting IL-13, some of the VHH antibodies of the present invention have about 100 fold higher affinity to the cytokine.

[0154] The binding data for the novel VHH antibodies of the present invention have been confirmed functionally in cell-based assays forblocking activation of human IL- 13 receptor and structurally by crystallography in complex with the human IL- 13 polypeptide, proposing the mode of binding of the VHH antibodies to their target.

[0155] To generate VHH antibodies that neutralize human IL- 13, the inventors designed and bacterially produced an IL-13 immunogen. The project further included alpaca immunizations, isolation of lymphocytes, amplification of VHH-encoding sequences by nested RT-PCR, immune library construction, phage display using immobilised human IL- 13 as a bait, sequence analysis of the hits obtained, classification according to sequence, as well as recombinant production and in-depth analysis of VHH lead candidates. Figure 1 shows a sequence alignment and a class definition of the leads. Table 1 summarizes their properties, Table 2 lists their complementaritydetermining regions (CDRs) sequences, and Table 3 their entire sequences. Figures 2A-2i, and 8B document affinities of selected VHH for IL- 13 as measured by biolayer interferometry (Abdiche et cd.. 2008), Figures 3A-3i show thermostabilities, measured by differential scanning fluorescence (Goldberg et al., 2011). Figures 4A-4D introduce the cell-based assays used for testing the IL-13 neutralization capacity and potency of the here disclosed VHH antibodies. IL-13 neutralization by various members of the three selected anti-IL-13 VHH classes is shown in Figures 5A-5F and 6A-6D.

[0156] Discovery, characterization, and optimization of class A anti-IL-13 binders

[0157] The Bml9 / Bm20 series were the first generation of anti-IL-13 VHH antibodies and included two major classes of genuine IL- 13 binders. Class A, represented here by the VHHs Bml9G08 and Bm20D05, proved to be the most relevant one, as it could not only bind IL- 13 (Table 1; Figures 2A-2i and 8B), but also block IL- 13 -signalling through the IL-13 / IL4 receptor complex (Figures 5A-5F and 6A-6D). Bm20C02 represents class B. It is a low nanomolar IL-13-binder (Table 1) but fails to neutralize IL- 13 (Figure 5 A), indicating that obtaining neutralizing binders was not a trivial result.

[0158] Indeed, IL- 13 was a challenging target for generating VHH antibodies, because the IL- 13 receptor interface is rather hydrophilic, which makes it very hard for an antagonist with a small footprint to bind very tightly. While Bml9G08 and Bm20D05 were promising leads, with KDs of 3 and 4 nM, they were still not optimal in terms of target affinity (Table 1). Bml9G08 and Bm20D05 were not fully thermostable either, but showed an onset of melting at a Tm of around 57°C (Table 1). Furthermore, they lost binding and neutralization activity during multiple cycles of freezing and thawing (Table 1). The inventors tested several formulations with freeze-protective additives and indeed found that the addition of 15% propylene glycol reduced this freeze-thaw sensitivity. The high osmolarity of such a formulation is, however, prohibitive for the use as an injectable drug. The inventors therefore searched further for better leads.

[0159] This involved a re-immunization of the alpaca with a human IL-13-Fc fusion produced in mammalian cells, the generation of a new immune library and another phage display selection, this time with a stringent off-rate selection, yielding the Bm41 series of anti-IL-13 VHH antibodies. The recovered sequences were again dominated by class A VHH antibodies, but this class had diversified and showed between 7 and 24 differences from the Bml9G08 sequence (Figure 1). These differences occurred in CDR1, CDR2, CDR3, and the VHH framework. Extensive testing of recombinantly produced Bm41 series VHHs identified Bm41 A08 as the mostpromising lead. Compared to Bml9G08, Bm41A08 showed an improved thermostability, with a melting temperature that had increased by 12°C to 69°C (Table 1, Figures 3A-3i). Likewise, its affinity for IL-13 had improved 10-fold to aKo of 320 pM (Table 1, Figures 2A-2i). Furthermore, it was a very potent inhibitor of IL-13 in cell-based reporter assays (Figures 5A-5F), showing complete neutralization at a stoichiometric ratio to the added IL-13 (1.6 nM), indicating that neutralization with Bm41 A08 was not affinity-limited in this experimental setup (IL-13 Bioassay reporter cell line). The alternative HEK-Blue IL- 13 reporter cell line can be used already with 10-fold less IL-13 (Figures 4C and 4D). Here, complete neutralization was evident at a Bm41 A08 concentration as low as 0.4 nM (Figures 6A-6D). This corresponds to 6 ng / ml and is 100-fold less than required to see the same effect with the approved therapeutic anti-IL-13 monoclonal antibody tralokinumab.

[0160] Still, Bm41 A08 progressively lost binding and neutralization activity during repeated freeze-thaw cycles (Table 1, see also below). Furthermore, this VHH contains an unpaired cysteine (C97), which might be subject to deleterious oxidation. Therefore, the inventors created another phage display library. The input included the entire pool of class A members obtained from the previous selection. Furthermore, the entire pool was randomly mutagenized at the C97 position and subjected to extensive DNA-shuffling and random mutagenesis. Stringent phage display selection retrieved then the Bm46 generation of anti-IL-13 VHH antibodies. These included excellent, picomolar IL-13 binders, including Bm46A10, Bm46H07, and Bm46G09 (Table 1 and Figures 2A-2i). In particular, Bm46A10 bound virtually irreversibly to IL-13, with an estimated KD of 10 pM or better (Figures 2A-2i). Plus, in terms of IL- 13 blockage, it appeared to have reached an optimum (Figures 6A, 6B), reaching an essentially full neutralisation at a stoichiometric ratio to the IL-13 concentration added in the assay (160 pM). However, the problem of freeze-thaw sensitivity and limited thermostability (Tm=65°C) still persisted.

[0161] The inventors performed at this point a crystallographic analysis of IL-13 complexed to either Bm46H07 or Bm46A10. This revealed not only that the VHH binding causes a frontal clash with the IL-13Ra, explaining the mode of action (shown for Bm46H07 in Figures 7A-7D). But also, it revealed a remarkable mode of interaction between CDR3 and its target. The long CDR3 loop contains a tightly bound cation - complexed through the negatively charged CDR3 sidechains. This cation-binding restrains the conformation of CDR3 in a target-complementary shape. This mode of binding can thus be considered a defining feature of the class A anti-IL-13 VHH antibodies.The x-ray diffraction pattern alone cannot discern which cation is bound, but Na+, K+, Ca2+or Mg2+would be compatible in terms of geometry and electron density. Of these, only Na+ was contained in the purification buffer and the precipitants. It seems therefore likely that the solved structures indeed show a Na+-complexed CDR3 loop. Yet, the structure probably allows for a cation exchange.

[0162] Nevertheless, the Bm46 generation still suffered from limited thermostability and sensitivity to freeze-thaw cycles. The inventors observed, however, that the presence of Ca2+ions increased their melting temperatures by up to 10°C but at the same time decreases their affinity for IL- 13. They reasoned that the ideal lead should bind IL- 13 very tightly also in the presence of Ca2+, and re-selected the Bm46 pool accordingly by another round of phage display in the presence of 2mM each Mg2+and Ca2+and a lowered Na+concentration, under conditions of on-rate selection. This yielded the Bm60 VHH generation.

[0163] A comprehensive testing of this generation finally identified Bm60Gll as a high-affinity, fully thermostable (Tm >95°C; Table 1, Figures 8A and 9A-9F) and highly freeze-tolerant anti-IL-13 lead (documented in Figures 8A-8B, Figures 9A-9F and Figure 10A-10B). Its CDR3 is identical to that of Bm46A10 (Figure 1; Table 2), however, it contains several stabilizing substitutions, including a T59Y exchange, which provides a tyrosine to pack against CDR3, restraining its conformation, as well as changes in the hydrophobic core that allow for a better packing of sidechains and an additional, buried hydrogen bond. Compared to Bm46A10, Bm60Gll shows a slight reduction in affinity for IL- 13 and in anti-IL-13 potency, but still, the Ko~50pM (Table 1; Figure 8B) and a near complete block of IL- 13 effects in the reporter cell assay at a concentration of 400 pM (Figure 6A) suggest that Bm60Gll is an excellent clinical candidate for treating conditions caused by IL- 13 dysregulation. In fact, a 100-fold higher concentration of the approved anti-IL-13 monoclonal antibody tralokinumab was required for a similar neutralizing effect (Figure 6B).

