EP4558526A1

EP4558526A1 - Binding molecules targeting il-35r

Info

Publication number: EP4558526A1
Application number: EP23758372.9A
Authority: EP
Inventors: Luc Van Rompaey; Giel Steven TANGHE; Rudi Beyaert; Savvas SAVVIDES; Harald Braun; Sammy DETRY; Ioannis SKORDOS
Original assignee: Universiteit Gent; Vlaams Instituut voor Biotechnologie VIB
Current assignee: Universiteit Gent; Vlaams Instituut voor Biotechnologie VIB
Priority date: 2022-07-21
Filing date: 2023-07-21
Publication date: 2025-05-28
Also published as: CA3262764A1; GB202210680D0; WO2024018242A1

Abstract

The present invention provides binding molecules, particularly antibodies which bind to the IL-12Rβ2 and gp 130 subunits of the IL-35 receptor (IL-35R). The binding molecules are preferably able to act as agonists of the IL-35R. The invention further relates to pharmaceutical compositions comprising the binding molecules. The binding molecules and pharmaceutical compositions of the invention may be used in therapy and diagnosis. They may be employed to treat or prevent autoimmune and inflammatory conditions.

Description

BINDING MOLECULES TARGETING IL-35R

Field of Invention

The present invention relates to binding molecules that bind both the IL-12Rβ2 and gp130 receptor subunits of the IL-35 receptor (IL-35R). The present invention further relates to the use of such binding molecules in treatment and diagnosis. The binding molecules are particularly useful in suppressing or reducing unwanted immune responses. They may also be used to induce immune tolerance.

Background of Invention

IL-35 is a member of the Interleukin- 12 (IL-12) family of cytokines. The IL-12 family of cytokines is unusual as the cytokines in the family are heterodimeric each consisting of two different polypeptide chains. Each member of the IL-12 family comprises an a chain, with a helical structure similar to type 1 cytokines like IL-6, and a β-chain structurally related to the extracellular region of Type 1 cytokine receptors like soluble IL-6 receptor. Different members of the IL- 12 family overlap with each other structurally in the sense that they have the same chain for one of the two chains of the heterodimer. Interleukin- 12 (IL-12) consists of an a chain (IL-12p35) and a P chain (IL-12p40) encoded respectively by two separate genes, IL-12A and IL-12B. Other IL-12 family members are IL- 23 (consisting respectively of the a and β chains IL-23pl9 and IL-12p40), IL-27 (consisting respectively of the a and β chains IL-27p28 and Ebi3) and IL-35 (consisting respectively of the a and β chains IL-12p35 and Ebi3). IL-35 therefore shares an a chain with IL-12 and a P chain with IL-27.

The IL- 12 family cytokines each recruit two receptor subunits to form a tripartite signalling assembly of the cytokine and the two receptor subunits. The receptor for the cytokine is effectively the two receptor subunits, but the two receptor subunits do not usually exist together in the absence of the cytokine, only being brought together by the cytokine. For example, IL-12 is thought to first bind the high affinity IL-12Rβ2 and then IL-12Rβ1 is recruited to create the tripartite signalling assembly of IL-12, IL-12Rβ1, and IL-12Rβ2. IL-23 recruits IL-12Rβ1 and IL-23Rα receptor subunits to form a tripartite signalling assembly of IL-23, IL-12Rβ1 and IL-23Rα. IL-27 recruits IL-27Rα and gp130 receptor subunits to form a tripartite signalling assembly of IL-27, IL-27Rα and gp130. IL-35 is unusual in that is able to form a tripartite signalling assembly with IL-12Rβ2 and gp130, but also tripartite signalling assemblies with IL-35 and two IL-12Rβ2 receptors subunits or IL-35 with two gp130 receptor subunits are reported. In addition, it can form a complex with the IL-12Rβ2 and IL- 27Ra receptor chains. The different IL-12 family cytokines therefore overlap in the receptor subunits recruited to the tripartite signalling assembly. The tripartite signalling assemblies formed by IL-12 and IL-23 receptors both include the IL-12Rβ1 subunit. The tripartite signalling assemblies formed by IL- 12 and IL-35 both include the IL-12Rβ2 subunit. Signalling via the assembled tripartite signalling assemblies involves Janus kinases (JAKs), with the different receptors varying in terms of which STAT proteins are involved in signalling.

The different cytokines in the IL- 12 family also have different functional roles. IL- 12, IL-23, and IL-27 are thought to promote immune responses. IL-35 is thought to have a role in inhibiting immune responses, tolerance induction and immune system maintenance mediated via regulatory B and T cells, such as iTr35 cells. IL-35 is reported to increase the expression of the largely anti- inflammatory cytokine IL- 10. IL-35 is endogenously produced as a non-covalent heterodimer, which proves difficult to produce and purify recombinantly, requiring co-expression of both receptor units in the same cell with formation of significant proportions of homodimers relative to the heterodimeric IL-35. IL-35 is often produced recombinantly as a single chain variant, fused to the Fc domain and extensive studies comparing the wild-type non-covalent untagged IL-35 versus the single chain variant are lacking.

Summary of Invention

IL-35 itself is difficult to produce and purify in large quantities meaning that the production of IL-35 and optimised variants for use in therapy is difficult. The present invention provides binding molecules that are able to act as IL-35 agonists which do not typically suffer such difficulties in their production. That is because, rather than being based on IL-35 itself, they bind to the receptor subunits that make up the IL-35 receptor. The present invention provides binding molecules that are able to bind both the IL-12Rβ2 receptor and the gp130 receptor subunit. IL-35 assembles both the IL-12Rβ2 receptor and the gp130 receptor subunit into atripartite signalling complex. Although the IL-12Rβ2 receptor and the gp130 receptor subunit are only brought together by the action of IL-35 usually, the IL-12Rβ2 receptor subunit and the gp130 receptor subunit can be thought of as together representing the IL-35 receptor (IL-35R). By binding both the IL-12Rβ2 receptor and the gp130 receptor subunits of the IL-35R, binding molecules of the present invention can modulate an IL-35R. It is believed that the inventors for the present application are the first to produce antibody binding molecules able to bind both gp130 and IL-12Rβ2, leading to downstream signalling.

The binding molecules of the present invention may display a number of advantages. For instance, the binding molecules typically have good EC₅₀ values. The binding molecules also may show potent activity in terms of phosphorylation of STAT protein, particularly in respect of phosphorylation of STAT3 protein which is part of the downstream activation pathway. Binding molecules of the present invention may also display the ability to induce a high level of IL- 10 secretion.

In an especially preferred embodiment, binding molecules of the present invention can act as agonists of the IL-35R. As IL-35 plays a role in suppressing or reducing immune responses, the binding molecules of the present invention which are agonists of the IL-35 receptor can be used to suppress or reduce immune responses. That can be helping in treating autoimmune and inflammatory responses. The binding molecules are also useful in inducing immune tolerance.

Accordingly, the present invention provides a binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising:

(i) at least one binding -site specific for the gp130 subunit of the IL-35 receptor; and

(ii) at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor.

The present invention also provides a pharmaceutical composition comprising a binding molecule of the present invention.

Also provided is a binding molecule or pharmaceutical composition of the present invention for use is a method of treatment of the human or animal body.

Further provided is the binding molecule or pharmaceutical composition of the present invention for use in treating or preventing an autoimmune or inflammatory disorder or inducing immune tolerance.

The present invention also provides a method of treating or preventing an autoimmune or inflammatory disorder or inducing immune tolerance comprising a binding molecule or pharmaceutical composition of the invention to a subject in need thereof.

The present invention provides a method for detecting association of the IL-12Rβ2 and gp130 receptor subunits comprising: (a) contacting a candidate molecule with a cell expressing modified versions of IL-12Rβ2 and gp130 receptor subunits which have been modified so when they associate a detectable signal is produced; (b) detecting the signal, if present, optionally when the magnitude of the signal is measured.

Brief description of the Figures

Figure 1: Shows the ability of selected anti-gp130 and anti-IL-12Rβ2 VHH antibodies to compete with IL-6 or IL-12 signaling. A: HEK-Blue IL-6 reporter cells were incubated with different dilutions of periplasmic extracts containing anti-gp130 VHHs prior to addition of recombinant human IL-6. One day later, IL-6 signaling activity was determined by measuring the levels of secreted embryonic alkaline phosphatase in the cell culture medium. The Figure corresponds to Table 7, here clones with various IL-6 competition properties are depicted as an example of the results obtained. B: HEK-Blue IL- 12 reporter cells were incubated with different dilutions of periplasmic extracts containing anti-IL-12Rβ2 VHHs prior to addition of recombinant human IL- 12. One day later, IL- 12 signaling activity was determined by measuring the levels of secreted embryonic alkaline phosphatase in the cell culture medium. The Figure corresponds to Table 8, here clones with various IL-12 competition properties are depicted as an illustrative example of the results obtained. Figure 2: Shows the ability of selected anti-gp130/IL-12Rβ2 bispecific VHHs to induce gp130 and IL-12Rβ2 heterodimerisation and STAT phosphorylation. A: HEK293T cells transiently transfected with expression plasmids for human gp130 and IL-12Rβ2 respectively fused at the C-terminus with NanoLuc LargeBiT and NanoLuc SmallBiT. After adding NanoLuc substrate the luciferase activity was measured over time. After 24 minutes background measurement, the cells were stimulated with indicated bsVHHs (timing indicated with arrow). B: HEK-Blue IL- 12 reporter cells, which express both gp130 and IL-12Rβ2, were incubated with purified bsVHH or IL-12 for 30 minutes. Then, cells were lysed and analysed by western blotting for phospho(Tyr705)-STAT3 (pSTAT3) activation. The graph depicts the quantified band intensity of pSTAT3 normalized to unstimulated cells.

Figure 3: Shows the ability of selected anti-gp130/IL-12Rβ2 bsVHH-Fc to induce gp130 and IL-12Rβ2 heterodimerisation and STAT phosphorylation. A: HEK293T cells were transiently transfected with expression plasmids for human gp130 and IL-12Rβ2 respectively fused at the C- terminus with NanoLuc LargeBiT and NanoLuc SmallBiT. After adding NanoLuc substrate the luciferase activity was measured over time. After background measurement, the cells were stimulated with different concentrations of the indicated bsVHHs. The graph represents a dose-response of the bsVHH-induced NanoBiT clustering measured 20 minutes after stimulation. B: HEK-Blue IL-12 reporter cells, which express both gp130 and IL-12Rβ2, were incubated with purified bsVHH-Fc, IL- 12, IL-6 for 30 minutes. Then, cells were lysed and analysed by western blotting for phospho (Tyr705)- STAT3 (pSTAT3) activation. The graph depicts the quantified band intensity of pSTAT3 scaled to IL- 6.

Figure 4: Evaluation of bispecific antibody formats - A: Overview of the bispecific antibody formats applied to construct bispecific anti-gp130/IL-12Rβ2 antibodies. B: pSTAT3 activation by bivalent “gp130-IL-12Rβ2” and “IL-12Rβ2-gp130” ordered bsVHH-Fc combining anti-gp130 VHH 18439 and anti-IL-12Rβ2 VHH 20422. HEK-Blue IL- 12 reporter cells were stimulated for 30 minutes with indicated bsVHH-Fc followed by pSTAT3 analysis by flow cytometry. C: pSTAT3 activation by bsVHH-Fc-3 constructed with varying number of GGGGS (G4S) repeats linking gp130 and IL-12Rβ2 VHH. HEK-Blue IL-12 reporter cells were stimulated for 30 minutes with indicated bsVHH-Fc followed by pSTAT3 analysis by flow cytometry. D: pSTAT3 activation by selected monovalent knob- into-hole bispecific VHH-Fc constructs, compared to bsVHH-Fc-3. HEK-Blue IL-12 reporter cells were stimulated for 30 minutes with indicated bsVHH-Fc followed by pSTAT3 analysis by flow cytometry.

Figure 5: Shows the ability of anti-gp130/IL-12Rβ2 VHH-Fc to induce STAT phosphorylation in a T cell line - Jurkat T cells with stable gp130-IRES-mCherry and IL-12Rβ2- IRES-ZsGreen expression were stimulated for 30 minutes with anti-gp130/IL-12Rβ2 bsVHH-Fcs. The percentage of pSTAT3 positive cells was analysed by flow cytometry within the cell population positive for ZsGreen and mCherry. A: The graph depicts the maximal pSTAT3 efficacy within the dose-range of 0.01-10 nM for anti-gp130/IL-12Rβ2 bsVHH-Fcs designed in a second design wave. B: Dose- response pSTAT3 activation by selected anti-gp130/IL-12Rβ2 VHH-Fcs.

Figure 6: Shows the ability of anti-gp130/IL-12Rβ2 to induce IL-10 in a T cell line - Jurkat T cells with stable gp130-IRES-mCherry and IL-12Rβ2-IRES-ZsGreen expression were stimulated for 24 hours with bsVHH-Fc-71, KiH-8 or control protein bsVHH-Fc-30 at different doses. As positive control, cells were stimulated with PMA and ionomycin. IL-10 secretion was measured by ELISA.

Figure 7: Shows dose-dependent STAT3 activation and IL-10 secretion for selected anti- gp130/IL-12Rβ2 bsVHH-Fc molecules in a T cell line - Jurkat T cells with stable gp130-IRES- mCherry and IL-12Rβ2-IRES-ZsGreen expression were stimulated for 30 minutes (A, C) or 24 hours (B) with anti-gp130/IL-12Rβ2 bsVHH-Fc molecules. A and C: The percentage of pSTAT3 positive cells was analysed by flow cytometry within the cell population positive for ZsGreen and mCherry. (GxS)n indicates linker length between the 2 VHHs. B: IL-10 secretion in the supernatants was measured by ELISA (positive control: PMA/Iono(mycin).

Figure 8: Shows the ability of biparatopic bispecific anti-gp130/IL-12Rβ2 antibodies to induce STAT3 activation. A and C: Jurkat T cells with stable gp130-IRES-mCherry and IL-12Rβ2- IRES-ZsGreen expression were stimulated for 30 minutes with indicated single or combined bsVHH- Fc or KiH molecules. The percentage of pSTAT3 positive cells was analysed by flow cytometry within the cell population positive for ZsGreen and mCherry. B: Schematic representation of the biparatopic bispecific antibody formats of KiH-71/77 and KiH-107/108.

Figure 9: Shows dose-dependent STAT3 activation and IL-10 secretion for selected anti- gp130/IL-12Rβ2 bsVHH-Fc molecules in primary activated CD4 T cells - Primary human CD4 T cells were enriched from three different human peripheral blood mononuclear cell donors and activated with anti-CD3/CD28 antibodies and recombinant IL-2 for 48 hours, followed by a 24 hour rest period without activation stimuli. Subsequently, the cells were stimulated with bsVHH-Fc-77 at different concentrations for 30 minutes (A). After stimulation STAT3 phosphorylation was analysed by flow cytometry (A) Graphs represent the mean ± SEM (n=3). (B) Depicts the expression of gp130 and IL-12Rβ2 in CD4+ CD25+ activated T cells at the start of bsVHH-Fc stimulation (in A).

Figure 10: Shows induction of IL-10 secretion by selected anti-gp130/IL-12Rβ2 bsVHH- Fc molecules in primary activated CD4 T cells - Primary human CD4 T cells were enriched from three different human peripheral blood mononuclear cell donors and activated with anti-CD3/CD28 antibodies and recombinant IL-2 for 48 hours, followed by a 24 hour rest period without activation stimuli. Subsequently, the cells were stimulated with bsVHH-Fc-77 at different concentrations for 3 and 6 days. IL-10 secretion in the culture supernatants was determined by IL-10 ELISA. Graphs represent the mean ± SEM (n=3), One-way ANOVA test (* : p < 0.05).

Figure 11: Provides the amino acid sequences of bsVHH-Fc-77, bsVHH-Fc-78, bsVHH- Fc-84, bsVHH-Fc-96, and bsVHH-Fc-71 but with constant regions of Durvalumab (comprising the CH2CH3 regions of that constant region, but not the CHI region). The CDR sequences of the VHH regions are shown underlined. Those five constructs and binding molecules based on the CDR combinations that they comprise represent preferred binding molecules of the present invention. For instance, versions where the VHH domains have been humanised, but retain the same CDRs represent preferred molecules of the present invention. Constructs based on the combination of CDRs in bsVHH-Fc-77 are especially preferred.

Figure 11: Provides the amino acid sequences of bsVHH-Fc-77, bsVHH-Fc-78, bsVHH- Fc-84, bsVHH-Fc-96, and bsVHH-Fc-71, but with the constant regions of Durvalumab (comprising the CH2CH3 regions of that constant region, but not the CHI region). The CDR sequences of the VHH regions are shown underlined. Those five constructs and binding molecules based on the CDR combinations that they comprise represent preferred binding molecules of the present invention. For instance, versions where the VHH domains have been humanised, but retain the same CDRs represent preferred molecules of the present invention. Constructs based on the combination of CDRs in bsVHH-Fc-77 are especially preferred.

Detailed description

Overview

The present invention provides binding molecules that can specifically bind the IL-12Rβ2. It also provides binding molecules that can specifically bind gp130. In an especially preferred embodiment though, a binding molecule of the present invention can bind both IL-12Rβ2 and gp130. In a further especially preferred embodiment, the binding molecule of the present invention can bind both IL-12Rβ2 and gp130, with the binding molecule able to act as an agonist of the IL-35 receptor. Such binding molecules able to bind both IL-12Rβ2 and gp130 may be said to be at least bispecific binding molecules. In particular, the molecules are preferably able to bind both IL-12Rβ2 and gp130 at the same time. In some embodiments, the binding molecule is biparatopic in respect of IL-12Rβ2. In some embodiments, the binding molecule is biparatopic in respect of gp130. In some embodiments, the binding molecule is biparatopic in respect of IL-12Rβ2 and is also biparatopic in respect of gp130. The binding molecules provided are not IL-35 itself or a variant thereof.

Binding molecule formats

Any suitable format binding molecule may be provided by, or employed in, the present invention. In an especially preferred embodiment, the binding molecule of the invention is an antibody. The term “antibody” as used herein is not limited to the “classical” structure of the four chain structure of an IgG antibody in humans comprising two light and two heavy chains. However, such structure antibodies are also provided, both in the case of providing either specificity individually or a bispecific antibody where each arm of the bispecific targets one of the IL-12Rβ2 and gp 130 subunits. The term “antibody” though covers non-naturally occurring antibody formats and those are specifically envisioned to form part of the invention. In embodiments where a binding molecule comprises antibody-based sequences, the overall binding molecule may be simply referred to as an antibody. Hence, reference to an antibody may be used to refer to the overall molecule, even if the binding molecule comprises a constituent which itself would be viewed as an antibody, for instance, a VHH binding domain. The term “antibody” specifically includes a single chain antibody and a binding molecule comprising such a single chain antibody. A binding molecule of the present invention may be or comprise a single chain antibody that is specific for IL-12Rβ2. A binding molecule of the present invention may be or comprise a single chain antibody that is specific for gp130. A binding molecule of the present invention may also comprise non-antibody sequences, for example it may comprise binding sites that are not antibody based.

In a preferred embodiment, the binding molecule is characterised as being, or comprising, a single domain binding regions. In a preferred embodiment, the binding molecules comprises at least a single domain binding region for IL-12Rβ2 or gp 130. In a more preferred embodiment, the binding molecule comprises at least one binding domain for IL-12Rβ2 and at least one binding domain for gp130, where those binding domains are single binding domain binders.

