WO2025133042A2 - Activatable fusion proteins and methods of use - Google Patents

Abstract

The invention provides activatable fusion proteins and methods of using the same. The invention further provides anti-huIL-2 antibodies and methods of using them.

Description

ACTIVATABLE FUSION PROTEINS AND METHODS OF USE

TECHNICAL FIELD

The present invention relates to activatable fusion proteins and methods of using the same.

BACKGROUND

Therapeutic antibodies have revolutionized modern medicine. With their high specificity, they have made it possible to target specific molecules in a subject with unprecedented precision. While initially monoclonal antibody therapeutics were mostly limited to blocking interaction of the target molecule with natural ligands and thus interrupting signaling pathways underlying certain disease mechanisms, technology quickly progressed from these simpler modes of action to more complex formats, such as bispecific antibodies that can target two different epitopes or targeted cytokines which aim to localize cytokine activity to target tissues in order to trigger an immune response locally without simultaneously impacting healthy tissues.

However, with many therapeutically active substances, such as cytokines or agonistic antibodies, targeting them to the desired tissue with the help of a targeting antibody is not sufficient to prevent unspecific and undesired activity of the active substance in the periphery. In particularly with potent cytokines such as Interferon alpha this could trigger unwanted side effects.

Different molecule formats have been proposed to address this. For example, various attempts have been made to mask therapeutically active agents such as cytokines or CD3-binding (i.e. T cell activating) antigen-binding domains with molecular masks binding to these agents (frequently antibodies or antibody-derived molecules, such as scFvs, but also peptide masks are common). These masks are typically attached to the therapeutically active agents via a peptide linker that is susceptible to cleavage by a target tissue specific protease (e.g. a tumor protease). Thus, the mask is cleaved off once the molecule reaches the target tissue and releases the therapeutically active agent at the site of interest. Dual binding masks have also been proposed that are capable of specifically binding the therapeutically active agent as well as an antigen in the target tissue in a mutually exclusive manner. These molecules have been described to mask the therapeutically active agent in the absence of the target antigen, but in the target tissue they will bind to the target antigen, thus releasing the activity of the therapeutically active agent.

Protease-activatable molecules depend on the presence of highly tissuespecific proteases. If the protease is expressed not only in the target tissue, but also in healthy tissue, this can lead to unwanted side effects in the periphery. Mutually exclusive dual binding antibodies overcome this issue, but they require a lot of effort to develop, and not every target combination may be amenable for dual binding.

Thus, there is a continuing need for versatile molecule formats that allow highly specific targeting of therapeutically active agents to defined cell populations or tissues, while at the same time preventing undesirable side effects of the therapeutically active agent in healthy tissue.

SUMMARY

The invention provides activatable fusion proteins and methods of using the same. It has been found that by fusing a ligand, such as a cytokine, and a masking moiety (e.g. an antibody or an antibody fragment) that is capable of binding to the ligand and preventing it from exerting its biological activity (by blocking the interaction between the ligand and the ligand-binding moiety) to the two chains of an antigen-binding moiety that binds to a target antigen, the ligand’s biological activity can be made dependent on the presence of that target antigen. This unmasking is achieved when the antigen-binding moiety binds to its target antigen so that the inhibitory interaction between the ligand (e.g. the cytokine) and the masking moiety is sterically interrupted, without the need for protease cleavable linkers.

As a result, the present activatable fusion protein platform is not reliant on the presence of tissue-specific proteases or other cleavage mechanisms that are specific to certain tissues. While the latter lead to irreversible changes in the biologically active molecule, the mechanism that the activatable fusion proteins according to the present invention rely on is reversible once there is no target antigen present in the environment. Thus, if the activatable fusion protein remains in the circulation for an extended period of time, the risk of off-target activity is reduced, compared to molecules that rely on mechanisms that rely on more destructive principles, such as cleavage or digest. The fusion proteins of the invention are also independent of other activation mechanisms such as ATP-activation or pH- dependent activation that rely on the presence of certain chemicals or physicochemical conditions in the environment.

The activatable fusion proteins according to the invention accomplish target antigen-dependent activity through a simple arrangement of readily available polypeptide domains, allowing for easy implementation and optimization for their intended purpose. By varying the affinities of the different components, the molecules can be tailor-made to differentiate specific surface expression levels of the target antigen. The activatable fusion proteins demonstrate excellent signal-to- noise ratio and sensitivity, for instance in comparison to other targeted molecule formats that rely on the attenuation of the the ligand (such as a cytokine). The activatable fusion proteins described herein thus also have a very good side effect profile, showing very little target activation in the periphery. In certain embodiments, the activatable fusion proteins shown herein also can act not only on surface molecules, but on target molecules in solution. They also allow making the activation reliant on the presence of two different receptors at the target tissue/cell, thus potentially increasing the specificity for the target tissue.

The invention described herein provides activatable fusion proteins comprising

(a) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(b) a ligand capable of specifically binding to a ligand binding moiety, and

(c) a masking moiety capable of specifically binding to the ligand, characterized in that the ligand is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker,

the first and the second peptide linker do not comprise a protease cleavage site.

The target-dependent binding of the ligand comprised in the activatable fusion proteins described herein to the ligand-binding moiety is in particular not dependent on any proteolytic activity, in particular on any proteolytic cleavage, of the activatable fusion protein. In one aspect, the activatable fusion protein is functional and shows target antigen-dependent activity in its intact form and/or in the absence of proteases.

One embodiment of the invention is an activatable fusion protein characterized in that the first antigen-binding moiety is an antibody or an antibody fragment.

One embodiment of the invention is an activatable fusion protein characterized in that the antigen-binding moiety is an antibody fragment that is selected from the group consisting of a Fab, a DutaFab, a DAF, an Fv, a Fab', a Fab’- SH, a F(ab')2, a diabody, a linear antibody, and a multispecific antibody formed from antibody fragments.

One embodiment of the invention is an activatable fusion protein characterized in that the ligand is selected from the group consisting of a growth factor, a cytokine, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof. In a particular embodiment, the ligand comprised in the activatable fusion protein is a cytokine.

Essentially, in the activatable fusion proteins herein, the ligand can be any molecule that is capable of specifically binding to another molecule functioning as ligand-binding moiety whereas the binding is desired to be target antigen-dependent. One embodiment of the invention is an activatable fusion protein characterized in that the ligand binding moiety is selected from the group consisting of a growth factor receptor, a cytokine receptor, an antigen, a ligand receptor, an enzyme substrate, a fluorescent label, a radioactive label, or a hormone receptor. In a particular embodiment, the ligand comprised in the activatable fusion protein is a cytokine receptor. One embodiment of the invention is an activatable fusion protein characterized in that the ligand is a cytokine selected from the group consisting of interferons, interleukins, chemokines, lymphokines, monokines, colony-stimulating factors, and tumour necrosis factors. In a particular embodiment, the activatable fusion protein is a cytokine selected from the group consisting of interferons and interleukins. In one embodiment, the cytokine is a member of the IL-1 family, the IL-2 subfamily, the interferon (IFN) subfamily or the IL- 10 subfamily. In one such embodiment, the ligand is a cytokine selected from the group consisting of BMP, CSF-1, insulin, GLP-1, HGH, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL- 20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-34, IL-35, IL-36, GM-CSF, FGF, EGF, G-CSF, IFNa, IFNP, IFNy, PDGF, TGFP, TNFa, TNFP, VEGF, or EPO. In another embodiment, the ligand is a cytokine selected from the group consisting of IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IFNa, IFNP, and IFNy. In a particular embodiment the cytokine is selected from the group consisting of IL-2, IL- 7, IL-21 and IFNa.

One embodiment of the invention is an activatable fusion protein characterized in that the masking moiety is selected from the group consisting of an antibody, an antibody fragment, a single-chain antigen-binding moiety, a peptide mask, an anti-idiotypic antibody or anti-idiotypic antibody fragment, a receptor, a protein inhibitor or a binding protein capable of binding specifically to the ligand. In a particular embodiment of the invention, when the ligand is an antibody or antibody fragment, the masking moiety is an anti-idiotypic antibody or anti-idiotypic antibody fragment (e.g. scFv, VHH) or an antigen of the antibody or antibody fragment used as ligand. In another embodiment of the invention, the activatable fusion protein is characterized in that the masking moiety is a antigen-binding moiety selected from the group consisting of scFv, VHH and single-domain antibodies (sdAb).

One embodiment of the invention is an activatable fusion protein characterized in that the masking moiety is reversibly bound to the ligand (e.g. the cytokine). One embodiment of the invention is an activatable fusion protein characterized in that the binding of the masking moiety to the ligand (e.g. the cytokine) sterically hinders the ligand from binding to the ligand-binding moiety (e.g. the cytokine receptor). One embodiment of the invention is an activatable fusion protein characterized in that the binding of the masking moiety to the ligand (e.g. the cytokine) sterically hinders the binding of the first antigen-binding moiety to the antigen. In other words, the binding of the first antigen-binding moiety to the target antigen competes sterically with the binding of the masking moiety to the ligand.

One embodiment of the invention is an activatable fusion protein characterized in that the affinity of the first antigen-binding moiety in the activatable fusion protein to the target antigen is decreased when compared to the affinity of the first antigen-binding moiety alone. One embodiment of the invention is an activatable fusion protein characterized in that the binding of the first antigenbinding moiety to the target antigen releases the ligand (e.g. the cytokine) from the masking moiety bound to the ligand. One embodiment of the invention is an activatable fusion protein characterized in that the binding of the first antigenbinding moiety to the target antigen prevents the masking moiety from (re-)binding the ligand (e.g. the cytokine).

One embodiment of the invention is an activatable fusion protein characterized in that it further comprises a second antigen-binding moiety In one embodiment, the second antigen-binding domain is an antibody, an antibody fragment (such as a Fab, a DutaFab, a DAF, an Fv, a Fab', a Fab’-SH, a F(ab')2, a diabody, a linear antibody, a multispecific antibody formed from antibody fragments an scFv, a nanobody, a VHH, or a variable domain of new antigen receptor (VNAR)), an antibody mimetic (such as a DARPIN, an affibody, a monobody or an anticalin), an endogenous protein domain that interacts with the target, an engineered TCR, or a peptide capable of specifically binding to the antigen. In one particular embodiment the second antigen-binding moiety comprises at least a second heavy chain polypeptide and at least a second light chain polypeptide. In one embodiment, the activatable fusion protein is characterized in that the second antigen-binding moiety is a Fab, a Dutafab, a DAF or a DBA. In another embodiment, the second antigenbinding moiety comprised in the activatable fusion protein described herein is a single-chain antigen-binding moiety, preferably a single-chain antigen-binding moiety selected from the group consisting of an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb).

One embodiment of the invention is an activatable fusion protein characterized in that it further comprises an Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide. One embodiment of the invention is an activatable fusion protein characterized in that the first antigen-binding moiety is covalently attached to the N- terminus or to the C-terminus of one of the two Fc domain heavy chain polypeptides. In one embodiment, the masking moiety is attached at its N-terminus to the N- terminus or the C-terminus of one of the two Fc domain heavy chain polypeptides. Fusion between the first antigen-binding domain and the Fc domain may be via peptide linkers, which may also comprise or consist of (part of) an immunoglobulin hinge region.

One embodiment of the invention is an activatable fusion protein characterized in that the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding moiety is covalently attached via its C- terminus a) to the N-terminus of the first Fc domain heavy chain polypeptide, or b) to the C-terminus of the first Fc domain heavy chain polypeptide.

One embodiment of the invention is an activatable fusion protein characterized in that the second antigen-binding moiety is covalently attached to the N-terminus or to the C-terminus of one of the two Fc domain heavy chain polypeptides. In one embodiment, the activatable fusion protein is characterized in that the second antigen-binding moiety is a Fab, a Dutafab, a DAF or a DBA and that the second heavy chain polypeptide or the second light chain polypeptide of the second antigen-binding moiety is covalently attached at its C-terminus a) to the N- terminus of the second Fc domain heavy chain polypeptide, or b) to the C-terminus of the first or the second Fc domain heavy chain polypeptide. In another embodiment, the activatable fusion protein is characterized in that the second antigen-binding moiety is a single-chain antigen-binding moiety selected from the group consisting of an scFv, an scFab, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb), a nanobody and a VHH. Fusion between the second antigen-binding domain and the Fc domain may be via a peptide linker, which may also comprise or consist of (part of) an immunoglobulin hinge region.

One embodiment of the invention is an activatable fusion protein characterized in that a) the first antigen-binding moiety is covalently attached to the N-terminus of the first Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached to the N-terminus of the second Fc domain heavy chain polypeptide, or b) the first antigen-binding moiety is covalently attached to the N-terminus of the first or the second Fc domain heavy chain polypeptide and the second antigenbinding moiety is covalently attached to the C-terminus of the first or the second Fc domain heavy chain polypeptide.

One embodiment of the invention is an activatable fusion protein characterized in that the second antigen-binding moiety is covalently attached to the N-terminus of the ligand (e.g. the cytokine) via a third peptide linker and that the third peptide linker does not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein characterized in that the N-terminus or the C-terminus of the first heavy chain polypeptide of the Fc domain is covalently attached to the N-terminus of the ligand (e.g. the cytokine) via a third peptide linker, that the second antigen-binding moiety is covalently attached at the N-terminus of its heavy chain polypeptide or light chain polypeptide to the N-terminus or C-terminus of the second heavy chain polypeptide of the Fc domain via a fourth peptide linker, and that the third and fourth peptide linkers do not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein wherein the masking moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus to the N-terminus of the heavy chain polypeptide of the first antigenbinding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand (e.g. the cytokine), fused at its C-terminus to the N-terminus of the light chain polypeptide of the first antigenbinding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

One embodiment of the invention is an activatable fusion protein, wherein the masking moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand (e.g. the cytokine) fused at its C-terminus to the N-terminus of the heavy chain polypeptide of the first antigenbinding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

One embodiment of the invention is an activatable fusion protein wherein the masking moiety comprises a single-chain antigen-binding moiety and the second antigen-binding moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus to the N-terminus of the heavy chain polypeptide of the first antigenbinding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand (e.g. the cytokine), fused at its C-terminus to the N-terminus of the light chain polypeptide of the first antigen- binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain.

One embodiment of the invention is an activatable fusion protein, wherein the masking moiety comprises a single-chain antigen-binding moiety and the second antigen-binding moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand (e.g. the cytokine) fused at its C-terminus to the N-terminus of the heavy chain polypeptide of the first antigenbinding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain.

One embodiment of the invention is an activatable fusion protein characterized in that the second antigen-binding moiety is capable of specifically binding to an antigen that is the same or a different antigen from the target antigen. Selecting a second antigen-binding moiety that is capable of specifically binding to the same antigen as the first antigen-binding moiety is particularly useful in cases where the target antigen, while showing specific expression in the desired target tissue, is not expected to be expressed in very high numbers. Selecting a second antigen-binding moiety that binds to a different antigen as the first antigen-binding moiety can for instance be useful in cases where the target antigen is also expressed in tissues other than the target tissues. Combining two different target antigens may increase the specificity of the activatable fusion protein of the invention, if only the desired target tissue shows expression for both antigens. One embodiment of the invention is an activatable fusion protein characterized in that the second antigen-binding moiety (a) binds to the target antigen and (b) binds to an epitope on the target antigen that is different from the epitope that is bound by the first antigen-binding moiety. Selecting a second antigen-binding moiety may be useful since the second antigen-binding moiety will not saturate the epitope on the target antigen that the first antigen-binding moiety binds to, and thus this epitope remains accessible for the first antigen-binding moiety to achieve unmasking of the ligand (e.g. the cytokine). By selecting a second antigen-binding moiety that binds to a different epitope on the same target antigen as the first antigenbinding moiety, the activation mechanism of the activatable fusion protein can be made intra- as opposed to intermolecular, provided that the linkers and orientation in the activatable fusion protein allow binding to both epitopes in the same antigen molecule at the same time. By this, the activatable fusion protein of the invention may be made activatable not only by membrane-bound target antigens, but also by soluble target antigens, and may further display enhanced unmasking.

One embodiment of the invention is an activatable fusion protein characterized in that the second antigen-binding moiety binds to the same epitope on the target antigen as the first antigen-binding moiety. By selecting two antigenbinding moieties that bind to the same epitope, the activatable fusion protein may be made more dependent on the expression level of target antigen molecules in the target tissue, since the ligand (e.g. the cytokine) can only be released by the first antigen-binding moiety if there is another target antigen molecule available in sufficiently close vicinity to the target antigen molecule that is bound by the second antigen-binding moiety.

One embodiment of the invention is an activatable fusion protein characterized in that the first antigen-binding moiety is an antibody of the IgG type. In a particular embodiment of the invention, the activatable fusion protein is characterized in that the first antigen-binding moiety, the second antigen-binding moiety and the Fc region together form an antibody of the IgG type. In some embodiments, the activatable fusion protein is characterized in that the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain of the activatable fusion protein is an IgGl Fc domain or an IgG4 Fc domain.