[0164] Class C IL-13-neutralizing VHH antibodies

[0165] The Bm41 selection identified a third class of IL- 13 binders, now referred to as class C, and exemplified by Bm41F06 and Bm41Hll. They have a second disulfide bond between CDR2 and CDR3 (Figure 1), which provides additional stabilization. Accordingly, class C members are hyperthermostable (Tm>95°C; Table 1 and Figures 3A-3i). Furthermore, they have picomolaraffinities for IL-13 (Table 1 and Figures 2A-2i), block IL-13 effect at sub-nanomolar concentration, and are thus 100-fold more potent than tralokinumab (Figures 6C-6D).

[0166] A non-limitative list of VHH antibodies of the present invention and their affinities and utility for IL-13 neutralization is shown in the Tables 1-3.

[0167] Table 1: Anti-IL13 VHH antibodies and their properties

[0168] <

[0169] >

[0170] >

[0171] >

[0172]

[0173] *Example 2 and Figures 2A-2i and 8B; ^Neutralization Example 4 and Figures 4-6, ***Thermostability Example 3 and Figures 3A-3i, 8A, 9; ****Figures 10A-10B.

[0174] #It was also found, in additional engineering and selection experiments, that to maintain the hydrophobic core of this VHH antibody, the unpaired cysteine residue at position 97 may be replaced with Vai (V), or Leu (L), or less preferred with He (I), Met (M), Thr (T) or Ala (A).

[0175] Therapeutic uses

[0176] The here disclosed IL- 13 -neutralizing VHH antibodies may be used for the treatment of any condition that is caused or aggravated by excessive IL-13 signalling. The use against atopic dermatitis represents one indication, based on the clinical success of anti-IL-13 antibody (tralokinumab) for treating atopic dermatitis (Silverberg et al., 2021; Wollenberg et al., 2021; Blair, 2022)). Similar results were obtained with another antibody to IL- 13, lebrikizumab (Silverberg etal., 2023).

[0177] The use of tralokinumab as a monotherapy against asthma bronchiale did not yield consistently favourable results (Panettieri et al. , 2018) but antagonizing IL- 13 for treating respiratory diseasesmight still be beneficial in combination therapies or with inhalation of nanobodies. More encouraging results were obtained using lebrikizumab against asthma (Corren et al., 2011). Inhaled antibodies against IL- 13 were previously suggested as an attractive alternative to systemic application for treating asthma (Burgess etal., 2018; Nair and O'Byrne, 2019).

[0178] Additional autoimmune and autoinflammatory diseases remain to be explored as to their susceptibility towards anti-IL-13 VHH antibodies. Examples include rheumatoid arthritis (Iwaszko et al., 2021), ulcerative colitis Hoving, 2018) and food allergy (Brandt et al., 2009; Noah et al., 2019).

[0179] Besides applications in autoimmune diseases, antagonizing IL- 13 by VHH antibodies may also prove useful when treating cancer. For instance, Hodgkin’s lymphoma has been described as fuelled by autocrine IL- 13 signalling (Skinnider etal., 2002), and a specific subtype of Hodgkin’s disease was found to depend on IL-13 signalling, further suggesting this lymphoma as a possible disease target for VHH antibodies directed against IL-13. Other malignant diseases may also be susceptible to such treatment (Suzuki et al., 2015)

[0180] Antagonizing IL- 13 may also reduce fibrosis-associated diseases (Bernstein etal., 2023), e.g. the fibrosis of the lung or the liver in response to chronic inflammation. IL- 13 mediates liver fibrosis in the context of Schistosomiasis (Mentink-Kane and Wynn, 2004). Concerning idiopathic pulmonary fibrosis, although previous clinical trials using antibodies to IL-13 against lung fibrosis were unsuccessful (Parker et al., 2018), this might change when using direct application of IL- 13 -neutralizers through respiratory delivery by inhalation and / or when using a VHH of higher neutralization potency such as the one disclosed here.

[0181] Another potential application of anti-IL-13-nanobodies is in the manipulation of wound healing. Since IL- 13 mediates (at times excessive) collagen production and skin fibrosis (Nguyen et al., 2020), VHH antibodies against IL- 13 might be useful to prevent scarring, keloids and other aberrant wound healing processes.

[0182] Nanobodies against IL- 13 may be useful for patients that have / had been treated with tralokinumab or other antibodies to IL- 13 and have developed anti-drug antibodies that renders this treatment ineffective. The so far best candidates are the class A member Bm60Gll and the class C member Bm41Hll, both of which outperform tralokinumab in terms of neutralisation potency and by their hyperthermostability.Hyperthermostability is a highly desired feature of therapeutic proteins. It correlates with stability during production, purification, and storage. This stability is not only required to minimize the loss of activity but also to avoid aggregation, which would make the protein drug highly immunogenic, thereby eliciting an anti-drug antibody response and in the worst case allergic or anaphylactic reactions. Aggregation-resistance and a very low level of immunogenicity is particularly relevant for drugs that have to be given repeatedly, in particular for treating chronic diseases.

[0183] Systemic application probably requires a fusion of a module that extends the plasma half-life of the VHHs. An exemplary format is a fusion to a human Fc fragment without effector function, for example a human IgG4 Fc fragment. For application by inhalation, for inflammatory conditions along the respiratory system, such as asthma, one would use the non-fusion VHH modules, whose superior stability will minimise the risk of denaturation at the air-water interface. Also, monomers are less likely than dimers to elicit anti-drug antibodies. The same holds true for topical application on the skin.

[0184] As it is of great therapeutic value to block IL-4, IL- 13 and TSLP signaling with a single biological molecule, the anti-IL-13 VHH antibodies of the present invention may also be part of a tandem of two or three fused single domain antibodies, blocking, in addition to IL-13, also IL-4, and / or TSLP

[0185] VHH antibodies

[0186] The sequences of the CDRs of selected VHH antibodies of the present invention are listed in the following Table 2 while Table 3 lists the sequences of the full-length VHH antibodies.

[0187] Table 2. CDR sequences of selected VHH antibodies

[0188]

[0189]

[0190] Table 3. Sequences of selected VHH antibodies

[0191]

[0192] The present invention relates to a VHH antibody, which is a monovalent heavy chain-only antibody comprising a CDR1 domain, a CDR2 domain and a CDR3 domain linked by framework regions including, but not being limited to, whole VHH antibodies, e.g. native VHH antibodies comprising framework regions derived from camelids, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, e.g. a fusion protein with an immunoglobulin or non-immunoglobulin peptide or polypeptide, as long as it shows the properties according to the invention, namely binding to human IL- 13 and neutralizing or inhibiting its activity.

[0193] There are several methods known in the art for determining the CDR sequences of a given antibody or VHH antibody, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy chain variable regions can be made according to any method known in the art, including, but not limited to, the methods known as KABAT, Chothia, and IMGT. Common determination methods like KABAT would exclude essential parts of the variable regions as a VHH antibody binding site (paratope) often includes residues of the scaffold and outside a narrowly defined CDR region. This is also a difference between nanobodies and traditional antibodies. Other methods of identifying a binding site and CDR sequences of VHH antibodies include the use of custom reference databases utilizing large collection of VHH antibody sequencing data. A selected set of CDRs may include sequences identified by more than one method. CDRs may also be defined through a multiple alignment (with many other VHH antibodies), to identify the hot-spots of variability and relate them to a standard VHH antibody structure. It is also possible to define CDRs by analyzing the structure of the VHH antibody and deciding what is a loop and what is the antibody’s scaffold. In some cases, CDR-adjacent residues are also variable, and are therefore included in the CDR definition. According to some embodiments of the present invention, the CDR sequences of the VHH antibodies variable regions are determined using custom reference database, containing sequencing data of >10000 VHH antibodies.