In one embodiment, a binding molecule may have more than one binding site for IL-12Rβ2. In one embodiment, all of the binding sites of the binding molecule specific for IL-12Rβ2 are the same. In another embodiment, the binding molecule may comprise different binding sites for IL-12Rβ2, for instance each recognising a different epitope of IL-12Rβ2, so be, for instance, biparatopic in respect of IL-12Rβ2. In one embodiment, a binding molecule may have more than one binding site for gp130. In one embodiment, all of the binding sites of the binding molecule specific for gp130 are the same. In another embodiment, the binding molecule may comprise different binding sites for gp130, for instance each recognising a different epitope of gp130, so be, for instance, biparatopic in respect of gp130. Single domain binders that may be employed as part of binding molecules of the present invention include, for instance, non-Ig engineered protein scaffolds such as darpins, affibodies, adnectins, anticalin proteins, or peptides and the like. So wherever reference is made to sdB, sdAb, HCAb, and VHH, it may be possible to also employ a darpin, affibody, adnectin, anticalin, or peptide that is able to bind the stated target and the term sdB encompasses such binding entities being employed. Examples of single binding domain binders (sdBs) include in particular single domain antibodies (sdAb), for example, heavy chain only antibodies (HCAb), particularly VHH domain antibodies. Employing VHH antigen-binding domains is an especially preferred embodiment. A single-domain antibody (sdAb) is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, sdAb is able to bind selectively to a specific antigen. sdAb may be antibody fragments that can be engineered from single monomeric variable domains of either camelids’ heavy-chain antibody (VHH) or cartilaginous fishes’ IgNAR (VNAR), or be developed from camelized human antibodies. Any such sdAbs may be employed. Especially preferred sdAbs are VHH domains. In one embodiment, a binding molecule of the present invention may comprise at least two sdAb domains, with at least one specific for IL-12β2 and at least one specific for gp 130. In one embodiment, the binding molecule may comprise at least two sdAb domains on the same polypeptide. In a preferred embodiment, an antibody of the present invention may be a molecule that comprises a single domain antibody or single domain antibodies where the overall valency of the binding molecule is at least two. For example, an antibody of the present invention may comprise two such single domain antibodies joined together as part of the overall antibody. In another preferred embodiment, a binding m olecule of the present m ay compri se VHH binding d omains as sdAbs. SdAbs from organisms such as Camelids, sharks, and other cartilaginous fish that produce heavy chain-only antibodies may be employed. The single-domain variable fragments of these heavy chain- only antibodies are termed VHHs or nanobodies or sdAb. VHHs retain the immunoglobulin fold shared by antibodies, using three hypervariable loops, CDR1, CDR2 and CDR3, to bind to their targets. A VHH fragment (e.g., NANOBODY®) is a recombinant, antigen-specific, single-domain, variable fragment derived from camelid heavy chain antibodies.

In an especially preferred embodiment, a binding molecule of the present invention is, or comprises, at least one VHH domain antibody specific for a IL-12Rβ2 subunit, and at least one VHH domain antibody specific for a gp130 receptor subunit. The present application sets out examples of preferred VHH domains and those VHH domains may be employed in any suitable binding molecule format set out herein. In another especially preferred embodiment, a binding molecule of the present invention comprises:

(i) at least one VHH binding domain specific for IL-12Rβ2 linked to an Fc region; and

(a) at least one VHH binding domain specific for gp130 linked to an Fc region.

Illustrative examples of preferred binding molecule formats are shown in Figure 4.

In one preferred embodiment, a binding molecule comprises two polypeptides where each polypeptide comprises at least a binding site for IL-12Rβ2 or gp 130. In one embodiment, a binding molecule comprises two polypeptides where each polypeptide comprises at least one binding site for IL-12Rβ2 and at least one binding sites for gp130. In one embodiment, one polypeptide comprises a single binding site for IL-12Rβ2 and a single binding site for gp130, with the second polypeptide also comprising a single binding site for IL-12Rβ2 and a single binding site for gp 130. In one embodiment, the binding site for IL-12Rβ2 is the most N-terminal binding site of the polypeptide. In another embodiment, the binding sites for gp130 is the most N-terminal binding site of the polypeptide. In any of the possible binding molecule formats referred to in this paragraphs, the binding sites are preferably VHH binding domains. In any of the possible binding molecule formats referred to in this paragraph, preferably each polypeptide comprises a CH2CH3 constant region. In another preferred embodiment, a binding molecule comprises two polypeptides, where each of the two polypeptides is different so that the binding molecule is heterodimeric. In one embodiment, one polypeptide comprises at least one IL-12Rβ2 binding site, but not a binding site for gp130, with the other polypeptide comprising at least one binding site for gp130, but not IL-12Rβ2. In one embodiment, one polypeptide comprises a single binding domain which is a binding domain specific for IL-12Rβ2 and the other polypeptide comprises a single binding domain which is a binding domain specific for gp 130. In another embodiment, each polypeptide comprises two such binding sites. In another embodiment, each polypeptide comprises three such binding sites. In any of the possible binding molecule formats referred to in this paragraphs, the binding sites are preferably VHH binding domains. In any of the possible binding molecule formats referred to in this paragraph, preferably each polypeptide comprises a CH2CH3 constant region. The binding molecule also preferably comprises a hinge region. In a particularly preferred embodiment, the two constant regions comprise a sequence modification either promoting heterodimer formation or allowing purification of the heterodimer.

In one embodiment, where a binding molecule comprises a polypeptide with at least two binding sites, the binding sites may be separated by a linker. For example, in binding molecules comprising two polypeptides where each polypeptide comprises a IL-12Rβ2 binding site and a gp130 binding site, the two may be separated by a linker. In one embodiment the linker comprises units of the sequence GGGGS. In one embodiment, the linker comprises (GGGGS)_X where x is a value from 1 to 8. In one embodiment, X has a value of from 2 to 8. In one embodiment, x has a value of from 3 to

5. In one embodiment, x has a value of 3, 4, or 5. In a preferred embodiment, x has a value of 4, 5, or

6. In one embodiment, x has a value of 5. In one embodiment, x has a value of 7.

In one especially preferred embodiment, a binding molecule of the invention comprises two polypeptides, with each polypeptide comprising two VHH domains and an Fc region so that the two polypeptides may form an Fc region dimer. In one preferred embodiment, each polypeptide comprises a VHH domain specific for IL-12Rβ2 and a VHH domain specific for gp 130. In one embodiment, the VHH domains specific for IL-12Rβ2 in each of the two polypeptides bind a different epitope meaning the binding molecule is biparatopic for IL-12Rβ2. In one embodiment, the VHH domains specific for gp130 in each of the two polypeptides bind a different epitope meaning the binding molecule is biparatopic for gp 130. In one preferred embodiment, a binding molecule is biparatopic for both gp130 and IL-12Rβ2. In one preferred embodiment, there may be a linker between the two VHH domains of each polypeptide such as the linkers outlined about. Figures 10 and 11 provide examples of preferred binding molecules. In one preferred embodiment, a binding molecule of the present invention corresponds to one of those shown in Figures 10 and 11, but where the VHH domains have been humanised, though with the CDRs shown retained. As explained herein, variants of such molecules also represent binding molecules of the present invention. A variant will retain the ability to bind to the IL-35 receptor. Preferably, a variant will retain the ability to activate pSTAT3 signalling. Preferrably a variant will be able to induce IL-12 secretion. Binding molecules specific for IL-35 receptor subunits

The present invention provides and employs binding molecules that are able to bind to the IL- 12Rβ2 receptor subunit. The present invention provides and employs binding molecules that are able to bind to the gp130 receptor subunit. In an especially preferred embodiment, the binding molecules are able to bind to the IL-12Rβ2 and gp130 subunits. Typically, the binding molecule will be able to bind to the IL-35R subunit or subunits on the surface of a cell.

In a particularly preferred embodiment, a binding molecule is able to bind to a human IL- 12Rβ2 receptor subunit. In one preferred embodiment, a binding molecule is able to bind to the mouse IL-12Rβ2 receptor subunit. The amino acid sequence of the human and mouse IL-12Rβ2 receptor subunits are provided below:

SEQ ID NO: 3128, sequence of the human IL-12Rβ2 (NP 001550.1, interleukin-12 receptor subunit beta-2 isoform a precursor [Homo sapiens]):

SEQ ID NO: 3129, sequence of mouse IL-12Rβ2 (NP 032380.1, interleukin- 12 receptor subunit beta-2 isoform 1 precursor [Mus musculus]):

In a particularly preferred embodiment, a binding molecule is able to bind to a human gp130 receptor subunit. In one preferred embodiment, a binding molecule is able to bind to the mouse gp130 receptor subunit. In one preferred embodiment, a binding molecule is able bind to the human and mouse gp130 receptor subunits. In an alternative embodiment, a binding molecule is able bind to the human gp130 receptor subunit, but not the mouse gp130 receptor. The amino acid sequence of the human and mouse gp130 receptor subunits are provided below:

SEQ ID NO: 3130, sequence of the human gp130 (NP 002175.2, Interleukin-6 receptor subunit beta [Homo sapiens]):

SEQ ID NO: 3131, sequence of mouse gp130 (NP 034690.3, Interleukin-6 receptor subunit beta [Mus musculus]):

In a preferred embodiment, a binding molecule does not compete with IL- 12 for binding to a IL-12R[32 receptor subunit. Hence, in such embodiments, the binding molecule of the present invention does not inhibit the ability of IL- 12 to form a tripartite signalling assembly with the IL-12Rβ2 and IL-12Rβ1 receptor subunits. In an alternative embodiment, a binding molecule of the present invention is able to compete with IL-12 for binding to the IL-12Rβ2 receptor subunit.

In a further preferred embodiment, a binding molecule does not compete for binding with IL- 6. In a preferred embodiment, a binding molecule does not compete for binding with IL-6 family members. In one preferred embodiment, the overall binding molecule does not compete for binding with IL-6. In a particularly preferred embodiment, it does not compete for binding with IL-6 family members in general.

The “valency” of a binding molecule as used herein denotes the number of antigen-binding sites that the binding molecule comprises. A binding molecule may be said to have a valency for a particular target. For instance, in one embodiment a binding molecule of the invention may have only one binding site for IL-12Rβ2. In another embodiment, it may have two binding sites for IL-12Rβ2. In another embodiment, it may have three binding sites for IL-12Rβ2. In one embodiment, a binding molecule of the invention may have only one binding site for gp130. In another embodiment, it may have two binding sites for gpI30. In another embodiment, it may have three binding sites for gpI30. In an especially preferred embodiment, a binding molecule of the present invention may have the same number of binding sites for IL-12Rβ2 as gp130. For example, it may have one binding site for each. In another embodiment, it may have two binding sites for each. In a further embodiment, it may have three binding sites for each. In a preferred embodiment, where a binding molecule of the invention has such numbers of binding sites, the binding sites are VHH domain binding sites.

The strength of binding of an individual antigen-binding site to an IL-12Rβ2 or gp130 polypeptide may be referred to as the “affinity” of the binding site for IL-12Rβ2 or gp130 polypeptide. Whilst the overall strength of binding of a binding molecule is often also referred to as the affinity of the binding molecule, where the binding molecule has more than one binding site, the strength of binding may be referred to using the term avidity, which reflects the overall strength of binding when all of the binding sites of the binding molecule are taken into account. In one embodiment, the affinity of an individual binding site of the overall binding molecule may be determined by measuring it for the individual VHH binding domain on its own.

A binding site of the present invention may be said to specifically bind an IL-12Rβ2 receptor subunit. A binding site of the present invention may be said to specifically bind a gp130 receptor subunit. Specific binding may constitute binding to an IL-12Rβ2 or gp130, but not significantly binding to other polypeptides. In one embodiment, a binding domain may have a KD affinity value for IL-12Rβ2 which is about 400 nM or smaller, 200 nM or smaller such as about 100 nM, 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM or smaller. In one embodiment, the KD is 50 pM or smaller. In one embodiment, the KD of an individual antigen-binding site of a binding molecule of the present invention may be less than 1 pM, less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0. 1 nM, less than 10 pM, less than 1 pM, or less than 0. 1 pM. In some embodiments, the KD is from about 0.1 pM to about 1 pM. It may be an individual antigen-binding domain has such KD. It may be that such a KD for IL-12Rβ2 is displayed by the overall binding molecule of the invention. A KD affinity value KD is the equilibrium dissociation constant, a calculated ratio of Koff/Kon, between the antibody and its antigen. The association constant (Kon) is used to characterise how quickly the antibody binds to its target. The dissociation constant (Koff) is used to measure how quickly an antibody dissociates from its target. In another embodiment, a binding molecule or individual binding domain may have any of the above KD values, but for gp130.

Binding, including the presence or absence of binding, can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). Binding to an IL-12Rβ2 or gp130 may be measured using cells expressing an IL-12Rβ2 or gp 130 on their surface as discussed later in relation to assays and also in the Examples of the present application.

The ability of a binding molecule of the present invention to bring together IL-12Rβ2 and gp130 may be measured. In one embodiment, an assay which uses modified IL-12Rβ2 and gp130 that produce a detectable signal when brought together may be employed, such as the luciferase-based assay used in the Examples of the present application.

Preferred IL-12Rβ2 binding molecules

In a particularly preferred embodiment, the binding molecule comprises a VHH binding- domain or domains. In one preferred embodiment, all of the binding domains are VHH domains. Table 1 of the present application provides examples of particularly preferred VHH antibodies, as well as particularly preferred CDR “sets” of three CDRs which may be employed in a binding molecule. As well as the specific VHH binding domains and CDR sets of Table 1, variants and competing VHH binding domains are also provided, or may be employed, as discussed further below. VHH domain antibodies comprise three CDRs, CDR1, CDR2, and CDR3. They do not typically comprise a light chain. Reference to a “set of CDRs” in relation to a VHH domain antibody refers to the CDR1, CDR2, and CDR3 of that VHH domain. Table 1 provides examples of VHH binding domains and CDR sets that are provided. For example, the first antibody described in Table 1 is a VHH binding domain of SEQ ID NO: 1, with the CDR1, CDR2, and CDR3 of the antibody provided as SEQ ID Nos: 211, 212, and 213 respectively. The CDR1, CDR, and CDR3 sequences of SEQ ID NOs: 211, 212, and 213 may be therefore referred to as a “set” of CDR sequences.

Also representing particularly preferred VHH domains are humanised versions of those shown in Table 1, with the CDR sequences either remaining unchanged or representing variant CDR sequences as defined herein.

The present invention describes a binding molecule comprising, or consisting of, a VHH domain as set out in Table 1, hence a VHH domain having, or comprising, the sequence of any one of SEQ ID NOs 1 to 209. The present invention also provides a binding molecule comprising, or consisting of, a variant or competing VHH binding domain of any those set out in Table 1. The present invention further provides a binding molecule comprising a humanised version of one of the VHH domains of Table 1, so a VHH domain where the framework regions have been modified or substituted so that they are human sequences. Variants and competing VHH domains of those set out in Table 1 may also be humanised.

The present invention also provides a binding molecule comprising a “set” of CDRs from Table 1, so the sets of CDR1, CDR2, and CDR3 provided in Table 1 which have the various sequences set out as SEQ ID NOs: 211 to 837. The present invention also provides a binding molecule comprising a VHH binding domain where the CDRs are a set of three CDRs from Table 1, but the framework regions of the VHH binding domain are human.

Examples of particularly preferred sets of three CDRs for a VHH domain specific for IL- 12Rβ2 include those of the clones 20422, 20432, 21053, and 21060. Also preferred are variants of those specific sets of CDRs. Also preferred are VHH binding domains that are able to compete for binding to IL-12Rβ2 with the CDR sets clones 20422, 20432, 21053, and 2106. Particularly preferred are the sets of three CDRs from the clones 20422 and 20432. An example of a preferred CDR set is one comprising SEQ ID Nos: 364/365/366. An example of a preferred CDR set is one comprising SEQ ID Nos: 412/413/414. An example of a preferred CDR set is one comprising SEQ ID Nos: 796/797/798. An example of a preferred CDR set is one comprising SEQ ID Nos: 835/836/837. An example of a particularly preferred CDR set is one comprising SEQ ID NOs: 364/365/366. An example of a particularly preferred CDR set is one comprising SEQ ID NOs: 412/413/414. Variants and competing versions to any of the CDR sets mentioned in this paragraph are also preferred.

Further examples of particularly preferred CDR sets are those of clones 20427, 20378, 20407, 20418, 20370, 20389, 20391, 20404, and 21061. Examples of particularly preferred CDR sets are those of clones 20427, 20418, 20407, and 20391. An especially preferred CDR set is that of 20391. An example of a preferred CDR set is one comprising SEQ ID Nos: 598/599/600. An example of a preferred CDR set is one comprising SEQ ID Nos: 604/605/606. An example of a preferred CDR set is one comprising SEQ ID Nos: 652/653/654. An example of a preferred CDR set is one comprising SEQ ID Nos: 655/656/657. An example of a preferred CDR set is one comprising SEQ ID Nos: 730/731/732. An example of a preferred CDR set is one comprising SEQ ID Nos:736/737/738. An example of a preferred CDR set is one comprising SEQ ID Nos: 739/740/741. An example of a preferred CDR set is one comprising SEQ ID Nos:772/773/774. An example of a preferred CDR set is one comprising SEQ ID Nos: 811/812/813. An example of a particularly preferred CDR set is one comprising SEQ ID Nos: 598/599/600. An example of a particularly preferred CDR set is one comprising SEQ ID Nos: 652/653.654. An example of a particularly preferred CDR set is one comprising SEQ ID Nos: 655/656/657. An example of a particularly preferred CDR set is one comprising SEQ ID Nos: 739/740/741. An example of an especially preferred CDR set is one comprising SEQ ID Nos: 739/740/741. Variants and competing versions to any of the CDR sets mentioned in this paragraph are also preferred.

In another preferred embodiment, a preferred VHH is that from clones 20422, 20432, 21053, and 2106. Particularly preferred are the VHHs from the clones 20422 and 20432. Examples of preferred VHHs are those of clones 20427, 20378, 20407, 20418, 20370, 20389, 20391, 20404, and 21061. Examples of particularly preferred VHHs are those of clones 20427, 20418, 20407, and 20391. An example of an especially preferred VHH is that of clone 20391. In one embodiment, preferred VHHs are those corresponding to the clones mentioned above, as well as variants, and competing versions thereof. In another preferred embodiment, a preferred VHH is that from clones 20407, 20432, or 20391 or a variant version and in particularly a humanised version. An especially preferred VHH is that from 20407 or a variant version and in particular a humanised version. In one particularly preferred embodiment, a binding molecule comprises the CDR set from one of clones 20407, 20432, or 20391 or a variant version of those CDRs. In one preferred embodiment, a VHH employed is from the 2032 clone, or a variant thereof, particularly a humanised version.

In an especially preferred embodiment, a VHH domain specific for IL-12Rβ2 of a binding molecule of the present invention comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos 652/653/654 or a variant set. In one preferred embodiment, variant sets have no more than three amino acid sequence changes per CDR. In a more preferred embodiment, they have no more than two such amino acid sequence changes. In an even more preferred embodiment, they have only a single amino acid sequence changes.

In one embodiment, where the binding molecule is biparatopic for IL-12Rβ2, it will comprise CDR set combination from one of the following pairs of clones 20407/20391, 20407/20432, and 20432/20391 or variant CDR sets. In one preferred embodiment, it will comprise VHH domains from one of the following pairs of clones 20407/20391, 20407/20432 which have been humanised. A particularly preferred combination is 20407/20391, as well a a binding molecule where both of the binding sites specific for IL-12Rβ2 have the CDR sets from the 20407/20391 combination.

As discussed further below, as well as the specific VHH domains and CDR sets provided herein, the present invention also provides variants of those specific sequences which may be also employed in binding molecules of the present invention. The present invention also provides binding molecules and VHH domains which are able to compete for target binding with binding molecules comprising the specific VHH domains or the specific sets of CDRs set out herein. Variants of specific sequences in relation to IL-12Rβ2 binding are discussed further below. Variants and competing binding sites to any of the above mentioned sequences may be employed in preferred embodiments.

The present invention provides a binding molecule comprising at least one of the specific IL- 12Rβ2 CDR sets, VHH domains, variants of any of thereof, or competing versions of any of thereof. The present invention provides such a binding molecule which is just specific for IL-12Rβ2. It also though provides binding molecules with such binding sites where they further comprise at least one binding site for gp 130.

Preferred gpl30 binding sites/antibodies

In a particularly preferred embodiment, the binding molecule comprises a VHH binding- domain or domains. In a particularly preferred embodiment, all of the binding domains are VHH domains. Table 2B of the present application provides examples of particularly preferred VHH antibodies, as well as particularly preferred CDR “sets” of three CDRs which may be employed in a binding molecule. As well as the specific VHH binding domains and CDR sets of Table 2B, variants and competing VHH binding domains are also provided, or may be employed, as discussed further below. VHH domain antibodies comprise three CDRs, CDR1, CDR2, and CDR3. They do not typically comprise a light chain. Reference to a “set of CDRs” in relation to a VHH domain antibody refers to the CDR1, CDR2, and CDR3 of that VHH domain. Table 2B provides examples of VHH binding domains and CDR sets that are provided. For example, the first antibody describe in Table 2B is a VHH binding domain of SEQ ID NO: 840, with the CDR1, CDR2, and CDR3 of the antibody provided as 1412, 1413, and 1414 respectively. The CDR1, CDR, and CDR3 sequences of SEQ ID NOs: 1412/1413/1414 may be therefore referred to as a “set” of CDR sequences.

The present invention provides a binding molecule comprising, or consisting of, a VHH domain as set out in Table 2B, hence a VHH domain having, or comprising, the sequence of any one of SEQ ID NOs: 840 to 1411. The present invention also provides a binding molecule comprising, or consisting of, a variant or competing VHH binding domain of any those set out in Table 2. The present invention further provides a binding molecule comprising a humanised version of one of the VHH domains of Table 2, so a VHH domain where the framework regions have been modified or substituted so that they are human sequences. Variants and competing VHH domains of those set out in 'fable 2 may also be humanised.