One embodiment of the invention is an activatable fusion protein characterized in that it comprises at least two full-length IgG antibody heavy chains and wherein the heavy chains of the antigen-binding moiety are of the y type (IgG), in particular of the yl type. One embodiment of the invention is an activatable fusion protein characterized in that the activatable fusion protein comprises at least two light chains and wherein the light chains of the antigen-binding moiety are selected from the kappa (K) and/or lambda (X) subtype.

One embodiment of the invention is an activatable fusion protein characterized in that the target antigen is selected from the group consisting of alpha- synuclein, Amyloid beta, BCMA, BTLA, CD3e, CD4, CD8, CD 14, CD 16 (FcgRIIIa), CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD44, CD47, CD52, CD70, CD109, CD123, CD137, CEACAM5, c-MET, CTLA4, DLL3, CXCR4, EDB-FN, EpCAM, epidermal growth factor receptor (EGFR), EPO Receptor, FAPa, FGFR2, FGFR3, GD-2, GP100, GITR, GLP-1 receptor, GM-CSF, GPC3, Grp78, Hedgehog, HER2, HER3, HLA-G, ICAM (ICAM-1, -2, -3, -4, -5), IGF-1R, IL-1R1, IL-4Ra, Integrin av, b7 integrin subunit, a4b7 integrin, a4 integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notchl, Notch3, NRP1, NRP2, 0X40, PAR-2, PD-1, PD-L1, PDGFR, PSA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-Cl, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1, TGFp, TGF-Y, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2, or 5T4. In a particular embodiment of the invention, the target antigen is selected from the group consisting of PD1, PD-L1, CD8 and CD 19.

One embodiment of the invention is an activatable fusion protein characterized in that the ligand is an antigen-binding moiety.

One embodiment of the invention is an activatable fusion protein characterized in that the antigen-binding moiety is an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of alpha-synuclein, Amyloid beta, BCMA, BTLA, CD3e, CD4, CD8, CD 14, CD 16 (FcgRIIIa), CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD44, CD47, CD52, CD70, CD109, CD123, CD137, CEACAM5, c-MET, CTLA4, DLL3, CXCR4, EDB-FN, EpCAM, epidermal growth factor receptor (EGFR), EPO Receptor, FAPa, FGFR2, FGFR3, GD-2, GP100, GITR, GLP-1 receptor, GM-CSF, GPC3, Grp78, Hedgehog, HER2, HER3, HLA-G, ICAM (ICAM-1, -2, -3, -4, -5), IGF-1R, IL-1R1, IL-4Ra, Integrin av, b7 integrin subunit, a4b7 integrin, a4 integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB,, NKG2D, NKp30, nKp46, Notchl, Notch3, NRP1, NRP2, 0X40, PAR-2, PD-1, PD-L1, PDGFR, PSA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-Cl, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1, TGFp, TGF-y, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2, or 5T4.

One embodiment of the invention is an activatable fusion protein characterized in that the antigen-binding moiety is an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of BCMA, CD3, CD20, CD 19, CD27, CD28, CD40, CD47, CD 123, CD 137, CEA, CTLA4, DLL3, EpCAM, GP100, GITR, HER2, HLA-G, IL-7, kynureninase, MICA, MICB, 0X40, PD-L1, PD-1, the extracellular domain of TGFBR2, TNF, or VEGF-C. In a particular embodiment of the invention, the target antigen is selected from the group consisting of PD1, PD-L1, CD8 and CD 19.

One embodiment of the invention is an activatable fusion protein characterized in that the ligand is a non-cytokine ligand selected from the group consisting of a growth factor, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof.

The invention provides an isolated nucleic acid encoding the activatable fusion protein as described herein.

The invention provides a host cell comprising such nucleic acid.

The invention provides an in vitro method of producing an activatable fusion protein as described herein comprising culturing the host cell under conditions suitable for the expression of the activatable fusion protein as described herein. In a particular embodiment, the method further comprises recovering the antibody from the host cell.

The invention provides an activatable fusion protein produced by such method.

The invention provides a pharmaceutical composition comprising a activatable fusion protein as described herein and a pharmaceutically acceptable carrier. The invention provides an activatable fusion protein or the pharmaceutical composition as described herein for use as a medicament.

The invention provides an activatable fusion protein as described herein or the pharmaceutical composition as described herein for use in treating cancer, viral infection or autoimmune disease.

The invention provides the use of the activatable fusion protein as described herein or the pharmaceutical composition as described herein in the manufacture of a medicament. In one embodiment the medicament is for treatment of cancer, viral infection or autoimmune disease.

One embodiment of the invention is an activatable fusion protein comprising (A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide, (B) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide, (C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and (D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus via a first peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus via a second peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein comprising (A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide, (B) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide, (C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and (D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a second peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a first peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site. One embodiment of the invention is an activatable fusion protein comprising (A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide, (B) a second antigen-binding moiety, (C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and (D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus via a first peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus via a second peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, wherein the first and the second peptide linker do not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein comprising (A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide, (B) a second antigen-binding moiety, (C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and (D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a second peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a first peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, and c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, wherein the first and the second peptide linker do not comprise a protease cleavage site.

The invention further provides antibodies that bind to IL-2 or a variant thereof. It has been found that the antibodies of the invention bind not only to human IL-2 but also to IL-2v, an attenuated variant of human IL-2 with abolished CD25 (IL-2Ra) binding. The anti IL-2 antibodies disclosed herein compete for IL-2/IL-2v binding with, at least, IL-2Rp. Masking IL-2 by interrupting interaction with the high affinity receptor IL-2RP (instead of the low affinity receptor IL-2Ry), they are thus able of reducing target-mediated drug disposition (TMDD) by not allowing significant binding to the cell surface and - as they do not increase local concentration since they do not cluster via IL-2RP - also increasing IL-2-masking efficiency. In some embodiments of the invention, the anti-IL-2 antibodies disclosed herein block not only binding of IL-2 from IL-2RPy by competing with IL-2RPy for IL-2 binding, but surprisingly also prevent IL-2 at the same time from binding to IL-2Ra (CD25). Such anti-IL-2 antibodies demonstrating potent IL-2Ra competitive binding are very useful as masks for IL-2 molecules, since they eliminate IL-2 activity so completely that wildtype IL-2 could be used therapeutically instead of attenuated variants such as IL-2v. This is an advantage since the introduction of amino acid substitutions to attenuate a cytokine has a risk of increasing immunogenicity in the patient. In the context of the activatable fusion proteins described herein, the anti-IL-2 antibodies disclosed herein, when being used as a masking moiety for IL-2, have the ability of binding to IL-2Ra on the cell surface once IL-2 is released, thus contributing to the overall tumor targeting of the activatable fusion protein. In the context of tumor treatment, the anti-IL2 antibody binding to IL-2Ra may prove helpful since it may prevent IL-2Ra from binding to Tregs and Bregs. Also, as IL-2Ra is a high affinity receptor, masking it may have a positive impact by decreasing TMDD, the mechanism by which a drug binds to such extent to its pharmacological target that its pharmacokinetic characteristics are influenced.

One embodiment of the invention is an antibody that binds to IL-2 or a variant thereof, wherein the antibody binds to an epitope of IL-2 within amino acid residues 8-17, amino acid residue 30 and/or amino acid residues 77-81 of SEQ ID NO:81; and thus inhibits the binding of IL-2 to IL-2RPy and to IL-2Ra. In one embodiment, the epitope comprises amino acid residues corresponding to K8, Q13, E15, H16, N30, N77, H79 and R81 of SEQ ID N0:81; and thus binding ofthe anti-IL2 antibody to IL-2 inhibits the binding of IL-2 to IL-2RPy and to IL-2Ra.

One embodiment of the invention is an antibody that binds to IL-2 or a variant thereof, wherein the antibody comprises

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NONO, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

One embodiment of the invention is an antibody that comprises

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NON, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NON, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NON, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NONO, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

In one particular embodiment of the invention the antibody comprises

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NONO, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

In another particular embodiment of the invention the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

In one embodiment of the invention the antibody is a monoclonal antibody. In one embodiment, the antibody is a human, humanized or chimeric antibody. In a particular embodiment, the antibody is an antibody fragment that binds IL-2 or a variant thereof.

One embodiment of the invention is an antibody that comprises

(A) a VH sequence of SEQ ID NON and a VL sequence of SEQ ID NO: 8;

(B) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13;

(C) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(D) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(E) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NON 1;

(F) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(G) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39; (H) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42;

(I) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or

(J) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

In one embodiment of the invention, an antibody that specifically binds to IL- 2 or a variant thereof comprises

(A) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;

(B) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13;

(C) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(D) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(E) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:31;

(F) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(G) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39;

(H) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42;

(I) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or

(J) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

In a particular embodiment of the invention, the antibody comprises

(A) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(B) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(C) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:31;

(D) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(E) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39;

(F) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42;

(G) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or (H) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

In another particular embodiment of the invention, the antibody comprises a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

In one embodiment of the invention, the antibody is a full length IgGl antibody or a Fab.

In one embodiment, the antibody binds IL-2 with an affinity of < 125 nM or

IL-2v with an affinity of < 15,4 nM as measured by SPR assay.

In one embodiment, the antibody binds IL-2 with an affinity of < 1,5 nM or IL-2v with an affinity of < 0,9 nM as measured by SPR assay.

In one embodiment, the antibody is a multispecific antibody.

In one embodiment, the antibody is a Fab and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID

NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID

NO:54;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:56;

(E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58; (F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 64;

(I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

In one particular embodiment, the antibody is a Fab and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:54;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:56;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60; (E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 62;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 64;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:66; or

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

In another particular embodiment, the antibody is a Fab and comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

In one embodiment, the antibody is a full length antibody and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:56; (E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 64;

(I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 68, and wherein the antibody further comprises a human Fc region which comprises two human Fc region polypeptides selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72. SEQ ID NO:73, SEQ ID NO74: and SEQ ID NO: 75.

In one particular embodiment, the antibody that is a full length antibody comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:56; (C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 60;

(E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 62;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 64;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 66; or

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 68, and further comprises a human Fc region which comprises two human Fc region polypeptides selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72. SEQ ID NO:73, SEQ ID NO74: and SEQ ID NO:75.

In another particular embodiment, the antibody that is a full length antibody comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68, and further comprises a human Fc region which comprises two human Fc region polypeptides selected from the group consisting of SEQ ID NO:69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72. SEQ ID NO: 73, SEQ ID NO74: and SEQ ID NO:75.

One embodiment of the invention is an antibody which competes for binding to IL-2 or a variant thereof with the antibody of any one of embodiments 58 to 72. One embodiment of the invention is an isolated nucleic acid encoding the antibody described herein. One embodiment of the invention is a host cell comprising the nucleic acid encoding the antibody described herein. One embodiment of the invention is a method of producing an antibody that binds to IL-2 or a variant thereof comprising culturing said host cell under conditions suitable for the expression of the antibody. In one embodiment, the method further comprises recovering the antibody from the host cell. One embodiment of the invention is an antibody produced by said method.

One embodiment of the invention is a pharmaceutical composition comprising the antibody as described herein and a pharmaceutically acceptable carrier.

One embodiment of the invention is an anti-IL-2 antibody as described herein or the pharmaceutical composition as described herein for use as a medicament.

One embodiment of the invention is an anti-IL-2 antibody as described herein or the pharmaceutical composition as described herein for use in treating cancer, viral infection or autoimmune disease.

One embodiment of the invention is the use of the antibody as described herein or the pharmaceutical composition as described herein in the manufacture of a medicament for cancer, viral infection or autoimmune disease.

One embodiment of the invention is a method of treating an individual having cancer, viral infection or autoimmune disease comprising administering to the individual an effective amount of the antibody of as described herein or the pharmaceutical composition as described herein. In one embodiment, the method further comprises administering an additional therapeutic agent to the individual. In one embodiment, the additional therapeutic agent is selected from the group consisting of anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.

One embodiment of the invention is the use of an anti IL-2 antibody described herein as a masking antibody for IL-2, IL-2v or a variant thereof. One embodiment of the invention is the use of the anti IL-2 antibody described herein as a masking moiety in an activatable fusion protein described herein. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A: Schematic illustration of an exemplary activatable fusion protein based on a Fab (“CISS Fab molecule”). The figure illustrates the reversible binding between ligand (here: a cytokine) and the masking moiety (here: an anti-cytokine mask “aCytokine”) and how it sterically blocks access of the antigen binding moiety (“aTarget”) to the target antigen (not depicted).

Figure 1 B: Schematic illustration of an exemplary activatable fusion protein comprising a “CISS Fab molecule” and a second antigen-binding moiety as additional targeting arm fused to a Fc domain, resulting in an IgG based molecule. The molecule shown here consists of four polypeptide chains.

Figure 1 C: Schematic illustration of an exemplary activatable fusion protein comprising a “CISS Fab molecule” and a second antigen-binding moiety as additional targeting arm fused to a Fc domain, wherein the second antigen-binding moiety is a single-chain antigenbinding moiety (specifically a VHH). The molecule shown here consist of three polypeptide chains.

Figure 2 A: Schematic illustration of a ligand Fab fusion used as CISS Fab precursor molecule in the examples.

Figure 2 B: Schematic illustration of a mask Fab fusion used as CISS Fab precursor molecule in the examples.

Figure 2 C: Schematic illustration of an exemplary complete CISS Fab molecule.

Figure 3 A: Schematic illustration of the mode of action of a CISS Fab molecule. In solution, in the absence of the target antigen, the masking moiety of the CISS Fab molecule is bound to the ligand, preventing the ligand from binding to the ligand-binding moiety. In the presence of the target antigen, the Fab binds to it, concomitantly preventing the masking moiety from binding to the ligand which is now free to bind to the ligand binding moiety. Figure 3 B: Schematic illustration of an activatable fusion protein of the

“targeted CISS molecule” type. (I) When in solution, the ligand (dotted oval) is masked by the masking moiety (dark dotted rectangle) and unable to bind to the ligand binding moiety expressed on the cell surface. When the interaction between the ligand and the masking moiety oscillates opens, the ligand binding moiety is generally at extremely low concentration relative to ligand as compared to the masking moiety, therefore the masking interaction is generally re-established. (II) In the presence of the target antigen, the activatable fusion protein binds to the cell surface via the second antigen-binding moiety. (Ill) The effective antigen concentration for the first antigen binding moiety is heightened after the second antigen-binding moiety has bound to the target antigen on the cell surface. When the interaction between the ligand and the masking moiety oscillates between open and closed now, it leaves the binding surface of the first antigen binding moiety open to bind to the target antigen. (IV) The first antigen-binding moiety binds to a second cell-surface target antigen, holding the ligand mask open. (V) The ligand interacts with the ligand binding moiety on the cell surface.

Figures 4 A-D: Results of HEK-Blue assays of CISS precursor molecules (Fab cytokine fusions) to identify suitable cytokine linker lengths. The x-axis shows the concentration of the molecule which the cells were contacted with during the assay (in [nM]) in a logarithmical scale. The y-axis shows the intensity of the signal (OD620nm).

Figures 5 A-E: Results of HEK-Blue assays of CISS Fab molecules to evaluate the masking capacity of the CISS Fab molecules. The x-axis shows the concentration of the molecule which the cells were contacted with during the assay (in [nM]) in a logarithmical scale. The y-axis shows the intensity of the signal (ODe20nm).

Figures 6 A-B: Example dataset of a PDl-IL-2v CISS Fab and precursors that have each of the desired properties for a CISS molecule.

Figure 6 A: HEK-Blue assays confirming that the cytokine linker is of sufficient length to reach the cytokine receptors. Figure 6 B: Results of SPR assays for selection of potentially functional CISS

Fab molecules.

Figure 7: Results of HEK-Blue IL-2 reporter cell assays assessing the IL-2 activity of two targeted CISS molecules in a PD1 dependent and independent context.

Figure 8: Results of HEK-Blue IL-2 reporter assays comparing PDl-IL-2v

CISS with and without targeting subunits binding to PD1.

Figure 9: Results of HEK-Blue IL-2 reporter assays demonstrating that mutually exclusive CISS switching enhances function relative to simple cytokine masking.

Figures 10A-B: Results of HEK-Blue IL-2 reporter assays showing the activation using CD8 IL-2v CISS Fab molecules (and precursor molecules) of CD8-expressing HEK-Blue IL-2 reporter cells in comparison with HEK-Blue IL-2 reporter cells that do not express CD8 on their surface.

Figures 11A-B: Schematic illustration of different exemplary T cell engager molecule formats that utilize a CISS Fab unit.

Figure HA: The antigen-binding moiety of this CISS T-cell engager format (“CD3 outside”) is directed against the target antigen. The ligand is an anti-CD3 single-chain Fv which is masked by an anti- idiotypic VHH. The second antigen-binding moiety (“targeting arm”) is also directed against the target antigen.

Figure 11B: The antigen-binding moiety of this CISS T-cell engager format (“CD3 inside”) is an anti-CD3 Fab arm. The ligand is an anti-target antigen scFv which is masked by an idiotypic VHH. The second antigen-binding moiety (“targeting arm”) binds to the target antigen.