[0194] The present invention is also directed to a covalent or non-covalent conjugate of a VHH antibody molecule to a heterologous moiety. In some embodiments, the heterologous moiety is a non-proteinaceous molecule, for example, a labeling group, a capture group such as a solid phasebinding group, or an effector group such as a toxin. For example, the heterologous moiety may be elected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, and a nucleic acid such as an oligonucleotide. In other embodiments, fusion polypeptides comprising at least one VHH antibody that targets IL- 13 anda heterologous polypeptide, are provided. The heterologous polypeptide, e.g., a Fc fragment, in particular a human IgG4 fragment, or a human serum albumin or a fragment thereof, may be used, according to some embodiments, to prolong the half-life of the active VHH antibody. Fusion proteins of a VHH antibody that targets IL- 13, with a different VHH antibody that targets IL-13 and / or with a VHH antibody that targets a different human protein are also provided. Human cytokines involved in inflammation or fibrosis, and those that utilize the IL-4R are particularly relevant as targets.

[0195] The VHH antibody of the present invention is particularly a monoclonal VHH antibody characterized by a specific amino acid sequence. The VHH antibody may be produced in a prokaryotic host cell, a yeast cell or a mammalian cell. In certain embodiments, the VHH antibody is non-glycosylated. In some embodiments, a glycosylation site in a parent VHH antibody sequence is mutated to eliminate a predicted glycosylation. In particular embodiments, the mutation comprises substitution of an Asparagine (Asn, N) residue in an N-glycosylation site to prevent potential glycosylation of the VHH antibody. According to some embodiments, the potential N-glycosylation site NxT in a VHH antibody selected from Bm41F06 and Bm41Hll (class C), is mutated to eliminate the glycosylation site.

[0196] In certain embodiments, the VHH antibody is glycosylated, wherein a carbohydrate structure may be derived from a glycosylation site present in or introduced into the VHH sequence and / or from a fusion partner.

[0197] A VHH antibody according to the present invention is structurally characterized by (i) a CDR3 sequence, (ii) a combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (iii) by a complete VHH sequence, or (iv) by competition with a specific reference antibody. Specific CDR and VHH sequences are provided in Tables 2 and 3, and in Figure 1.

[0198] According to the present invention, sequences related to the above sequences are encompassed. These related sequences are defined by having a minimum identity to a specifically indicated amino acid sequence, e.g., a CDR or VHH sequence. This identity is indicated over the whole length of the respective reference sequence and may be determined by using well-known algorithms such as BLAST.

[0199] In particular embodiments, a related CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated CDR3 sequence, e.g., a substitution of 1, 2, or 3 amino acids.In particular embodiments, a related combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, e.g., a substitution of 1, 2, 3, 4, 5 or 6 amino acids by different amino acids.

[0200] In particular embodiments, a related VHH sequence has an identity of least 70%, at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence, e.g., a substitution of 1, 2, 3, 4, 5 or up to 20 amino acids.

[0201] Further, the invention refers to a VHH antibody, which competes with a specific VHH antibody disclosed herein for the binding to a human IL- 13 polypeptide. In certain embodiments, a competing VHH antibody binds the same or an overlapping epitope on the human IL- 13 polypeptide. Competition may be determined by label-free biolayer interferometry performed as a cross-competition or epitope binning assay using a label-free detection system, e.g., an Octet® system from Sartorius, according to the manufacturer's instructions.

[0202] In particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1, CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by another amino acid, while preserving structural integrity and epitope-binding of the VHH antibody. These exchanges can be conservative (i.e., by a similar amino acid) or nonconservative.

[0203] In further particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1, CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by a conservative amino acid substitution, i.e. a substitution of an amino acid by another amino acid with similar biochemical properties, for example a substitution of an aliphatic amino acid, e.g. Gly, Ala, Vai, Leu, or He, for another aliphatic amino acid; a substitution of a basic amino acid, e.g. His, Lys or Arg, against another basic amino acid or against Met; a substitution of an acidic amino acid or an amide thereof, e.g., Asp, Glu, Asn or Gin, against another acidic amino acid or an amide thereof; a substitution of an aromatic amino acid, e.g., Phe, Tyr or Trp, against another aromatic amino acid.

[0204] In further particular embodiments, the VHH antibody comprises a sequence selected from SEQ ID NOs: 21-30 and 32-33, or a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of any of these sequences are replaced by another amino acid.The invention provides two classes of VHH antibodies based on sequence similarity (80% or higher sequence identity for VHH antibodies of the same class) and on data gained from crystal structures of complexes of class A VHH antibodies with human IL-13.

[0205] Further, the present invention relates to a nucleic acid molecule, e.g., a DNA molecule, encoding a VHH as indicated above, or a vector, comprising said nucleic acid molecule as indicated above in operative linkage with an expression control sequence, particularly with a heterologous expression control sequence. Furthermore, the invention relates to a cell comprising a nucleic acid molecule or a vector as described above. Vectors for the recombinant production of VHH antibodies are well-known in the art. In certain embodiments, the vector is an extrachromosomal vector. In other embodiments, the vector is a vector for genomic integration. The cell may be a known host cell for producing antibodies or antibody fragments, e.g., a prokaryotic cell such as an E. coli or a Bacillus sp. cell, a yeast cell, particularly a Pichia yeast cell, an insect cell or a mammalian cell, e.g., a CHO cell, or a plant cell. In certain embodiments, the cell comprises the nucleic acid or the vector extrachromosomally. In other embodiments, the cell comprises the nucleic acid or the vector integrated into the genome, e.g., as a genomically integrated expression cassette.

[0206] Still a further aspect of the present invention is a method of recombinantly producing a VHH antibody by growing a cell as described above in a culture medium and obtaining the VHH antibody from the cell or the culture medium. Suitable culture media and culture conditions are well known in the art.

[0207] Binding to and neutralization of human IL-13

[0208] The VHH antibodies of the present invention bind to the human IL-13 polypeptide. The inventors have identified VHH antibodies, which bind with high affinity to human IL- 13 polypeptides as shown in Table 1.

[0209] In the context of the present disclosure the term “human IL-13” encompasses a human IL-13 identified by UniProt Accession No. P35225, corresponding to a polypeptide of 146 amino acids having the sequence:

[0210] MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQ KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQ FSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN (SEQ ID NO: 44).It should be noted, however, that the term “human IL-13” also encompasses naturally occurring variants of human IL-13 polypeptide and genetically modified constructs as described herein, and that the actual IL- 13 protein lacks the secretion signal (residues 1-24 of the above-mentioned UniProt sequence) that is cleaved during translocation into the endoplasmic reticulum and thus during an early stage of the secretion process.

[0211] The inventors have performed their immunization, selection, binding experiments and crystallographic analyses with IL- 13 constructs, including genetically modified constructs, using the following sequences or fusions thereof:

[0212] human IL-13 polypeptide:

[0213] GSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSIDLTAGMYCAALESLINVS GCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGR FNGSGTEGSEGGEGGEGGEGGEDEDED (SEQ ID NO: 45);

[0214] human IL-13 fused to human Fc domain, an avidin tag (Avi-tag) and a Histidine-6 tag (Hise-tag):

[0215] ETSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINV SGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG RFNGTENLYFQGPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDV SQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCK VHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVE WEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHN HYTQKSISRSPGKASGSEGSDGLNDIFEAQKIEWHEGSGTSHHHHHH (SEQ ID NO: 46);

[0216] Signal peptide-cleaved human IL- 13 fragment:

[0217] LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAA LESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLK KLFREGQFN (SEQ ID NO: 47).

[0218] In certain embodiments of the present invention, the VHH antibody binds to human IL- 13 with a binding affinity expressed as dissociation constant KD of about 1 nM or less, about 100 pM or less, or about 50 pM or less. The binding affinity and the methods of their measurements may be determined using methods known in the art, or as described herein in detail in the Examples and Figures. Particular measurement methods use biolayer interferometry (BLI) instruments. According to some embodiments, biotinylated VHH antibodies are immobilized on the sensorchips and the binding of the human IL-13 polypeptide (that is not fused to an Fc domain) to them is measured. In another setup, IL- 13 is immobilized on the chip and binding of monomeric VHH antibodies to them is measured. According to particular embodiments, the binding affinity of a monomeric VHH, expressed as a dissociation constant KD, to an immobilized human IL- 13 is determined.