The present invention also provides a binding molecule comprising a “set” of CDRs from Table 2, so the sets of CDR1, CDR2, and CDR3 provided in Table 2 which have the various sequences set out as SEQ ID NOs: 1412 to 3127. The present invention also provides a binding molecule comprising a VHH binding domain where the CDRs are a set of three CDRs from fable 2B, but the framework regions of the VHH binding domain are human.

Preferred sets of three CDRs include those from the 18406, 19556, 18439, 19557, 18442, and 19528 clones. Particularly preferred sets of CDRs include those from the 18406 and 18439 clones. An especially preferred set of CDRs is that from the 18439 clone. Such a set of CDRs, a variant set, or a competing set may be employed in the present invention. The CDRs from the 18439 clone are particularly effective as they appear to give good results with a number of different IL-12Rβ2 binding sites. In one embodiment a preferred set of CDRs is that of SEQ ID NOs: 2226/2227/2228. In one embodiment a preferred set of CDRs is that of SEQ ID NOs: 2734/2735/2736. In one embodiment a preferred set of CDRs is that of SEQ ID NOs: 1983/1984/1985. In one embodiment a preferred set of CDRs is that of SEQ ID Nos: 2704/2705/2706. In one embodiment a preferred set of CDRs is that of SEQ ID NOs: 2274/2275/2276. In one embodiment a preferred set of CDRs is that of SEQ ID NOs: 284/2843/2844. A particularly preferred set of CDRs is that of SEQ ID NOs: 2226/2227/2228. A particularly preferred set of CDRs is that of SEQ ID NOs: 1983/1984/1985. An especially preferred set of CDRs is that of SEQ ID NOs: 1983/1984/1985. Variants CDRs and CDRs sets resulting in a VHH that is able to compete with a VHH with one of those specific CDRs are also preferred.

In another embodiment, preferred VHH domains are those of 18406, 19556, 18439, 19557, 18442. In another embodiment, particularly preferred VHH domains are those of the 18406 and 18439 clones. An especially preferred VHH domain is that of the 18439 clone. Hence, preferred VHH domains include those of SEQ ID NOs: 1112, 1281, 1031, 1271, 1128 and 1317. Particularly preferred VHH domains are those of SEQ ID NOs: 1112 and 1031. An especially preferred VHH domain is that of SEQ ID NO: 1031. Variant VHHs of any of those specific VHH domains are also preferred, as are competing VHH domains.

In another preferred embodiment, a preferred VHH specific for gp130 is that from clones 18416, 18420, 19519, 18400 and 18439 or a variant version and in particularly a humanised version. An especially preferred VHH is that from 18416 or a variant version and in particular a humanised version. In one particularly preferred embodiment, a binding molecule comprises the CDR set from one of clones 18416, 18420, 19519, 18400 and 18439 or a variant version of those CDRs. In one preferred embodiment, a VHH employed is from the 18416 clone, or a variant thereof, particularly a humanised version.

In an especially preferred embodiment, a VHH domain of a binding molecule of the present invention comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos 2217/2218/2219 or a variant set. In one embodiment, a VHH domain of a binding domain of the present invention comprises the CDR1/CDR2/CDR3 set of 2217/2218/2219; 2011/0212/2013; 2746/2747/2748; 2130/2131/2132; or 1983/1984/1985 or a variant set thereof. In one preferred embodiment, variant sets have no more than three amino acid sequence changes per CDR. In a more preferred embodiment, they have no more than two such amino acid sequence changes. In an even more preferred embodiment, they have only a single amino acid sequence change.

In one embodiment, where the binding molecule is biparatopic for IL-12Rβ2, it will comprise CDR set combination from one of the following pairs of clones 19537/19539 or variant CDR sets. In one preferred embodiment, it will comprise VHH domains from one of the following pairs of clones 18416/18420; 18416/19519; 18416/18400; 18416/18439; 18420/19519; 18420/18400; 18420/18439; 19519/18400; 19519/18439 or 18400/18439 which have been humanised. Also preferred are binding molecules that comprise the CDR sets from such pairings, as well as variants thereof. The present invention provides a binding molecule comprising at least one of the specific gp130 CDR sets, VHH domains, variants of any of thereof, or competing versions of any of thereof. The present invention provides such a binding molecule which is just specific for gp130. It also though provides binding molecules with such binding sites where they further comprise at least one binding site for IL-12Rβ2.

As discussed further below, as well as the specific VHH domains and CDR sets provided herein, the present invention also provides variants of those specific sequences which may be also employed in binding molecules of the present invention. The present invention also provides binding molecules and VHH domains which are able to compete for target binding with binding molecules comprising the specific VHH domains or the specific sets of CDRs set out herein. Variants of specific sequences in relation to gp130 and gp130 binding are discussed further below. In one embodiment, a variant sequence will retain at least 50% of the ability to bind IL-12Rβ2 or gp 130 in comparison to the specific sequence, particularly to bind IL-12Rβ2 or gp30 on the cell surface. In one embodiment, a variant will retain at least 60%, 70%, or 80% of the ability to bind. In another embodiment, it will retain at least 90% of the ability to bind in comparison to the specific sequence. In another embodiment, it will retain at least 95% of the ability to bind. In one embodiment, an individual binding domain may retain that degree of binfing.

Preferred combinations of IL-12Rβ2 and gp130 binding sites/antibodies

Tables 11 and 15 set out combinations of different IL-12Rβ2 and gp130 binding sites. In a preferred embodiment, a binding molecule of the invention comprises at least one VHH domain comprising the CDR set for the IL-12Rβ2 binding site and at least one VHH domain comprising the CDR set for the gp130 CDR set from those specific combinations present in the VHH binding domains of a binding molecule in Tables 11 and 15. Variant and competing CDR sets may be also employed.

Preferred combinations of CDR sets from Table 11 include those of 18493 x 20422, 1840 x 20422, 18406 x 20422, and 18406 x 20432. Particularly preferred combinations are 18439 x 20422 and 18439 x 20432. An especially preferred combination is 18439 x 2042. Variant and competing versions of those CDR sets may be also employed. Preferred combinations of those from Table 15 are those of 18439 x 20427, 18439 x 20418, 18439 x 20407, and 18439 x 20391. An especially preferred combination is 18439 x 20391. Variants and competing versions of the CDR sets may be employed. In a preferred embodiment, such CDR sets are in a binding molecule of the format bsVHH-Fc. In a preferred embodiment, the binding molecule comprises two polypeptides where each polypeptide has one VHH domain of each specificity, as well as a constant region for the Fc. In an especially preferred embodiment, the Fc lacks Fc effector function.

In another preferred embodiment, the binding molecule has the CDR sets from the 18406 and 20432. In a preferred embodiment, the binding molecule is in the format of two polypeptides, each with the VHH for one specificity, but not the other, where the constant region comprises a knob-into- hole modification as discussed herein.

Particularly preferred binding molecules are those that use the CDR sets from clone 18439 for the gp130 specificity and those from one of clones 20427, 20418, 20407, and 20391 for the IL-12Rβ2 specificity. Variant CDRs and competing CDRs may be employed. The binding molecule format for those combinations may be any of those set out herein. In a particularly preferred embodiment, the format of such a binding molecule is either of the bivalent formats shown in Figure 4A. Hence, the binding molecule comprises two polypeptides, each polypeptide having a VHH specific for gp130 and a VHH specific for IL-12Rβ2, with the two VHHs optionally joined by a linker, with the polypeptides each further comprising a hinge region, CH2 and CH3 domain. The constant region may be any of those set out herein. In a preferred embodiment, the binding molecule has any of the possible constant regions set out herein. An especially preferred combination the CDR set from clone 18439 for the gp130 specificity and the CDR set from clone 20391 for the IL-12Rβ2 specificity. Variant CDR sets and competing CDR sets of those specific ones may be also employed.

In another preferred embodiment, the CDR sets from clone 18406 for the gp130 specificity and those from one of clone 20432 for the IL-12Rβ2 specificity are employed. Variant CDR sets or competing CDR sets may be also employed. In a preferred embodiment, the binding molecule is in the monovalent KiH format shown in Figure 4A. Hence, the binding molecule comprises two polypeptides, each with the VHH for one of the specificities, with a constant region consisting of a hinge, CH2 and CH3 region, where the constant regions of the two polypeptides comprise knob-into- hole mutations favouring heterodimerization over homodimerization.

Illustrative preferred binding molecules

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for IL-12Rβ2, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 652/653/654 as the CDR1, CDR2, and CDR3 respectively, or variants thereof. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for IL- 12Rβ2, wherein the binding site comprises a VHH of clone 20407. In a preferred embodiment, a binding site specific for IL-12Rβ2 is a humanised version of the VHH clone 20407.

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2217/2218/2219 as the CDR1, CDR2, and CDR3 respectively. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the binding site comprises a VHH of clone 18416. In a preferred embodiment, a binding site specific for gp130 is a humanised version of the VHH clone 18416.

Further preferred combinations of gp130 and IL-12Rβ2 antigen-binding sites

As part of identifying further preferred binding molecules, the present inventors screened the combinations of VHH binding domains shown in Table 17 of the present application. A polypeptide was designed comprising a VHH specific for gp130 and a VHH specific for IL-12Rβ2 and a Fc region so when expressing a binding molecule comprising two such polypeptides was generated. All of the permutations shown in Table 17 were generated and the permutations all represent part of the present invention. In particular, the present invention provides a binding molecule comprising two polypeptides, each polypeptide comprising a VHH specific for gp130 comprising a set of three CDRs corresponding to one of those indicated in Table 17 and a VHH specific for IL-12Rβ2 having a set of three CDRs that come from the same binding molecule is Table 17. So, for example, in one embodiment, a binding molecule comprises two polypeptides, wherein each polypeptide comprises a VHH domain specific for gp130 comprising the CDRs of the 18033 clone and a VHH domain specific for IL-12Rβ2 comprising the CDRs of the 20407 clone, so effectively having the CDRs from the bsVHH-Fc-73. The same goes for all of the binding molecules set out in Table 17. Variant CDRs may also be employed so long as the ability to bind to gp130 and IL-12Rβ2 is retained. VHH domains able to compete with the specific ones used in Table 17 may also be employed. In one preferred embodiments, variants with up to 3 amino acid sequence changes per CDR are preferred. In a more preferred embodiment, up to 2 amino acid sequence changes per CDR are possible. In a still more preferred embodiment, a single amino acid sequence change per CDR is possible.

Preferred binding molecules based on the combinations identified in Table 17 and in particular those based on the CDRs used in the bsVHH-Fc-77, bsVHH-Fc-78, bsVHH-Fc-84, and bsVHH-Fc-96 molecules from Table 17 are discussed in the sections that follow as well as those based on bsVHH-Fc-71. Figures 10 and 11 also provide preferred examples of CDR sets that be employed in combination with each other.

Illustrative preferred binding molecules based on the 20391 and 18439 clones

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for IL-12Rβ2, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 739/740/741 as the CDR1, CDR2, and CDR3 respectively, or variants thereof. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for IL- 12Rβ2, wherein the binding site comprises a VHH of clone 20407. In a preferred embodiment, a binding site specific for IL-12Rβ2 is a humanised version of the VHH clone 20407.

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 1983/1984/1985 as the CDR1, CDR2, and CDR3 respectively. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the binding site comprises a VHH of clone 17116. In a preferred embodiment, a binding site specific for gp130 is a humanised version of the VHH clone 18439.

In one preferred embodiment, the present invention provides a binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising:

(i) at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 1983/1984/1985 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each; and

(ii) at least one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 739/740/741 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each.

In one preferred embodiment, the binding molecule comprises a polypeptide comprising at least one antigen-binding-site specific for the gp130 subunit as defined in (i) and at least one antigen- binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one particularly preferred embodiment, the polypeptide further comprises an Lc region, such as an Lc region as defined herein. In one particularly preferred embodiment the binding molecule comprises two such polypeptides.

In one preferred embodiment, the present invention provides a binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising two polypeptides, where each polypeptide comprising: (i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 1983/1984/1985 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(ii) one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 739/740/741 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites; and

(iii) an Fc region, wherein the two polypeptides associate via the Fc regions to form the binding molecule.

In one embodiment, (i) to (iii) are present in that order going from N to C terminus of the polypeptide.

In one particularly preferred embodiment, the binding site of (i) and the binding site of (ii) are joined to each other in the polypeptide sequence via a linker. Any suitable linker may be employed, such as any set out herein. In one particularly preferred embodiment, the linker is a GS linker. Examples of linkers include those of (G4S)n, wherein n is a value of 1 to 10. Preferred linkers include those where n has a value of 2 to 8. More preferred linkers are those where n has a value of 3 to 8. In one preferred embodiment where the binding molecule comprises CDR sets from clones 20391 and 18439 and particularly where the molecule is bsVHH-Fc71 n has a value of 7.

In one preferred embodiment, the present invention provides a binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising two polypeptides, where each polypeptide comprises:

(i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 1983/1984/1985 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen-binding sites;

(ii) an antigen-binding -site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 739/740/741 as the CDR1, CDR2, and CRD3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(iii) optionally a linker, preferably of sequence (G4S)n wherein n is 2 to 10, more preferably 3 to 8; and (iv) a Fc region, preferably which is a Durvalumab constant region, preferably lack the CHI region, wherein the two polypeptides associate via the Fc regions to form the binding molecule.

In one preferred embodiment, the binding molecule is, or comprises two polyeptides each comprising the CDR sets present in bsVHH-Fc-71. Variants of those CDRs may also be employed as may antigen-binding sites that can compete with those of bsVHH-Fc-71 for binding to gp130 and IL- 12Rβ2. Preferably each polypeptide comprises humanised versions of the 18439 and 20407 VHH regions.

An example of a particularly preferred binding molecule is one comprising two polypeptides, wherein each polypeptide comprises: a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 1983, 1984, and 1985 respectively; a linker; a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 739, 740 and 741 respectively, and a Fc region derived from the Durvalumab constant region lacking the CHI region.

Illustrative preferred binding molecules based on the 20407 and 18416 clones

Especially preferred binding molecules are those comprising CDR sets based on the 20407 VHH clone specific for IL-12Rβ2. Further, especially preferred binding molecules are those comprising CDR sets based on the 18416 VHH clone specific for IL-12Rβ2. Further, especially preferred binding molecules are based on those comprising CDR sets from both. Variants and binding molecules able to compete with antigen-binding sites comprising those CDRs are also preferred.

(i) at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2217/2218/2219 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each; and

(ii) at least one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 652/653/654 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each.

In one preferred embodiment, the binding molecule comprises a polypeptide comprising at least one antigen-binding-site specific for the gp130 subunit as defined in (i) and at least one antigen- binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one particularly preferred embodiment, the polypeptide further comprises an Fc region, such as an Fc region as defined herein. In one particularly preferred embodiment the binding molecule comprises two such polypeptides.

In one preferred embodiment, the present invention provides a binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising two polypeptides, which each polypeptide comprising:

(i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2217/2218/2219 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(ii) one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 652/653/654 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites; and

(iv) an Fc region, wherein the two polypeptides associate via the Fc regions to form the binding molecule. In one embodiment, (i) to (iii) are present in that order going from N to C terminus of the polypeptide.

In one particularly preferred embodiment, the binding site of (i) and the binding site of (ii) are joined to each other in the polypeptide sequence via a linker. Any suitable linker may be employed, such as any set out herein. In one particularly preferred embodiment, the linker is a GS linker. Examples of linkers include those of (G4S)n, wherein n is a value of 1 to 10. Preferred linkers include those where n has a value of 2 to 8. Particularly preferred linkers are those where n has a value of 3 to 8.

(ii) one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 652/653/654 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(iii) optionally a linker, preferably of sequence (G4S)n wherein n is 2 to 10, more preferably 3 to 8; and

(iv) a F c region, preferably which is a Durvalumab constant region, preferably lacking the CHI region, wherein the two polypeptides associate via the Fc regions to form the binding molecule.

In one preferred embodiment, the binding molecule is, or comprises two polyeptides each comprising the CDR sets present in bsVHH-Fc-77. Variants of those CDRs may also be employed as may antigen-binding sites that can compete with those of bsVHH-Fc-77 for binding to gp130 and IL- 12Rβ2. Preferably each polypeptide comprises humanised versions of the 18416 and 20407 VHH regions.

An example of a particularly preferred binding molecule is one comprising two polypeptides, wherein each polypeptide comprises: a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 2217, 2218, and 2219 respectively; a linker; a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 652, 653 and 654 respectively, and a Fc region derived from the Durvalumab constant region lacking the CHI region. Illustrative preferred binding molecules based on the 20407 and 18420 clones

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2011/2012/2013 as the CDR1, CDR2, and CDR3 respectively. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the binding site comprises a VHH of clone 18420 In a preferred embodiment, a binding site specific for gp130 is a humanised version of the VHH clone 18420.

(i) at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2011/2012/2013 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each; and

In one preferred embodiment, the binding molecule comprises a polypeptide comprising at least one antigen-binding-site specific for the gp130 subunit as defined in (i) and at least one antigen- binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one preferred embodiment, the binding molecule comprises a polypeptide comprising one antigen-binding-site specific for the gp130 subunit as defined in (i) and one antigen-binding -site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one particularly preferred embodiment, the polypeptide further comprises an Lc region, such as an Lc region as defined herein. In one particularly preferred embodiment the binding molecule comprises two such polypeptides.

(i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2011/2012/2013 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(iv) a Fc region, preferably which is a Durvalumab constant region, preferably lack the CHI region, wherein the two polypeptides associate via the Fc regions to form the binding molecule.

In one preferred embodiment, the binding molecule is, or comprises two polyeptides each comprising the CDR sets present in bsVHH-Fc-78 Variants of those CDRs may also be employed as may antigen-binding sites that can compete with those of bsVHH-Fc-78 for binding to gp130 and IL- 12Rβ2. Preferably each polypeptide comprises humanised versions of the 18416 and 20407 VHH regions.

An example of a particularly preferred binding molecule is one comprising two polypeptides, wherein each polypeptide comprises: a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 2011, 2012, and 2013 respectively; a linker; a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 652, 653 and 654 respectively, and a Fc region derived from the Durvalumab constant region lacking the CHI region. Variant CDRs may also be employed.

Illustrative preferred binding molecules based on the 20407 and 19519 clones

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2746/2747/2748 as the CDR1, CDR2, and CDR3 respectively. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the binding site comprises a VHH of clone 19519. In a preferred embodiment, a binding site specific for gp130 is a humanised version of the VHH clone 19519.

(i) at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2746/2747/2748 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each; and

In one preferred embodiment, the binding molecule comprises a polypeptide comprising at least one antigen-binding-site specific for the gp130 subunit as defined in (i) and at least one antigen- binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one preferred embodiment, the binding molecule comprises a polypeptide comprising one antigen-binding-site specific for the gp130 subunit as defined in (i) and one antigen-binding -site specific for the IL-12Rβ2 subunit of the IL-35 receptor as defined in (ii). In one particularly preferred embodiment, the polypeptide further comprises an Fc region, such as an Fc region as defined herein. In one particularly preferred embodiment the binding molecule comprises two such polypeptides.

(i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2746/2747/2748 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

In one particularly preferred embodiment, the binding site of (i) and the binding site of (ii) are joined to each other in the polypeptide sequence via a linker. Any suitable linker may be employed, such as any set out herein. In one particularly preferred embodiment, the linker is a GS linker. Examples of linkers include those of (G4S)n, wherein n is a value of 1 to 10. Preferred linkers include those where n has a value of 2 to 8. Particularly preferred linkers are those where n has a value of 3 to 8

(iii) optionally a linker, preferably of sequence (G4S)n wherein ni is 2 to 10, more preferably 3 to 8; and

In one preferred embodiment, the binding molecule is, or comprises two polyeptides each comprising the CDR sets present in bsVHH-Fc-84 Variants of those CDRs may also be employed as may antigen-binding sites that can compete with those of bsVHH-Fc-84 for binding to gp130 and IL- 12Rβ2. Preferably each polypeptide comprises humanised versions of the 19519 and 20407 VHH regions.

An example of a particularly preferred binding molecule is one comprising two polypeptides, wherein each polypeptide comprises: a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 2746, 2747, and 2748 respectively; a linker; a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 652, 653 and 654 respectively, and a Fc region derived from the Durvalumab constant region lacking the CHI region.

Illustrative preferred binding molecules based on the 20407 and 18400 clones

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for IL-12Rβ2, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 412/413/414 as the CDR1, CDR2, and CDR3 respectively, or variants therefof. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for IL- 12Rβ2, wherein the binding site comprises a VHH of clone 20432. In a preferred embodiment, a binding site specific for IL-12Rβ2 is a humanised version of the VHH clone 18400.