Figure 12: A schematic illustration of the mode of action of an “CD3 outside”

CISS T-cell engager molecule. Figure 13: A schematic illustration of an exemplary CISS molecule format that is activated via a intramolecular mechanism and its mode of action. The format comprises a first antigen-binding moiety, a second antigen-binding moiety, a ligand and a masking moiety. Both the second-antigen-binding moiety and the masking moiety are single-chain antigen-binding moieties in this example. The ligand is covalently attached via a peptide linker to the N-terminus of either the heavy chain or the light chain polypeptide of the first antigen-binding moiety. The masking moiety is covalently attached at its C-terminus via a peptide linker to the N-terminus of the other polypeptide of the first antigen-binding moiety. The second antigen-binding moiety is bound at its C-terminus or its N- terminus via a peptide linker to the masking moiety.

Figures 14A-C: A schematic illustration of exemplary alternative targeted CISS molecule formats. These formats contain, in addition to the CISS module, a Fc domain and a second antigen-binding moiety.

Figure 14A: The first antigen-binding domain of the CISS module is not directly linked to the Fc domain, but is covalently attached at the N-terminus of one of its heavy chain polypeptides via a peptide linker to the C-terminus of the masking moiety, which is itself covalently attached at its N-terminus via another peptide linker to the C-terminus of one of the two Fc domain heavy chain polypeptides, whereas the second antigen-binding domain is covalently attached via one of its N-termini to the C-terminus of the other of the two Fc domain heavy chain polypeptides with another peptide linker. The second antigen-binding moiety in this example is a CrossFab (indicated by the different coloring of the variable domains) to promote correct heavy-chain/light-chain pairing during manufacture.

Figure 14 B: The first antigen-binding moiety of the CISS module is covalently attached at the C-terminus of its heavy chain polypeptide via a peptide linker to the N-terminus of the Fc domain, while the second antigen-binding moiety is covalently attached at its N-terminus via another peptide linker to the C-terminus of one of the Fc domain heavy chain polypeptides.

Figure 14 C: Schematic illustration of the mode of action of the molecule shown in Figure 14 A. The molecule binds to the target antigen on the cell surface via the second antigen-binding moiety (“targeting arm”). When the mask temporarily releases the cytokine fused to the first antigen-binding moiety, the latter can bind to a second epitope on the same target antigen molecule on the cell surface, thus interrupting the mask-cytokine interaction and releasing the cytokine to bind to the cytokine receptor on the cell surface (not shown).

Figure 15: SPR sensorgrams for molecules P1AJ7955, P1AI4373 (1040int

Fab), P1AJ7982 (Fc containing bispecific molecule featuring a non- functional Fab (DP47) fused to one Fc polypeptide and the G05 VHH to the other Fc polypeptide) and Pl AJ6652. The affinity of P1AJ7955 is significantly higher than either of PD1 binders alone. This is indicative of a capacity for P1AJ7955 to bind to the PD1 twice resulting in improved binding. The 221 scFv contained within molecule P1AJ7955 retains good binding to IL-2v indicating no fusion intolerance induced by the G05 VHH. SPR results for P1AJ6652 confirm that the masking interaction appears to be functional (as seen by the absence of IL-2v or IL-2Rbg binding).

Figure 16: Results of HEK-Blue IL-2 reporter cell assays comparing the use of the attenuated IL-2 variant Q126T in a targeted PD1 IL-2 CISS molecule versus using IL-2v, which has intact IL-2R signaling, as cytokine.

Figure 17: Results of HEK-Blue IL-2 reporter cell assays comparing the use of masking moieties with different affinities for the cytokine in a PD1 IL-2 CISS molecule.

Figure 18: Schematic illustration of the SPR assay set up used to determine competition for IL-2 binding of the anti IL-2 Fabs that were generated with IL-2RPy .

Figures 19A-C: Schematic illustration of the SPR-based competitive binding assay used to determine whether binding of the generated anti-IL-2 Fabs to IL-2 competed with binding of IL-2 to IL-2Rbg.

Figures 20A-B: Schematic illustration of the SPR-based competitive binding assay used to determine whether binding of the generated anti-IL-2 / IL- 2v specific Fabs competed with binding of IL-2 to IL-2Ra (CD25). Figure 21: Results of the SPR analysis to determine the binding affinities of the generated anti IL-2/IL-2v Fabs using a Biacore 8K or 8K+ instrument (Cytiva). Binding kinetics of binding to human IL-2v (right column), human IL-2 (middle column) and to CD79B dimer (control, left column) were analyzed in this assay.

Figure 22: Results of the SPR-based competitive binding assay used to determine whether binding of the generated anti-IL-2 Fabs to IL-2 competed with binding of IL-2 to IL-2Rbg.

Figures 23A-B: Results of the SPR-based competitive binding assay used to determine whether binding of the generated anti-IL-2 Fabs to IL-2 competed with binding of IL-2 to IL-2Ra (CD25).

Figures 24A-F: Results of SPR analysis used to determine the affinity of affinity- matured anti IL-2 Fabs.

Figures 25A-B: Results of SPR analysis used to assess IL-2Rbg and IL-2Ra competition of 221-derived point-mutated anti IL-2 Fabs.

Figure 26A-C: Results of X-ray diffraction anaylsis of IL-2 binding to affinity- matured anti-IL-2 Fab P029-221.AM2 and the impact on binding to IL-2RaPy.

Figure 26A: Structure of P029.221.AM2 IL-2 complex.

Figure 26B: IL-2 bound to P029-221.AM2 overlayed to the IL-2 / IL-2RaPy complex (PDB 2erj).

Figure 26 C: P029-221. AM2 overlayed to the IL-2 / IL-2aPy complex.

Figure 27A-F: Structural alignment of IL-2 bound to 221.AM2 and IL-2 bound to the trimeric receptor complex (PDB 2erj) to elucidate IL-2 binding competition of P029.221.AM2 with IL-2Ra.

Figure 27 A: IL-2 / P029.221.AM2 complex overlayed to unbound IL-2 (PDB 2erj, front).

Figure 27 B: IL-2 / P029.221.AM2 complex overlayed to unbound IL-2 (PDB erj, back).

Figure 27 C: Overlay of IL-2 bound to P021.221.AM2 and unbound IL-2 (PDB 2erj, front).

Figure 27 D: Overlay of IL-2 bound to P021.221.AM2 and unbound IL-2 (PDB 2erj, detail).

Figure 27 E: IL-2 (PDB 2erj) / IL-2Ra complex overlayed to IL-2 bound to P021.221.AM2.

Figure 27 F: IL-2 (PDB 2erj) / IL-2Ra complex overlayed to 11-2 bound to P021.221.AM2, detail.

Figures 28 A-B: Results of HEK-Blue IFNalpha reporter assays comparing the activation by PD-Ll-IFNa CISS molecules with and without a second antigen-binding moiety targeting the molecule to PD-L1 on the cell surface.

Figure 28 A: The graphs show the IFNa activation in the parental HEK-Blue™ IFNa/p reporter cell line which has relatively low PD-L1 expression (“HEK-Blue IFNa PD-L1 low”).

Figure 28 B: The graphs show the IFNa activation in the high expressing PD-L1 positive IFNa reporter cells (clone 45, CL022702, “HEK-Blue IFNa PD-Ll high”).

Figure 29: Results of HEK-Blue IL-2 reporter assays comparing the IL-2 activation of CD8 IL-2v CISS molecules (a CISS Fab and a targeted CISS molecule) in CD8a negative (parental) HEK-Blue IL-2 reporter cells (marked “HEK-Blue IL-2”, solid line and circles) and the PD1 and CD8a positive IL-2 reporter cell line (CL023901; marked “HEK-Blue IL-2 CD8a”, dotted lines and triangles). Additionally, a condition in which the CD8a molecules on the PD1 and CD8a positive IL-2 reporter cells (CL023901) are pre-blocked with an OKT8 variant (P1AF4823) was used (marked “HEK-Blue IL-2 CD8a block”, dashed line and squares).

Figure 30: Schematic illustration of the STAT-5P assay used to determine IL-

2R signaling in cis (activation of a IL-2 receptor on the same cell) and in trans (activation of a IL-2 receptor on another cell). For this assay, CD4 T cells from healthy donor PBMCs were sorted and activated for 3 days using anti-CD3 and anti-CD28 antibodies to induce PD-1 expression. After 3 days, cells were either labeled with Cell Trace Violet (CTV) or left unlabeled, then the unlabeled cells were blocked with an anti-PD-1 antibody. These PD-1 preblocked cells were co-cultured with PD-1+ CTV-labeled cells and treated with various concentrations of immunoconjugates. Phosphorylation of STAT-5 was assessed using flow cytometry, and data analysis provided insights into the potency of the molecules in signaling through the IL-2R, both dependent and independent of PD-1 expression.

Figure 31 A-C: Results of the IL-2R Signaling Assay.

Figure 32: Schematic Illustration of a STAT-5-P assay used to determine IL-

7R activation. CD4 T cells from healthy donor PBMCs were sorted and activated for 3 days using anti-CD3 and anti-CD28 antibodies to induce PD-1 expression. After 3 days, cells were either labeled with Cell Trace Violet (CTV) or left unlabeled, then the unlabeled cells were blocked with an anti-PD-1 antibody. These PD-1 preblocked cells were co-cultured with PD-1+ CTV-labeled cells and treated with various concentrations of immunoconjugates. Phosphorylation of STAT-5 was assessed using flow cytometry, and data analysis provided insights into the potency of the molecules in signaling through the IL-7R, both dependent and independent of PD-1 expression.

DETAILED DESCRIPTION

I. DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art, unless otherwise defined herein.

Unless otherwise defined herein the term “comprising of’ shall include the term “consisting of’.

The term “about” as used herein in connection with a specific value (e.g. temperature, concentration, time and others) shall refer to a variation of +/- 1 % of the specific value that the term “about” refers to.

As used herein, the term "activatable" refers to the ability of a ligand (e.g. a cytokine), that is part of a fusion protein as described herein, to bind the ligand binding moiety (e.g. a cytokine receptor) conditionally upon the presence of a target antigen in close proximity to the fusion protein, in particular conditionally upon the first antigen-binding moiety binding to the target antigen.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody, a ligand) and its binding partner (e.g., an antigen, a ligand binding moiety). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following.

As used herein, the term "antigen" refers to a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigens can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins referred to as antigens herein (e.g. alpha-synuclein, Amyloid beta, BCMA, BTLA, CD3e, CD4, CD8, CD 14, CD 16 (FcgRIIIa), CD 19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD44, CD47, CD52, CD70, CD109, CD123, CD137, CEACAM5, c-MET, CTLA4, DLL3, CXCR4, EDB-FN, EpCAM, epidermal growth factor receptor (EGFR), EPO Receptor, FAPa, FGFR2, FGFR3, GD-2, GP100, GITR, GLP-1 receptor, GM-CSF, GPC3, Grp78, Hedgehog, HER2, HER3, HLA-G, ICAM (ICAM-1, -2, -3, -4, -5), IGF-1R, IL-1R1, IL-4Ra, Integrin av, b7 integrin subunit, a4b7 integrin, a4 integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notchl, Notch3, NRP1, NRP2, 0X40, PAR-2, PD-1, PD-L1, PDGFR, PSA, PSMA, SLAMF6, SR-A1, SR- A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-Cl, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1, TGFp, TGF-y, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VC AM, VEGF, VEGFR1,VEGFR2, or 5T4) can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. The antigen may also be a variant of a naturally occurring polypeptide carrying one or more amino acid substitutions. In a particular embodiment the antigen is a human protein.

As used herein, the term "antigen-binding moiety" or "antigen-binding domain" refers to a polypeptide molecule that specifically binds to an antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. In one embodiment, the antigen-binding moiety comprises a heavy chain and a light chain. In certain embodiments, the heavy chain and the light chain are fused via a peptide linker. In some embodiments, the antigen-binding moiety comprises two polypeptide chains. Particular antigen-binding moieties of the activatable fusion proteins described herein include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding moieties may comprise all, or part, of at least one, antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: a, 5, a, y, or p. Useful light chain constant regions include any of the two isotypes: K and X. In some embodiments, the antigen-binding moiety is a singlechain antigen-binding moiety, such as an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), or a single-domain antibody (sdAb). In some embodiments, the single-chain antigen-binding moiety is an antibody mimetic, such as a DARPin, a monobody, an affibody or an anticalin.

As used herein, the terms "first" and "second" with respect to antigen-binding moieties, peptide linkers, Fab fragments etc., are used for convenience of distinguishing when there is more than one of each type of moiety, e.g. within a single fusion protein. Use of these terms is not intended to confer a specific order or orientation of the bi specific antigen binding molecule unless explicitly so stated.

The terms “anti-target antigen (TA) antigen-binding moiety” and “an antigen-binding moiety that binds to target antigen (TA)” refer to an antigen-binding moiety that is capable of specifically binding a target antigen (TA) with sufficient affinity such that the antigen-binding moiety is useful as a diagnostic and/or therapeutic agent in targeting the TA. In one aspect, the extent of binding of an anti- TA antigen-binding moiety to an unrelated, non-TA protein is less than about 10% of the binding of the antigen-binding moiety to TA as measured, e.g., by surface plasmon resonance (SPR). In certain aspects, an antigen-binding moiety that binds to TA has a dissociation constant (KD) of < IpM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'⁸ M or less, e.g., from 10'⁸ M to 10'¹³ M, e.g., from 10'⁹ M to 10'¹³ M). An antigen-binding moiety is said to “specifically bind” to TA when the antigen-binding moiety has a KD of 1 pM or less. In certain aspects, an anti-TA antigen-binding moiety binds to an epitope of target antigen that is conserved among target antigens from different species.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005). The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antigen-binding moiety or any other antigen-binding moiety binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antigen-binding moiety after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

Competitive binding can be used to easily determine whether an antigenbinding moiety binds to the same epitope of a target antigen (TA) as, or competes for binding with, a reference anti-TA antigen-binding moiety. For example, an “antigen-binding moiety that binds to the same epitope” as a reference anti-TA antigen-binding moiety refers to an antigen-binding moiety that blocks binding of the reference anti-TA antigen-binding moiety to its antigen in a competition assay by 50% or more, and conversely, the reference antigen-binding moiety blocks binding of the antigen-binding moiety to its antigen in a competition assay by 50% or more. Also for example, to determine if an antigen-binding moiety binds to the same epitope as a reference anti-TA antigen-binding moiety, the reference antigenbinding moiety is allowed to bind to TA under saturating conditions. After removal of the excess of the reference anti-TA antigen-binding moiety, the ability of an anti- TA antigen-binding moiety in question to bind to TA is assessed. If the anti-TA antigen-binding moiety is able to bind to TA after saturation binding of the reference anti-TA antigen-binding moiety, it can be concluded that the anti-TA antigenbinding moiety in question binds to a different epitope than the reference anti-TA antigen-binding moiety. But, if the anti-TA antigen-binding moiety in question is not able to bind to TA after saturation binding of the reference anti-TA antigen-binding moiety, then the anti-TA antigen-binding moiety in question may bind to the same epitope as the epitope bound by the reference anti-TA antigen-binding moiety. To confirm whether the antigen-binding moiety in question binds to the same epitope or is just hampered from binding for steric reasons routine experimentation can be used (e.g., peptide mutation and binding analyses using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art). This assay should be carried out in two set-ups, i.e. with both of the antibodies being the saturating antibody. If, in both set-ups, only the first (saturating) antibody is capable of specifically binding to TA, then it can be concluded that the anti-TA antibody in question and the reference anti-TA antibody compete for binding to TA.

In some aspects, two antibodies are deemed to bind to the same or an overlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some aspects, two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

The term “CISS” as used herein means “competitive interaction space switching”, a mode of action for conditionally activatable molecules described herein that relies on a specific arrangement of at least a ligand (e.g. a cytokine), a masking moiety and an antigen-binding moiety within a polypeptide complex that renders the state of the ligand being masked or unmasked dependent on the presence of a target antigen. The term “CISS molecule” is used herein as shorthand for, and interchangeably with, the term “activatable fusion protein”. It refers to any molecule that comprises an arrangement of at least a ligand, a masking moiety and an antigenbinding moiety as described herein that renders masking and de-masking of the ligand dependent on the presence of a target antigen. A “CISS Fab” or “CISS Fab molecule” refers to a CISS molecule that consists essentially of a Fab as antigenbinding moiety to which the ligand and the masking moiety are both covalently attached to via peptide linkers. The linker and the masking moiety are preferably covalently attached to the N-termini of the Fab heavy and light chains in such a CISS molecule. The term “CISS molecule” as used herein refers to any molecule that comprises a CISS Fab to convey CISS functionality to the entire molecule. A CISS molecule can comprise further polypeptide domains, such as Fc domains, further Fab domains, antibody fragments, or other polypeptides such as cytokines.