[0219] Crystal structures of VHH antibody-IL-13 complexes (Figures 7A-7D) document a deep understanding of the mode of binding of the VHH antibodies to their target and to elucidate their mode of action in neutralizing the activity of IL-13.

[0220] The VHH antibodies of the present invention are capable of neutralizing the binding of human IL- 13 to a human IL- 13 receptor and are capable of preventing or inhibiting activation of the receptor. The inventors have identified VHH antibodies, which neutralize human IL- 13 as shown in Table 1 and Example 4.

[0221] Stability

[0222] For the intended therapeutic application, the anti-IL- 13 VHH antibodies should not only be highly potent in neutralization, but also, they should be developable as biological drugs. This includes that they are stable enough to survive a lengthy, large-scale production process as well as transportation and storage (ideally for years in liquid, semi-liquid or solid formulations) without aggregation or loss of activity.

[0223] A good predictor for stability is thermostability, which can be measured, e.g., by thermal shift assays or specifically by differential scanning fluorimetry. The inventors have identified several thermostable or hyperthermostable VHH antibodies as shown in Table 1.

[0224] In a particular embodiment, the invention relates to a VHH antibody, which is stable, particularly thermostable, or hyperthermostable. Preferably, the VHH antibody has a melting point (melting temperature; Tm) of at least about 65°C, of at least about 80°C, of at least 90°C or of at least about 95°C, and / or an aggregation temperature of at least about 50°C, of at least about 60°C, of at least 70°C or of at least about 80°C when measured under non-reducing conditions.

[0225] Melting and aggregation temperatures are determined as described herein. According to some embodiments, a stable VHH antibody has a melting point (melting temperature) of at least about 65°C and / or having an aggregation temperature of at least 50°C, and an hyperthermostable VHH antibody has a melting point (melting temperature) of at least 95°C, and / or an aggregation temperature of at least about 80°C when measured under non-reducing conditions. It should benoted, however, that the outcome of a stability measurement depends on the conditions. Aggregation, for example, is favored by a high protein concentration, and / or by a pH close to the isoelectric point of the protein. The stability is also influenced by the buffer composition and additives used with the tested ingredient.

[0226] Sets of VHH antibodies

[0227] In a further aspect, the present invention relates to a set comprising at least 2, 3, 4 or more of the above VHH antibodies. In such a set, the individual VHH antibodies are present in suitable molar ratios. Typically, the molar ratios are in the range of about 2:1 to about 1:2, particularly about 1.5 : 1 to about 1:1.5, even more particularly about 1 : 1. In certain embodiments, the VHH antibody set may comprise a single composition wherein the VHH antibodies in said set consist of a predetermined number of different species of VHH antibodies as described above. The VHH antibody set may comprise a plurality of compositions each comprising a different species of VHH antibody as described above.

[0228] Monovalent, bivalent and multivalent VHH antibodies

[0229] In certain embodiments, the VHH antibody of the present invention is in a monovalent format, i.e., it has a single binding site for a human IL-13 polypeptide. In these embodiments, the VHH antibody may be present as such or covalently or non-covalently attached to a heterologous moiety, e.g., a peptidic or non-peptidic moiety. Several identical VHH antibodies, or a different VHH antibody that recognizes the same epitope of human IL-13, or that recognizes different epitopes of IL- 13 may be coupled together.

[0230] In further embodiments, the VHH antibody of the present invention is in a multimeric, e.g., dimeric, or trimeric format. In these embodiments, several VHH antibody units may be covalently or non-covalently attached to each other together via a linker and / or a multimerization, e.g., dimerization or trimerization moiety. In some embodiments, the VHH antibodies may be attached to each other in a tandem format, namely one terminus of a VHH antibody coupled to a terminus to another (identical, similar, or different) VHH antibody, directly or through a linker, i.e., the C-terminus of one VHH is fused to the N-terminus of the next VHH. In other embodiments, the VHH antibodies are couples in a branched multimeric format.

[0231] In certain embodiments, the VHH antibody is a homodimeric VHH antibody, wherein a VHH antibody unit is covalently attached to a dimerization moiety, e.g., an immunoglobulin Fc fragment. In other embodiments, the VHH antibody is in a multimeric form comprising at leasttwo identical or different VHH antibodies, in a tandem, branched or other format, wherein at least one of the VHH antibodies binds human IL-13. The VHH antibodies in the dimeric or multimeric format are attached directly or through a linker or a heterologous moiety. According to some embodiments, all VHH antibodies of the dimer or multimer are capable of binding human IL- 13. According to other embodiments, at least one of the VHH antibodies is capable of binding a polypeptide other than IL- 13, e.g., to a human cytokine. According to some specific embodiments, the polypeptide capable of binding polypeptide other than IL-13 is selected from IL-4, TSLP, IL- 17 and human serum albumin.

[0232] In certain embodiments, the VHH antibody is a heterodimeric or heteromultimeric VHH antibody, particularly a covalently linked VHH heterodimer or heterotrimer comprising a first VHH antibody, a second VHH antibody, and optionally a third VHH antibody, wherein the first VHH antibody and the second and / or third VHH antibody bind to different epitopes on IL-13 or wherein the first VHH antibody binds to IL-13 and the second and third VHH antibody binds to a different target, for example a human cytokine involved in inflammation, autoimmunity or fibrosis. The heterodimeric or heteromultimeric antibodies may be in branched (for example using an IgG Fc fragment), or in tandem format. Heterodimeric or heteromultimeric VHH antibodies in tandem format may also be fused to an Fc fragment or to a different carrier molecule.

[0233] Nucleic acid sequences

[0234] In some embodiments of the present invention, a recombinant construct comprising a nucleic acid sequence encoding a VHH antibody specific to IL- 13 is provided, wherein the nucleic acid sequence is operatively linked to at least one transcription control element to form a recombinant molecule, also referred to herein as a recombinant construct.

[0235] The nucleic acid sequence may include DNA, RNA, or their derivatives. An isolated nucleic acid sequence encoding a VHH antibody specific to IL- 13 may be obtained from its natural source, either as an entire (i.e., complete) gene or a portion thereof. A nucleic acid molecule can also be produced using recombinant DNA technology (employing well-known nucleic acid cloning techniques such as polymerase chain reaction (PCR) amplification) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and / or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional VHH antibody that targets IL- 13 or an active fragment or a conjugate thereof.A nucleic acid sequence homolog can be purchased as a gene synthesis or produced using a number of methods known to those skilled in the art. For example, nucleic acid sequences can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, (PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid.

[0236] The phrase "operatively linked" refers to linking a nucleic acid sequence to a transcription control sequence in such a manner that the encoded protein molecule is able to be expressed when the nucleic acid sequence is transfected (i.e., transformed, transduced or transfected) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.

[0237] Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Transcription control sequences include those which function in animals, yeast, bacteria, and preferably in animal cells. Particular transcription control sequences include, but are not limited to RSV control sequences, CMV control sequences, retroviral LTR sequences, SV-40 control sequences and beta-actin control sequences as well as other sequences capable of controlling gene expression in eukaryotic cells.

[0238] It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transfected nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of secretion. Post-translational modifications, related to secretion, include cleavage of the signal peptide and formation of a disulfide bond. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include,but are not limited to, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals, modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts, or operatively linking nucleic acid molecules to high-copy number plasmids in E. coli. In the case of mammalian expression, one would introduce an intron that enhances transcript stability and export from the cell nucleus.

[0239] The DNA molecules and constructs of the present invention encode according to some embodiments, antibodies that bind to and target human IL-13, or a conjugate thereof, wherein the VHH antibody or conjugate comprises a set of 3 CDR sequences. Representative CDR sequences are presented in Table 2. Sequences of VHH antibodies encoded by the DNA constructs of the present invention are provided in Table 3. Non-limiting examples of DNA sequences encoding the VHH antibodies are provided in SEQ ID NOs: 34-43.

[0240] Production of VHH antibodies

[0241] VHH antibodies may be produced using any method known in the art for producing proteins, antibodies and nanobodies. VHH antibodies including monomeric and multimeric VHH antibodies may be produced as described in WO 2022 / 023483, WO 2022 / 023484 and WO 2024 / 141567, the contents of which are herein incorporated by reference, or by other methods well known in the art.