In a particularly preferred embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2130/2131/2132 as the CDR1, CDR2, and CDR3 respectively. In one embodiment, each variant CDR has at most three amino acid sequence changes compared to those specific sequences. In a preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In a more preferred embodiment, the variant CDRs each have at most two amino acid sequence changes compared to the specific sequences. In one embodiment, a binding molecule comprises an antigen-binding site specific for gp130, wherein the binding site comprises a VHH of clone 18400. In a preferred embodiment, a binding site specific for gp 130 is a humanised version of the VHH clone 20391.

(i) at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2130/2131/2132 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each; and

(ii) at least one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 412/413/414 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each.

(i) a VHH comprising an antigen-binding-site specific for the gp130 subunit of the IL-35 receptor, wherein the at least one antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 2130/2131/2132 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(ii) one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 412/413/414 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites; and

(iii) an Fc region, wherein the two polypeptides associate via the Fc regions to form the binding molecule. In one embodiment, (i) to (iii) are present in that order going from N to C terminus of the polypeptide.

In one particularly preferred embodiment, the binding site of (i) and the binding site of (ii) are joined to each other in the polypeptide sequence via a linker. Any suitable linker may be employed, such as any set out herein. In one particularly preferred embodiment, the linker is a GS linker. Examples of linkers include those of (G4S)n, wherein n is a value of 1 to 10. Preferred linkers include those where n has a value of 2 to 8. Particularly preferred linkers are those where n has a value of 4 to 8. In one more preferred embodiment, n has a value of 7.

(ii) one antigen-binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor, wherein the antigen-binding site comprises a set of three CDRs which are those of SEQ ID Nos: 412/413/414 as the CDR1, CDR2, and CDR3 respectively or variant versions thereof with at most three amino acid sequence changes each or an antigen-binding site which is able to compete for binding with one of the preceding antigen binding sites;

(iii) optionally a linker, preferably of sequence (G4S)n wherein ni is 2 to 10, more preferably 4 to 8; and

(iv) a F c region, preferably which is a Durvalumab constant region, preferably lack the CHI region, wherein the two polypeptides associate via the Fc regions to form the binding molecule.

In one preferred embodiment, the binding molecule is, or comprises two polyeptides each comprising the CDR sets present in bsVHH-Fc-96 Variants of those CDRs may also be employed as may antigen-binding sites that can compete with those of bsVHH-Fc-96 for binding to gp130 and IL- 12Rβ2. Preferably each polypeptide comprises humanised versions of the 18400 and 20432 VHH regions.

An example of a particularly preferred binding molecule is one comprising two polypeptides, wherein each polypeptide comprises: a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 2130, 2131, and 2132 respectively; a linker; a VHH comprising a CDR1, CDR2, and CDR3 of SEQ ID Nos 412, 413 and 414 respectively, and a Fc region derived from the Durvalumab constant region lacking the CHI region. Variant CDRs may also be employed. Biparatopics

In one preferred embodiment, a binding molecule of the invention is biparatopic for gp130 that comprises two different antigen binding sites specific for gp130, wherein each binding site binds a different epitope of g 130.

In one preferred embodiment, a binding molecule of the invention is biparatopic for IL-12Rβ2 that comprises two different antigen binding sites specific for IL-12Rβ2, wherein each binding site binds a different epitope of IL-12Rβ2.

In one particularly preferred embodiment, a binding molecule is biparatopic for gp130 and biparatopic for IL-12Rβ2. In one embodiment, the binding molecule comprises two polypeptides with two antigen-binding sites on each polypeptide. In one preferred embodiment, each polypeptide has one binding site for gp130 and one binding site for IL-12Rβ2.

In one preferred embodiment, the binding molecule comprises:

(i) a first polypeptide comprising a first binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2755, 2756, and 2757 respectively and a first binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 412, 413, and 414 respectively or alternatively the first binding site for gp130 and/or first binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maximum of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen; and

(ii) the second polypeptide comprising a second binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2692, 2693, and 2694 respectively and a second binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 412, 413, and 414 respectively or alternatively the second binding site for gp130 and/or second binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen.

In one preferred embodiment, the binding molecule comprises:

(i) a first polypeptide comprises a first binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 1983, 1984, and 1985 respectively and a first binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 739, 740, and 741 respectively or alternatively the first binding site for gp130 and/or first binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen; and

(ii) the second polypeptide comprises a second binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2217, 2218, and 2219 respectively and a second binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 652, 653, and 654 respectively or alternatively the second binding site for gp130 and/or second binding site for IL- 12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen. In one preferred embodiment, the binding molecule in only biparatopic for one of gp130 and IL-12Rβ2, with the binding molecule being monoparatopic for the other. In one embodiment, a binding molecule comprises:

(i) a first polypeptide comprises a first binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2130, 2131, and 2132 respectively and a first binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2755, 2756, and 2757 respectively or alternatively the binding site for gp130 and/or first binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen; and

(ii) the second polypeptide comprises a first binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2130, 2131, and 2132 respectively and a second binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 412, 413, and 414 respectively or alternatively the second binding site for gp130 and/or second binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen.

In any of the embodiments wherein the binding molecule is biparatopic for gp130 and/or IL- 12Rβ2 and comprises two polypeptides, preferably the polypeptides comprise an Fc region, such as any discussed herein. In a particularly preferred embodiment, the Fc region of the polypeptides comprises sequences that favour heterodimer formation over homodimer formation. A preferred modification is the knobs-in-holes. A particularly preferred Fc region is based on Durvalumab. In one preferred embodiment, the Fc region is the Durvalumab region minus the CHI region and with knobs- in-holes modification.

Examples of preferred biparatopic molecules include those of KiH-71/77 and KiH-107/108. As well as variant versions. In a preferred embodiment, the binding molecule corresponds to KiH- 71/77 except that the VHH domains have been humanised and in particular the framework regions have been humanised. In a further preferred embodiment, the binding molecule corresponds to KiH- 71/77 except that the VHH domains have been humanised and in particular the framework regions have been humanised.

Further preferred binding molecules

Examples of particularly preferred binding molecules are shown in Figures 10 and 11. In one preferred embodiment, a binding molecule comprises the same CDR sets and same number of binding sites in the same format as shown in those Figures, but where the VHH domains have been humanised. Figure 11 shows examples of particularly preferred binding molecules in conjunction with a durvulumab constant region which are is a particular preferred embodiment. Variant binding sites and molecules

As well as the specific binding molecules, VHH binding domains, CDR sets, and other sequences set out herein variant forms of those may be also employed in the present invention. Variants may be defined, for example, in terms of having a particular level of sequence identity or number of sequence changes in comparison to a specific VHH domain or set of CDR sequences from Table 1 or 2B. The sequence identity may be over the entire length of a sequence, such as over the entire length of a VHH domain or just over the length of the set of CDR sequences. A variant may have such a level of sequence identity or modification and retain the function of the specific sequence. Hence, for instance, a variant IL-12Rβ2 binding site will typically retain the ability to bind IL-12Rβ2. A variant gp130 binding site will typically retain the ability to bind gp130. In a preferred embodiment, a variant binding molecule will retain the ability to bind to IL-12Rβ2 and gp130. Preferably, the variant will also retain the ability to act as an agonist of IL-35R. In one preferred embodiment, a variant binding molecule will retain the ability to activate STAT3, for instance when measured using an assay as described in the Examples of the present application. It may retain the ability to stimulate IL- 10 production, for instance as measured in the Examples of the present application. In one embodiment, activation may be measured by measuring phosphorylation of STATE In another embodiment, activation of STAT4 will be measured. In a particularly preferred embodiment though, activation as measured by phosphorylation of STAT3 protein will be measured and a variant will retain the ability to bring about such activation.

Sequence identity can be defined in terms of over the entire length of the polypeptide in question or over the regions discussed above, such as over the CDR set or VHH domain. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST™ software available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al., 1996, Meth. Enzymol. 266: 131-141; Altschul, S.E et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656). “Identity”, as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity", as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);

- lysine, arginine and histidine; - aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having amide side chains); and

- cysteine and methionine (amino acids having sulphur-containing side chains).

In one embodiment, a variant VHH binding domain may have at least 80% amino acid identity, for example 85% or greater, such as 90% or greater, in particular 95%, 96%, 97%, 98% or 99% or greater identity to one of the specific VHH binding domains set out in Table 1 or 2B. In one embodiment a sequence may have at least 95% sequence identity to at least one of those sequences. In another embodiment, a variant VHH binding domain may have such percentages values in relation to the degree of amino acid sequence similarity that they display to the specific sequence. The variant will be still able to bind the relevant IL35R subunit, i.e. IL-12Rβ2 or gp130. Typically, the overall binding molecule will still be able to act as an agonist of the receptor. In a preferred embodiment, such levels of sequence identity are over the overall length of the specific CDR set.

In another embodiment, a binding molecule of the invention may comprise a variant set of CDRs which are a variant of one of the specific CDR sets of Table 1 or 2B. In one embodiment, only one of the three CDRs shows sequence variation in comparison to the corresponding CDR of the set of three CDRs. In another embodiment, two of the CDRs show sequence variation in comparison to the specific set of three CDRs. In another embodiment, all three CDRs may show sequence variation compared to the specific CDRs of the set. In one embodiment, the sequence variation is only in the CDR3 of the CDR set. In one embodiment, the level of sequence identity over the total length of the three CDRs in comparison to the set of three specific CDR sequences from Table 1 or 2B is at least 80%. In another embodiment, it is at least 85%. In a further embodiment, the level of identity is at least 90%. In a preferred embodiment, the level of sequence identity is at least 95%. The variant will still be able to bind the relevant IL-35R subunit, i.e. IL-12Rβ2 or gp130. Typically, the overall binding molecule will be still able to act as an agonist of the receptor.

In one embodiment, a variant may have a set of three CDRs comprising from one to twenty, such as from one to ten, for example as one, two, three, four, five or up to those values of amino acid sequence changes or at least those values, or up to those values compared to the set of CDRs from Table 1 or 2B, so long as the variant is still able to bind the relevant IL-35R subunit, i.e. IL-12Rβ2 or gp130. Typically, the overall binding molecule will still be able to act as an agonist of the receptor.

In another embodiment, a variant of the present invention may have at least five, six, seven, eight, nine, ten, eleven or twelve amino acid sequence changes compared to the CDRs of one of the specific antibodies set out herein, for example it may have that number of sequence changes in a set of CDRs making up a VHH domain. A binding molecule of the present invention may have that number of sequence changes in a set of three CDRs compared to the sequence of the set of CDRs identified in Table 1 or 2B. In one embodiment, a set of three CDRs may have from five to ten, ten to fifteen, or fifteen to twenty amino acid sequence changes compared to a specific set of three CDRs set out herein. Variant binding molecules will typically retain the ability to specifically bind IL-12Rβ2 or gp130. They may also retain one of the other functions set out herein. The overall binding molecule will be typically still able to act as an agonist of the IL-35R.

In one embodiment, the binding molecules, are mutated to provide improved affinity for the relevant IL-35R subunit. Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. colt (Low et al J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al Nature, 391, 288-291, 1998). Vaughan et al discusses these methods of affinity maturation (Vaughan et al., Nat. Biotech., 16, 535-539, 1998). Where not specifically for VHH domains such approaches may be adapted for them. Improving the affinity of binding of individual binding sites will typically also improve the overall avidity for the target where the binding molecule has more than one binding site.

The present invention also provides, and may employ, binding molecules which are able to compete with the specific binding molecules set out herein. The assays section of the present application sets out various binding and competition assays that may be performed and such assays may be used to confirm a given binding molecule is one able to compete with one of the specific binding molecules set out herein. Hence, the present invention also provides a binding molecule that is able to compete for binding with one of the VHH binding domains of Table 1 or 2B. The present invention also provides a binding molecule that is able to compete for binding to IL-12Rβ2 or gp130 with a binding molecule having a VHH binding-domain comprising a set of three CDRs from Table 1 or 2B. In one embodiment, variant antibodies may be identified by identifying such antibodies that are able to cross-block specific antibodies set out herein. Cross-blocking binding molecules, in particular antibodies, can be identified using any suitable method in the art, for example by using competition ELISA or BIAcore assays where binding of the cross-blocking antibody to antigen prevents the binding of an antibody of the present invention or vice versa. Such cross-blocking assays may use cells expressing IL-12Rβ2 and/or gp130 as a target and preferably both. In one embodiment, flow cytometry is used to assess binding to cells expressing them. In embodiments where a binding site is defined by competition to a binding site with a specific sequence, it may be that the assay for competition is performed with just the individual VHH binding domains for that specificity rather than the overall binding molecule.

The skilled person may generate binding molecules using any suitable method known in the art. Antigen polypeptides, for use in generating antibodies for example for use to immunize a host or for use in panning, such as in phage display, may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems or they may be recovered from natural biological sources. In one embodiment, the host may be immunised with a cell expressing an IL-12Rβ2. In a particularly preferred embodiment, a VHH domain of the present invention is obtained by immunising a camelid and in particular a llama. In another embodiment gp130 may be used as the immunogen. In a preferred embodiment, VHH binding domains specific for IL-12Rβ2 and gpI30 may be generated and assessed individually and then assembled into a binding molecule for both specificities, which can in turn be assessed.

In one example, the antigen-binding sites, and in particular the VHH regions, of the antibodies according to the invention are humanised. Humanised (which include CDR-grafted antibodies) as employed herein refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g., US 5,585,089; WO91/09967 which are incorporated by reference). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In a preferred embodiment though, the whole CDR or CDRs is/are transplanted. Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived. As used herein, the term “humanised antibody molecule” refers to an antibody molecule wherein one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a murine monoclonal antibody) are grafted into a framework of an acceptor antibody (e.g., a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment, the whole binding molecule may be humanised, so the CDRs are introduced into a molecule which is otherwise human.

When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate, and human framework regions. Suitably, the humanised antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein. Examples of human frameworks which can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM. For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used; these are available at:

In a humanised binding molecule of the present invention, particularly a VHH domain based antibody, the acceptor framework does not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains. The framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91/09967. Derivatives of frameworks may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids replaced with an alternative amino acid, for example with a donor residue. Donor residues are residues from the donor antibody, i.e., the antibody from which the CDRs were originally derived, in particular the residue in a corresponding location from the donor sequence is adopted. Donor residues may be replaced by a suitable residue derived from a human receptor framework (acceptor residues).

The Kabat et al numbering system is referred to herein. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al. (supra)”). This numbering system is used in the present specification except where otherwise indicated. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard Kabat numbered sequence. The CDRs of the heavy chain variable domain are typically located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Uesk, A.M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR- H1 extends from residue 26 to residue 32. Thus, unless indicated otherwise “CDR-H1” as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia’s topological loop definition. The CDRs of the light chain variable domain are typically located at residues 24-34 (CDR-U1), residues 50-56 (CDR-U2) and residues 89-97 (CDR- U3) according to the Kabat numbering system.

The skilled person is able to test variants of CDRs or humanised sequences in any suitable assay such as those described herein to confirm activity is maintained.

Further provided, and which may be employed, are binding molecules that bind the same epitope on IL-12Rβ2 or gp130 as one of the specific antibodies set out herein. For instance, the binding molecule may be an antibody that binds to the same epitope.

A further preferred embodiment, is the change of the first amino acid of the IL-12Rβ2 VHH sequence from glutamine (Q) to glutamic acid (E). This AA change is present in bsVHH-Fc 55 to the last bsVHH-Fc (including the biparatopic bsVHH-Fc). It may though also be introduced into any of bsVHH-Fc 1 to 54 as well, as well as in the IL-12Rβ2 VHH domains set out herein. Constant regions

In one particularly preferred embodiment, a binding molecule of the present invention will comprise a constant region. In a particularly preferred embodiment, the constant region will have been modified so that the effector functions of the Fc region have been reduced or eliminated. For example, in a preferred embodiment for a binding molecule of the present invention that can bind both IL- 12Rβ2 and gp130, binding to both or either will not result in antibody effector functions such as ADCC, ADCP, or CDC. Thus, the binding molecule will be preferably able to act as an agonist of IL- 35, but not kill the target cell.

In a preferred embodiment, a binding molecule of the present invention does not bind Fc receptors and in particular does not bind to FcyR receptors. In one preferred embodiment, the binding molecule of the present invention is an antibody and it does not bind to Fc receptors, either because it does not comprise an Fc region or alternatively as it is has an Fc region modified so that it does not bind Fc receptors. Fc domain as employed herein generally refers to -(CFLCFL^, unless the context clearly indicates otherwise, where CH2 is the heavy chain CH2 domain, CH3 is the heavy chain CH3 domain, and there are two CH2CH3 with one from each heavy chain. In one embodiment, an antibody of the present invention does not comprise a -CH2CH3 fragment. In one embodiment, an antibody of the present invention does not comprise a CH2 domain. In one embodiment, an antibody of the present invention does not comprise a CH3 domain.

In one embodiment, a binding molecule of the present invention binds to an Fc gamma receptor but to a substantially decreased extent relative to binding of an identical antibody comprising an unmodified Fc region to the FcgR (e.g., a decrease in binding to a FcyR by at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to binding of the identical antibody comprising an unmodified Fc region to the FcyR as measured). In a particularly preferred embodiment though the binding molecule has no detectable binding to an FcyR at all. Binding, including the presence or absence of binding, can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORE™ analysis or Octet™ analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g. gel filtration).

In one embodiment, where an Fc region is present, the Fc region employed is mutated, in particular comprising a mutation described herein. In one embodiment the mutation is to remove binding to Fc receptors and in particular FcyR. In one embodiment, an antibody may comprise an aglycosylated Fc region, for example to bring about reduced Fc function and in particular a nearly Fc-null phenotype. In one embodiment, an antibody has a modification at N297 and in particular N297A. In one embodiment an antibody has modifications at F243 and/or F244 of the constant region, in particular ones that mean that the antibody comprises a glycosylated constant region. In one embodiment, an antibody may comprise the F243A and/or F244A heavy chain modifications. In another embodiment, one or more of F241, F243, V262 and V264 may be modified and particularly to amino acids that influence glycosylation. In one embodiment, an antibody may have modifications at F241A, F243A, and/or V262E. In one embodiment, it may have the modification V264E. Yu et al (2013) Journal of American Chemical society, 135(26): 9723-9732, which is incorporated by reference in its entirety, particularly in relation to the modifications discussed therein. In one particular preferred embodiment, an antibody of the present invention may comprise the LALA modification, Leu234Ala/Leu235Ala. In another particularly preferred embodiment, an antibody of the present invention may comprise the LFLEPS modification, Leu234Phe/Leu235Glu/Pro331/Ser. Further, a binding molecule, in particular an antibody, of the present invention may be produced in a cell type that influences glycosylation as a further approach for sugar engineering. In one embodiment, the fucosylation, sialylation, galactosylation, and/or mannosylation may be altered either by sequence modifications and/or via the type of cell used to produce the binding molecule, and in particular antibody.

In one embodiment, an antibody has modifications at position 297 and/or 299. For example, in one embodiment, an antibody of the present invention comprises a N297A modification in its heavy chains, preferably N297Q or mutation of Ser or Thr at 299 to other residues.

In one embodiment, a binding molecule of the present invention, and in particular an antibody, comprises two different heavy chains where the heavy chains comprise modifications that allow the different heavy chains to preferentially associate compared to heavy chains associating with identical heavy chains. Such an approach may be in particular employed where the antibody different antigen-binding sites on each of the polypeptides comprising the binding molecule, for instance where one polypeptide comprises at least one antigen-binding site specific for IL-12Rβ2 and the other polypeptide comprises at least one antigen binding site specific for gp130, wherein each polypeptide does not comprise an antigen binding site for the other specificity. In one embodiment, the two different heavy chains comprise knob-in -hole mutations. In certain embodiments, the knob-into-hole mutations are a T366W mutation in one heavy chain constant region and a T366S, L368A, and a Y407V mutation in the other domain. In one preferred embodiment, the knob-into-hole mutations employed are E357K/E399K and/or K392D/K409D. In one particularly preferred embodiment, the knob-into-hole modifications employed are E357K/E399K and K392D/K409D. In other embodiments, the knob-in hole mutations are a T366W mutation in one heavy chain constant region and a T366S, L368A, and a Y407V mutation in the other domain. In certain aspects, the modifications comprise charge-pair mutations. In certain aspects, the charge-pair mutations are a T366K mutation in one of the heavy chain constant regions and a corresponding L35 ID mutation in the other domain. In an alternative embodiment, rather than have modifications that result in preferential pairing of different heavy chains, the heavy chains comprise modifications that mean a heterodimer comprising the two heavy chains can be purified preferentially from the homodimers only comprising one type of heavy chain. For example, the modifications may alter affinity for Protein A, with one heavy chain still able to bind Protein A, whilst the modified heavy chain does not do so, meaning that heterodimers of the two different heavy chains can be purified based on their affinity for Protein A.