A “targeted CISS molecule” refers to a CISS molecule that comprises a CISS-type arrangement of a ligand (e.g. a cytokine), a masking moiety and an antigen-binding moiety and at least one additional antigen-binding moiety that binds either to the same or a different antigen as the antigen-binding moiety of the CISS module, thus contributing to the targeting of the CISS molecule to the desired target tissue/molecule. Targeted CISS molecules may comprise additional polypeptide domains, such as an Fc domain.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgG?, IgG₄, IgAi, and IgA?. In certain aspects, the antibody is of the IgGi isotype. In certain aspects, the antibody is of the IgGi isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function. In other aspects, the antibody is of the IgG? isotype. In certain aspects, the antibody is of the IgG₄ isotype with the S228P mutation in the hinge region to improve stability of IgG₄ antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

The terms “covalently attached” or “fused” are used interchangeable herein. As used herein, “(covalently) attached to the N-terminus of a polypeptide” means that a moiety is linked to polypeptide via a covalent bond at or near the N-terminus of said polypeptide. In one particular aspect, the moieties (e.g. a Fab and an Fc domain) are linked by a peptide bond between the C-terminus (i.e. the free carboxy group at the C-terminal end of the polypeptide chain) of one moiety and the N- terminus (i.e. the free amino group at the N-terminal end of the polypeptide) of the other moiety, either directly by an amide bond between the carboxy and the amino group, or via one or more peptide linkers. In another aspect, one moiety is linked to a polypeptide via an amino acid side chain of said polypeptide. Fusion between the various domains of the activatable fusion molecule may be via peptide linkers, which may also comprise or consist of (part of) an immunoglobulin hinge region, in particular when a Fab as (first and/or second) antigen-binding domain is fused to an Fc domain.

The term "cytokine" as used herein refers to small, secreted regulatory proteins ranging from around 5 kDa to 20 kDa that are crucial for cell signaling, particularly within the immune system. Cytokines include in particular molecules such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines, and colony-stimulating factors, and are produced by an array of cell types, including (but not limited to) immune cells (macrophages, B and T lymphocytes, mast cells, monocytes), as well as non-immune cells (endothelial cells, fibroblasts, interstitial cells). Cytokines regulate the immune response to inflammation and infection and modulate various cellular functions such as survival, growth, and gene expression, and can be classified into pro-inflammatory and antiinflammatory cytokines. Some cytokines are also capable of mobilizing the immune system to fight cancer. Cytokines function through specific receptors, playing a pivotal role in balancing humoral and cellular immune responses and regulating the maturation, growth, and responsiveness of cell populations. The cytokines herein may be naturally occurring (e.g. have a wild-type sequence) or carry specific mutations to alter their function, activity, or specificity. The matching cell- surface receptor to a cytokine is herein also referred to as “cytokine receptor”. Binding of the cytokine to the cytokine receptor may trigger cascades of intracellular signaling which may then alter cell functions, including the upregulation and/or downregulation of genes or transcription factors, resulting in the production of other cytokines or an increase in the number of surface receptors for other molecules. Examples of cytokine families include bone morphogenetic proteins (BMP), chemokine ligands (CCL), C-X-C motif ligands (CXCL), growth/differentiation factors (GDF), growth hormones, interferons (IFN), interleukins (IL), and tumor necrosis factors (TNF). Interleukins, in particular, are synthesized by CD4 helper T cells, monocytes, macrophages, and endothelial cells, and they promote the development and differentiation of T and B lymphocytes and hematoblasts. In particular aspects of the invention disclosed herein, the cytokine may be an interleukin or an interferon. In one aspect, the cytokine may be a member of the IL-1 family, or the IL-2 subfamily, the interferon (IFN) subfamily or the IL- 10 subfamily.

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The terms “Fc region”, "Fc", "Fc fragment" and “Fc domain” are used interchangeably herein and relate to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post- translational cleavage of one or more, particularly one or two, amino acids from the C -terminus of the heavy chain. Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Typically, an Fc region comprises two heavy chain polypeptides. Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal glycinelysine dipeptide if not indicated otherwise. In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Exemplary sequences of Fc regions with and without amino acid modifications are shown in Table D (SEQ ID NO:69 - 75).

The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The term “fusion protein”, as used herein, refers to a fusion polypeptide molecule comprising two or more, in particular three or more, moieties, wherein the components of the fusion protein are linked to each other either directly or through peptide linkers. One or more moieties of the fusion proteins disclosed herein may be antibodies or antibody fragments comprising two or more polypeptide chains. To be clear, “fusion protein” as described herein comprises also fusion protein complexes that comprise more than one polypeptide chain wherein at least one polypeptide chain is a fusion polypeptide comprising two or more moieties that are linked to each other directly or through peptide linkers. For example, a “fusion protein” as described herein may also be an IgG class antibody wherein a ligand (e.g. a cytokine) and a masking moiety are covalently attached to the N-termini of the heavy and light chain, respectively, of one of the two Fab arms of said IgG class antibody via peptide linkers. In this example of a fusion protein as used herein, only two of the four polypeptide chains comprise fused moieties. For clarity, the individual peptide chains of the antibody or antibody fragment moieties of the fusion protein may be linked to each other non-covalently, e.g. by disulfide bonds.

The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).

Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR- H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 3 l-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

The term "interleukin-2" or "IL-2" as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 81. Unprocessed human IL-2 additionally comprises an N-terminal 20 amino acid signal peptide having the sequence of SEQ ID NO: 83, which is absent in the mature IL-2 molecule. "Full-length" when used in reference to IL-2 is intended to mean the mature, natural length IL-2 molecule. For example, full-length human IL-2 refers to a molecule that has 133 amino acids (see e.g. SEQ ID NO: 81).

As used herein, a “wild-type” form of IL-2 is a form of IL-2 that is otherwise the same as the mutant IL-2 polypeptide except that the wild-type form has a wildtype amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is the full-length IL-2 (i.e. IL-2 not fused or conjugated to any other molecule), the wild-type form of this mutant is full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g. an antibody chain) the wild-type form of this IL-2 mutant is IL-2 with a wild-type amino acid sequence, fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (the mutated or modified sequence within the non-truncated portion of IL-2) then the wild-type form of this IL-2 mutant is a similarly truncated IL-2 that has a wild-type sequence. For the purpose of comparing IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants to the corresponding wild-type form of IL-2, the term wild-type encompasses forms of IL-2 comprising one or more amino acid mutation that does not affect IL-2 receptor binding compared to the naturally occurring, native IL-2, such as e.g. a substitution of cysteine at a position corresponding to residue 125 of human IL-2 to alanine.

The term “IL-2 mutant” or “mutant IL-2 polypeptide” as used herein is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms where IL-2 is linked to another molecule such as by fusion or chemical conjugation. The various forms of IL-2 mutants are characterized in having a at least one amino acid mutation affecting the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL-2 mutant may be referred to herein as a mutant IL-2 peptide sequence, a mutant IL-2 polypeptide, a mutant IL-2 protein or a mutant IL-2 analog. Designation of various forms of IL-2 is herein made with respect to the sequence shown in SEQ ID NO: 81. Various designations may be used herein to indicate the same mutation. For example a mutation from phenylalanine at position 42 to alanine can be indicated as 42A, A42, A42, L42A, or Phe42Ala.

In some embodiments, wild-type IL-2 for the purpose of the present invention comprises the amino acid substitution Cl 25 A.

The term “IL-2 variant”, “IL-2v” or “Interleukin-2v” as used herein refers to an Interleukin-2 variant with abolished CD25 binding. While there are different IL-2 variants available with abolished CD25 binding, the variant designated IL-2v herein is of the SEQ ID NO: 82 with the amino acid exchanges T3A, F42A, Y45A, L72G, Cl 25 A and is commonly used during the examples in this text.

The terms "CD25", "alpha-subunit of the IL-2 receptor", "a-subunit of the IL-2 receptor", “IL-2Ra” or “IL-2Ra” are used interchangeably herein and refer to any native CD25 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length", unprocessed CD25 as well as any form of CD25 that results from processing in the cell. The term also encompasses naturally occurring variants of CD25, e.g. splice variants or allelic variants. In certain embodiments CD25 is human CD25. The amino acid sequence of an exemplary human CD25 (with signal sequence, Avi-tag and His-tag) is shown in SEQ ID NO: 85 and is found e.g. in UniProt entry no. P01589 (version 185).

The term "IL-2RPy" or "IL-2Rbg" as used herein refers to the heterodimeric form of the IL-2 receptor, consisting of the receptor y- subunit (also known as common cytokine receptor y-subunit, yc, or CD132, and herein also referred to as “IL-2Ry”) and the receptor P-subunit (also known as CD122 or p70, and herein also referred to as “IL-2RP”) (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)). IL-2 functions as a lymphocyte growth and stimulatory factor, signaling via activation of the heterodimeric IL-2 receptor complex, or the heterotrimeric IL-2 receptor complex that further includes IL-2Ra (CD25).

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term "masked" refers to a polypeptide domain, e.g., an antigen-binding domain of an antibody, that is sterically hindered from binding to a target sequence, or a ligand that is sterically hindered from binding to a ligand-binding moiety, e.g., its respective cytokine receptor.

The term “nucleic acid molecule” or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 Bl).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-TA antibody” refers to one or more nucleic acid molecules encoding anti-TA antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

As used herein, the term "ligand" as used herein refers to a molecule (e.g. a cytokine) capable of specifically binding to one or more specific sites of another polypeptide molecule (e.g. a cytokine receptor), which is referred to herein as “ligand-binding-moiety”. In one aspect, a ligand may be a growth factor, a cytokine, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof, preferably a cytokine.

For instance, in ligand binding, the ligand is frequently a signal-triggering molecule, which binds to a site on a target protein. This binding can result in a change of conformation (or shape) and/or function of the protein, influencing biological processes.

As used herein, the term "ligand-binding moiety" refers to a molecule, in particular to a polypeptide molecule, that comprises one or more specific sites capable of being bound by a ligand. In one aspect, the ligand binding moiety is a membrane-bound molecule. In a particular aspect, the ligand binding moiety is selected from the group consisting of a growth factor receptor, a cytokine receptor, an antigen, a ligand receptor, an enzyme substrate, a fluorescent label, a radioactive label, or a hormone receptor, preferably a cytokine receptor.

As used herein, “masking moieties”, sometimes also shortly referred to as “masks”, “protein masks” or “antibody masks”, refer to polypeptides that are able to bind to another polypeptide, for example a ligand, and to reduce or entirely eliminate the ability of the ligand (e.g. a cytokine) to specifically bind to the ligand binding moiety (e.g. a cytokine receptor). In some aspects, the masking moiety comprises the paratope of an antibody or antibody fragment. In other aspects, the masking moiety is a polypeptide that is a natural binding partner of the ligand (e.g. a receptor of the ligand). In one aspect, the masking moiety comprising an antibody, an antibody fragmenta single-chain antigen-binding moiety, a peptide mask (anti-idiotypic antibody, an anti-idiotypic antibody fragment (e.g. scFv, VHH) or a receptor, protein inhibitor or binding protein capable of binding specifically to the ligand.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain. The term “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 20, but sometimes up to 40, amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic peptide linkers are, for example, (GG)_n, (GsS)_n (SEQ ID NO: 91) or (G4S)_n (SEQ ID NO: 92) peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4, in particular 2. Peptide linkers of particular interest are GG, GGGG (SEQ ID NO:86), SGSGSG (SEQ ID NO:87), (GSGGS)_n (SEQ ID NO: 93), (GGGS)n (SEQ ID NO: 91), (GSGGG)_n (SEQ ID NO: 94), (GGGSG)_n (SEQ ID NO: 95), (GSSSG)_n (SEQ ID NO: 96), (GGGGS)_n (SEQ ID NO: 92) and (GGSGG)_n (SEQ ID NO: 97), where n represents an integer of at least 1, preferably from 2 to 6. Table A shows different peptide linkers that may be used to covalently attach the different moieties within a CISS molecule.

Table A - peptide linkers for use in CISS molecules

In one aspect of the invention, the first peptide linker connecting the ligand (e.g. a cytokine) to the first antigen binding moiety is of sufficient length to enable the interaction with the masking moiety and not cause such tension that the interaction is poor and multimers occur. The length of the first peptide linker further enables an interaction between the ligand and the masking moiety that is close enough to the paratope of the first antigen-binding moiety to demonstrate near or complete mutual exclusivity with the binding of the ligand to the ligand-binding moiety. In another aspect, the first peptide linker is of sufficient length to reach the ligand-binding moiety (e.g. on the target cell surface) when the first antigen-binding moiety is simultaneously bound to the target antigen.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.

Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www. ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global proteimprotein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. As used herein, the term "polypeptide", also sometimes referred to as “polypeptide chain” refers to a

As used, the term “target antigen”, for ease of reading sometimes also referred to herein as TA (particularly in the context of anti-target antigen antigenbinding moieties), refers to an antigen or portion thereof present in or on a target cell or tissue. Target antigens may be selected for their usefulness in targeting a ligand, such as e.g. a cytokine or a receptor-binding ligand, to a desired target cell or tissue, e.g. for the purpose of target-dependent activation or blocking of the ligand-binding moiety (e.g. a cytokine receptor or a cell surface receptor). Target antigens can be bound by the antigen-binding domain of an antigen-binding moiety, such as an antibody or antibody fragment. The term “target antigen” encompasses, for example, cell surface molecules present on effector cells such as T cells or NK cells. In some embodiments, the target antigen is CD3. The term “target antigen” also encompasses tumor antigens, as described supra. In certain embodiments, target antigens may be located on functionalized surfaces that may be introduced into diseased tissues or organs (e.g. by injection) in order to activate systematically administered activatable fusion proteins active in that area.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigenbinding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

II. COMPOSITIONS AND METHODS

In one aspect, the invention is based, in part, on the finding that by arranging antigen-binding moieties, ligands (e.g. cytokines) and masking moieties that bind said ligand in the way disclosed herein, conditionally active fusion proteins may be obtained which demonstrate mutually exclusive binding to the target antigen and the ligand, and moreover show conditional binding of the ligand to the ligand-binding moiety (e.g. a cytokine receptor), i.e. the ligand binding to the ligand-binding moiety depends on the presence of the target antigen in the proximity of the ligand-binding moiety, without having to resort to using a mutually exclusive bispecific binder (“switch binder” or “flip binder”) which are difficult to generate and may not be possible to make for every target antigen/ligand combination in the desired quality.

The activatable fusion proteins herein are based on a format that allows for great versatility and broad applicability, as the only building blocks required for making such an activatable fusion proteins are a) antigen-binding moieties specific for the desired target antigen and b) masking moieties for the desired ligand which are capable of preventing the ligand from binding to the ligand-binding moiety and exerting its biological function, molecules that are easily obtainable using state of the art methods. The effect achieved by the activatable fusion proteins reported herein relies on the first antigen-binding moiety binding to the target antigen in such a way that the distance between the masking moiety and the ligand significantly increases. As a result, the masking moiety and the ligand which are both fused to the first antigen-binding moiety via peptide linkers are no longer able to reach each other, effectively blocking the masking moiety from binding to the ligand. The ligand is thus set free to bind to the ligand-binding moiety. The activatable fusion proteins as described herein do not rely on specific requirements with regard to the target tissue, such as presence of tissue-specific proteases. Also, the mechanism for activating the activatable fusion proteins described herein are reversible and non- destructive. In the absence of target-antigen, the masking moiety re-attaches to the ligand and the masking effect is restored. Thus, if the fusion protein remains in the circulation for an extended time, off-target activity is significantly reduced.

The present fusion proteins thus differ from known molecule platforms in that the ligand (e.g. the cytokine) that is fused to the first antigen-binding moiety is the (primarily) biologically active entity of the activatable fusion proteins described herein, and in that binding of the first antigen-binding moiety to the target antigen releases the ligand to exert its biological function by sterically hindering the interaction between the ligand and the masking moiety, without having to rely on mechanisms that cleave or digest the masking moiety, such as proteases.

Excellent target-dependent binding and activation of the target cell expressing the ligand-binding moiety can be achieved using the activatable fusion proteins of the invention, particularly if said activatable fusion proteins of the invention comprise a second antigen-binding moiety that functions as a targeting arm localises the molecule to the surface of the target cell. The antigen bound by the second antigen-binding moiety can be the same or a different antigen than the one bound by the first antigen-binding moiety.

In one aspect, the ligand binding activity of the activatable fusion protein is attenuated, e.g. if the ligand is a cytokine, it has attenuated cytokine receptor binding activity. In one embodiment, if the ligand is a cytokine, the cytokine-receptor activating activity of the activatable fusion protein is at least about 10 times less than the cytokine-receptor activating activity of a fusion protein that contains the cytokine polypeptide but no masking moiety.

Activatable fusion proteins of the invention are useful, e.g., for the diagnosis or treatment of diseases such as cancer, viral infection or autoimmune disease.

In certain aspects, an activatable fusion protein as described herein

• shows target-dependent binding of the ligand to the ligand binding moiety, and in particular - if the ligand is a cytokine - target-dependent activation of the cytokine receptor or - if the ligand is an enzyme - target-dependent enzyme activity (e.g. protease activity, phosphorylation, etc.),

• shows decreased or inhibited binding of the ligand to the ligand binding moiety if the levels of the target antigen are below a certain threshold or if there is no target antigen present at all, and/or • does not rely on the presence of a (target- specific) protease to release the ligand from the masking moiety in order for the ligand to bind the ligand binding moiety.