[0242] Typically, the VHH antibodies are produced recombinantly in a prokaryotic or eukaryotic host cell system or host organism, such as a bacterium, a yeast, a plant cell or in a mammalian cell. For this purpose, a nucleic acid molecule encoding the VHH antibody is introduced into the host cell or host organism and expressed in the host cell or host organism. The nucleic acid molecule may encode a monomeric VHH antibody or a subunit of a multimeric VHH antibody.

[0243] In a particular embodiment, the VHH antibody is recombinantly produced in a bacterium, e.g., E. coli or Bacillus. Expression in a bacterium may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or secretory expression and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression to the periplasm and / or into the culture medium.In a further particular embodiment, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism, preferably in yeast, e.g., in Pichia pasloris. Saccharomyces cerevisiae. or Hansenula polymorpha, or in an animal cell, particularly in a mammalian, e.g., human or a hamster cell. For example, expression in a eukaryotic host cell or host organism, e.g., yeast may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or preferably secretion from the host cell and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression into the culture medium. Therapeutic applications and methods of use

[0244] Still a further aspect of the present invention is the use of a VHH antibody as described above in medicine, particularly for therapeutic and / or in vitro or vivo diagnostic use. In certain embodiments, the VHH antibody is used in human medicine.

[0245] The VHH antibody of the present invention is useful in the prevention or treatment of a disorder or a pathology caused by and / or associated with dysregulation of IL-13. According to some embodiments, the disorder or pathology is caused by or associated with an overexpression or overactivity of IL-13.

[0246] Several disorders associated with IL-13 and IL-4 might become amenable to treatment with the IL-13 antagonists of the present invention. These include but are not limited to Th2 diseases such as asthma, and eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, and different types of cancers, including but not limited to Hodgkin’s lymphoma. In addition, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-IL-4Ra, may be amendable with the anti-IL-13 VHH antibodies of the present invention, optionally together with anti TSLP, and / or anti-IL-4.

[0247] According to some embodiments, the disease or disorder is an inflammatory and / or fibrotic disorder.

[0248] According to some embodiments, the disease or disorder is a skin fibrosis or disorder. According to some embodiments, the skin fibrosis or disorder is selected from atopic dermatitis skin eczema and aberrant wound healing process, e.g., scarring or keloids.According to some embodiments, the disease or disorder is asthma, particularly allergic asthma, moderate asthma or severe asthma.

[0249] According to some embodiments, the disease or disorder is pulmonary or liver fibrosis, for example in response to chronic inflammation, or liver fibrosis in the context of schistosomiasis. According to some embodiments, the disease is idiopathic pulmonary fibrosis.

[0250] In therapeutic applications, the VHH antibody is administered in an effective amount to a subject in need thereof, particularly to a human subject. The dose will depend on the specific type of agent, e.g., monovalent VHH antibody or multimeric VHH antibody, the type of disease, and the route of administration.

[0251] Typically, the VHH antibody is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable diluent, salt or excipient. Examples of suitable diluents and excipients for formulating antibodies or antibody fragments are well-known in the art.

[0252] The pharmaceutical compositions for use, according to the present invention may be administered as part of a regimen comprising administration or applying of at least one additional treatment. The present invention further provides methods of treating diseases and disorders associated with dysregulation, expression, overexpression activity or overactivity of human IL-13, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising an effective amount of a VHH antibody that neutralizes IL- 13 receptor activation.

[0253] Diseases and disorders associated with IL-13 and IL-4 that are amenable to treatment with the IL-13 antagonists of the present invention include but are not limited to Th2 diseases such as asthma, and eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, and different types of cancers, including but not limited to Hodgkin’s lymphoma. In addition, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-IL-4Ra, may be amendable with the anti-IL-13 VHH antibodies of the present invention, optionally together with anti-TSLP, and / or anti-IL-4.

[0254] According to some embodiments, the disease or disorder is an inflammatory and / or fibrotic disorder.According to some embodiments, the disease or disorder is a skin fibrosis or disorder. According to some embodiments, the skin fibrosis or disorder is selected from atopic dermatitis skin eczema and aberrant wound healing process, e.g., scarring or keloids.

[0255] According to some embodiments, the disease or disorder is allergic asthma, moderate asthma or severe asthma.

[0256] Any mode of administration may be used in the methods of the present invention, depending on the disorder and subject to be treated. According to some embodiments, administration is systemic, by injection or infusion. According to other embodiments, the administration is locally to a site of a subject in need of such treatment. This includes but is not limited to topical administration and pulmonary or nasal administrations.

[0257] The term “respiratory delivery” refers to administration of a pharmaceutical composition to the respiratory track, for example by nasal and pulmonary administration. Pharmaceutical compositions administered by these routes are typically formulated as mist, aerosol, spray or suspension, but additional formulations are possible. Administration is typically aided using a device such as metered-dose inhaler, sprayer, nebulizer and the like.

[0258] The terms “administering locally”, or “local administration” mean that the VHH antibody is not administered systematically. The terms include in particular topical application on the surface of the skin, particularly on a defined area of the skin, or on a wound.

[0259] The treatment period is ideally of sufficient time to provide an improvement in the disease or disorder treated. The treatment period can be at least 1 day, at least 3 days, 1 week, at least one month, at least 2, 3, 4, 5 or 6 months, or more. The treatment period may extend over multiple weeks, months or multiple years. The pharmaceutical composition may be administered daily, once every other day, once every three days, once a week, a month, a year, or as long as an improvement is achieved.

[0260] While the subject for the pharmaceutical methods may be of any suitable age, the subject is, in some embodiments, a child, an adult or a geriatric subject.

[0261] The terms “treat”, "treatment" and "treating" may be used interchangeably herein refers to amelioration or reversal of at least one measurable physical parameter related to the amendable disease or disorder. This includes but is not limited to inhibiting, preventing or arresting the development of a disease or disorder and / or causing the reduction, remission, or regression of a disease or disorder.The phrase "inflammatory disease or disorder" used herein refers to a disease, condition or disorder associated with inflammation. The term "inflammation" as used herein refers the process by which a subject's immune system coordinates a response to tissue damage, infection, antigenic challenge, etc. Inflammation may be associated with an increased blood supply to the tissue, increased capillary permeability in the tissue and / or increased leukocyte migration to the tissue. The term “fibrotic disease or disorder” refers to a pathological feature defined by the accumulation of excess extracellular matrix (ECM) components, such as collagen and fibronectin, in and around inflamed or damaged tissue (Wynn and Ramalingam, 2012). Fibrosis affects nearly every tissue in the body and can lead to permanent scarring, organ malfunction and, ultimately, death, as seen in end-stage liver disease, kidney disease, idiopathic pulmonary fibrosis (IPF) and heart failure. Fibrosis is also a major pathological feature of many chronic autoimmune diseases, including scleroderma, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus. Fibrosis also influences tumor invasion and metastasis, chronic graft rejection and the pathogenesis of many progressive myopathies.

[0262] Pharmaceutical compositions

[0263] The present invention provides pharmaceutical compositions comprising as an active agent at least one VHH antibody that neutralize IL-13, or a conjugate or fusion thereof, and a pharmaceutically acceptable diluent, salt or excipient.

[0264] The VHH antibodies of the present invention may be formulated in pharmaceutical compositions of any form, according to the intended use. Suitable forms of pharmaceutical compositions according to the present invention include liquid, semi liquid, solid and semi solid formulations. Pharmaceutical composition according to the present invention may be formulated in a form selected from the group consisting of a solution, suspension, spray, aerosol, powder, and a patch. Each possibility represents a separate embodiment of the invention.

[0265] According to some embodiments, the VHH antibodies are formulated for systemic administration, e.g., by injection or infusion.

[0266] According to other embodiments, the VHH antibodies are formulated for administration by respiratory delivery, for example inhalation as mist, aerosol or spray.

[0267] According to yet other embodiments, the VHH antibodies are formulated for local or topical administration.According to some embodiments, the pharmaceutical composition further comprises one or more excipient or buffer. According to some embodiments, the pharmaceutical composition comprises an excipient selected from the group consisting of pH buffering agents, preservatives, chelating agents, tonicity agents, humectants, salts, and antioxidants.