In other embodiments, heavy and light chains may comprise modifications that change whether or not a disulphide bridge is formed between them. In a variety of embodiments, the modifications comprise mutations that generate engineered disulfide bridges between light and heavy chains. As described herein, “engineered disulfide bridges” are mutations that provide non- endogenous cysteine amino acids in two or more polypeptides such that a non-native disulfide bond forms when the two or more domains associate. Engineered disulfide bridges are described in greater detail in Merchant et al. (1998) Nature Biotech., 16:677-681, the entirety of which is hereby incorporated by reference. In a particular embodiment, the mutations that generate engineered disulfide bridges are a K392C mutation in one of a first or second CH3 domains, and a D399C in the other CH3 domain. In a preferred embodiment, the mutations that generate engineered disulfide bridges are a S354C mutation in one of a first or second CH3 domains, and a Y349C in the other CH3 domain. In another preferred embodiment, the mutations that generate engineered disulfide bridges are a 447C mutation in both the first and second CH3 domains that are provided by extension of the C-terminus of a CH3 domain incorporating a KSC tripeptide sequence.

In one embodiment, a binding molecule, and in particular an antibody, of the present invention has Fc region modification(s) that alter the half-life In one embodiment, binding molecules, and in particular antibodies, of the present invention may comprise modifications that alter serum half-life. Such modifications may be present as well as those that alter Fc functions. In one particularly preferred embodiment, a binding molecule, and in particular an antibody, of the present invention has modification(s) that alter its serum half-life compared to in the absence of such modifications. In one embodiment, the modifications result in increased serum half-life. In another embodiment, they result in decreased serum half-life. In another preferred embodiment, an antibody comprises one or more modifications that collectively both silence the Fc region and decrease the serum half-life of the antibody compared to an antibody lacking such modifications.

Illustrative examples of constant region modifications that may be included in particular embodiments of the invention include:

• N297A - Asn297Ala which confers Fc silencing;

• LALA - Leu234Ala/Leu235Ala which decreases binding to Fc receptors;

• LFLEPS - Leu234Phe/Leu235Glu/Pro331/Ser which decreases binding to Fc receptors; • PG - Pro329Gly which decreases binding to Clq;

• LALA-PG - Leu234Ala/Leu235Ala/Pro329Gly which decreases binding to Fc receptors and Clq;

• TM - Pro33 lSer/Leu234Glu/Leu235Phe - which decreases binding to Fc receptors and Clq;

• DA - Asp265Ala - which decreases binding to Fc receptors;

• GRLR - Gly236Arg/Leu328Arg - which decreases binding to Fc receptors; and

• cFAE - K409R/F405L which promote heterodimer formation.

The LALA-PG, and cFAE modifications are particularly preferred, for instance in one embodiment the constant regions will include all of those modifications. In one preferred embodiment, the LALA modifications are present.

In an especially preferred embodiment, the antibody has a constant region with minor to no effector functions, such as an antibody derived from the FDA-approved antibody Durvalumab with Fc modifications L234F/L235E/P331S. The Fc modifications in Durvalumab help eliminate Fc functions and so the use of the light and heavy chain constant regions or Durvalumab is a particularly effective way to provide a constant region with the desired lack of Fc functions. As Durvalumab has gained clinical approval that further represents a reason why the use of its constant regions represents a particularly preferred embodiment. Hence, in one preferred embodiment, the binding molecule comprises the Fc region of Durvalumab.

In any of the embodiments, where the binding molecule comprises constant region sequences derived from Durvalumab, they may comprise any of the constant region modifications discussed herein. Durvalumab has a human IgGl backbone. Hence, in a further preferred embodiment, where the binding molecule comprises a constant region, it may comprise a human IgG region. In a preferred embodiment, it may comprise a human IgGl region. Such regions may be modified to eliminate Fc function. In one embodiment, the constant regions may be modified to delete the CHI region, particularly where the antigen-binding domains are sdBrs.

The heavy and light chain constant region sequences of Durvalumab are provided respectively as SEQ ID NOs 838 and 839. In one preferred embodiment a binding molecule of the present invention comprises such light and heavy chain variable sequences or a variant of such a sequence. In one preferred embodiment, the variant sequence or sequences have at least 90% sequence identity to the relevant specific sequence. In another embodiment, the variant has at least 95% sequence identity. In one embodiment, a binding molecule of the present invention comprises the heavy and light chain constant region sequences of SEQ ID Nos: 838 and 839, but with one or more of the constant region sequence modifications discussed herein. In one preferred embodiment, such heavy and light chain constant regions are employed in binding molecules provided herein.

A variant of the heavy chain constant region of Durvalumab sequence with the CHI region deleted is provided as SEQ ID NO: 210. In one preferred embodiment, such a constant region is employed where the binding molecule comprises sdAb based antigen binding sites, preferably with VHH antigen-binding sites. Such a sequence, but with one or more of the other constant region modifications discussed herein may be employed. In one embodiment, a binding molecule of the present invention may comprise the sequence of SEQ ID NO: 210 or a variant thereof with at least 90% sequence identity. In another embodiment, the variant may have at least 95% sequence identity. A variant in such embodiments will still be CHI deleted. Such heavy chain constant regions with a CHI deletion is preferably employed in one embodiment.

Table A below sets out the full Durvalumab heavy and light chain constant region sequences, as well as the heavy region sequence lacking the CHI region.

Table A: Durvalumab constant region sequences Conjugates, fusion proteins, effector molecules, and labels

Binding molecules of the present invention may be conjugated to other molecules. In one embodiment, the binding molecule is conjugated to a label. In another embodiment, a binding molecule, particularly an antibody, of the invention is not conjugated to an effector molecule. In one embodiment, a binding molecule, particularly an antibody, of the invention is not conjugated to a toxin. In another embodiment, a binding molecule, particularly an antibody, of the invention is not conjugated to a radioisotope. In another embodiment, it is not conjugated to an agent for imaging.

Assays

In one embodiment, an assay may be employed to determine if a given binding molecule has a particular property or properties, or the level of an activity of interest a binding molecule has. Such assays may detect or measure the activity of interest. One or more of the assays described in the Examples of the present application may be employed to assess a particular binding molecule and whether it has a desired property or properties. The assays in the Examples may be employed, for instance, to determine the ability of a binding molecule to bind IL-12Rβ2. They may be used to determine the ability to bind to gp130. They may also be used to determine the ability of a given molecule to act as an agonist of an IL-35 receptor. Any suitable method for measuring binding may be employ ed, such as those used in the Examples of the present application. The ability to bind IL-12Rβ2 or gp130 may be assessed by employing techniques like surface plasmon resonance using IL-12Rβ2, gp 130, or a portion of either thereof, bound to a chip. A binding molecule of the present invention will be typically able to bind to IL-12Rβ2 or gp130, when present on the cell surface and preferably both when present on the surface of the cell.

In one embodiment, where an antibody, or VHH domain, is defined by its ability to compete with another, the ability to compete for binding to just IL-12Rβ2 or gp130 is measured. In one embodiment, the ability of individual VHH binding domains will be measured, rather than looking at competition between molecules comprising both IL-12Rβ2 and gp130 binding domains. Hence, where a binding domain is said to have at least one VHH domain specific for IL-12Rβ2 and at least one VHH binding domain specific for gp130, where both of those binding domains are defined by their ability to compete with one of the specific VHH binding domains of the present invention, the binding assays to determine the ability to compete will be performed on the individual VHH binding domain for IL-12Rβ2 and the individual binding domain for gp130 separately. The preferred assay for measuring competition is flow cytometry.

In one embodiment, the ability of a candidate binding molecule to bind to IL-12Rβ2 is assessed in an assay comprising: (a) contacting a candidate binding molecule with a cell expressing IL-12Rβ2 on its surface; and (b) detecting any binding of the candidate binding molecule to the cells with IL-12Rβ2 on their surface. In a particularly preferred embodiment, the method is a flow cytometry method. In one embodiment, the cells express human IL-12Rβ2 on their surface. In one embodiment, the cells employed are HEK293T cells transiently transfected with a human IL-12Rβ2 expression plasmid. In another embodiment, the cells used are a stable cell line expressing IL-12Rβ2. In one embodiment, the cells are a stable HEK293T cell line expressing IL-12Rβ2 on their surface. In a further preferred embodiment, either the candidate binding molecule is itself labelled or is detected using a secondary antibody. In one embodiment, the ability of a candidate binding molecule to bind to IL-12Rβ2 is assessed in a flow cytometry assay comprising: (a) contacting a candidate binding molecule with a stable HEK293T cell line expressing IL-12Rβ2 on its surface; and (b) detecting by flow cytometry any binding of the candidate binding molecule to the cells, wherein the candidate binding molecule comprises HA, with binding to the cell detected using a mouse anti-HA antibody and an anti -mouse PE antibody. Such assays may also be performed for gp130 binding for the VHH domains which are specific for gp130 using cell lines expressing gp130. They may be performed with both IL-35 receptor subunits.

The present invention also provides a novel assay for detecting, and preferably measuring, the ability of a given molecule to bring about the association of the IL-12Rβ2 and gp130 receptor subunits of the IL-35R. In particular, the present invention provides a method of detecting the ability of a test molecule to bring about the association of the IL-12Rβ2 and gp130 receptor subunits comprising: (a) contacting a test binding molecule with a cell expressing modified versions of IL- 12Rβ2 and gp130, wherein IL-12Rβ2 and gp130 have been modified to produce a detectable signal when associated; and (b) detecting for the detectable signal, if present, resulting from the association of IL-12Rβ2 and gp130. The method may comprise measuring the detectable signal. It may comprise comparing the detectable signal to a control. In one embodiment, the control may be a positive control known to lead to the association of IL-12Rβ2 and gp130. In another the control may a control known not to lead to association. In one embodiment, the control may be the assay performed without any test molecule. The detectable signal may be any suitable signal resulting from association of the two. In one embodiment, the detectable signal is the activity of an enzyme which only becomes active when IL-12Rβ2 and gp130 associate. A preferred assay for measuring the ability of a binding molecule to bring about gp130 and IL-12Rβ2 dimerization is a NanoLuc Binary Technology assay (NanoBiT), for instance that employed in Example 2.

In one embodiment, the invention provides a method for determining whether a test molecule brings about the association of the IL-12Rβ2 and gp130 receptor subunits comprising: (a) contacting a test binding molecule with a cell expressing modified versions of IL-12Rβ2 and gp130 where IL- 12Rβ2 and gp130 have been modified so that when the two associate an enzyme becomes activated; and (b) measuring for the activity of the enzyme. In one embodiment, the method comprises: (a) contacting a test binding molecule with a cell expressing modified versions of IL-12Rβ2 and gp130, where when the two associate luciferase becomes activated; and (b) measuring for the activity of the enzyme. In a particularly preferred embodiment a luciferase-based gp130-IL-12Rβ2 assay is employed where gp130 is fused to one of Large BiT (LgBiT; 17.6kDa) and Small BiT (SmBiT; 11 amino acids) and IL-12Rβ2 to the other, IL-12Rβ2 and gp130 dimerising triggers luciferase activity in the form of the Nanoluc enzyme. Hence, the present invention further provides a method for determining whether a test molecule brings about the association of the IL-12Rβ2 and gp130 receptor subunits comprising: (a) contacting a test binding molecule with a cell expressing modified versions of IL-12Rβ2 and gp130 where gp130 is fused to one of Large BiT (LgBiT; 17.6kDa) and Small BiT (SmBiT; 11 amino acids) and IL-12Rβ2 to the other, so that IL-12Rβ2 and gp130 dimerising triggers luciferase activity in the form of the Nanoluc enzyme; and (b) measuring for luciferase activity, if present. Hence, in one embodiment, a binding molecule will be assessed for its ability to dimerise gp130 and IL-12Rβ2 receptor chains with the assay using either transiently transfected cells or cell lines stably transfected to express both the IL-12Rβ2 and gp130 by contacting the cells with the candidate binding molecule and measuring luciferase activity.

Competition assays may be used to determine whether a candidate binding molecule or individual VHH binding domain is able to compete for binding to IL-12Rβ2 with another binding molecule. In one embodiment, a variant binding molecule has the ability to compete for binding to IL- 12Rβ2 with one of the specific binding molecules of the present invention. In one embodiment, an assay to assess competition may comprise: (a) contacting a candidate binding molecule, a labelled binding molecule of the invention, and a cell expressing IL-12Rβ2 on its surface; (b) determining the amount of binding of the labelled binding molecule of the present invention to the cells compared to the amount of binding for the same assay performed without the candidate binding molecule. In one embodiment, rather than a competition assay, the ability of a candidate binding molecule and a specific binding molecule of the present invention to bind to cells expressing IL-12Rβ2 is measured individually using the same assay and the two results obtained are compared. Any of the assays and methods set out herein to measure binding to cells may also be used to measure competition or to compare binding of two binding molecules to IL-12Rβ2. The same binding assay may be performed to look for competition for gp130 binding sites. Such assays for competition may be performed with individual binding domains. They may also be performed with the overall binding molecule.

In one preferred embodiment, a binding molecule of the present invention does not compete with IL-12 for binding to IL-12Rβ2 or does not significantly do so. Hence, an assay may be used to determine the ability of a given binding molecule to compete with IL- 12 for binding. In one embodiment, the assay may comprise: (a) contacting HEK-Blue IL- 12 reporter cells with IL- 12 in the presence and absence of the binding molecule; and (b) measuring secreted embryonic alkaline phosphatase in the cell culture medium to determine if the presence of the binding molecule decreases the amount of secreted embryonic alkaline phosphatase indicating that the binding molecule is competing with IL- 12 for binding to the IL- 12 receptor. In one preferred embodiment, the assay is performed with different dilutions of the binding molecule to determine the effect of increasing binding molecule concentration on the ability of IL- 12 to bind to its receptor. In one embodiment, a binding domain for IL-12Rβ2, rather than the whole molecule, is assessed for its ability to compete with IL- 12.

In one preferred embodiment, a binding molecule of the present invention does not compete with IL-6 for binding to gp 130 or does not significantly do so. Hence, an assay may be used to determine the ability of a given binding molecule to compete with IL-6 for binding to gp 130. In one embodiment, the assay may comprise: (a) contacting HEK-Blue IL- 12 reporter cells with IL- 12 and IL-6 in the presence and absence of the binding molecule; and (b) measuring for STAT3 phosphorylation in lysates of the cells. In one embodiment, phospho (Tyr705)-STAT3 is measured in the antibody, for instance using an antibody against the phosphorylated STAT3. In another preferred embodiment, the assay will be performed with a number of IL-6 family members to check that a given binding molecule does not compete with them.

In one embodiment, such competition assays may be used to determine the ability of a binding molecule, or an individual binding domain, to compete with IL-6. In other embodiments, such assays may be used to assess ability to compete with other IL-6 family members.

In one particular embodiment, a binding molecule of the present invention will be able to bring about activation of STAT3. Illustrative assays for measuring STAT3 activation are set out in the Examples of the present application. For example a anti-phospho(Tyr705)-STAT3 antibody may be employed to measure ability to induce phosphorylation of STAT3. In a preferred embodiment, the HEK-Blue -IL- 12 SEAP report cell line may be employed, for instance in the assay described in Example 2 of the present application.

The efficacy of binding molecule, in particular of an antibody, may be assessed in an in vivo system, such as in an animal model of a disease condition. In one embodiment, an animal model of any of the conditions mentioned herein may be employed to assess a binding molecule of the present invention. Such animal models may be used to assess whether a given binding molecule is able to treat or prevent the condition in question or to reduce the degree of severity of the disease. As the binding molecules of the invention may be used to reduce unwanted immune responses, such assays may look at the size of an immune response, for example, before during or after treatment. The assay may look for inflammatory markers.

Diagnosis and Therapy

The binding molecules of the invention are preferably used as agonists of the IL-35 receptor. Activation of the IL-35 receptor is generally associated with suppressing immune response. Hence, the binding molecules of the invention may be used to modulate an immune response. Preferably a binding molecule of the invention may be used to suppress or reduce an immune response. Hence, in one embodiment, the present invention provides a method of suppressing or reducing an immune response comprising administering a binding molecule of the present invention to a subject in need thereof. The invention also provides a binding molecule of the present invention for use in a method of suppressing or reducing an immune response. A binding molecule of the present invention may also be used to induce immune tolerance.

In one particularly preferred embodiment, the disorder to be treated or prevented is an autoimmune disorder. In another particularly preferred embodiment, the disorder is an inflammatory condition. Examples of disorders which may be treated or prevented include allergic airway disorders, for example eosinophilia and allergic rhinitis. The disorder may be arthritis, preferably rheumatoid arthritis. The disorder may be colitis. In one embodiment, the disorder may be multiple sclerosis. In another embodiment, the disorder may be multiple sclerosis, diabetes, viral myocarditis, SLE, or atherosclerosis. In a particularly preferred embodiment, the condition is graft versus host disease (GVHD). In one embodiment, the disorder to be treated or prevent may be selected from GVHD, multiple sclerosis, chronic obstructive pulmonary disorder (COPD), allergic rhinitis, ulcerative colitis, crohn’s disease, immune thrombocytopenia (ITP), fetomatemal tolerance, Hashimoto’s thyroiditis, atherosclerosis, coronary artery disease, metabolic syndrome and Type 2 Diabetes (T2D). The subject matter show a reduction in inflammatory markers. In another embodiment, the disease to be treated or prevented is selected from allergic airway (eosinophilia, allergic rhinitis), rheumatoid arthritis (RA), colitis, multiple sclerosis (MS), diabetes, Graft Versus Host Disease (GVHD), viral myocarditis, Systemic Lupus Erythematosus (SLE), primary Sjogren syndrome, psoriasis, dermatomyositis, systemic sclerosis, heart disease, atherosclerosis, and atherosclerotic heart disease.

In one embodiment, the condition to be treated is one characterised by reduced IL-35 levels, particularly reduced circulating IL-35 levels in the serum. Reduced circulating IL-35 levels are seen in many human autoimmune diseases including Multiple sclerosis (MS), Chronic Obstructive Pulmonary Disease (COPD), allergic rhinitis, Ulcerative Colitis (UC), Crohn’s Disease (CD), Immune Thrombocytopenic Purpura (ITP), fetomatemal tolerance, Hashimoto’s thyroiditis, atherosclerosis, and coronary artery disease. In one embodiment, the condition to be treated or prevented is one of those conditions.

Also provided is a binding molecule, in particular an antibody, of the present invention for use as a medicament. The present invention also provides for the use of a binding molecule of the present invention for the manufacture of a medicament for treating or preventing one of the conditions mentioned herein. In another embodiment a binding molecule, in particular an antibody, of the present invention is provided for use in a method of therapy of the human or animal body. Please note that, in the various therapeutic uses set out herein where reference is made to a binding molecule or an antibody of the present invention, a pharmaceutical composition comprising it may be also employed and vice versa unless stated otherwise, as may be a composition encoding an antibody of the invention. A binding molecule, in particular an antibody, of the present invention may also be used in in vitro diagnosis, for example such diagnosis performed on a sample from a subject. A binding molecule, in particular an antibody, of the present invention may be employed to treat a condition. As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

Pharmaceutical compositions

In one embodiment, the present invention provides a pharmaceutical composition comprising: (a) a binding molecule of the present invention; and (b) a pharmaceutically acceptable carrier, diluent, and/or excipient. The particularly preferred binding molecule for any of the pharmaceutical compositions of the present invention is an antibody of the present invention. In one embodiment, a pharmaceutical composition of the present invention comprises a binding molecule of the present invention as well as a carrier, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, a preservative and/or an adjuvant. In one embodiment, a pharmaceutical composition of the present invention is in solid or liquid form. In one embodiment, the pharmaceutical composition may be in the form of a powder, a tablet, a solution or an aerosol. In one embodiment, a pharmaceutical composition of the present invention is provided in a frozen form. In one embodiment, a pharmaceutical composition of the present invention is provided in lyophilized form.

A pharmaceutical composition of the present invention will usually be supplied as a sterile, pharmaceutical composition. A pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable adjuvant. In another embodiment, no such adjuvant is present in a pharmaceutical composition of the present invention. The present invention also provides a process for preparation of a pharmaceutical or medicament composition comprising adding and mixing binding molecule of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Such carriers may be used, for example, so that the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient. The term “pharmaceutically acceptable excipient” as used herein typically refers to a pharmaceutically acceptable formulation carrier, solution or additive to enhance the desired characteristics of the compositions of the present invention. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates, and benzoates.