In one aspect of the invention, the activatable fusion protein comprises a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide, a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and a masking moiety capable of specifically binding to the ligand. The activatable fusion protein is characterized in that the ligand is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker, and the first and the second peptide linker do not comprise a protease cleavage site. The target-dependent binding of the ligand comprised in the activatable fusion proteins described herein to the ligand-binding moiety is in particular not dependent on any proteolytic activity, in particular on any proteolytic cleavage, of the activatable fusion protein, e.g. in the target tissue. In one aspect, the activatable fusion protein is functional and shows target antigen-dependent activity in its intact form and/or in the absence of proteases.

A. Exemplary CISS molecule formats

In some aspects of the activatable fusion protein, the first antigen-binding moiety is an antibody or an antibody fragment.

For making a functional CISS molecule the antigen-binding moiety, the ligand and the masking moiety need to be arranged in the way described herein, i.e. the ligand is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker and the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker. Neither the first nor the second peptide linker is susceptible to protease cleavage, i.e. they do not comprise a protease cleavage site. To achieve activatable binding of the ligand (e.g. a cytokine) to the ligand-binding moiety (e.g. a cytokine receptor), an antigen-binding moiety is selected as the first antigen-binding moiety wherein the N-termini of the heavy and the light chain are in close proximity, e.g. as in a Fab. Thus, in another aspect of the activatable fusion protein described herein, the first antigen-binding moiety is an antibody fragment which is selected from the group consisting of a Fab, a DutaFab, a DAF, an Fv, a Fab', a Fab’-SH, a F(ab')2, a diabody, a linear antibody, and a multispecific antibody formed from antibody fragments. In another aspect, the first antigen-binding moiety is an engineered T-Cell Receptor (TCR), such as a soluble TCR (that lacks a transmembrane domain) or a single-chain TCR wherein the TCR variable a region and the TCR variable P region are linked by a peptide linker.

It will be appreciated that CISS molecules may also be generated based on a first antigen-binding moiety that comprises only one polypeptide chain, such as a DARPin. In such an embodiment, the ligand and the masking moiety would be attached via peptide linkers to the N- and the C-termini of the first antigen-binding moiety.

In one aspect of the activatable fusion protein described herein, the ligand is selected from the group consisting of a growth factor, a cytokine, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof, preferably a cytokine.

In one aspect of the activatable fusion protein described herein, the ligand binding moiety which the ligand is capable of specifically binding to is selected from the group consisting of a growth factor receptor, a cytokine receptor, an antigen, a ligand receptor, an enzyme substrate, a fluorescent label, a radioactive agent, a radioactive label, or a hormone receptor, preferably a cytokine receptor.

In one aspect, the masking moiety comprised in the activatable fusion protein described herein is selected from the group consisting of an antibody, an antibody fragment, a single-chain antigen-binding moiety (the single-chain antigen-binding moiety may also be an antibody mimetic, e.g. a DARPin, monobody, affibody, or anticalin), a peptide mask, an anti-idiotypic antibody, an anti-idiotypic antibody fragment (e.g. scFv, VHH, dAb, VNAR) (only if the ligand is an antibody/antibody fragment), or a receptor binding specifically to the ligand.

In some aspects of the activatable fusion protein, the masking moiety is a single-chain antigen-binding moiety selected from the group consisting of an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb). In one aspect of the activatable fusion protein provided herein, the masking moiety is reversibly bound to the ligand. In some aspects, the binding of the masking moiety to the ligand sterically hinders the binding of the first antigen-binding moiety to the antigen. In another aspect, the affinity of the first antigen-binding moiety in the activatable fusion protein is decreased when compared to the affinity of the first antigen-binding moiety by itself. In yet another aspect, the first antigen-binding moiety of the activatable fusion protein is hindered from binding to the antigen when the masking moiety is bound to the ligand. In a further aspect, the interaction between the ligand and the masking moiety is interrupted and the masking moiety is prevented from binding the ligand when the first antigen-binding moiety is bound to the target antigen. In yet another aspect, the ligand is released from the ligand-binding moiety to bind to the ligand-binding moiety when the antigen-binding moiety is bound to the target antigen.

The ligand and the masking moiety are covalently attached, preferably at their C-termini, to the N-termini of the heavy and the light chain polypeptide of the first antigen-binding moiety via peptide linkers. Peptide linkers are commonly short peptides comprising one or more amino acids, typically about 2 to 20, but sometimes up to 40 and more, amino acids. The peptide linkers of the activatable fusion proteins described herein do not contain protease cleavage sites, i.e. neither the ligand nor the masking moiety will be shed when the activatable fusion protein exerts its therapeutic effect in the target tissue. The molecules described herein will retain their conditional therapeutic activity even after having reached their physiological targets. This allows for the use of molecules with a long half-life. Even if those molecules continue circulating in the body after having reached their target tissue, they will demonstrate very low undesired activity in the periphery since the masking moiety will bind to the ligand again once the target antigen is no longer present.

In another aspect, the activatable fusion protein further comprises a second antigen-binding moiety. In a particular embodiment, the second antigen-binding moiety comprises at least a second heavy chain polypeptide and at least a second light chain polypeptide. In some aspects, the second antigen-binding domain is selected from the group consisting of a Fab, a DutaFab, a scFab, a DAF, an Fv, a Fab’, a Fab’-SH, or a F(ab’)2 fragment, in particular a Fab fragment. In another embodiment, the second antigen-binding moiety is a single-chain antigen-binding moiety, e.g. an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb). In some aspects, the second antigen-binding moiety is covalently attached to the first antigen-binding moiety, either directly or via a peptide linker. The first and the second antigen-binding moiety may also form a F(ab')2 fragment.

In yet another aspect, the activatable fusion protein further comprises an Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide. It will be appreciated that including an Fc domain in the activatable fusion proteins described herein may be useful in many aspect, for example for providing attachment sites for additional domains (e.g. antigen-binding moieties), but also for conveying desirable properties to the molecule, such as tailor- made half life or Fcy-dependent cytotoxicity.

In one aspect, the first antigen-binding moiety comprised in the activatable fusion protein described herein is covalently attached to the N-terminus or to the C- terminus of one of the two Fc domain heavy chain polypeptides. In particular aspects, the first antigen-binding moiety is covalently attached to the N-terminus or to the C- terminus of one of the two Fc domain heavy chain polypeptides. In a particular aspect, the first antigen-binding moiety is covalently attached to the N-terminus of one of the two Fc domain heavy chain polypeptides. In some aspects, the first antigen-binding moiety of the activatable fusion protein is a Fab, a Dutafab, a DAF or a DBA and the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding moiety is covalently attached via its C-terminus (a) to the N-terminus of the first Fc domain heavy chain polypeptide, or (b) to the C-terminus of the first Fc domain heavy chain polypeptide. In another aspect, the first antigenbinding moiety of the activatable fusion protein is a Fab, a Dutafab, a DAF or a DBA and the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding moiety is covalently attached via its N-terminus (a) to the N- terminus of the first Fc domain heavy chain polypeptide, or (b) to the C-terminus of the first Fc domain heavy chain polypeptide, in particular to the C-terminus fo the first Fc domain heavy chain polypeptide.

In some aspects, the second antigen-binding moiety comprised in the activatable fusion protein is covalently attached to the N-terminus or to the C- terminus of one of the two Fc domain heavy chain polypeptides. In some aspects, the second antigen-binding moiety is a Fab, a Dutafab, a DAF or a DBA and that the second heavy chain polypeptide or the second light chain polypeptide of the second antigen-binding moiety is covalently attached via its C-terminus (a) to the N- terminus of the second Fc domain heavy chain polypeptide, or (b) to the C-terminus of the first or the second Fc domain heavy chain polypeptide. In another embodiment, the second antigen-binding moiety is a single-chain antigen-binding moiety, e.g. an scFv, an scFab, a VHH or a single-domain antibody.

In one aspect, the first antigen-binding moiety comprised in the activatable fusion protein described herein is covalently attached either (a) to the N-terminus of the first Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached to the N-terminus of the second Fc domain heavy chain polypeptide, or (b) the first antigen-binding moiety is covalently attached to the N- terminus of the first or the second Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached to the C-terminus of the first or the second Fc domain heavy chain polypeptide.

By way of illustration, Figure 1 shows two examples of the activatable fusion proteins described herein, an exemplary CISS Fab (Figure 1A) and an exemplary targeted CISS molecule (Figure IB). The molecule shown in Figure 1 A is based on an anti-TA Fab wherein the ligand (here: a cytokine) and the masking moiety (here: a scFv capable of specifically binding to the cytokine) are fused to the N-termini of the heavy and the light chain of the Fab, respectively. The molecule shown in Figure IB has a structure similar to an IgG. One Fab arm of the IgG molecule is the basis for the CISS module, with the ligand and the masking moiety covalently attached to the N-termini of the heavy and the light chain of said Fab, respectively. The other Fab arm serves as a second antigen-binding moiety, providing the molecule with additional targeting to the target tissue (by either binding to TA or another antigen located in the target tissue).

An exemplary format of an activatable fusion protein that may be activated via a intramolecular mechanism is shown in Figure 13, comprising a first antigenbinding moiety, a second antigen-binding moiety, a ligand (e.g. a cytokine) and a masking moiety. In this example, both the second-antigen-binding moiety and the masking moiety are single-chain antigen-binding moieties. The term “intramolecular” in the context of a targeted CISS molecule means that the first and the second antigen-binding moieties bind to different epitopes on the same target antigen in such a way that both antigen-binding moieties binding to one and the same target antigen molecule result in release of the ligand (e.g. a cytokine) to bind the ligand-binding moiety (e.g. a cytokine receptor). (If the first and second antigenbinding moieties are selected such that they are not capable of specifically binding to one and the same target antigen molecule at the same time, they rely on binding two different target antigen molecules at the same time, a mechanism that is herein referred to as “intermolecular”.) The ligand is covalently attached at its C-terminus, via a peptide linker, to the N-terminus of either the heavy chain or the light chain polypeptide of the first antigen-binding moiety. The masking moiety is covalently attached at its C-terminus, via a peptide linker, to the N-terminus of the other polypeptide chain of the first antigen-binding moiety. The second antigen-binding moiety is bound either at its C-terminus or its N-terminus, via a peptide linker, to the masking moiety.

Figure 14 shows further exemplary formats of the activatable fusion proteins according to the invention. Figure 14 A and B show alternative targeted CISS molecules. These have alternative formats that, in addition to the CISS module, contain a Fc domain and a second antigen-binding moiety. In the format shown in Figure 14 A, the first antigen-binding moiety of the CISS module is not directly linked to the Fc domain, but is covalently attached at the N-terminus of one of its polypeptide chains, via a peptide linker, to the C-terminus of the masking moiety, which is itself covalently attached at its N-terminus via another peptide linker to the C-terminus of one of the two Fc domain heavy chain polypeptides, whereas the second antigen-binding moiety is covalently attached via its N-terminus to the C- terminus of the other of the two Fc domain heavy chain polypeptides with yet another peptide linker. In the format shown in Figure 14 B, the first antigen-binding moiety of the CISS module is covalently attached at the C-terminus of its heavy chain polypeptide via a peptide linker to the N-terminus of the Fc domain, while the second antigen-binding moiety is covalently attached at its N-terminus via another peptide linker to the C-terminus of one of the Fc domain heavy chain polypeptides. It will be appreciated that the attachment sites for the peptide linkers can be varied between the different polypeptide chains of the Fabs and the Fc domains used for assembling the CISS molecules.

Figure 14 C exemplarily illustrates the mode of action of the molecule shown in Figure 14 A. The molecule binds to the target antigen on the cell surface via the second antigen-binding moiety (“targeting arm”). When the mask temporarily releases the cytokine fused to the first antigen-binding moiety, the latter can bind to another epitope of the same target antigen molecule on the cell surface, thus interrupting the mask-cytokine interaction and releasing the cytokine to bind to the cytokine receptor on the cell surface (not shown here).

In another aspect, the second antigen-binding moiety comprised in the activatable fusion protein described herein is covalently attached to the N-terminus of the ligand (e.g. a cytokine) via a third peptide linker which does not comprise a protease cleavage site. In a particular aspect, the first and the second antigen-binding moiety are capable of specifically binding to two different epitopes on the same target antigen molecule concomitantly. The proximity of the second antigen-binding moiety to the ligand, the masking moiety and the first antigen-binding moiety allows intramolecular activation of the ligand that can lead to a release of the ligand even if the target antigen is soluble. In some aspects, the second antigen-binding moiety that is covalently attached to the N-terminus of the masking moiety is a single-chain antigen-binding moiety selected from the group consisting of an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb).

When the second antigen-binding moiety binds to the target antigen, the effective concentration of the target antigen in the proximity of the activatable fusion protein is increased, promoting the binding of the first antigen-binding moiety to the target antigen, thus leaving the masking moiety open so that the ligand (e.g. a cytokine) is free to bind to the ligand binding moiety (e.g. a cytokine receptor). Intramolecular unmasking will generally increase unmasking efficiency since it is independent of the cell surface concentration of the receptor and instead should make use of the much higher effective concentration achieved by intramolecular binding. This could be advantageous in situations where the entire population with a given receptor is to be targeted or where an in-solution or immobile target is desired.

In some of the exemplary formats of the activatable fusion protein described above, the N-terminus or the C-terminus of the first heavy chain polypeptide of the Fc domain is covalently attached to the N-terminus of the masking moiety via a third peptide linker (Figure 14 A), and in some the second antigen-binding moiety is covalently attached via the N-terminus of its heavy chain polypeptide or light chain polypeptide to the N-terminus or C-terminus of the second heavy chain polypeptide of the Fc domain via a fourth peptide linker (Figure 14 A and B). To be clear, said third and fourth peptide linkers do not comprise a protease cleavage site.

Activatable fusion proteins described herein that contain antibody-derived domains such as Fc domains or Fabs will frequently comprise more than one polypeptide chain. Some formats described herein comprise three or four polypeptide chains. In one aspect of the activatable fusion proteins described herein, the masking moiety comprises a single-chain antigen-binding moiety, and the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus, via a peptide linker, to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigenbinding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand (e.g. a cytokine), fused at its C-terminus, via a peptide linker, to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

In one aspect of the activatable fusion proteins described herein, the masking moiety comprises a single-chain antigen-binding moiety and the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand (e.g. a cytokine) fused at its C-terminus, via a peptide linker, to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigenbinding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus, via a peptide linker, to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

In one aspect of the activatable fusion proteins described herein, the masking moiety comprises a single-chain antigen-binding moiety, and the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus, via a peptide linker, to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigenbinding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand (e.g. a cytokine), fused at its C-terminus, via a peptide linker, to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety which is a single-chain antigen-binding moiety, fused at its C-terminus to the N- terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain.

In one aspect of the activatable fusion proteins described herein, the masking moiety comprises a single-chain antigen-binding moiety and the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand (e.g. a cytokine) fused at its C-terminus, via a peptide linker, to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigenbinding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus, via a peptide linker, to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety which is a single-chain antigen-binding moiety, fused at its C-terminus to the N- terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain.

In another aspect, the activatable fusion protein comprises a second antigenbinding moiety that is capable of specifically binding to an antigen that is the same or a different antigen from the target antigen. In some aspects, the second antigenbinding moiety (a) binds specifically to the target antigen and (b) binds specifically to an epitope on the target antigen that is different from the epitope that is bound by the first antigen-binding moiety. In some aspects, the second antigen-binding moiety binds specifically to the same epitope on the target antigen as the first antigenbinding moiety.

Many times, the second antigen-binding moiety will be selected to be capable to bind specifically to the same target antigen, or even the same epitope on the target antigen, as the first antigen-binding moiety. These embodiments have been found to be particularly useful if a target antigen shows high specificity for the desired target tissue, e.g. a tumor tissue or a certain organ tissue. Using a first and a second antigenbinding moiety that bind specifically to the same epitope on the target antigen or even using the same antigen-binding moiety as first and second antigen-binding moiety can lead to a higher specificity of the activatable fusion protein for target cells with a particularly high concentration of the target antigen on its surface, as the fusion protein is only activated when there are at least two target antigens on the cell surface in sufficient proximity that the first and the second antigen-binding moiety can bind to them simultaneously (as they cannot bind the same target antigen at the same time, since one would block the binding of the other).

It can be particularly useful for an activatable fusion protein to comprise a second antigen-binding moiety that is capable of specifically binding an antigen that is different from the target antigen, particularly of the concentration of the desired target antigen is not high enough, or if the expression of the target antigen is not specific enough for the target tissue or target cells for the desired mode of action. In such cases, a second antigen can be targeted with the help of the second antigenbinding moiety, in order to increase specificity for a certain target cell/tissue.