[0268] The term “pharmaceutical composition” as used herein refers to a composition suitable for preventing or treating a disease or disorder, caused by and / or associated with an activity or overactivity of IL-13. Particularly, the pharmaceutical compositions are useful in the prevention or treatment of an inflammatory, fibrotic, autoimmune, malignant and other diseases and disorders as disclosed above.

[0269] The term “pharmaceutical acceptable” as used herein mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans. The constituents of the pharmaceutical compositions of the present invention are all pharmaceutically acceptable agents.

[0270] The composition, if desired, can also contain minor amounts of or pH buffering agents such as acetates, citrates or phosphates. Preservative such as benzyl alcohol or methyl parabens; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or trehalose or sucrose, are also envisioned.

[0271] Other optional excipients include, but are not limited to, humectants such as water-soluble liquid polyols, e.g., glycerin, sorbitol, propylene glycol, and mixtures thereof; antioxidants and mixtures thereof.

[0272] Depot formulations of the VHH antibodies of the present invention are therefore included within its scope.

[0273] For injection, the pharmaceutical composition can be formulated as a liquid such as a solution. According to preferred embodiments, the pharmaceutical composition is a sterile solution. Acceptable solvents include, but are not limited to, water, Ringer's solution and isotonic sodium chloride.

[0274] In some embodiments, the active agent, with or without excipients, are maintained in a separate state from the solvent, for example, as a dry powder. The intermixing of the desired amount of the active agent with the desired amount of the solvent, performed by the subject immediately prior to application of the pharmaceutical composition, ensures that the active agent will retainits maximum efficacy, and will also permit the potency of the composition to be tailored to the individual needs of the subject.

[0275] Depending on the stage and the severity of the disorder, the pharmaceutical composition may be administered once or several times during the course of the disorder. For example, it may be administered once or several times daily, each second day, two times weekly, weekly, monthly or every few months, for a suitable period of time.

[0276] In certain embodiments, the pharmaceutical composition is administered parenterally, e.g., by intravenous, subcutaneous, or intramuscular injection or by infusion. In certain embodiments, the pharmaceutical composition is administered by injection. According to certain embodiments, the pharmaceutical composition administered by injection or infusion is in the form of a solution. In further embodiments, the pharmaceutical composition is administered locally, e.g., topically, orally, nasally or intrapulmonary, for example by inhalation as an aerosol.

[0277] The VHH antibody may be administered alone or together with a further active agent or treatment, particularly with a further agent that is useful in the prevention and / or treatment of a disorder caused by and / or associated with IL- 13 expression, activity or overactivity.

[0278] Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disease or disorder of the subject being treated.

[0279] The term “effective amount” is that amount of the active agent which is sufficient to provide a beneficial effect to the subject to which the composition is administered.

[0280] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0281] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0282] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The dataobtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0283] Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to achieve the minimal effective concentration (MEC). The MEC will vary for each preparation but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma or tissue concentrations.

[0284] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved.

[0285] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0286] The present invention also provides kits comprising the compositions of the present invention, means for applying the composition and instructions of use. According to some embodiments, the kits comprise pharmaceutical compositions suitable for injection and a syringe or an injection device.

[0287] Diagnostic applications

[0288] Diagnostic applications include in-vitro and ex-vivo methods wherein a VHH antibody is used for detecting human IL- 13 in a sample, e.g., in a body fluid such as saliva, blood, serum or plasma, stool samples or in a tissue or biopsy sample. For diagnostic applications, the VHH antibody may carry a label for direct detection or used in combination with secondary detection reagents, e.g., antibodies, including conventional antibodies or VHH antibodies, for indirect detection according to established techniques in the art.

[0289] A diagnostic composition comprising at least one VHH antibody that recognizes human IL- 13, and at least one excipient, diluent or carrier are therefore included within the scope of the invention, as well as their use in diagnosis, prognoses, disease staging and in research.Kits comprising a VHH antibody according to the invention, a standard or a reference sample comprising a known amount or concentration of human IL-13, and instructions of use, are also provided.

[0290] Evaluating the activity and safety of the VHH antibodies for use in therapy

[0291] The VHH antibodies and sets of VHH antibodies of the present invention may be tested in-vitro, in-vivo and ex-vivo for their activity in IL-13 related disease models.

[0292] In vitro assays that may be used to evaluate the activity of the VHH antibodies include the method detailed in Example 4 below and additional methods known in the art (e.g., Octet assay) to measure binding or biological activity.

[0293] To assess safety in humans, a phase Ella, randomized, double blind, placebo-controlled trial may be conducted. Tolerability, immunogenicity, pharmacokinetics, pharmacodynamics and efficacy of multiple ascending doses of anti -IL- 13 VHH antibodies of the present invention are evaluated. In vivo models that are used for the VHH antibodies of the present invention may include but are not limited to asthma and atopic dermatitis animal models well known in the art. For example, a pulmonary asthma model tests inhaled or systemic treatment with one or more anti-IL-13 VHH antibodies described above in ovalbumin-induced allergic airway inflammation in mice or hamsters (Casaro et al., 2019, Zhu et al., 2024, Yu 2018). Another example is an atopic dermatitis model in which human skin is engrafted into SCID mice, disease is induced by adding PBMCs from atopic dermatitis patients to the skin graft and treating with VHH antibodies according to the present invention (Paus et al., 2018, Waldron-Lynch et al., 2012, Mougel et al., 2025). This mice model is further used to evaluate the biodistribution of the anti-IL-13 VHH antibodies. The phrase "subject in need thereof' used herein refers to a mammalian male or female subject (e.g., human being) who is diagnosed with an inflammatory disease or disorder. In a specific embodiment, this term encompasses individuals who are at risk to develop an inflammatory disease or disorder. The subject may be of any gender or at any age including neonatal, infant, juvenile, adolescent, adult and elderly adult.

[0294] The terms "comprises", "comprising", "includes", "including", and “having” mean "including but not limited to".

[0295] The term “consisting of means “including and limited to”.The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0296] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0297] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0298] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0299] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0300] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in thecontext of various embodiments are not to be considered essential features of those embodiments unless the embodiment is inoperative without those elements.

[0301] The term “about” refers to a value which is 10%, 5% or 1% above or below the indicated value. It is noted that each possibility disclosed throughout the specification represents a separate embodiment of the present invention.

[0302] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0303] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0304] EXAMPLES

[0305] Example 1: Expression and purification of VHH antibodies.

[0306] VHH antibodies were expressed with an N-terminal DsbA signal sequence and a C-terminal Hisn tag from a Kan-ColEl plasmid harboring a T5 / lac promoter in E. coli NEBExpress (New England Biolabs). A 125-ml preculture in Terrific Broth (TB) containing 50 pg / ml Kanamycin was grown overnight at 28 °C to early stationary phase. The culture was then diluted with fresh medium (500 ml, pre-warmed to 37 °C). After 30 minutes of growth at 37 °C, protein expression was induced with 0.05 mM IPTG (isopropyl P-D-l-thiogalactopyranoside) and growth was continued for 2 hours at 37 °C, whereby the culture reached a final ODeoo of ~8.

[0307] Bacteria were harvested by centrifugation and lysed by osmotic shock lysis: cell pellets were resuspended in 7 ml 65 mM Tris / HCl pH 8.0, 5 mM EDTA, 40% (w / v) sucrose. After gentle mixing at 23 °C for 60 min, 100 ml of ice-cold water were added and mixing was continued at 4 °C for 30 min. 20 mM Tris / HCl pH 7.5, 100 mM NaCl and 15 mM imidazole were added to the cell suspension. Periplasmic extract was then recovered as the supernatant of two consecutive centrifugation steps at 4 °C: a low-speed spin at 4000 g (20 min, F13 rotor, Thermo Fisher Scientific) and a high-speed spin at 38000 rpm (~1 hour, T647.5 rotor). The VHH antibody waspurified at 4 °C via Ni2+EDTA-amide chelate affinity chromatography (0.6 ml matrix). Beads were washed with ten column volumes of 50 mM Tris / HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.2% (w / v) Triton X-100 and ten column volumes of buffer lacking detergent. After elution with 50 mM Tris / HCl pH 7.5, 300 mM NaCl, 500 mM imidazole, the buffer was exchanged to 50 mM Tris / HCl pH 7.5, 300 mM NaCl, 250 mM sucrose via a PD-10 desalting column (GE Healthcare). Concentrations were determined by measuring the optical density at 280 nm, and division by the calculated s280 extinction coefficient. Aliquots were frozen in liquid nitrogen and stored at -80 °C.