In certain embodiments, the pharmaceutical composition may contain formulation materials for the purpose of modifying, maintaining or preserving certain characteristics of the composition such as the pH, osmolarity, viscosity, clarity, color, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991). Additional pharmaceutical compositions include formulations involving the antibody of the present invention in sustained or controlled delivery formulations. Techniques for formulating a variety of sustained- or controlled-delivery means are known to those skilled in the art. A binding molecule, in particular antibody, of the present invention may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems, or in macroemulsions. Such techniques are also disclosed in Remington's Pharmaceutical Sciences.

A subject will be typically administered a therapeutically effective amount of a pharmaceutical composition and hence of a binding molecule, in particular an antibody, of the present invention. The term “therapeutically effective amount” typically refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, a therapeutically effective amount will be from 0.01 mg/kg to 50 mg/kg, for example 0.1 mg/kg to 20 mg/kg per day. Alternatively, the dose may be 1 to 500 mg per day, such as 10 to 100, 200, 300 or 400 mg per day. In one embodiment, the amount in a given dose is at least enough to bring about a particular function.

In one embodiment, a binding molecule, in particular an antibody, of the present invention may be given in combination with another treatment for the condition being treated. For example, a binding molecule, in particular an antibody, of the present invention may be provided simultaneously, sequentially, or separately with such a further agent. In another embodiment, an antibody of the present invention may be provided in the same pharmaceutical composition as a second therapeutic agent.

In one preferred embodiment, the therapeutic agent of the invention, when in a pharmaceutical preparation, may be present in unit dose forms. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of 0.01 to 20 mg/kg, for example 0.1 to 20 mg/kg, for example 1 to 20 mg/kg, for example 10 to 20 mg/kg or for example 1 to 15 mg/kg, for example 10 to 15 mg/kg. To effectively treat conditions of use in the present invention in a human, suitable doses may be within the range of 0.001 to 10 mg, 0.01 to 1000 mg, for example 0. 1 to 1000 mg, for example 0. 1 to 500 mg, for example 500 mg, for example 0. 1 to 100 mg, or 0.1 to 80 mg, or 0. 1 to 60 mg, or 0. 1 to 40 mg, or for example 1 to 100 mg, or 1 to 50 mg, of a dual targeting protein of this invention, which may be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such a dose may be, if necessary, repeated at appropriate time intervals selected as appropriate by a physician. A binding molecule, and in particular an antibody, of the present invention may be, for instance, lyophilized for storage and reconstituted in a suitable carrier prior to use. Lyophilization and reconstitution techniques can be employed.

The binding molecules, in particular antibodies, and pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO 98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. In one preferred embodiment, administration is via intravenous administration. In another preferred embodiment, administration is via subcutaneous administration, for example via subcutaneous injection. The compositions can also be administered into a specific tissue of interest. In some embodiments, administration is via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody molecule or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application.

Dosage treatment may be a single dose schedule or a multiple dose schedule. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulary agents, such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the pharmaceutical may be in dry form, for reconstitution before use with an appropriate sterile liquid. In one embodiment, a pharmaceutical composition comprising an antibody of the present invention is provided in lyophilised form. If a composition is to be administered by a route using the gastrointestinal tract, the composition will typically need to contain agents which protect the binding molecule, in particular antibody, from degradation but which release the binding molecule once it has been absorbed from the gastrointestinal tract. In another embodiment, a nebulisable formulation according to the present invention may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 ml, of solvent/solution buffer.

A pharmaceutical composition of the present invention may be provided in a receptacle that provides means for administration to a subject. In one embodiment, a pharmaceutical composition of the present invention may be provided in a prefdled syringe. The present invention therefore provides such a loaded syringe. It also provides an auto-injector loaded with a pharmaceutical composition of the present invention.

In one embodiment the formulation is provided as a formulation for topical administrations including inhalation. Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the invention containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient. These inhalable powders may include monosaccharides (e.g., glucose or arabinose), disaccharides (e.g., lactose, saccharose, maltose), oligo- and polysaccharides (e.g., dextranes), polyalcohols (e.g., sorbitol, mannitol, xylitol), salts (e.g., sodium chloride, calcium carbonate) or mixtures of these with one another. Mono- or disaccharides are suitably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns for example from 1 to 5 pm. The particle size of the active ingredient such as the antibody or fragment is of primary importance. The propellant gases which can be used to prepare the inhalable aerosols are known in the art. Suitable propellant gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The above mentioned propellent gases may be used on their own or in mixtures thereof. Particularly suitable propellent gases are halogenated alkane derivatives selected from among TG 11, TG 12, TG 134a and TG227. Of the above-mentioned halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable. The propellent-gas- containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface -active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0. 1 to 2 % by weight, 0.5 to 2 % by weight or 0.5 to 1 % by weight of active ingredient.

Alternatively topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

Nebulisable formulation according to the present invention may be provided, for example, as single dose units (e.g., sealed plastic containers or vials) packed in foil envelopes. Each vial contains a unit dose in a volume, e.g., 2 mL, of solvent/solution buffer. The present invention also provides a syringe loaded with a composition comprising an antibody of the invention. In one embodiment, a pre-fdled syringe loaded with a unit dose of an antibody is provided. In another embodiment, an autoinjector loaded with a binding molecule, in particular an antibody, of the invention is provided. In a further embodiment, an IV bag loaded with a pharmaceutical composition of the invention is provided.

It is also envisaged that an antibody of the present invention may be administered by use of gene therapy. In order to achieve this, DNA sequences encoding the binding molecule, in particular antibody, under the control of appropriate DNA components are introduced into a patient such that the binding molecule, in particular antibody chains and so antibody, are expressed from the DNA sequences and assembled in situ.

Once formulated, the compositions of the invention can be administered directly to the subject. By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. In a preferred embodiment, the subject to be treated is a mammal. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. The subjects to be treated can be animals. However, in one or more embodiments the compositions are adapted for administration to humans. In a particularly preferred embodiment, the subject is human. In another embodiment, the subject is an animal model of one of the conditions recited herein.

Kits

The present invention also extends to a kit comprising a binding molecule of the invention, in particular an antibody, of the invention, optionally with instructions for administration. In yet another embodiment, the kit further comprises one or more reagents for performing one or more functional assays. In another embodiment, a kit containing single -chambered or multi -chambered pre-fdled syringe is provided which is pre-fdled with a pharmaceutical composition of the invention. The invention also provides a kit for a single-dose administration unit which comprises a pharmaceutical composition of the invention. In another embodiment, the kit comprises packaging.

Further numbered embodiments

The following represent further numbered embodiments of the invention, but do not at present represent claims.

[1]. A binding molecule specific for the IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising:

(i) at least one binding-site specific for the gp130 subunit of the IL-35 receptor; and

(iii) at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor.

[2], The binding molecule of [1], wherein:

(a) the binding sites for gp130 and IL-12Rβ2 are VHH domain binding sites; and/or

(b) the binding molecule is an antibody with at least one binding-site specific for the gp130 subunit of the IL-35 receptor and at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor.

[3] . The binding molecule of [ 1] or [2] , wherein the binding molecule comprises a binding site specific for the IL-12Rβ2 subunit of the IL-35 receptor which comprises:

(a) at least one VHH antigen-binding domain that binds IL-12Rβ2 comprising a set of three CDRs (CDR1, CDR2, and CDR3) selected from the sets of three CDRs of Table 1;

(b) at least one VHH antigen-binding domain that binds IL-12Rβ2 comprising a set of three CDRs (CDR1, CDR2, and CDR3) that correspond to a set of three CDRs of Table 1 apart from a maximum of ten amino acid sequence changes;

(c) at least one VHH antigen-binding domain that binds IL-12Rβ2 comprising a set of three CDRs (CDR1, CDR2, and CDR3) that have at least 90% sequence identity to a set of three CDRs of Table 1 ; or

(d) a VHH antigen-binding domain that can compete for binding to IL-12Rβ2 with a VHH antigen-domain of any of (a) to (c).

[4] . The binding molecule of [3], wherein the binding molecule comprises one or more of the following VHH antigen-domains:

(a) a VHH antigen-binding domain that binds IL-12Rβ2 and is selected from the VHH antigen-binding domain having the sequence of one of the VHH antigen-binding domains of Table 1; (b) a VHH antigen-binding domain that binds IL-12Rβ2 and has at least 80% sequence identity to one of the VHH antigen-binding domains of Table 1;

(c) a VHH antigen-binding domain that binds IL-12Rβ2 and is a humanized version of one of VHH antigen-binding domains of Table 1; or

[5], The binding molecule of any one of [1] to [4], wherein the binding molecule comprises a binding site specific for the gp130 subunit of the IL-35 receptor which comprises:

(a) at least one VHH antigen-binding domain that binds gp130 comprising a set of three CDRs (CDR1, CDR2, and CDR3) selected from the sets of three CDRs of Table 2B;

(b) at least one VHH antigen-binding domain that binds gp130 comprising a set of three CDRs (CDR1, CDR2, and CDR3) that correspond to a set of three CDRs of Table 2B apart from a maximum of ten amino acid sequence changes;

(c) at least one VHH antigen-binding domain that binds gp130 comprising a set of three CDRs (CDR1, CDR2, and CDR3) that have at least 90% sequence identity to a set of three CDRs of Table 2B; or

(d) at least one VHH antigen-binding domain that can compete for binding to gp130 with an a VHH antigen-domain of any of (a) to (c).

[6] . The binding molecule of [5], wherein the binding molecule comprises one or more of the following VHH antigen-domains:

(a) a VHH antigen-binding domain that binds gp130 and is selected from the VHH antigen-binding domain having the sequence of one of the VHH antigen-binding domains of Table 2B;

(b) a VHH antigen-binding domain that binds gp130 and has at least 80% sequence identity to one of the VHH antigen-binding domains of Table 2B;

(c) a VHH antigen-binding domain that binds gp130 and is a humanized version of one of VHH antigen-binding domains of Table 2B; or

(d) a VHH antigen-binding domain that can compete for binding to gp130 with a VHH antigen-domain of any of (a) to (c).

[7], The binding molecule of any one of [1] to [6] which is a bispecific binding molecule wherein: (a) the binding molecule comprises a VHH comprising the same set of three CDRs for the IL-12Rβ2 binding site and a VHH comprising the same set of three CDRs for the gp130 binding site as one of the bispecific binding molecules of Table 11 or 15;

(b) the binding molecule comprises a VHH comprising the same set of three CDRs for the IL-12Rβ2 binding site apart from a maximum of ten amino acid sequence changes and the same set of three CDRs for the gp130 binding site apart from a maximum of ten amino acid sequence changes as one of the binding molecules of Table 11 or 15;

(c) the binding molecule comprises a VHH specific for IL-12Rβ2 comprising a set of three CDRs for the IL-12Rβ2 binding site with at least 95% sequence identity to a set of three CDRs for an IL-12Rβ2 binding site of one of the bispecific binding molecules of Table 11 or 15 and a VHH specific for gp130 comprising a set of three CDRs with at least 95% sequence identity to a set of three CDRs for a gp130 binding site of the same bispecific binding molecules of Table 11 or 15; or

(d) the VHH antigen-binding domain specific for IL-12Rβ2 can compete for binding with one of the VHH antigen binding domains specific for IL-12Rβ2 of a bispecific binding molecule of Table 11 or 15 and the VHH antigen-binding domain specific for gp130 can compete for binding with the gp130 binding site of the same bispecific binding molecule of Table 11 or 15.

[8] . The binding molecule of [7], wherein the binding molecule:

(a) has the CDRs for the IL-12Rβ2 and gp130 binding site from the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15;

(b) has sets of CDRs for the IL-12Rβ2 and gp130 binding site with a maximum of ten amino acid sequence changes per set compared to the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15;

(c) has sets of CDRs for the IL-12Rβ2 and gp130 binding site with at least 95% sequence identity compared to one of the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15; or

(d) has a binding site for IL-12Rβ2 that is able to compete for binding to one of the binding sites specific for IL-12Rβ2 of the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15 and a binding site for gp130 which is able to compete for binding with the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15.

[9], The binding molecule of any one of [1] to [8], wherein the binding molecule comprises VHH binding domains and an Fc region.

[10] . The binding molecule of [9], wherein the binding molecule: (a) comprises two polypeptide chains each with two VHH binding domains, with one of the two VHH binding domains specific for IL-12Rβ2 and the other specific for gp130; or

(b) comprises two polypeptide chains each comprising a VHH binding domain and an Fc region, wherein the VHH binding domain of one of the polypeptides is specific for IL-12Rβ2 and the other is specific for gp130, wherein the Fc regions of the polypeptides comprise amino acid sequences favouring heterodimer formation or allowing preferential purification of heterodimers.

[11]. The binding molecule of [ 10] , wherein the binding molecule is :

(a) bsVHH-Fc-3, bsVHH-Fc-64, bsVHH-Fc-68, bsVHH-Fc-69, bsVHH-Fc-70, or bsVHH-Fc-71;

(b) a bsVHH-Fc with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (a);

(c) a bsVHH-Fc with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (a), apart from up to a maximum of 10 amino acid sequence changes in each set;

(d) a bsVHH-Fc with sets of CDRs have at least 95% sequence identity to the sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (a); or

(e) a bsVHH-Fc with VHH binding domains that are able to compete for binding to IL- 12Rβ2 and gp130 in comparison to the VHH binding domains for the specificities of the same bispecific molecule of (a).

[12], The binding molecule of [ 10] , wherein the binding molecule is :

(a) KiH8;

(b) a KiH format binding molecule with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains as KiH8;

(c) a KiH format binding molecule with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of KiH, apart from up to a maximum of 10 amino acid sequence changes in each set;

(d) a KiH format binding molecule with sets of CDRs have at least 95% sequence identity to the sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of KiH; or

(e) a KiH format binding molecule with VHH binding domains that are able to compete for binding to IL-12Rβ2 and gp130 in comparison to the VHH binding domains of KiH.

[13], The binding molecule of any one of [1] to [10], wherein the binding molecules comprises a linker or linkers separating antigen binding domains, preferably wherein the linkers are GGGGS or linkers comprising more than one copy of GGGGS. [14], The binding molecule of any one of [1] to [10], wherein the binding molecule comprises:

(a) a constant region modified to reduce or eliminate Fc regions;

(b) the Durvalumab constant region comprising:

(i) the heavy and light chain sequences of SEQ ID Nos: 838 and 839 respectively; or

(ii) the Durvalumab constant region comprising the heavy chain sequence of SEQ ID NO: 210 lacking the CHI sequence and light chain sequence of SEQ ID NO: 839;

(iii) a variant of the Durvalumab constant region with at least 90% sequence identity to the heavy and light chain sequences of 838 and 839 or the heavy and light chain sequences of SEQ ID NOs: 210 and 839.

[15], A pharmaceutical composition comprising a binding molecule according to any one of [1] to

[14].

[16], The binding molecule of any one of [1] to [14] or pharmaceutical composition of [15] for use is a method of treatment of the human or animal body.

[17], The binding molecule of any one of [1] to [14] or pharmaceutical composition of [15] for use in treating, preventing an autoimmune or inflammatory disorder, or inducing immune tolerance.

[18], The binding molecule or pharmaceutical composition for use of claim 17, wherein the disorder is selected from graft versus host disease (GVHD), multiple sclerosis, chronic obstructive pulmonary disorder (COPD), allergic rhinitis, ulcerative colitis, Crohn’s disease, immune thrombocytopenia (ITP), atherosclerosis, and Diabetes.

[19], A method of treating or preventing an autoimmune or inflammatory disorder, or inducing immune tolerance comprising a binding molecule of any one of [1] to [14] or pharmaceutical composition of [15] to a subject in need thereof.

[20], The method of [19] wherein the disorder is selected from graft versus host disease (GVHD), multiple sclerosis, chronic obstructive pulmonary disorder (COPD), allergic rhinitis, ulcerative colitis, Crohn’s disease, immune thrombocytopenia (ITP), atherosclerosis, and Diabetes.

[21], A method for detecting association of the IL-12Rβ2 and gp130 receptor subunits comprising: (a) contacting a candidate molecule with a cell expressing modified versions of IL-12Rβ2 and gp130 receptor subunits which have been modified so when they associate a detectable signal is produced;

(b) detecting the signal, if present, optionally when the magnitude of the signal is measured.

[22], The method of [21], wherein:

(a) the detectable signal is the activation of an enzyme; or

(b) the detectable signal is the activation of luciferase.

EXAMPLES

The invention will be further understood with reference to the following non-limiting Examples.

Example 1: Generation of bispecific antibodies targeting gp130 and IL-12Rβ2

A: Llama immunization with recombinant gp130 and IL-12Rβ2

For gp 130 immunization, a male and female llama were subcutaneously injected on days 0, 14, 28 and 42, each time with a mixture of recombinant human gp130 fused to hlgGl Fc-His6 at the C- terminus (hgp130-Fc) (R&D Systems, Cat. No. 671-GP-100), recombinant mouse gp130 fused to hlgGl Fc-His6 at the C-terminus (mgp130-Fc) (R&D Systems, Cat. No. 468-MG), tagless human gp130 (produced in-house), and tagless mouse gp130 (produced in-house) in combination with Gerbu adjuvant P.

For IL-12Rβ2 immunization, a male and female llama were subcutaneously injected on days 0, 14, 28 and 42, each time with a mixture of recombinant human IL-12Rβ2 fused to hlgGl Fc-His6 at the C-terminus (hIL-12Rβ2-Fc) (R&D Systems, Cat. No. 1959-B2B-050), recombinant mouse IL- 12Rβ2 fused to hlgGl Fc-His6 at the C-terminus (mIL-12Rβ2-Fc) (R&D Systems, Cat. No. 7406-MR- 050), tagless human IL-12Rβ2 (produced in-house), and tagless mouse IL-12Rβ2 (produced in-house) in combination with Gerbu adjuvant P.

Four and 8 days after last immunization (4 d.p.i. and 8 d.p.i.), each time about 100 ml anticoagulated blood was collected from each llama for lymphocyte preparation.

B: Selection of VHHs binding to gp130 and IL-12Rβ2

Individual libraries of VHH regions of llama heavy chain-only antibodies were constructed from each llama’s lymphocytes to screen for the presence of antigen-specific VHHs. To this end, total RNAs from peripheral blood lymphocytes from 4 d.p.i. & 8 d.p.i. were pooled per animal and used as template for cDNA synthesis. Then, the VHH encoding sequences were amplified by PCR and cloned into the pMECS phagemid vector. Phagemid libraries were separately panned in solution on either Avi- tagged, site specifically biotinylated human or mouse gp130-avi-his (produced in-house), or random biotinylated human or mouse IL-12Rβ2 (produced in-house) for 3 rounds. Colonies from each panning set were analyzed by ELISA for the presence of antigen-specific VHHs in their periplasmic extracts. The screening ELISA was performed on the same human and mouse avi -tagged, biotinylated gp 130/IL- 12Rβ2, using streptavidin-coated blocked wells as negative control. Based on sequence data of the positive colonies, the number of unique full length VHHs were determined and categorized in different CDR3 groups. For anti-IL-12Rβ2, 209 unique anti-IL-12Rβ2 VHHs were identified in 62 CDR3 groups, of which 19 showed signs of human/mouse cross-reactivity based on ELISA results. 572 unique anti-gp130 VHHs were identified in 146 CDR3 groups. From this collection, 107 anti-gp130 VHHs showed signs of human/mouse cross-reactivity based on ELISA results. (Table 1, Table 2, Table 3, Table 4).

Dissociation off-rates of the human-specific and human-mouse cross-reactive anti-gp130 and anti-IL-12Rβ2 VHH clones (based on ELISA binding study) were analyzed using Bio-Layer Interferometry (BLI). Biotinylated human or mouse recombinant gp130 or IL-12Rβ2 proteins (10 pg/ml) were immobilized on a Fortebio streptavidin-coated biosensor tips. Next, 200 pl VHH- containing P.E. were mixed with 2 pl of 10% Tween20-PBS in a 96-well plate. pMECS vector P.E. was used as negative control. The P.E. plate was loaded into a Fortebio Octet Red and brought into contact with antigen-coated Octet tips. Consequently, the binding profile for each clone was determined. Using the Fortebio Data Analysis Software, the blanks were subtracted and the curves were aligned. Based on these curves, the off-rates were calculated using a 1: 1 binding model. Data are shown in Table 3 and Table 4.