In one aspect of the activatable fusion proteins described herein the first antigen-binding moiety is, or is part of, an antibody of the IgG type. In some aspects, one arm of the IgG type antibody comprises the first antigen-binding moiety and the other arm of the IgG type antibody comprises the second antigen-binding moiety. In one aspect, the first antigen-binding moiety, the second antigen-binding moiety and the Fc region of the activatable fusion protein together form an antibody of the IgG type. In some aspects, the Fc domain is an IgG Fc domain, particularly an IgGl Fc domain or an IgG4 Fc domain. In some aspects, the activatable fusion protein comprises at least two full-length IgG antibody heavy chains and the heavy chains of the antigen-binding moiety are of the y type (IgG), in particular of the yl type. In another aspect, the activatable fusion protein comprises at least two light chains and the light chains of the antigen-binding moiety are selected from the kappa (K) and/or lambda ( ) subtype.

In one aspect, the activatable fusion proteins described herein comprise an Fc domain that comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular towards Fey receptor. In another aspect, the Fc domain is of the human IgGl subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

In one aspect, the activatable fusion proteins described herein comprise an Fc domain comprising a modification promoting the association of the first and second Fc domain heavy chain polypeptide. In some aspects, the first Fc domain heavy chain polypeptide comprises knobs and the second Fc domain heavy chain polypeptide comprises holes according to the knobs into holes method. In some aspects, the first Fc domain heavy chain polypeptide comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second Fc domain heavy chain polypeptide comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

Molecule complexes that comprise more than one different heavy chain/light chain pairing are at risk of forming undesired heavy chain/light chain pairings during manufacture. In order to prevent such wrong pairings, the CrossMab technology may be used in the activatable fusion proteins herein. Accordingly, in one aspect, the first and the second antigen-binding moieties of the activatable fusion proteins described herein are Fabs and in one of the Fabs the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain, i.e. one of the Fabs is a “cross-Fab fragment”. In some aspects, in the constant domain CL of one of the two Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular aspect, in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In one aspect of the activatable fusion proteins described herein, the target antigen that the first antigen-binding moiety specifically binds to is selected from the group consisting of alpha-synuclein, Amyloid beta, BCMA, BTLA, CD3e, CD4, CD8, CD14, CD16 (FcgRIIIa), CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD44, CD47, CD52, CD70, CD109, CD123, CD137, CEACAM5, c-MET, CTLA4, DLL3, CXCR4, EDB-FN, EpCAM, epidermal growth factor receptor (EGFR), EPO Receptor, FAP a, FGFR2, FGFR3, GD-2, GP100, GITR, GLP-1 receptor, GM-CSF, GPC3, Grp78, Hedgehog, HER2, HER3, HLA-G, ICAM (ICAM-1, -2, -3, -4, -5), IGF-1R, IL-1R1, IL-4Ra, Integrin av, b7 integrin subunit, a4b7 integrin, a4 integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notchl, Notch3, NRP1, NRP2, 0X40, PAR-2, PD-1, PD-L1, PDGFR, PSA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR- A6, SR-B, dSR-Cl, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1, TGFp, TGF-y, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1,VEGFR2, or 5T4. In a particular aspect, the target antigen may be selected from the group consisting of PD1, PD-L1, CD8 and CD19.

In one aspect of the activatable fusion proteins described herein, the ligand comprised in the activatable fusion protein is a cytokine selected from the group consisting of interferons, interleukins, chemokines, lymphokines, monokines, colony-stimulating factors, and tumour necrosis factors. In one aspect, the cytokine is a member of the IL-1 family, the IL-2 subfamily, the interferon (IFN) subfamily or the IL- 10 subfamily. In a particular aspect the ligand is a cytokine selected from the group consisting of interferons and interleukins. In some aspects, the ligand is a cytokine selected from the group consisting of BMP, CSF-1, insulin, GLP-1, HGH, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL- 36, GM-CSF, FGF, EGF, G-CSF, IFNa, IFNP, IFNy, PDGF, TGFp, TNFa, TNFp, VEGF, or EPO. In one such aspect, the ligand is a cytokine selected from the group consisting of IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 11, IL- 12, IL- 13, IL- 14, IL- 15, IL- 16, IL- 17, IL- 18, IL- 19, IL-20, IL-21, IL- 22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL- 34, IL-35, IL-36, IFNa, IFNP, and IFNy. In some aspects, the ligand is a cytokine is selected from the group consisting of IL-2, IL-7, IL-21 and IFNa. In one aspect the cytokine may be a mutein, a variant, an active subunit, an active fragment and/or an attenuated variant of a naturally occurring cytokine.

In one particular aspect, the activatable fusion protein comprises

(a) a first antigen-binding moiety that is a Fab capable of specifically binding to a target antigen,

(b) a cytokine capable of specifically binding to a cytokine receptor, and

(c) a masking moiety that is a VHH or an scFv capable of specifically binding to the cytokine, characterized in that the cytokine is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker, and the first and the second peptide linker do not comprise a protease cleavage site.

In another aspect, the activatable fusion protein comprises

(a) a first antigen-binding moiety that is a Fab capable of specifically binding to a first target antigen,

(b) a second antigen-binding moiety, preferably a Fab, capable of specifically binding to the first or a second target antigen,

(c) an Fc domain comprising a first and a second Fc domain heavy chain polypeptide.

(d) a cytokine capable of specifically binding to a cytokine receptor, and

(e) a masking moiety that is a VHH or an scFv capable of specifically binding to the cytokine, characterized in that the cytokine is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker, the heavy chain polypeptide of the first antigen-binding moiety is covalently attached to the N-terminus of the first Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached to the N-terminus of the second Fc domain heavy chain polypeptide, and the first and the second peptide linker do not comprise a protease cleavage site. Where the second antigen-binding moiety is a Fab, it is linked to the Fc domain at the C-terminus of its heavy chain or its light chain, in particular its heavy chain. Fusion between the various domains of the activatable fusion molecule may be via peptide linkers, which may also comprise or consist of (part of) an immunoglobulin hinge region, in particular when a Fab as (first and/or second) antigen-binding domain is fused to an Fc domain.

In some aspects of the activatable fusion proteins disclosed herein, it is not (only) the ligand but the first antigen-binding moiety that has therapeutic activity and/or activates a response in the patient that triggers the contemplated mode of action.

In one aspect of the activatable fusion proteins described herein, the ligand is an antigen-binding moiety. In some aspects, the antigen-binding moiety may be an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of BCMA, GPRC5D, FcRH5, CD38, CS-l/SlamF7, CD20, CD19, CD22, CD27, CD28, CD40, CD47, CD123, CD137, CEACAM5, DLL3, EpCAM, HLA-G, GITR, HER2, HER3, MICA, MICB„ PD-L1, PSMA, STEAP-1, TROP-2, EpCAM, HER3 and cMet. It will be appreciated that if an antigen-binding moiety is used as the ligand comprised in the activatable fusion protein described herein, the masking moiety will typically be an anti-idiotypic antigen-binding moiety that is capable of specifically binding to the antigen-binding moiety that is being used as the ligand.

The CISS molecule format described herein may be used to realize different types of modes of action. In one aspect, an activatable T-cell or NK-cell engager is disclosed. The activatable T-cell or NK-cell engager comprises a first antigenbinding moiety that is capable of specifically binding a target antigen, a ligand that is a antigen-binding moiety capable of specifically binding to a T-cell or NK-cell antigen, such as CD3, TCR or CD28, and a masking moiety that is capable of specifically binding to the antigen-binding moiety that is the ligand, i.e. an anti- idiotypic antibody or antibody fragement capable of specifically binding to the antiT-cell or anti-NK-cell antigen-binding moiety. One advantage of this format is that different target antigens can be used without having to generate a new anti-idiotypic mask for the antigen-binding moiety binding to the target antigen. Figure 11 A shows a schematic depiction of such an exemplary activatable T-cell engager consisting essentially of an IgG type antibody capable of specifically binding to a target antigen, wherein the ligand, an anti-CD3s scFv, and the masking moiety, an anti-CD3 VHH, are bound to one of the two Fab arms of the IgG antibody. The other arm serves as the second antigen-binding moiety in this example. In the absence of a target expressing cell, the anti-CD3s sc Fv is bound by the anti-CD3 VHH masking moiety (Figure 12). Once the activatable T-cell engager binds to the target antigen with both antigen-binding moieties, the anti-CD3s scFv is released from the mask and free to bind to its antigen, CD3s, on a T-cell, thus recruiting the T cell to the tumor and inducing T-cell activation and tumor cell elimination.

In another aspect, an activatable T-cell or NK-cell engager as disclosed herein can also comprise a first antigen-binding moiety that is capable of specifically binding to a T-cell or NK-cell antigen, such as CD3, TCR, CD28, CD16a or NKG2D, while the ligand is capable of specifically binding to a second target antigen and the mask is an anti-idiotypic antibody or antibody fragment (e.g. a VHH) capable of specifically binding to the antigen-binding moiety that serves as the ligand here. In a particular aspect, the ligand is a antigen-binding moiety binding to a target antigen. Figure 11 B shows a schematic depiction of such an exemplary activatable T-cell engager consisting essentially of a bispecific IgG type antibody comprising two Fab arms, of which one is capable of specifically binding to CD3s while the other arm is capable of specifically binding to the target antigen. The ligand in this example, an anti-TA scFv, and the masking moiety, an anti-anti-TA idiotypic VHH, are covalently attached to one Fab arm of the IgG antibody that is capable of specifically binding to CD3s. In the absence of a target expressing cell, the anti-target scFv is bound by the anti-anti-TA VHH masking moiety. Once the activatable T-cell engager binds to the target antigen with the anti-TA scFv and the anti-TA Fab arm, the anti-CD3s scFv is no longer sterically hindered from binding to its antigen, CD3s, on a T-cell, thus recruiting the T cell, y5 T-cell or other innate immune cell e.g. NK cell, macrophage/monocyte, neutrophile and inducing T-cell activation and tumor cell elimination.

The activatable T-cell engagers described herein are in particular able to tune affinity and avidity to select between cells expressing target at low and high density, in particular if combined with two antigen-binding moieties binding to two different target antigens. They further strongly decrease the binding to CD3s of T-cells in circulation by „masking“ CD3 and reduce potential mode-of-action driven cytokine release syndrome by „masking“ the anti-CD3 antigen-binding domain. As with the other activatable fusion proteins described herein, the second antigen-binding moiety can either bind to the same or a different antigen as the first-antigen-binding moiety or ligand. In cases where the activatable T-cell or NK-cell engager comprises a second antigen-binding moiety that binds to a different antigen than the first-antigen- binding moiety, this may result in increased specificity to a subset of target cells expressing both these antigens. In some embodiments, the activatable T cell engager described herein is a CD3 bispecific or CD3 trispecific T-cell engager.

In another aspect, the present disclosure provides an activatable fusion protein that is an activatable antagonist, wherein the first antigen-binding moiety is an antagonistic binder capable of specifically binding e.g. to an essential membrane transporter or channel.

In another aspect, the ligand may be a non-cytokine ligand, i.e. a polypeptide other than a cytokine, selected from the group consisting of a growth factor, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof.

In one aspect, an activatable fusion protein is provided comprising

(A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide

(B) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide,

(C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and

(D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, and characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus via a first peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus via a second peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site.

In one aspect, an activatable fusion protein is provided comprising

(A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(B) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide,

(C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and

(D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, and characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a second peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a first peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein comprising

(A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(B) a second antigen-binding moiety,

(C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and

(D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus via a first peptide linker to the N-terminus of the heavy chain polypeptide of the first - n - antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus via a second peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, and c) a third polypeptide, comprising (cl) the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, wherein the first and the second peptide linker do not comprise a protease cleavage site.

One embodiment of the invention is an activatable fusion protein comprising

(A) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(B) a second antigen-binding moiety comprising (or consisting of) a singlechain antigen-binding moiety,

(C) a ligand (e.g. a cytokine) capable of specifically binding to a ligand binding moiety (e.g. a cytokine receptor), and

(D) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a second peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a first peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, and c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, wherein the first and the second peptide linker do not comprise a protease cleavage site.

In a further aspect, an activatable fusion protein according to any of the above aspects may incorporate any of the features, singly or in combination, as described in Sections 1-8 below:

/. Antibody Fragments

In certain aspects, an activatable fusion protein provided herein comprises an antibody fragment. The antibody fragment may be comprised in particular in an antigen-binding moiety or in the ligand.

In one aspect, the antibody fragment is a Fab, DutaFab, scFab, DAF, Fv, Fab’, Fab’-SH, or F(ab’)2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI). The term “Fab” or “Fab fragment” thus refers to an antibody fragment comprising a light chain polypeptide comprising a VL domain and a CL domain, and a heavy chain polypeptide comprising a VH domain and a CHI domain. “Fab’ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody. “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

In a further aspect, the antibody fragment is a single chain Fab fragment. A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1- linker-VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL- CH1 -linker- VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

In another aspect, the antibody fragment is single-chain variable fragment (scFv). A “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Spring er- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single-domain antibody. “Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single- domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

2. Multispecific Antibodies

In certain aspects, an activatable fusion protein provided herein comprises a multispecific antibody, e.g., a bispecific antibody. “Multispecific antibodies” are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for the target antigen and the other specificity is any other antigen. In certain aspects, bispecific antibodies may bind to two (or more) different epitopes of the same target antigen. Multispecific antibodies may be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731, 168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to the target antigen as well as another different antigen, or two different epitopes of the target antigen (see, e.g., US 2008/0069820 and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term “cross- Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.

Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95- 106).

A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activating, invariant component of the T-cell receptor (TCR) complex, such as CD3, for retargeting of T-cells or NK-cells to kill target cells. Hence, in certain aspects, an antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is for the target antigen and the other is for CD3.

Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261, and WO 2008/119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C- terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are whole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) el203498.

3. Molecule Variants

In certain aspects, amino acid sequence variants of the activatable fusion proteins provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of the antigen-binding moieties. Amino acid sequence variants of an antigen-binding moiety may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

4. Fc region variants

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an activatable fusion protein provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGi, IgG?, IgGs or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain aspects, the invention contemplates an activatable fusion protein that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the activatable fusion protein in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the activatable fusion protein lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat ’I Acad. Sci. USA 83:7059- 7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non- radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the activatable fusion protein is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738- 2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’L Immunol. 18(12): 1759-1769 (2006); WO 2013/120929 Al).

Fc regions with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Certain Fc regions with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).) In certain aspects, an activatable fusion protein comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In certain aspects, an activatable fusion protein comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the activatable fusion protein further comprises D265 A and/or P329G in an Fc region derived from a human IgGi Fc region. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgGi Fc region. (See, e.g., WO 2012/130831). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgGi Fc region.

In some aspects, alterations are made in the Fc region that result in altered (z.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (See, e.g., US Patent No. 7,371,826; Dall'Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524) and may be used in the activatable fusion proteins described herein.

Fc region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues 1253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined.

In certain aspects, an activatable fusion protein comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or 435 of the Fc-region (EU numbering of residues). In certain aspects, the activatable fusion protein comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435. In one aspect, the substitutions are 1253 A, H310A and H435A in an Fc region derived from a human IgGl Fc-region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

In certain aspects, an activatable fusion protein comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the activatable fusion protein comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436. In one aspect, the substitutions are H310A, H433 A and Y436A in an Fc region derived from a human IgGl Fc-region. (See, e.g., WO 2014/177460 Al).

In certain aspects, an activatable fusion protein comprises an Fc region with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues). In certain aspects, the activatable fusion protein comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgGi Fc-region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

Where the activatable fusion protein as reported herein comprises an Fc region, the C-terminus of the heavy chain of the Fc region can be a complete C- terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending PG. In one aspect of all aspects as reported herein, an activatable fusion protein comprising a Fc domain heavy chain polypeptide including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one aspect of all aspects as reported herein, an activatable fusion protein comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).

5. Exemplary anti-IL-2 antibodies

In one aspect, the invention provides antibodies that bind to human IL-2 (huIL-2). In one aspect, provided are isolated antibodies that bind to human IL-2 of SEQ ID NO:81. In one aspect, provided are isolated antibodies that bind to the attenuated variant IL-2v (SEQ ID NO: 82). In one aspect, provided are isolated antibodies that bind both to human IL-2 of SEQ ID NO: 81 and to the attenuated variant IL-2v (SEQ ID NO: 82). In one aspect, the invention provides antibodies that specifically bind to human IL-2. In one aspect, the invention provides antibodies that specifically bind to human IL-2 and to human IL-2v. In certain aspects, an anti-huIL-2 antibody binds to the surface comprising helices A and C, and to the loop between helices B' and C (Stauber D. J., et al., PNAS 2005) of human IL-2 and/or IL-2v. In certain aspects, an anti-huIL-2 antibody blocks interaction of IL-2 and/or IL-2v with IL-2RPy. In one aspect, an anti-huIL-2 antibody additionally blocks binding of IL-2 and/or IL-2v to IL-2Ra. In another aspect, an anti-huIL-2 antibody binds to human IL-2 and/or IL- 2v with an affinity of < 1.1 nM, in particular of < 0.8 nM.