[0308] Alternatively, class A VHH antibodies were cytoplasmically expressed as Hisi4-NEDD8 fusions, captured on a Ni2+ chelate column, and eluted by NEDP1 -mediated tag-cleavage (Frey and Gbriich, 2014; Pleiner et al., 2015). The structural disulfide bond was introduced after purification by treatment with DsbA, DsbC and oxidized glutathione, as disclosed in WO2024 / 256467.

[0309] Example 2: Kinetic measurements by biolayer interferometry (BLI)

[0310] For affinity measurements, human IL-13 was produced as a secreted protein in human Expi293F cells (Thermo Fisher Scientific). IL- 13 had been fused at its N-terminus to a secretion signal, and at its C-terminus with a Tev-cleavage site, followed by a rabbit Fc fragment, an avidin tag (Avi-tag), and 6 Histidine residues tag (Hise tag). The Hise tag was used for purification by Ni(II) chelate chromatography. The Avi-tag was enzymatically biotinylated with BirA, biotin and ATP (Beckett et al., 1999).

[0311] 130 nM biotinylated IL-13 fusion in assay buffer (20mM Tris / HCl pH 7.5, 150mM NaCl, 0.02% Tween 20 and 0.1% BSA) was bound to a binding threshold of 3nm to High Precision Streptavidin biosensors of an Octet RED96e instrument (ForteBio / Sartorius). After washing in assay buffer, assay buffer + 350 mM NaCl, and assay buffer, 100, 50, 20, 10, or 5 nM of the indicated VHH antibodies (Bm41 A08, Bm46A10, Bm46H07, Bm46G09, Bm46D05, Bm46E09, andBm46H02 of class AandBm41F06 andBm41Hll of class C), were allowed to bind for 600-1200 seconds, followed by a dissociation step of 600-1800 seconds. Binding and dissociation were recorded as wavelength shifts (in nm). Baselines were recorded by measuring a ‘minus VHH control’ in parallel. On-rates (kon), off-rates (kog), and dissociation constants (KDS) were calculated using the Octet Data Analysis HT 12.0 software by fitting a mass transport model to the data. The results are demonstrated in Figures 2A-2i and Table 1.Example 3. Measurement of VHH thermostability

[0312] To obtain the thermostability data listed in Table 1 and shown in Figures 3A-3i and 8A, VHH antibodies were subjected to Differential Scanning Fluorimetry (DSF), which exploits the fact that thermal unfolding exposes aromatic / hydrophobic residues, which then bind and enhance the fluorescence of the added SYPRO Orange dye. Assays were performed in a volume of 20 pl, at a VHH concentration of 1 mg / ml in 50 mM Tris / HCl, 150 mM NaCl (pH 8.0 at 20°C) and lx dye (diluted from a 5000x stock; Life Technologies). Sample were pipetted into a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal foil (Bio-Rad), briefly centrifuged to remove air bubbles, and placed into a CFX96 Real-Time System (Cl 000 Thermal Cycler, BioRad). The samples were first incubated at 20°C for 5 minutes. Then the temperature was increased to 95°C in 1°C steps of 45 seconds each. At the end of each step, fluorescence was measured after excitation at 532 nm using a 555 nm long-pass filter. Melting temperatures were defined as the inflection point of the first melting peak. A VHH antibody was considered stable if its melting peak was lower than its initial fluorescence at 20°C. As shown in Figures 3 A-3i, all class A members show a clear melting peak between 61 °C and 69°C, while the class C members are inherently stable. Furthermore, as shown in Figure 8 A, Bm46A10 showed a clear melting peak with a Tm of 65°, while Bm60Gll was fully resistant to thermal unfolding throughout the entire temperature range (20°C-95°C).

[0313] Example 4. Cell-based IL-13 stimulation assay and measurement of reporter activity. HEK-Blue™ IL-4 / IL-13 cells are HEK293 cells stably expressing the human STAT6 gene. The other genes of the STAT6 signaling pathway are endogenously expressed to constitute a fully active STAT6 signaling pathway. HEK-Blue™ IL-4 / IL-13 cells further encode a secreted embryonic alkaline phosphatase (SEAP) reporter controlled by an Interferon-P minimal promoter with four STAT6 binding sites. Upon binding of the IL- 13 ligands to its receptors on the cell surface STAT6 gets activated, SEAP is produced and secreted into the cell supernatant. The amount of SEAP can be measured colorimetrically, using QUANTI-Blue™ as a substrate.

[0314] Cells were cultivated in DMEM medium supplemented with 10% (v / v) fetal bovine serum, 4.5 g / L Glucose, 2 mM L-Glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin and 100 pg / mL Normocin™. Selection antibiotics (10 pg / mL Blasticidin and 100 pg / mL Zeocin) wereintroduced after the second passage and the growth medium was renewed at least twice per week. Cells were passaged at 70-80% confluency by scraping in PBS and not cultivated longer than 20 passages.

[0315] To evaluate the cellular response, titration curves were conducted with IL- 13. For this, HEK-Blue™ IL-4 / IL-13 cells were seeded in 96-well plates in medium without selection antibiotics and stimulated with serial three-fold dilutions of IL-13 (produced in CHO cells, SinoBiological 10369-HNAC). Medium without cells and untreated cells served as negative controls. After overnight incubation, 20 pl cell supernatant from each well was mixed with 180 pl QUANTL Blue™ solution in a separate 96-well plate and incubated for 30 min at 37°C. SEAP levels in the supernatant were determined by measuring the absorbance at 620 nm (OD620). The OD620 of the medium-only control served as the background and was subtracted from all sample wells. Each condition was assayed in triplicate. Induction was calculated from the ratio OD620 (sample) / OD620 (untreated). Based on the titration curve (Figures 4C and 4D), a concentration of 2 ng / ml IL-13 was used to achieve a comparable range of induction in the neutralization assays shown in Figures 6A-6D.References

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Claims

1. CLAIMS1. A VHH antibody recognizing a human IL-13 polypeptide, the VHH antibody is selected from:i. a class A VHH antibody comprising a CDR3 sequence selected from:X7ADLDFTIX8TIX9GDX11X10W (SEQ ID NO: 1) wherein X7is selected from L, V, and C, X8is T or K X9is Q or E, X11is D or E, and X10is Y or F, and ii. a class C VHH antibody comprising the CDR3 sequence STTICSVVAGLISNKYDSR (SEQ ID NO: 2),wherein the sequence identity between different VHH antibodies within a specific class is 80% or more.

2. The class A VHH antibody according to claim 1 comprising a CDR3 sequence selected from:LADLDFTITTIQGDEYW (SEQ ID NO: 3), CADLDFTIKTIEGDEFW (SEQ ID NO: 4), and VADLDFTIKTIEGDEFW (SEQ ID NO: 48).

3. The class A VHH antibody according to any one of claims 1 and 2, comprising a CDR1 selected from TNSGRTFNSYGM (SEQ ID NO: 7), VASGRTFNSFGM (SEQ ID NO: 9) and ANSGRTFNSYAM (SEQ ID NO: 11), a CDR2 selected from AISWDSGTTY (SEQ ID NO: 8), AISWSSGETS (SEQ ID NO: 10) and SISWSEGRVT (SEQ ID NO: 12), and a CDR3 selected from LADLDFTITTIQGDEYW (SEQ ID NO: 3), VADLDFTIKTIEGDEFW (SEQ ID NO: 48), and CADLDFTIKTIEGDEFW (SEQ ID NO: 4).

4. The class A VHH antibody according to any one of claims 1 to 3, wherein the VHH antibody comprises:i. a CDR1 comprising TNSGRTFNSYGM (SEQ ID NO: 7), a CDR2 comprising AISWDSGTTY (SEQ ID NO: 8), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);ii. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising CADLDFTIKTIEGDEFW (SEQ ID NO: 4);iii. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3);iv. a CDR1 comprising VASGRTFNSFGM (SEQ ID NO: 9), a CDR2 comprising AISWSSGETS (SEQ ID NO: 10), and a CDR3 comprising VADLDFTIKTIEGDEFW (SEQ ID NO: 48); andv. a CDR1 comprising ANSGRTFNSYAM (SEQ ID NO: 11), a CDR2 comprising SISWSEGRVT (SEQ ID NO: 12), and a CDR3 comprising LADLDFTITTIQGDEYW (SEQ ID NO: 3).