C: Binding of anti-gp130/IL-12Rβ2 VHH clones to gp130/IL-12Rβ2 expressed on cells

First, the ability of anti-gpI30 or anti-IL-12Rβ2 VHHs to bind human cell membrane-expressed gpI30 or IL-12Rβ2 was analyzed using HEK293T cells transiently transfected with an expression plasmids coding for the human gp130/IL-12Rβ2 receptor. 48 hours after transfection cells were harvested and reseeded at 100,000 cells/well of a 96-plate, washed with FACS buffer, and incubated with different dilutions of anti-gp130 or anti-IL-12Rβ2 VHH-containing P.E. in FACS buffer at 4°C for 40 minutes. After washing the cells with FACS buffer, cells were incubated with a mouse anti-HA antibody (Biolegend, clone HA.11), to bind the C-terminal hemagglutinin (HA) tag fused to the VHHs, for 40 minutes at 4°C. Cells were washed again with FACS buffer and stained with anti -mouse IgG-PE detection antibody (Biolegend, 405307) for 30 minutes at 4°C. Stained cells were analyzed on an LSR HTS (BD Biosciences). Dead cells were excluded from the analysis based on forward and side scatter properties. The data for the results obtained can be found in Table 5 and Table 6. D: Competition of anti-gp130 VHHs with IL-6 signaling

The ability of human -specific anti-gp130 VHHs to compete with IL-6 cytokine signaling was determined using the HEK-Blue IL-6 reporter cell line (Invivogen, #hkb-il6), these cells express endogenous levels of gp130 and overexpress IL-6R, STAT3 and a STAT3 -dependent secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cell culture was performed according to the manufacturer’s protocol. Cells were seeded at 50,000 cells/well of a 96-well plate, and pre-incubated with three dilutions of anti-gp130 VHH-containing P.E. (1/5-1/50-1/500), or 1 pg/ml anti-gp130 blocking antibody (clone B-R3), for 30 minutes at room temperature. Subsequently, cells were stimulated with 0.1 ng/ml human recombinant IL-6 (in-house produced). After 24 hours culturing at 37°C in a CO2 incubator the SEAP levels were measured by adding culture supematans to Quanti-Blue substrate. Colorimetric changes were measure at O.D. 650 nm using a iMark Microplate Absorbance Reader. The inhibitory capacity of VHH clones was determined as percent decreased activity compared to IL-6 stimulated cells treated with P.E. that does not contain a VHH. VHH clones were considered to block IL-6 signaling when they show more than 50% reduction of IL-6 activity. CDR3 families were categorised as IL-6 competing if 50% or more of the clones show 50% or more reduced IL-12 activity. The results are summarized in Table 7, data of selected clones can be found in Figure 1. 26 out of 62 tested CDR3 groups were considered to be IL-6 competing.

E: Competition of anti-IL-12Rβ2 VHH with IL-12 signaling

The ability of anti-IL-12Rβ2 VHH, which show binding to human-only or human and mouse IL-12Rβ2 in the ELISA screening, to compete with IL- 12 cytokine signaling was determined using the HEK-Blue IL-12 reporter cell line (Invivogen, #hkb-il 12), these cells overexpress IL-12Rβ1, IL-12Rβ2 and a STAT4-dependent secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cell culture was performed according to the manufacturer’s protocol. Cells were seeded at 50,000 cells/well of a 96-well plate, and pre-incubated with three dilutions of anti-IL-12Rβ2 VHH-containing P.E. (1/5-1/50- 1/500) for 30 minutes at room temperature. Subsequently, cells were stimulated with 2.6 ng/ml human recombinant IL- 12 (produced in-house). After 24 hours culturing at 37°C in a CO2 incubator the SEAP levels were measured by adding culture supematans to Quanti-Blue substrate (Invivogen). Colorimetric changes were measured at O.D. 650 nm using an iMark Microplate Absorbance Reader. The inhibitory capacity of anti-IL-12Rβ2 VHHs was determined as percent decreased activity compared to IL- 12 stimulated cells treated with P.E. that does not contain a VHH. VHH clones were considered to block IL- 12 signaling when they showed more than 50% reduction of IL- 12 activity. CDR3 families were categorised as IL-12 competing if 50% or more of the clones show 50% or more reduced IL-12 activity. These results are summarized in Table 8, with data of selected clones shown in Figure 1. 16 out of 32 tested CDR3 groups were considered to be IL-12 competing. Table 1: Amino acid sequence of anti-IL-12Rp2 VHHs

TABLE 2A (BELOW)

Table 2B

Table 3: Binding characteristics of anti- gp130 VHHs

Table 4: Binding characteristics of anti-IL-12Rβ2 VHHs

Table 5 - Binding of anti-gp130 VHHs to gp130 expressing cells

Table 6 - Binding of anti-IL-12Rβ2 VHH antibodies to IL-12Rβ2 expressing cells

Table 7 - IL-6 competition activity of anti-gp130 VHH CDR3 families

Table 8 - IL-12 competition activity of anti-IL-12Rβ2 VHH CDR3 families

Example 2: Construction and characterization of bispecific VHHs targeting gp130 and IL-12Rβ2

A: Construction of bispecific VHHs targeting gp130 and IL-12Rβ2

In a first design wave of bispecific VHH (referred to as ‘bsVHHs’) generation 6 VHH clones specific forgp130 and 4 clones specific for IL-12Rβ2 were selected (Table 9 and Table 10). The gp130 VHH was C-terminally fused to the IL-12Rβ2 VHH by a (G₄S)₇ linker, and C-terminal to the IL-12Rβ2 VHH a HA and His-tag was added. As controls we constructed bsVHHs where either the gp130 or IL- 12Rβ2 VHH was replaced by an irrelevant VHH targeting BCII10, the subunit 10 of the P-lactamase Bell enzyme of Bacillus cereus (Conrath et al, 2001. DOI: 10. 1128/AAC.45.10.2807-2812.2001). Table 11 summarizes the bsVHH constructs with their identifier. The bispecific VHH constructs were produced in bacteria, and purified by Immobilized Metal Affinity Chromatography (IMAC).

Table 9: Binding and competition characteristics of selected anti-gp130 VHH clones for bsVHH construction

Table 10: Binding and competition characteristics of selected anti-IL-12Rβ2 VHH clones for bsVHH construction Table 11: Overview of the first design wave of bispecific VHH(-Fc) constructs

B: bispecific anti-gpl3O/IL-12Rβ2 VHHs induce gp130 and IL-12Rβ2 dimerization

In order to analyze the ability of bsVHH molecules to induce gp130 and IL-12Rβ2 heterodimerization, NanoLuc Binary Technology (NanoBiT) was adopted to develop a luciferase-based gp130-IL-12Rβ2 dimerization assay. The Large BiT (LgBiT; 17.6kDa) and Small BiT (SmBiT; 11 amino acids) subunits were fused to either gp130 or IL-12Rβ2, and when expressed, the gp130-IL- 12Rβ2 protein-protein interaction brings the LgBit and SmBit in close proximity to form the functional NanoLuc enzyme that generates a luminescent signal. For this, the cDNA sequence of human gp130 (NM_002184.4) and human IL-12Rβ2 (NM_001374259.2) were subcloned into the pBiTl.l-C (containing the ‘LgBiT’) and pBiT2.1-C (containing the ‘SmBiT’) vectors. We found that the pBiTl .1- C-gp130 together with pBiT2.1-C-IL-12Rβ2 results in the highest assay window. The sequences encoding gp130 fused to the Large BiT and of IL-12Rβ2 fused to the Small BiT are provided below.

Full length human gpl30 fused to LgBiT NanoLuc - SEQ ID NO: 3132:

Full length IL12RB2 fused to SmBiT NanoLuc - SEQ ID NO: 3133:

The bispecific anti-gp130/IL-12Rβ2 VHHs were tested in the gp130-IL-12Rβ2 NanoBiT dimerization assay. For this, 150,000 HEK293T cells were seeded in a clear-bottom black 96 well culture plate in 100 pl of ‘complete DMEM culture media’ (DMEM media (Gibco, 41965) supplemented with 10% FCS and L-glutamin). Twenty-four hours later the cells were transfected with 50 ng pBiTl.l-C-gp130 and pBiT1.2-C-IL-12Rβ2 using 3 pl of FugeneHD (Promega, Cat#E2311) per pg of DNA in Opti-MEM (Gibco, Cat# 31985062). Forty-eight hours later the cells were refreshed with 100 pl Opti-MEM supplemented with 20 mM HEPES and subsequently 25 pl Nano-Gio Live cell Reagent was added to the cells. Luminescent signal was measured for around 20 minutes with a Glomax instrument to assess background signal, after which the bsVHHs were added to the cells and luminescence measurement was continued for another 60 minutes. Results of selected bsVHH are depicted in Figure 2. BsVHH-1, -3, -8 are able to induce gp130 and IL-12Rβ2 dimerization which results complementation of the intracellular fused NanoBiT enzyme. BsVHHs were compared to the corresponding single VHHs as negative control.

C: bispecific anti-gp130/IL-12Rβ2 VHHs induce STAT activation

Next gp130-IL-12Rβ2 dimerization induced by bispecific anti-gp130/IL-12Rβ2 VHH binding and triggering of downstream STAT activation was assessed. For this aHEK-Blue IL-12 SEAP reporter cell line (Invivogen, #hkb-il 12) was used as the cells have endogenous gp130 expression and had been engineered to express IL-12Rβ2. HEK-Blue IL- 12 cells were seeded in a 24-well plate in complete DMEM medium and incubated for 24 hours. The cells were stimulated with 2 pg/ml anti-gp130/IL- 12Rβ2 VHHs or 100 ng/ml rhIL-6 or rhIL-12 (produced in-house) for 30 minutes and subsequently washed with ice cold PBS and lysed in laemmli buffer. Lysates were boiled for 10 minutes at 95°C before separating by SDS-PAGE and were subsequently immunoblotted to a nitrocellulose membrane. The membrane was blocked (TBS-Tween-20 with 5% milk powder) and probed with the primary anti- phospho(Tyr705)-STAT3 antibody (Cell Signaling Technologies, Cat#9138). After washing, the immunoblots were detected with anti-mouse-HRP antibody. The band intensity of the immunoblots were quantified using Image Studio software. The band intensity of pSTAT3 was normalized to that of untreated conditions and scaled to the percentage of pSTAT3 induction of IL-12. Results are depicted in Figure 2, and show that BsVHH-3 (18439-20422) is the most potent bsVHH with a pSTAT3 signal in the range of IL-12 stimulation. Note that IL-12 has been described to most potently induce pSTAT4, however HEK-Blue IL- 12 cells have very low STAT4 expression, which results in a skewed activation to pSTATl and pSTAT3.

Example 3: Construction and characterization of bispecific VHH antibodies targeting gp130 and IL-12Rβ2

A: Construction of bispecific antibodies targeting gp130 and IL-12Rβ2

To increase the avidity of the bsVHH molecules, bivalent bispecific anti-gp130/IL-12Rβ2 antibodies were generated. The gp130 VHH-(G4S)7-IL-12Rβ2 VHHs were fused C-terminally through the IgGl hinge sequence to a human IgGl Fc domain (Figure 4). Antibody molecules were produced by transient transfection in HEK293T cells and purified from cell supernatant by protein A affinity chromatography.

Table 12 - Sequences used to generate bispecific anti-gp130/IL-12Rβ2 VHH-Fc molecules

B: gp130 and IL-12Rβ2 dimerization by bispecific antibodies targeting gp130 and IL-12Rβ2

The ability of bispecific anti-gp130/IL-12Rβ2 VHH-Fc antibodies to induce gp130 and IL- 12Rβ2 dimerization was assessed by making use of the NanoBiT dimerization assay (as described in example 2). Briefly, HEK293T cells transfected with the pBiTl.l-C-gp130 and pBiT1.2-C-IL-12Rβ2 plasmids were stimulated with approximately 2-4 pg/ml of batch purified bsVHH-Fc proteins. The results are listed in table 13. Further, we assessed the ability to induce gp130 and IL-12Rβ2 dimerization by bsVHH-1, -3 and -8 and bsVHH-Fc-1, -3, -8 in a dose-response. The fusion of bsVHH to an Fc domain enhanced the efficacy of the bsVHHs, and has the potential to increase the potency (Figure 3).

Table 13 - Nanobit clustering characteristics of selected anti-gp130/IL-12Rβ2 bsVHHs C: STAT activation by bispecific antibodies targeting gp130 and IL-12Rβ2

The bsVHH-Fc clones were tested for the ability to activate STAT3 phosphorylation downstream of the gp130 and IL-12Rβ2 heterodimers making use of the HEK-Blue IL- 12 cells as described in Example 2. HEK-Blue IL-12 cells were stimulated for 30 minutes with purified anti- gp130/IL-12Rβ2 bsVHHs, 100 ng/ml rhIL-6 or rhIL-12 and subsequently analyzed by western blotting for STAT3 phosphorylation. It was found that fusion of the bsVHH to an Fc domain enhanced the efficacy of bsVHHs to trigger STAT3 activation (Figure 3).

Example 4: Geometry evaluation of bispecific anti-gp130/IL-12Rβ2 antibodies

In order to study the impact of bsVHH-Fc geometry on the agonistic properties, the effect of shortening the linker length joining the gp130 and IL-12Rβ2 VHHs, changing the VHH order (“gp130- IL-12Rβ2” versus “IL-12Rβ2-gp130” - N.B. the most N terminal VHH is mentioned first) and the antibody format (bivalent bispecific versus monovalent bispecific) were assessed (Figure 4).

An orientation variant of bsVHH-Fc-3 was generated in which the gp130 and IL-12Rβ2 VHH domains were switched from the gp130 VHH-(G4S)7-IL-12Rβ2 VHH-Fc format to an IL-12Rβ2 VHH- (G₄S)₇-gp130 VHH-Fc format (referred to as bsVHH-45). The potency of both “gp130-IL-12Rβ2” and “IL-12Rβ2-gp130” bsVHH-Fc geometries to induce STAT3 phosphorylation was compared in HEK- Blue IL-12 cells, which was analysed by flow cytometry. Briefly, 300,000 cells/well were seeded in a 96-well plate in complete RPMI culture media and stimulated with 10 nM of bsVHH-Fc-3 and bsVHH- Fc-45 or 10-fold dilutions thereof for 30 minutes at 37°C. Cells were then fixed with paraformaldehyde (2% final concentration) for 10 minutes at room temperature, washed with FACS buffer, and incubated with BD Phosflow Perm Buffer III (BD Biosciences) for 30 minutes on ice. After washing with FACS buffer the cells were stained with anti-phospho(Tyr705)-STAT3-alexa fluor 647 detection antibody (BD Biosciences, Cat#562071) for 1 hour at room temperature. Dead cells were excluded from the analysis by using a Fixable Viability Dye eFluor780 (FVD780, eBioscience, Cat# 65-0865-18). Flow cytometric measurements were performed on a BD LSR flow cytometer (BD Biosciences). Results are represented in Figure 4. It was found that the order of the anti-gp130 VHH clone 18406 and anti-IL- 12Rβ2 VHH clone 20422 in the bsVHH-Fc format did not affect the potency to induce STAT3 phosphorylation.

Next, the linker length of bsVHH-Fc3 was varied from 7 (G4S) repeats to GGGGS (G₄S)₁ and analyzed again the potency to induce STAT3 activation in HEK-Blue IL- 12 cells using flow cytometry. Data is depicted in Figure 4. Shortening the linker of bsVHH-Fc-3 to (G₄S)₅ increases the efficacy of STAT3 activation in HEK-Blue IL-12 cells, however efficacy is lost when further shortening to (G₄S)₁.

Finally, a monovalent knob-into-hole format presenting one anti-gp130 VHH on the ‘knob’ Fc and one anti-IL-12Rβ2 VHH on the ‘hole’ Fc was evaluated. For this, the anti-gp130 or anti-IL-12Rβ2 VHHs from selected bsVHHs were fused to human IgGl hinge plus Fc region. The Fc domains comprised either the E357K and E399K or K392D and K409D amino acid substitutions (EU numbering) to generate respectively a ‘knob’ or ‘hole’ Fc chains to allow heterodimeric knob-into-hole Fc assemble based on inverted charges (Gunasekaran et al. 2010, DOI: 10.1074/jbc. M110. 117382). The knob-into-hole antibodies were produced by transient co-transfection of plasmids expressing both the knob and hole chain in HEK293T cells and were purified from the supernatant by protein A affinity chromatography. Knob-into-hole versions of bsVHH- 1 (VHH clones 18406-20422), bsVHH-3 (VHH clones 18439-20422), bsVHH-8 (VHH clones 18406-20432), and bsVHH-10 (VHH clones 184369- 20432), respectively KiH-1, KiH-3, KiH-8 and KiH-10, were tested as describe above in HEK-Blue IL- 12 cells for their ability to induce STAT3 activation by flow cytometry. Data is depicted in Figure 4. Only KiH-8 was able to induce significant STAT3 activation.

Example 5: Broadening of bsVHH-Fc repertoire - second design wave

The bispecific VHH-Fc combinations were expanded by selecting anti-gp130 VHH clones that generated active bsVHH-Fc molecules and combining these with new anti-IL-12Rβ2 clones. Specifically, anti-gp130 clones 18406 (used for bsVHH-Fc-1 and -8) and clone 18439 (used for bsVHH-Fc-3 and -10) were each combined with 9 new anti-IL-12Rβ2 VHH clones in the bsVHH-Fc format with the VHHs in the order ‘gp130-IL-12Rβ2’ starting from the mostN-terminal (Table 14 and 15).

Table 14: Binding and competition characteristics of selected anti-IL-12Rβ2 VHH clones for bsVHH construction Table 15: Overview of the second design wave of bispecific VHH(-Fc) constructs

We assessed the ability of these bispecific anti-gp130/IL-12Rβ2 antibodies to induce STAT3 phosphorylation using a Jurkat T cell line that expresses gp130 and IL-12Rβ2. Wildtype Jurkat cells were engineered to stably express human gp130 and human IL-12Rβ2 by transduction with pLVX- gp130-IRES-mCherry and pLVX-IL-12Rβ2-IRES-ZsGreen derived lentiviral particles. The cells were subsequently sorted for mCherry and ZsGreen positivity, and referred to as ‘Jurkat-gp130-IL-12Rβ2’ cells. 300,000 Jurkat-gp130-IL-12Rβ2 cells/well were seeded in a 96-well plate in complete RPMI culture media and stimulated the cells with 10 nM of anti-gp130/IL-12Rβ2 bsVHH-Fc antibodies or 10-fold dilutions thereof for 30 minutes at 37°C. We included bsVHH-Fc-3 and -8 as reference controls. Cells were then fixed with paraformaldehyde (2% final concentration) for 10 minutes at room temperature, washed with FACS buffer, and incubated with BD Phosflow Perm Buffer III (BD Biosciences) for 30 minutes on ice. After washing with FACS buffer the cells were stained with anti- phospho(Tyr705)-STAT3-alexa fluor 647 detection antibody (BD Biosciences, Cat#562071) for 1 hour at room temperature. Dead cells were excluded from the analysis by using a Fixable Viability Dye eFluor780 (FVD780, eBioscience, Cat# 65-0865-18). Flow cytometric measurements were performed on a BD Symphony A3 flow cytometer (BD Biosciences). The percentage of pSTAT3 positive cells was analysed within the mCherry and ZsGreen double positive cells. Data is depicted in Figure 5. We identified bsVHH-Fc-68, -69, -70 and -71 as molecules with significant higher efficacy compared to bsVHH-Fc-3.

Example 6: Induction of IL-10 in gp130-IL-12Rβ2 positive T cell line

The potency of selected anti-gp130/IL-12Rβ2 bispecific VHH-Fc antibodies to induce production of the immune suppressive cytokine IL-10 was analyzed in the ‘Jurkat-gp130-IL-12Rβ2’ cells (described in example 5). The cells were seeded at 500,000 cells/well of a 96 well plate in complete RPMI media and stimulated with different doses of bsVHH-Fc-3 and KiH-8 for 24 hours. As negative control, the anti-IL-12Rβ2 VHH subunit of bsVHH-Fc-3 was substituted for the irrelevant BCII10 VHH, referred to as bsVHH-Fc-30. The cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin as positive control. The supernatants were collected 24 hours later and the levels of IL- 10 cytokine was determined by ELISA (Invitrogen, Cat# 88-7106). Figure 6 shows the ability of bsVHH- Fc-71 and KiH-8 to induce IL- 10 secretion at a similar level as PMA/ionomycin, while bsVHH-Fc-30 fails to induce IL-10.

Example 7

The bispecific VHH-Fc combinations were expanded in a new design wave by selecting anti-IL-12Rβ2 VHH clones that generated potent bsVHH-Fc molecules in design wave 1 and 2 and combining these with new anti-gp130 clones from new CDR3 VHH families to introduce more diversity. Specifically, anti-IL-12Rβ2 clones 20407 (used for bsVHH-Fc-70), clone 20432 (used for KiH-8), and clone 20391 (used for bsVHH-Fc-71) were each combined with 19 new anti-gp130 VHH clones in the bsVHH-Fc format with the VHHs in the order ‘gp130-IL-12Rβ2’ starting from the N-terminal side (Table 16 and 17).