In one aspect, the invention provides an anti-huIL-2 antibody comprising

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

In any of the aspects provided herein, an anti-huIL-2 antibody is humanized. In one aspect, an anti-huIL-2 antibody further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

In a further aspect, an anti-huIL-2 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of

(A) the VH domain of SEQ ID NON and the VL domain of SEQ ID NO:8;

(B) the VH domain of SEQ ID NO: 12 and the VL domain of SEQ ID NO: 13; (C) the VH domain of SEQ ID NO: 18 and the VL domain of SEQ ID NO: 19;

(D) the VH domain of SEQ ID NO:24 and the VL domain of SEQ ID NO:25;

(E) the VH domain of SEQ ID NO:30 and the VL domain of SEQ ID NO:31;

(F) the VH domain of SEQ ID NO:35 and the VL domain of SEQ ID NO:36;

(G) the VH domain of SEQ ID NO:38 and the VL domain of SEQ ID NO: 39;

(H) the VH domain of SEQ ID NO:41 and the VL domain of SEQ ID NO:42;

(I) the VH domain of SEQ ID NO:44 and the VL domain of SEQ ID NO:45; or

(J) the VH domain of SEQ ID NO:47 and the VL domain of SEQ ID NO:48.

In one aspect, the anti-huIL-2 antibody comprises

(A)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8;

(B)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12 and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13; (C)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19;

(D)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25;

(E)(a) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:30, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31;

(F)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:35, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36;

(G)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39;

(H)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:41, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:42;

(I)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:44, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:45; or

(J)(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:47, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:48.

In one aspect, the anti-huIL-2 antibody comprises

(A) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8;

(B) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13;

(C) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19;

(D) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:24 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:25;

(E) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:30 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:31; (F) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:35 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:36;

(G) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:38 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:39;

(H) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:42;

(I) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:44 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:45; or

(J) a VH domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:47 and a VL domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:48.

In one aspect, the anti-huIL-2 antibody comprises

(A) a VH domain that comprises the amino acid sequence of SEQ ID NO: 7 and a VL domain that comprises the amino acid sequence of SEQ ID NO:8;

(B) a VH domain that comprises the amino acid sequence of SEQ ID NO: 12 and a VL domain that comprises the amino acid sequence of SEQ ID NO: 13;

(C) a VH domain that comprises the amino acid sequence of SEQ ID NO: 18 and a VL domain that has comprises the amino acid sequence of SEQ ID NO: 19;

(D) a VH domain that comprises the amino acid sequence of SEQ ID NO:24 and a VL domain that comprises the amino acid sequence of SEQ ID NO:25;

(E) a VH domain that comprises the amino acid sequence of SEQ ID NO:30 and a VL domain that comprises the amino acid sequence of SEQ ID NO: 31 ; (F) a VH domain that comprises the amino acid sequence of SEQ ID NO:35 and a VL domain that comprises the amino acid sequence of SEQ ID NO: 36;

(G) a VH domain that comprises the amino acid sequence of SEQ ID NO:38 and a VL domain that comprises the amino acid sequence of SEQ ID NO: 39;

(H) a VH domain that comprises the amino acid sequence of SEQ ID NO:41 and a VL domain that comprises the amino acid sequence of SEQ ID NO:42;

(I) a VH domain that comprises the amino acid sequence of SEQ ID NO:44 and a VL domain that comprises the amino acid sequence of SEQ ID NO:45; or

(J) a VH domain that comprises the amino acid sequence of SEQ ID NO:47 and a VL domain that comprises the amino acid sequence of SEQ ID NO:48.

In one aspect, the anti-huIL-2 antibody comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 56;

(E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58; (F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

In one aspect, an anti-huIL-2 antibody is provided, wherein the antibody is a Fab and comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO:24 and SEQ ID NO:25, SEQ ID NO:30 and SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, or SEQ ID NO:47 and SEQ ID NO:48, respectively, including post- translational modifications of those sequences. In one aspect, the antibody comprises the heavy chain and light chain sequences in SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO 55 and SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, or SEQ ID NO:67 and SEQ ID NO:68, respectively, including post-translational modifications of those sequences.

In a further aspect of the invention, an anti-huIL-2 antibody according to any of the above aspects is a monoclonal antibody, including a chimeric, humanized or human antibody. In one aspect, an anti-huIL-2 antibody is an antibody fragment, e.g., an Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In one aspect, the anti-huIL-2 antibody is a Fab.

In one aspect, an anti-huIL-2 antibody according to any of the above aspects is a DutaFab. In one aspect, an anti-huIL-2 antibody comprises a human IL-2 paratope and a non-binding paratope (i.e. a paratope that binds to no epitope) within one cognate pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the non-binding paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antigen-binding moiety, and wherein the IL-2 paratope comprises amino acid residues from the CDR-H1, CDR- H3 and CDR-L2 of the antigen-binding moiety.

In another aspect, the antibody is a full-length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.

In a further aspect, the antibody as described herein comprises an Fc region polypeptide of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or the human heavy chain constant region of SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80. In a further aspect, the antibody as described herein is of IgGl isotype/subclass and comprises an Fc region polypeptide of SEQ ID NO: 69, SEQ ID NO:71, SEQ ID NO: 72, or SEQ ID NO:73, or the human heavy chain constant region of SEQ ID NO:78, or SEQ ID NO:79. In one aspect, additionally the C-terminal glycine (Gly446) is present. In one aspect, additionally the C-terminal glycine (Gly446) and the C-terminal lysine (Lys447) is present.

In one aspect, the antibody as described herein comprises at least one Fab fused at the C-terminus of its heavy chain polypeptide, either directly or via a peptide linker, to the N-terminus of a Fc region polypeptide, wherein the Fab has a amino acid sequence as disclosed above, and wherein the Fc region polypeptide has an amino acid sequence as disclosed above. In a particular aspect, the antibody as described herein comprises two Fabs, each covalently attached at the C-terminus of its heavy chain polypeptide, either directly or via peptide linker, to one of the two N-termini of the two Fc region polypeptides, wherein the Fab has an amino acid sequence as disclosed above, and wherein the Fc region polypeptide has an amino acid sequence as disclosed above. In one aspect, an anti-huIL-2 antibody is provided that comprises a heavy chain having the amino acid sequence of SEQ ID NO: 67 and a light chain having the amino acid sequence of SEQ ID NO:68.

Table B: SEQ ID NO: of anti IL-2 Fab CDRs

Table C: Description of amino acid sequences of anti-IL-2 antibodies

In a further aspect, an anti-huIL-2 antibody according to any of the above aspects may incorporate any of the features, singly or in combination, as described in Sections 3-8 below: 6. Antibody Affinity

In certain aspects, an antibody provided herein has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM or < 0.1 nM, (e.g., 10'⁸ M or less, e.g., from 10'⁸ M to 10'¹⁰M, e.g., from 10’⁹M to 10’¹⁰ M).

In one aspect, KD is measured using a Biacore® surface plasmon resonance assay. For example, an assay using a Biacore®8K or 8K+ instrument (Cytiva) is performed at 25°C using an anti-Fab capture setup. In one aspect, a CM5 carboxymethylated dextran biosensor chips (Cytiva; catalog number 29149604) are activated with N- ethyl-A’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier’s instructions. Anti human Fab antibody (Cytiva; Catalog number 28958325) is diluted with 10 mM sodium acetate, pH 5, to 10 pg/ml before injection at a flow rate of 10 pl/minute for 500 s to achieve approximately 5000 response units (RU) of coupled protein. Following the injection of the Anti human Fab antibody, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, 100 nM Fab is injected in HBS-EP+ buffer lx (Cytiva, catalog number BRI 00669) at 25°C at a flow rate of 10 pl/min for 60 seconds. 0 nM, 10 nM, 50 nM and 150 nM of antigen (IL-2, IL-2v or CD79B) diluted in HBS-EP+ buffer lx was flown at 30 pl/min for 120 sec followed by a 240 second dissociation window at a flow rate of 30 pl/min. The surface was regenerated by injecting 10 mM Glycine pH 2 for 60s at a flow rate of 30 pl/min. Association rates (k_on) and dissociation rates (k_off) are calculated using a simple one-to-one Langmuir binding model (Biacore 8K Control Software version 4.0.8.20368) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k_off/k_on. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

7. Antibody Fragments

In certain aspects, a fusion protein provided herein comprises one or more antibody fragments. In certain aspects, an antibody provided herein is an antibody fragment.

In one aspect, the antibody fragment is a Fab, Fab’, Fab’-SH, or F(ab’)2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CHI domain. “Fab’ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigenbinding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

In certain aspects, the antibody fragment is a DutaFab. A DutaFab is a Fab wherein a single pair of a VH domain and a VL domain specifically binds to two different epitopes, wherein one paratope comprises amino acid residues from CDR-H2, CDR- L1 and CDR-L3 and the other paratope comprises amino residues from CDR-H1, CDR-H3 and CDR-L2. In one aspect, the DutaFab comprises two non-overlapping paratopes within a cognate VH/VL pair and binds to the two different epitopes in a mutually exclusive manner (“Dutaflip”).

In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody. “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

In a further aspect, the antibody fragment is a single chain Fab fragment. A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL-CH1 -linker- VH- CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

In another aspect, the antibody fragment is single-chain variable fragment (scFv). A “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., ( Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. - I l l -

In another aspect, the antibody fragment is a single-domain antibody. “Singledomain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

8. Chimeric and Humanized Antibodies

In certain aspects, an antibody provided herein is a chimeric antibody. In certain aspects, a fusion protein provided herein comprises a chimeric antibody or a fragment of a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851- 6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non- human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain aspects, a chimeric antibody is a humanized antibody. Typically, a non- human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat ’I Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Set. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).

9. Human Antibodies

In certain aspects, an antibody provided herein is a human antibody. In other aspects, a fusion protein provided herein comprises a human antibody or a fragment of a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075, 181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3): 927- 937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

/ 0. Library-Derived Antibodies

In certain aspects, an antibody or antigen-binding moiety as described herein is derived from a library. In other aspects, a fusion protein provided herein comprises an antibody, in particular an antibody fragment, such as a Fab or a DutaFab derived from a library. Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods is known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8: 1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury m Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. n Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. vn EMBO Journal 12: 725-734 (1993). Furthermore, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and 2007/0292936.

Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. m Methods in Molecular Biology 503: 135-56 (2012) and in Cherf et al. n Methods in Molecular biology 1319:155-175 (2015) as well as in Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132- 5134 (1997) and in Hanes et al. m PNAS 94:4937-4942 (1997).

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. 11. Multispecific Antibodies

In certain aspects, an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. “Multispecific antibodies” are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for human IL-2 and the other specificity is for any other antigen. In certain aspects, bispecific antibodies may bind to two (or more) different epitopes of human IL-2. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells which express human IL-2. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731, 168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen-binding fragment thereof also includes a “Dual Acting Fab” or “DAF” comprising an antigen-binding site that binds to human IL-2 as well as another different antigen, or two different epitopes of human IL-2 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a CrossFab. The term “CrossFab” or “xFab” or “crossover Fab” refers to a Fab, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A CrossFab comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.

Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell and/or an immune cell in the tumor micro environment, and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting of T cells to kill target cells. Hence, in certain aspects, an antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is for human IL-2 and the other is for CD3.

Examples of bi specific antibody formats that may be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261, and WO 2008/119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C- terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are whole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) el203498.

12. Antibody and Activatable Fusion Protein Variants

In certain aspects, amino acid sequence variants of the activatable fusion protiens and antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of the antibody or of an antigen-binding moiety comprised in the activatable fusion protein. Amino acid sequence variants may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or the activatable fusion protein, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody or the activatable fusion protein. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. a) Substitution, Insertion, and Deletion Variants

In certain aspects, antibody (or antigen-binding domain) variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table D under the heading of “preferred substitutions”. More substantial changes are provided in Table D under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more. CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some aspects of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

TABLE D

In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody or antigen- binding moiety that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081- 1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigenantibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody. b) Glycosylation variants

In certain aspects, an activatable fusion protein or antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated, in particular if the activatable fusion protein comprises an Fc domain. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the activatable fusion protein or the antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties. In one aspect, antibody and activatable fusion protein variants are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e. no fiicosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues, relative to the sum of all oligosaccharides attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2006/082515, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcyRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies or activatable fusion proteins with reduced fucosylation include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fiicosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., BiotechnoL Bioeng., 94(4): 680-688 (2006); and WO 2003/085107), or cells with reduced or abolished activity of a GDP-fucose synthesis or transporter protein (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

In a further aspect, antibody variants or variants of activatable fusion proteins are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764. c) Fc region variants

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of a fusion protein or antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGi, IgG?, IgGs or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain aspects, the invention contemplates a fusion protein or antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, nonradioactive assays methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929 Al).

Antibodies or fusion proteins with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain aspects, a fusion protein or antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In certain aspects, a fusion protein or antibody variant comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265 A and/or P329G in an Fc region derived from a human IgGi Fc region. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgGi Fc region. (See, e.g., WO 2012/130831). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgGi Fc region. In some aspects, alterations are made in the Fc region that result in altered (z.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (See, e.g., US Patent No. 7,371,826; Dall'Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524).

Fc region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues 1253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined.

In certain aspects, a fusion protein or antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or 435 of the Fc-region (EU numbering of residues). In certain aspects, the fusion protein or antibody variant comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435. In one aspect, the substitutions are 1253 A, H310A and H435A in an Fc region derived from a human IgGl Fc-region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508. In certain aspects, a fusion protein or antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436. In one aspect, the substitutions are H310A, H433A and Y436A in an Fc region derived from a human IgGl Fc-region. (See, e.g., WO 2014/177460 Al).

In certain aspects, a fusion protein or antibody variant comprises an Fc region with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues). In certain aspects, the fusion protein or antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgGi Fc-region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

The C-terminus of the heavy chain of the antibody as reported herein can be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending PG. In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions). d) Cysteine engineered antibody variants

In certain aspects, it may be desirable to create cysteine-engineered antibodies or activatable fusion proteins, e.g., THIOMAB™ antibodies, in which one or more residues of an antibody or an activatable fusion protein are substituted with cysteine residues. In particular aspects, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody or the activatable fusion protein and may be used to conjugate the antibody or the activatable fusion protein to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856. e) Antibody Derivatives

In certain aspects, a fusion protein or antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

B. Recombinant Methods and Compositions

Activatable fusion proteins or antibodies described herein may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an activatable fusion protein are provided.

In case of an activatable fusion protein that is based solely on a first antigenbinding moiety that comprises a heavy chain and a light chain polypeptide, one of which is covalently attached to the ligand and the other one is covalently attached to the masking moiety, two nucleic acids are required, one for the light chain polypeptide and one for the heavy chain polypeptide. Similarly, in case of a native antibody or native antibody fragment two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the activatable fusion protein (e.g., the light and/or heavy chain(s) of the activatable fusion protein). These nucleic acids can be on the same expression vector or on different expression vectors.

In case of an activatable fusion protein further comprising a second antigenbinding moiety and an Fc domain with heterodimeric heavy chains, four nucleic acids may be required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc-region polypeptide. Similarly, in case of a bispecific antibody with heterodimeric heavy chains four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc-region polypeptide. The four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors. Such nucleic acid(s) encode an amino acid sequence comprising the first VL and/or an amino acid sequence comprising the first VH including the first heteromonomeric Fc-region and/or an amino acid sequence comprising the second VL and/or an amino acid sequence comprising the second VH including the second heteromonomeric Fc- region of the activatable fusion protein (e.g., the first and/or second light and/or the first and/or second heavy chains of the activatable fusion protein). These nucleic acids can be on the same expression vector or on different expression vectors, normally these nucleic acids are located on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chain comprises the so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises the so-called “hole mutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73) according to EU index numbering.

In one aspect of the invention, an activatable fusion protein or an antibody according to the invention comprises an Fc domain comprising a modification promoting the association of the first and second Fc domain heavy chain polypeptide. In one aspect, the first Fc domain heavy chain polypeptide comprises knobs and the second Fc domain heavy chain polypeptide comprises holes according to the knobs into holes method. In a particular aspect of the invention, the activatable fusion protein is characterized in that the first Fc domain heavy chain polypeptide comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second Fc domain heavy chain polypeptide comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

In one aspect of the invention, the activatable fusion protein or the antibody described herein comprises two Fabs as antigen-binding moieties wherein one of them is a cross-Fab, i.e. the activatable fusion protein is characterized in that the first and the second antigen-binding moieties are Fabs and that in one of the Fabs the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In one aspect, the amino acid at position 124 in the constant domain CL of one of the two Fab fragments is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular aspect of the invention, the amino acid at position 124 in the constant domain CL of the first antigen-binding moiety is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In one aspect, isolated nucleic acids encoding an activatable fusion protein or an antibody as reported herein are provided. In one aspect, isolated nucleic acids encoding an activatable fusion protein as used in the methods as reported herein are provided. In one aspect, a host cell comprising the nucleic acid encoding an activatable fusion protein as reported herein is provided.

In one aspect, a method of making an activatable fusion protein or an antibody is provided, wherein the method comprises culturing a host cell comprising nucleic acid(s) encoding the activatable fusion protein or the antibody in vitro, as provided above, under conditions suitable for expression of the activatable fusion protein or the antibody, and optionally recovering the activatable fusion protein or the antibody from the host cell (and/or from the host cell culture medium). In one aspect, an activatable fusion protein or an antibody produced by the methods reported herein is provided.