5. The class A VHH antibody according to any one of claims 1-4 comprising a sequence selected from SEQ ID NOs: 21-24 and 29-30, or a variant thereof comprising at least 89% sequence identity.

6. The class A VHH antibody variant according to claim 5, wherein framework (FW) position 6 is Q or E, FW position 10 is G or S, FW position 11 is S or L, FW position 14 is A or P, FW position 16 is E or G, FW position 61 is A or G, FW position 62 is D or E, FW position 72 is R or S, FW position 75 is T or A, FW position 76 is N or K, FW position 77 is T or N, FW position 78 is A or T, and FW position 85 is N or S.

7. The class A VHH antibody or variant according to any one of claims 1-6 comprising, a combination of the FWs of SEQ ID NOs: 52, 53, 54 and 55 and a set of CDR1, CDR2 and CDR3 sequences selected from: SEQ ID NOs: 7, 8 and 3; SEQ ID NOs: 9, 10, and 4; SEQ ID NOs: 9, 10, and 3; SEQ ID NOs: 9, 10, and 48; SEQ ID NOs: 11, 12, and 3; and SEQ ID NOs: 11, 19, and 20.

8. The class A VHH antibody according to claim 6 comprising the sequence QVQLVESGGX12LVQX13GGSLRLSCANSGRTFNSYAMGWFRQAPGKEREFVAAISW SGGRTTYAESVKGRFIISSDNAKNTVYLQMNSLKPEDTAVYYCVADLDFTITTIEDD DYWGQGTQVTVSS, wherein X12is G or S and X13is A or P (SEQ ID NO: 51), or a sequence having at least 90% identity.

9. The class C VHH antibody according to claim 1 comprising TASGSGFTLNNEDI (SEQ ID NO: 13) as CDR1, CLSFKNNKTYTY (SEQ ID NO: 15) as CDR2, and STTICSVVAGLISNKYDSR (SEQ ID NO: 2) as CDR3.

10. The class C VHH antibody according to claim 9, comprising a sequence having at least 90% identity to SEQ NO. 32.

11. The VHH antibody according to any one of the preceding claims, wherein the VHH antibody is selected from Bm60Gl 1 (class A, SEQ ID NO: 30), Bm41Hl 1 (class C, SEQ ID NO: 33), Bm46A10 (class A, SEQ ID NO: 24), Bm46H07 (class A, SEQ ID NO: 29), Bm41F06 (class C, SEQ ID NO: 32), Bm41A08 (class A, SEQ ID NO: 23), Bml9G08 (class A, SEQ ID NO: 21), Bm20D05 (class A, SEQ ID NO: 22), Bm46A10 (class A, SEQ ID NO: 24).

12. The VHH antibody according to any one of the preceding claims, having in a monomeric form, a binding affinity to human IL-13 expressed as a dissociation constant KD, to an immobilized human IL-13 of 5 nM, 1 nM, 500 pM, 300 pM, 100 pM, 50 pM, or less.

13. The VHH antibody according to any one of the preceding claims, that neutralizes human IL- 13 and thus signaling through the IL-13Ra and IL-4Ra receptors, at a concentration of about 10 nM or less, of about 3 nM or less, of about 1 nM or less, of about 0.3 nM or less, of about 0.1 nM or less, when tested in an appropriate cell-based assay under affinity-limited test conditions.

14. The VHH antibody according to any one of the preceding claims, which is thermostable or hyperthermostable having a melting temperature of at least about 65°C, of at least about 80°C, of at least 90°C or of at least about 95°C when measured under non-reducing conditions and / or an aggregation temperature of at least about 60°C, of at least 70°C, of at least about 80°C, of at least about 90°C, or of at least about 95°C, when measured under non-reducing conditions.

15. A conjugate comprising a VHH antibody according to any one of the preceding claims and at least one heterologous moiety.

16. The conjugate of claim 15, wherein the heterologous moiety is selected from a constant region of an antibody, or a fragment thereof, a human serum albumin or a fragment thereof, and collagen or a fragment thereof.

17. The conjugate of claim 15, wherein the heterologous moiety is a different VHH antibody.

18. The conjugate of claim 17, wherein the different VHH antibody targets a human cytokine selected from the group consisting of TSLP, IL-4, and IL-17.

19. A dimeric, trimeric, or multimeric VHH antibody comprising at least one VHH antibody according to any one of claims 1 to 14, and at least one VHH antibody that targets a human cytokine selected from TSLP, IL-4, and IL- 17.

20. The dimeric, trimeric, or multimeric VHH antibody of claim 19, wherein the VHH antibodies are conjugated in a tandem format.

21. A set of two or more different VHH antibodies recognizing a human IL- 13 polypeptide, comprising at least one VHH antibody or conjugate according to any one of claims 1 to 18.

22. A nucleic acid molecule encoding a VHH antibody, or a conjugate according to any one of claims 1 to 20.

23. A recombinant cell or cells or a non-human organism transformed or transfected with the nucleic acid molecule of claim 22 or with a vector comprising said nucleic acid molecule.

24. The recombinant cell of claim 23 selected from a bacterium cell, a yeast cell, an insect cell, a mammalian cell, and a plant cell.

25. A method for recombinant production of a VHH antibody, or a conjugate according to any one of claims 1 to 20, comprising cultivating cells or an organism in a suitable medium and obtaining the VHH antibody, or conjugate from the cells or organism or from the medium.

26. A pharmaceutical composition comprising a VHH antibody, conjugate, or set according to any one of claims 1 to 21, and a pharmaceutically acceptable excipient, or diluent.

27. The pharmaceutical composition of claim 26, formulated for parenteral administration.

28. The pharmaceutical composition of claim 26, formulated for local administration.

29. The pharmaceutical composition of claim 28, formulated for pulmonary or nasal delivery.

30. The pharmaceutical composition of claim 29, formulated as spray, mist, or aerosol.

31. The pharmaceutical composition of any one of claims 26 to 30, for use in the prevention or treatment of a disease or disorder caused by or associated with an activity or overactivity of IL-13.

32. The pharmaceutical composition for use of claim 31, wherein the disease or disorder is an inflammatory disease or disorder and / or a fibrotic disease or disorder.

33. The pharmaceutical composition for use of claim 31, wherein the disease or disorder is selected from asthma, eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, and different types of cancers, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-ZL-4Ra.

34. The pharmaceutical composition for use of claim 31, wherein the use comprises treatment with an anti -IL-4, anti-TSLP and / or anti-IL-17 agent.

35. A diagnostic composition comprising a VHH antibody or conjugate according to any one of claims 1 to 16, and an acceptable excipient, or diluent.

36. A method for the prevention or treatment of a disorder caused by or associated with an activity or overactivity of IL- 13, comprising administering to a subject in need thereof a VHH antibody, or conjugate according to any one of claims 1 to 20, or a pharmaceutical composition of any one of claims 26 to 30.

37. The method of claim 36, wherein the disease or disorder is an inflammatory disease or disorder and / or a fibrotic disease or disorder.

38. The method of claim 36, wherein the disease or disorder is selected from asthma, eosinophilic COPD, chronic sinusitis, nasal polyps, rhinitis conjunctivitis, atopic dermatitis, idiopathic pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, eosinophilic esophagitis, and different types of cancers, autoimmune and autoinflammatory diseases such as ulcerative colitis, rheumatoid arthritis and food allergy, and diseases treated with an anti-IL-4Ra.

39. The method of claim 36, comprising combination treatment with an anti-IL-4, anti-TSLP and / or anti-IL-17 agent.

40. The method according to any one of claims 36 to 39, wherein the composition is administered by injection or infusion.

41. The method according to any one of claims 36 to 39, wherein the composition is administered by pulmonary or nasal delivery.

42. A method of detection or quantitation of human IL-13 using a VHH antibody or conjugate according to any one of claims 1 to 16.

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