Table 16: Binding and competition characteristics of selected anti-gp130 VHH clones for bsVHH construction.

P.E. : Periplasmic extract

Table 17:

We assessed the ability of the bispecific antibodies bsVHH-Fc-73 to -132 to induce STAT3 phosphorylation using the above described Jurkat T cell line that expresses gp130 and IL-12Rβ2, and referred to as ‘Jurkat-gp130-IL-12Rβ2’ cells. 300,000 Jurkat-gp130-IL-12Rβ2 cells/well were seeded in a 96-well plate in complete RPMI culture media and stimulated with different concentrations of anti- gp130/IL-12Rβ2 bsVHH-Fc antibodies or 10-fold dilutions thereof for 30 minutes at 37°C. We included bsVHH-Fc-71 as reference controls. We assessed STAT3 phosphorylation by flow cytometry as described in Example 5. The percentage of pSTAT3 positive cells was analysed within the mCherry and ZsGreen double positive cells, and thus expressing both gp130 and IL-12Rβ2 receptors. In Figure 7A we depict the dose-response data for the screened bsVHH-Fc molecules with higher potency and/or efficacy compared to bsVHH-Fc-71. Particulary, bsVHH-Fc-77 demonstrated to have the highest potency and efficacy in inducing pSTAT3 upon receptor activation.

Next we assessed the potency of bsVHH-Fc-71, -77, -78, -84, and -96 to induce IL-10 secretion in the Jurkat-gp130-IL-12Rβ2 cells, as described in Example 5. All tested bsVHH-Fc molecules demonstrated a dose-dependent induction of IL- 10 secretion, with bsVHH-Fc-77 being the most potent and efficacious bsVHH-Fc molecules (Figure 7B).

In an attempt to optimize the sequence of bsVHH-Fc-77 we generated variants with different linker lengths separating the anti-gp130 and anti-IL-12Rβ2 VHH sequences from 7 to 1 (G4S) repeats, next to introducing the L234F/L235E/P331 S mutations from the Durvalumab constant region to reduce effector functions of the Fc domain. The functionality of the latter molecules was assessed by measuring the ability to induce pSTAT3 in Jurkat-gp130-IL-Rβ2 T cells as described above. We observed that the efficacy of the bsVHH-Fc antibodies increased with increasing G4S linker length to (G₄S)7 (Figure 7C).

Example 8

We investigated the possibility to induce a synergistic response by co-stimulation of two different bsVHH-Fc molecules. We tested multiple combinations of bsVHH-Fc to induce pSTAT3 activation in Jurkat-gp130-IL-12Rβ2 cells. We found that co-stimulation of bsVHH-Fc- 107 and -108, which have on their own little activity, resulted in a strong pSTAT3 induction, albeit with less potency compared to bsVHH-Fc-77 (Figure 8A). BsVHH-Fc-107 and -108 share the same anti-IL-12Rβ2 VHH clone but differ in the anti-gp130 VHH clone from different CDR3 groups. We reason that the combination of bsVHH-Fc- 107 and 108 could possibly result in a higher-order receptor clustering due to binding of different epitopes, and result in increased downstream receptor activation. To further exploit this, we designed biparatopic bispecific anti-gp130/IL-12Rβ2 binding molecules by making use of the knob- into-hole technology as described in Example 4. For example we generated a KiH-107/108 molecule, which constitutes the monovalent bispecific arm of bsVHH-Fc-107 on the ‘knob’ Fc and the monovalent bispecific arm of bsVHH-Fc-108 on the ‘hole’ Fc. In a similar way, we generated KiH- 71/77 (Figure 8B), which combines the a-gp130/IL-12RB2 VHH clones from bsVHH-Fc-71 and -77. Using the Jurkat-gp130-IL-12Rβ2 cells we demonstrated that the biparatopic bispecific KiH-71/77 and -107/108 molecules are able to induce pSTAT3 activation downstream of the IL-35 receptor (Figure 8C).

The amino acid sequences for the polypeptides of the bsVHH-Fc-71 knob, bsVHH-Fc-77 hole, bsVHH- Fc-107 hole and bsVHH-Fc-108 polyptides used to form the antigen binding proteins studied are set out in Table 18 below. In order to allow for purification C-terminal His tags were employed, but are not indicated in the sequences. Such His tags are optional in the sense that they will only be included where purification by His tags is being used.

TABLE 18 Example 9

We assessed the ability of bsVHH-Fc-77 to induce STAT activation downstream of the IL-35 receptor in primary human CD4 T cells. CD4 T cells were enriched from different PBMC donors and seeded at 10⁶ cells/ml in presence of anti-CD3/CD28 antibodies (Ab clones, HIT3a and CD28.2, respectively, both at 2 pg/ml) to activate the cells. The latter T cell receptor stimulation is required to upregulated the expression of IL12RB2, which is not expressed in naive CD4 T cells. Forty-eight hours after TCR activation the T cells were depleted of a-CD3/CD28 antibodies and IL-2 and rested overnight. The cells were stimulated with different doses of bsVHH-Fc-77 and incubated for 30 minutes. Cells were then stained with Fixable Viability Dye eFluor™ 780 fixed with paraformaldehyde (2% final concentration) for 10 minutes at room temperature, washed with FACS buffer, and incubated with BD Phosflow Perm Buffer III (BD Biosciences) for 30 minutes on ice. After washing with FACS buffer the cells were stained with anti-CD4-FITC (Biolegend, Cat# 980802), anti-CD25-PE-Cy7 (Biolegend, Cat# 356107), anti-phospho(Tyr701)-STATl-Briliant Violet 421 (BD Biosciences, Cat# 568925), anti- phospho(Tyr705)-STAT3-alexa fluor 647 (BD Biosciences, Cat# 562071) detection antibodies for 1 hour at room temperature. Dead cells were excluded from the analysis by using a Fixable Viability Dye eFluor780 (FVD780, eBioscience, Cat# 65-0865-18). The data is depicted in Figure 9A. We demonstrate a dose-dependent activation of STAT3 by bsVHH-Fc-77 in activated CD4+ CD25+ T cells, with an EC₅₀ of 0.007 nM. Figure 9B depicts the percentage of gp130 and IL-12RB2 single and double positive cells among the CD4+ CD25+ T cells at the moment of bsVHH-Fc-77 stimulation. In addition, we also monitored the secretion of IL-10 upon long-term stimulation with bsVHH-Fc-77 in the pre-activated CD4 T cells. We found that bsVHH-Fc-77 significantly induces IL-10 production at 0.1 nM (Figure 10), which correlates with the dose inducing a maximal efficacy in the pSTAT3 assay (Figure 9A).

Claims

1. A binding molecule specific for an IL-35 receptor, wherein the binding molecule is an agonist of the IL-35 receptor, with the binding molecule comprising:

2. The binding molecule of claim 1, wherein:

(b) the binding molecule is an antibody with at least one binding -site specific for the gp130 subunit of the IL-35 receptor and at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor.

3. The binding molecule of claim 1 or 2, wherein:

(i) the at least one binding -site specific for the gp130 subunit of the IL-35 receptor:

(a) is an antigen-binding site that is able to bind gp130, with the antigen comprises a CDR1, CDR2, and CDR3 having the sequence of SEQ ID NOs: 2217, 2218, and 2219, or variant sequences having at most three amino acid sequence changes per CDR; or

(b) is an antigen-binding site able to compete for binding to gp130 wih an antigen-binding site of (a);

(aa) is an antigen-binding site that is able to bind IL-12Rβ2, with the antigen comprises a CDR1, CDR2, and CDR3 having the sequence of SEQ ID NOs: 652, 653, and 654, or variant sequences having at most three amino acid sequence changes per CDR; or

(bb) is an antigen-binding site able to compete for binding to gp130 wih an antigen-binding site of (aa);

4. The binding molecule of claim 1 or 2, wherein:

(i) the at least one binding -site specific for the gp130 subunit of the IL-35 receptor has a set of three CDRs for the CDR1, CDR2, and CDR3 selected from one of the following sets:

(a) SEQ ID NOs: 2217/2218/2219;

(b) SEQ ID NOs: 2011/2012/2013;

(c) SEQ ID NOs: 20746/2747/2748;

(d) SEQ ID NOs: 2130/2131/2132; (e) SEQ ID NOs: 1983/1984/1985;

(f) a variant set of three CDRs of one of the CDR sets of (a) to (e), wherein each CDR has, at most, three amino acid sequence changes compared to the specific sequence of the CDR set off (a) to (e) and the antigen binding site is still able to bind gp130; and

(g) a CDR set that means the antigen-binding site can compete for binding to gp130 with an antigen-binding site having one of the CDR sets of (a) to (f), and/or

(ii) the at least one binding -site specific for the IL-12Rβ2 subunit of the IL-35 receptor has a set of three CDRs for the CDR1, CDR2, and CDR3 selected from one of the following sets:

(aa) SEQ ID Nos: 652/653/654;

(bb) SEQ ID Nos: 412/413/414;

(cc) SEQ ID Nos : 739/740/741 ;

(dd) a variant set of three CDRs of one of the CDR sets of (aa) to (cc), wherein each CDR has at most three amino acid sequence changes compared to the specific sequence of the CDR set off (aa) to (cc) and the antigen binding site is still able to bind gp130; and

(ee) a CDR set that means the antigen-binding site can compete for binding to gp130 with an antigen-binding site having one of the CDR sets of (aa) to (dd).

5. The binding molecule of claim 4, wherein:

(i) the at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 2217, 2218, and 2219 respectively and the at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor is at least one antigen- binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 2217, 2218, and 2219 respectively;

(ii) the at least one antigen-binding -site specific for the gp130 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 2011, 2012 and 2013 respectively and the at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor is at least one antigen- binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 652, 653, and 654 respectively;

(iii) the at least one antigen-binding-site specific for the gp130 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 2746, 2747, and 2748 respectively and the at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor is at least one antigen- binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 652, 653, and 654 respectively;

(iv) the at least one antigen-binding -site specific for the gp130 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 2130, 2131, and 2132 respectively and the at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 412, 413, and 414 respectively; and

(v) the at least one antigen-binding -site specific for the gp130 subunit of the IL-35 receptor is at least one antigen-binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nosl983, 1984, and 1985 respectively and the at least one binding-site specific for the IL-12Rβ2 subunit of the IL-35 receptor is at least one antigen- binding site comprising a set of three CDRs where the CDR1, CDR2, and CDR3 of the set have the sequences of SEQ ID Nos: 739, 740, and 741 respectively;

(vi) the binding molecule is a variant of a binding molecule as defined in any of (i) to (v) above with a maxium of 3 amino acid sequence changes in each CDR, wherein the binding molecule is still able to bind both gp130 and IL-12Rβ2; and

(vii) the binding molecule is a binding molecule able to compete for binding to both gp130 and IL-12Rβ2 with one of the binding molecvules of (i) to (vi).

6. The binding molecule of claim 1 or 2, wherein the binding molecule comprises a binding site specific for the IL-12Rβ2 subunit of the IL-35 receptor which comprises:

7. The binding molecule of any one of claims 1, 2 or 6, wherein the binding molecule comprises one or more of the following VHH antigen-domains: (a) a VHH antigen-binding domain that binds IL-12Rβ2 and is selected from the VHH antigen-binding domain having the sequence of one of the VHH antigen-binding domains of Table 1;

(b) a VHH antigen-binding domain that binds IL-12Rβ2 and has at least 80% sequence identity to one of the VHH antigen-binding domains of Table 1;

8. The binding molecule of any one claims 1,2, 6 or 7, wherein the binding molecule comprises a binding site specific for the gp 130 subunit of the IL-35 receptor which comprises:

9. The binding molecule of claim 8, wherein the binding molecule comprises one or more of the following VHH antigen-domains:

10. The binding molecule of any one of claims 1, 2, or 6 to 9 which is a bispecific binding molecule wherein: (a) the binding molecule comprises a VHH comprising the same set of three CDRs for the IL-12Rβ2 binding site and a VHH comprising the same set of three CDRs for the gp130 binding site as one of the bispecific binding molecules of Table 17, 11 or 15;

(b) the binding molecule comprises a VHH comprising the same set of three CDRs for the IL-12Rβ2 binding site apart from a maximum of ten amino acid sequence changes and the same set of three CDRs for the gp130 binding site apart from a maximum of ten amino acid sequence changes as one of the binding molecules of Table 17, 11 or 15;

(c) the binding molecule comprises a VHH specific for IL-12Rβ2 comprising a set of three CDRs for the IL-12Rβ2 binding site with at least 95% sequence identity to a set of three CDRs for an IL-12Rβ2 binding site of one of the bispecific binding molecules of Table 11 or 15 and a VHH specific for gp130 comprising a set of three CDRs with at least 95% sequence identity to a set of three CDRs for a gp 130 binding site of the same bispecific binding molecules of Table 17, 11 or 15; or

(d) the VHH antigen-binding domain specific for IL-12Rβ2 can compete for binding with one of the VHH antigen binding domains specific for IL-12Rβ2 of a bispecific binding molecule of Table 11 or 15 and the VHH antigen-binding domain specific for gp130 can compete for binding with the gp130 binding site of the same bispecific binding molecule of Table 17, 11 or 15.

11. The binding molecule of claim 10, wherein the binding molecule:

(a) has the sets of CDRs forthe IL-12Rβ2 and gp130 binding sites from one of bispecific molecules 77, 78, 84, 96 and 71 of Table 17;

(b) has the sets of CDRs forthe IL-12Rβ2 and gp130 binding sites with a maximum of ten amino acid sequence changes per set compared to one of bispecific molecules 77, 78, 84, 96 or 71 of Table 17;

(c) has sets of CDRs for the IL-12Rβ2 and gp130 binding sites with at least 95% sequence identity compared to one of bispecific molecule 77, 78, 84, 96 or 71 of Table 17; or

(d) has a binding site for IL-12Rβ2 that is able to compete for binding to one of the binding sites specific for IL-12Rβ2 of the bispecific molecule 77, 78, 84, 96 or 71 of Table 17 and a binding site for gp130 which is able to compete for binding with the bispecific molecule 77, 78, 84, 96 or 71 of Table 17.

(e) has the CDRs forthe IL-12Rβ2 and gp130 binding site from the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15;

(f) has sets of CDRs for the IL-12Rβ2 and gp130 binding site with a maximum of ten amino acid sequence changes per set compared to the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15;

(g) has sets of CDRs for the IL-12Rβ2 and gp130 binding site with at least 95% sequence identity compared to one of the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15; or (h) has a binding site for IL-12Rβ2 that is able to compete for binding to one of the binding sites specific for IL-12Rβ2 of the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15 and a binding site for gp130 which is able to compete for binding with the bispecific molecule 1, 3, 5, 8 or 10 from Table 11 or the bispecific molecule 57, 61, 64, or 66 to 71 of Table 15.

12. The binding molecule of any one of the preceding claims wherein the binding molecule comprises VHH binding domains and an Fc region.

13. The binding molecule of claim 12, wherein the binding molecule:

(a) comprises two polypeptide chains each with two VHH binding domains, with one of the two VHH binding domains specific for IL-12Rβ2 and the other specific for gp130; or

14. The binding molecule of claim 13, wherein the binding molecule is:

(a) bsVHH-Fc-77, bsVHH-Fc-78, bsVHH-Fc-84, bsVHH-Fc-96;

(b) bsVHH-Fc-3, bsVHH-Fc-64, bsVHH-Fc-68, bsVHH-Fc-69, bsVHH-Fc-70, or bsVHH-Fc-71;

(c) a bsVHH-Fc with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (c);

(d) a bsVHH-Fc with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (a) or (b), apart from up to a maximum of 10 amino acid sequence changes in each set;

(e) a bsVHH-Fc with sets of CDRs have at least 95% sequence identity to the sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains of one of the bispecific molecules of (a) or (b);

(f) a bsVHH-Fc with VHH binding domains that are able to compete for binding to IL- 12Rβ2 and gp130 in comparison to the VHH binding domains for the specificities of the same bispecific molecule of (a) or (b); or

(g) a bispecific molecule as defined in any of (a) to (f), but where the VHH domains of the binding molecule have been humanized

15. The binding molecule of claim 14, wherein the binding molecule is:

(a) KiH8; (b) a KiH format binding molecule with the same sets of CDRs for the VHH IL-12Rβ2 and VHH gp130 binding domains as KiH8;

16. The binding molecule of any one of claims 1 to 15, wherein the binding molecules comprises a linker or linkers separating antigen binding domains, preferably wherein the linkers are GGGGS or linkers comprising more than one copy of GGGGS.

17. The binding molecule of any one of claims 1 to 16, wherein the binding molecule comprises:

(a) a constant region modified to reduce or eliminate Fc regions;

(b) the Durvalumab constant region comprising:

18. The binding molecule of claim 1 or 2, wherein the binding molecule:

(i) comprises at least two different binding-site specifics for the gp130 subunit of the IL- 35 receptor;

(ii) comprises at least two binding-sites specific for the IL-12Rβ2 subunit of the IL-35 receptor.

19. The binding molecule of claim 18, wherein the binding molecule comprises:

(i) a first polypeptide comprises a first binding site for gp130 and a first binding site for IL-12Rβ2; and

(ii) a second polypeptide comprises a second binding site for gp130 and a second binding site for IL-12Rβ2, wherein the first and second binding sites for gp130 bind different epitopes of gp130 meaning that the binding molecule is biparatopic for gp130 and wherein the first and second binding sites for IL-12Rβ2 bind different epitopes of IL-12Rβ2 meaning that the binding molecule is biparatopic for IL-12Rβ2.

20. The binding molecule of claim 19, wherein:

(i) a first polypeptide comprises a first binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2755, 2756, and 2757 respectively and a first binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 412, 413, and 414 respectively or alternatively the first binding site for gp130 and/or first binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen; and

(ii) the second polypeptide comprises a second binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2692, 2693, and 2694 respectively and a second binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 412, 413, and 414 respectively or alternatively the second binding site for gp130 and/or second binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen.

21. The binding molecule of claim 19, wherein:

(ii) the second polypeptide comprises a second binding site for gp130 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 2217, 2218, and 2219 respectively and a second binding site for IL-12Rβ2 comprising a CDR1, CDR2 and CDR3 set of SEQ ID Nos: 652, 653, and 654 respectively or alternatively the second binding site for gp130 and/or second binding site for IL-12Rβ2 comprise a variant set of three CDRs with a maxium of three amino acid sequence changes per CDR, with the binding site still able to bind the antigen.

22. The binding molecule of any one of claims 19 to 21, wherein the first and second polypeptides each further comprise an Fc region allowing the two polypeptides to form an Fc region dimer.

21. The binding molecule of claim 22, wherein the Fc regions are those of the Durvalumab constant region modified to remove the CHI region as well as to include complementary knob-into-hole modifcations to allow the two polypeptides to preferentially associate to form heterodimers, rather than homodimers.

22. The binding molecule of any one of claims 18 to 21, wherein the binding molecules comprises a linker or linkers separating antigen binding domains, preferably wherein the linkers are GGGGS or linkers comprising more than one copy of GGGGS.

23. A pharmaceutical composition comprising a binding molecule according to any one of the preceding claims.

24. The binding molecule of any one claims 1 to 22 or pharmaceutical composition of claim 23 for use is a method of treatment of the human or animal body.

25. The binding molecule of any one claims 1 to 22 or pharmaceutical composition of claim 23 for use in treating, preventing an autoimmune or inflammatory disorder, or inducing immune tolerance.

26. The binding molecule of any one claims 1 to 22 or pharmaceutical composition of claim 23 for use of claim 15, wherein the disorder is selected from graft versus host disease (GVHD), multiple sclerosis, chronic obstructive pulmonary disorder (COPD), allergic rhinitis, ulcerative colitis, Crohn’s disease, immune thrombocytopenia (ITP), atherosclerosis, psoriasis, and Diabetes.

27. A method of treating or preventing an autoimmune or inflammatory disorder, or inducing immune tolerance comprising administering a binding molecule of any one claims 1 to 22 or pharmaceutical composition of claim 23 to a subject in need thereof.

28. The method of claim 27 wherein the disorder is selected from graft versus host disease (GVHD), multiple sclerosis, chronic obstructive pulmonary disorder (COPD), allergic rhinitis, ulcerative colitis, Crohn’s disease, immune thrombocytopenia (ITP), atherosclerosis, and Diabetes.

29. A method for detecting association of the IL-12Rβ2 and gp130 receptor subunits comprising:

(a) contacting a candidate molecule with a cell expressing modified versions of IL-12Rβ2 and gp130 receptor subunits which have been modified so when they associate a detectable signal is produced;

30. The method of claim 29 wherein:

(a) the detectable signal is the activation of an enzyme; or

(b) the detectable signal is the activation of luciferase.