For recombinant production of an activatable fusion protein or an antibody, nucleic acids encoding the activatable fusion protein or the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the activatable fusion protein) or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expression of vectors encoding the polypeptides of an activatable fusion protein or an antibody include prokaryotic or eukaryotic cells described herein. For example, activatable fusion proteins or antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the activatable fusion protein or the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the polypeptides of an activatable fusion protein or an antibody, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of an activatable fusion protein with a partially or fully human glycosylation pattern. See Gerngross, T.U, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of (glycosylated) activatable fusion protein or antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959, 177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS- 7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for production of antibodies and other proteins, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

C. Assays

Activatable fusion proteins and antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

/. Binding assays and other assays

In one aspect, an activatable fusion protein of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc. Target and Ligand Binding Affinity.

In certain aspects, any antigen-binding domain of the activatable fusion proteins provided herein has a dissociation constant (KD) of < IpM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'⁸M or less, e.g., from 10’⁸M to 10’¹³ M, e.g., from 10’⁹ M to IO’¹³ M).

In one aspect, KD is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or a BIACORE ®- 3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). In one aspect, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with A-ethyl-A’- (3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (~0.2 pM) before injection at a flow rate of 5 pl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 pl/min. Association rates (k_on) and dissociation rates (k_off) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k_off/k_on. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M'¹ s'¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO ™ spectrophotometer (Thermo Spectronic) with a stirred cuvette.

In an alternative method, KD is measured by a radiolabeled antigen binding assay (RIA). In one aspect, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti- Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN- 20®) in PBS. When the plates have dried, 150 pl/well of scintillant (MICRO SCINT- 20 ™; Packard) is added, and the plates are counted on a TOPCOUNT ™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

2. Activity assays

In one aspect, assays are provided for identifying activatable fusion proteins having biological activity. Biological activity may include cytokine activity or enzymatic activity. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain aspects, an activatable fusion protein of the invention is tested for such biological activity, e.g. in cell-based assays such as HEK-Blue™ reporter cell assays (Invivogen). Reporter cell lines may be generated for the testing of activatable fusion proteins in a target dependent or independent manner. HEK-Blue™ IL-2 cells recapitulate the human IL-2R signaling pathway via expression of the IL-2R chains (IL-2Ra, P and y subunits) and effectors of the downstream signaling cascade (JAK3 and STAT5) and a STAT5-inducible secreted alkaline phosphatase (SEAP) reporter system. Upon addition of Quanti-BlueTM substrate (Invivogen rep-qbs), absorbance at 650 nm is measured, correlating with SEAP levels and IL-2R activity. A HEK- Blue™ IL-2 reporter cell line can be modified to assess activation of the IL-2 pathway in a target-dependent manner, by geneticall engineering a HEK-Blue™ IL 2 reporter cell line (Invivogen hkb-il2) to express the desired target antigen, e.g. PD1. For modification, a transposon vector system was used, which consists of two plasmids: transposon vector, in which the target antigen gene of interest is flanked by two inverted/direct repeats IR/DR, and a vector encoding the Sleeping Beauty transposase SBIOOx. Full-length cDNA encoding the target antigen is subcloned into the transposon vector, carrying a neomycin-resistance. In the final plasmid the target antigen expression is under the control of a UBC promoter. The transposon and the transposase vectors are co-transfected into HEK-Blue IL-2 reporter cells (Invivogen, #hkb-IL2) using Lipofectamine 2000 Reagent (Invitrogen, #11668019) according to the manufacturer’s protocol. HEK-Blue IL-2 cells are maintained in DMEM media (PAN, #P04-03609) supplemented with 10% FCS (Gibco, #10500), 2 mM L- Glutamine (PAN, #P04-80100), lx HEK Blue CLR selection solution containing blasticidin, hygromycin, zeocin (Invivogen, #hb-csm) and Ipg/mL puromycin (Gibco, #A11138-03).

HEK-Blue IL-2 cells stably expressing the target antigen are isolated by single cell sorting with a BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Stable cell clones are screened for target antigen expression and expanded. The expression level and stability may be confirmed by flow cytometry analysis using anti-human TA-antibodies over a period of 3 weeks.

In addition, a QUANTI-Blue assay (Invivogen, #rep-qbs2) with human IL-2 stimulation (Miltenyi Biotec, #130-097-748) may be performed according to the manufacturer's protocol in order to confirm that the reporter activation via the IL-2 pathway is not affected by stable transfection of the transgenic target antigen.

In certain aspects, an activatable fusion protein of the invention is tested for such biological activity, e.g. in cell-based assays such as a p-Stat5 assay. Upon induction of the cytokine (e.g. IL-2) signaling pathway a protein called STAT5 is phosphorylated in activated donor T cells. Phospho-STAT5 (p-STAT5) can be fluorescently stained using antibodies and populations of cells can thereby be assessed for cytokine (e.g. IL-2) activation.

D. Pharmaceutical Compositions

In a further aspect, provided are pharmaceutical compositions comprising any of the activatable fusion proteins or antibodies provided herein, e.g., for use in any of the below therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

Pharmaceutical compositions of an activatable fusion protein or antibody as described herein are prepared by mixing such activatable fusion protein or antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized compositions are described in US Patent No. 6,267,958. Aqueous compositions include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter compositions including a histidineacetate buffer.

The pharmaceutical composition herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Pharmaceutical compositions for sustained-release may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the activatable fusion protein or antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

The pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

E. Therapeutic Methods and Routes of Administration

Any of the activatable fusion proteins or antibodies provided herein may be used in therapeutic methods.

In one aspect, an activatable fusion protein or antibody for use as a medicament is provided. In further aspects, an activatable fusion protein or antibody for use in treating a disease is provided. In certain aspects, an activatable fusion protein or antibody for use in a method of treatment is provided. In certain aspects, the invention provides an activatable fusion protein or antibody for use in a method of treating an individual having a disease comprising administering to the individual an effective amount of the activatable fusion protein or antibody. The disease may be selected from the group consisting of cancer, hyperproliferative diseases, autoimmune diseases, neurodegenerative disease, liver diseases, kidney diseases, lung diseases and heart diseases. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In further aspects, the invention provides an activatable fusion protein or antibody for use in activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T-cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for killing of tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis . In certain aspects, the invention provides an activatable fusion protein or antibody for use in a method of activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T cells, y5 T- cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for killing of tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis in an individual comprising administering to the individual an effective amount of the activatable fusion protein or antibody to activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis. An “individual” according to any of the above aspects is preferably a human.

In a further aspect, the invention provides for the use of an activatable fusion protein or antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treatment of a disease, in particular a disease selected from the group consisting of cancer, hyperproliferative diseases, autoimmune diseases, neurodegenerative disease, liver diseases, kidney diseases, lung diseases and heart diseases. In a further aspect, the medicament is for use in a method of treating a disease, in particular a disease selected from the group consisting of cancer, hyperproliferative diseases, autoimmune diseases, neurodegenerative disease, liver diseases, kidney diseases, lung diseases and heart diseases, comprising administering to an individual having said disease an effective amount of the medicament. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further aspect, the medicament is for activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis. In a further aspect, the medicament is for use in a method of activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis in an individual comprising administering to the individual an effective amount of the medicament to activate cytokine or chemokine response, inhibit (tumor) cell proliferation, recruit T-cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, induce cell death, and activate various cellular functions, such as promotion of antibody secretion from B cells, activation of T cells, modification of antigen presentation from APCs, promotion of proliferation and differentiation of immune cell subsets, promotion of immune regulatory functions, stimulation of further cytokine and chemokine secretion, promotion of cell survival or apoptosis, promotion of cytotaxis, promotion or regulation of inflammatory responses, promotion of angiogenesis, or promotion of haematopoiesis. An “individual” according to any of the above aspects may be a human.

In a further aspect, the invention provides a method for treating a a disease, in particular a disease selected from the group consisting of cancer, hyperproliferative diseases, autoimmune diseases, neurodegenerative disease, liver diseases, kidney diseases, lung diseases and heart diseases. In one aspect, the method comprises administering to an individual having such a disease, in particular a disease selected from the group consisting of cancer, hyperproliferative diseases, autoimmune diseases, neurodegenerative disease, liver diseases, kidney diseases, lung diseases and heart diseases, an effective amount of an activatable fusion protein or antibody. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

An “individual” according to any of the above aspects may be a human.

In a further aspect, the invention provides a method for activating cytokine or chemokine response, inhibiting (tumor) cell proliferation, recruiting T cells, y5 T- cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, inducing cell death, and activating of various cellular functions, such as promoting antibody secretion from B cells, activating T cells, modificating antigen presentation from APCs, promoting proliferation and differentiation of immune cell subsets, promoting immune regulatory functions, stimulating further cytokine and chemokine secretion, promoting cell survival or apoptosis, promoting cytotaxis, promoting or regulating inflammatory responses, promoting angiogenesis, or promoting haematopoiesis in an individual. In one aspect, the method comprises administering to the individual an effective amount of an activatable fusion protein or antibody to activate cytokine or chemokine response, inhibit (tumor) cell proliferation, recruit T cells, y5 T-cells or other innate immune cells e.g. NK cells, macrophages/monocytes, neutrophiles for tumor cells, induce cell death, and activate various cellular functions, such as promotion of antibody secretion from B cells, activation of T cells, modification of antigen presentation from APCs, promotion of proliferation and differentiation of immune cell subsets, promotion of immune regulatory functions, stimulation of further cytokine and chemokine secretion, promotion of cell survival or apoptosis, promotion of cytotaxis, promotion or regulation of inflammatory responses, promotion of angiogenesis, or promotion of haematopoiesis. In one aspect, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical compositions comprising any of the activatable fusion proteins or antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and at least one additional therapeutic agent, e.g., as described below. Activatable fusion proteins or antibodies of the invention can be administered alone or used in a combination therapy. For instance, the combination therapy includes administering an activatable fusion protein or antibody of the invention and administering at least one additional therapeutic agent (e.g. one, two, three, four, five, or six additional therapeutic agents). In certain aspects, the combination therapy comprises administering an activatable fusion protein or antibody of the invention and administering at least one additional therapeutic agent.

An activatable fusion protein or antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. An activatable fusion protein or antibody of the invention can also be delivered as mRNA, via a gene therapy vector by an injected cell line. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous, subcutaneous or intratumoral injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Activatable fusion proteins or antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The activatable fusion protein or antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of activatable fusion protein or antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g., O.lmg/kg-lOmg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

EMBODIMENTS OF THE INVENTION

In the following specific embodiments of the invention are listed:

1. An activatable fusion protein comprising

(a) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(b) a ligand capable of specifically binding to a ligand binding moiety, and

(c) a masking moiety capable of specifically binding to the ligand, characterized in that the ligand is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker, and the first and the second peptide linker do not comprise a protease cleavage site.

2. The activatable fusion protein of embodiment 1 characterized in that the first antigen-binding moiety is an antibody or an antibody fragment.

3. The activatable fusion protein of embodiment 2 characterized in that the antibody fragment is selected from the group consisting of a Fab, a DutaFab, an scFab, a DAF, an Fv, a Fab', a Fab’-SH, a F(ab')2, a diabody, a linear antibody, and a multispecific antibody formed from antibody fragments.

4. The activatable fusion protein of any one of embodiments 1 to 3, characterized in that the ligand is selected from the group consisting of a growth factor, a cytokine, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof, preferably a cytokine.

5. The activatable fusion protein of any one of embodiments 1 to 4, characterized in that the ligand binding moiety is selected from the group consisting of a growth factor receptor, a cytokine receptor, an antigen, a ligand receptor, an enzyme substrate, a fluorescent label, a radioactive label, or a hormone receptor, preferably a cytokine receptor.

6. The activatable fusion protein of any one of embodiments 1 to 5, characterized in that the masking moiety is selected from the group consisting of an antibody, an antibody fragment, an antibody mimetic, a single-chain antigen-binding moiety, a peptide mask, an anti-idiotypic antibody or an anti-idiotypic antibody fragment (only if the ligand is an antibody/antibody fragment) or a receptor, protein inhibitor or binding protein capable of binding specifically to the ligand.

7. The activatable fusion protein of embodiment 6, characterized in that the masking moiety is a single-chain antigen-binding moiety selected from the group consisting of an scFv, an scFab, a VHH, VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb).

8. The activatable fusion protein of any one of embodiments 1 to 7, characterized in that the masking moiety is reversibly bound to the ligand.

9. The activatable fusion protein of any one of embodiments 1 to 8, characterized in that the binding of the masking moiety to the ligand sterically hinders the binding of the first antigen-binding moiety to the antigen.

10. The activatable fusion protein of any one of embodiments 1 to 9, characterized in that the affinity of the first antigen-binding moiety in the activatable fusion protein is decreased when compared to the affinity of the first antigen-binding moiety alone.

11. The activatable fusion protein of any one of embodiments 1 to 10, characterized in that the first antigen-binding moiety is hindered from binding to the antigen when the masking moiety is bound to the ligand.

12. The activatable fusion protein of any one of embodiments 1 to 11, characterized in that it further comprises a second antigen-binding moiety, wherein the second antigen-binding moiety comprises a) at least a second heavy chain polypeptide and at least a second light chain polypeptide, or b) a single-chain antigen-binding moiety, preferably a single-chain antigenbinding moiety selected from the group consisting of an scFv, an scFab, a VHH, a VNAR, a domain antibody (dAb), a DARPin, an affibody, a monobody, an anticalin and a single-domain antibody (sdAb).

13. The activatable fusion protein of any one of embodiments 1 to 12, characterized in that it further comprises an Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide.

14. The activatable fusion protein of embodiment 13, characterized in that the first antigen-binding moiety is covalently attached at the C-terminus of the first heavy chain polypeptide to the N-terminus or to the C-terminus of one of the two Fc domain heavy chain polypeptides. 15. The activatable fusion protein of embodiment 14, characterized in that the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding moiety is covalently attached at its C-terminus a) to the N-terminus of the first Fc domain heavy chain polypeptide, or b) to the C-terminus of the first Fc domain heavy chain polypeptide.

16. The activatable fusion protein of any one of embodiments 13 to 15, characterized in that the second antigen-binding moiety is covalently attached to the N-terminus or to the C-terminus of one of the two Fc domain heavy chain polypeptides.

17. The activatable fusion protein of embodiment 16, characterized in that the second antigen-binding moiety is a Fab, a Dutafab, a DAF or a DBA and that the second heavy chain polypeptide or the second light chain polypeptide of the second antigen-binding moiety is covalently attached at its C-terminus a) to the N-terminus of the second Fc domain heavy chain polypeptide, or b) to the C-terminus of the first or the second Fc domain heavy chain polypeptide.

18. The activatable fusion protein of embodiment 13, characterized in that a) the first heavy chain polypeptide of the first antigen-binding moiety is covalently attached at its C-terminus to the N-terminus of the first Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached at its C-terminus to the N-terminus of the second Fc domain heavy chain polypeptide, or b) the first heavy chain polypeptide of first antigen-binding moiety is covalently attached to the N-terminus of the first or the second Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached, preferably at its C-terminus (in particular the C-terminus of the second heavy chain polypeptide if the second antigen-binding moiety has a heavy chain polypeptide and a light chain polypeptide) to the C-terminus of the first or the second Fc domain heavy chain polypeptide.

19. The activatable fusion protein of any one of embodiments 12 to 15, characterized in that the second antigen-binding moiety is covalently attached at its C -terminus (in particular the C-terminus of the second heavy chain polypeptide if the second antigen-binding moiety has a heavy chain polypeptide and a light chain polypeptide) to the N-terminus of the ligand via a third peptide linker and that the third peptide linker does not comprise a protease cleavage site.

20. The activatable fusion protein of embodiment 13 characterized in that a) the second antigen-binding domain is covalently attached at the N- terminus of its heavy chain polypeptide or light chain polypeptide to the N- or C- terminus of the second heavy chain polypeptide of the Fc domain via a third peptide linker, and/or b) the first heavy chain polypeptide of the Fc domain is covalently attached at the N- or C-terminus to the N-terminus of the ligand via a fourth peptide linker, and that the third and (if present) fourth peptide linker do not comprise a protease cleavage site.

21. The activatable fusion protein of embodiment 13, wherein the masking moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigenbinding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

22. The activatable fusion protein of embodiment 13, wherein the masking moiety comprises a single-chain antigen-binding moiety, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C- terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety.

23. The activatable fusion protein of any one of embodiments 12 to 22 characterized in that the second antigen-binding moiety is capable of specifically binding to an antigen that is the same or a different antigen from the target antigen.

24. The activatable fusion protein of embodiment 23 characterized in that the second antigen-binding moiety (a) is capable of specifically binding to the target antigen and (b) is capable of specifically binding to an epitope on the target antigen that is different from the epitope that is bound by the first antigen-binding moiety.

25. The activatable fusion protein of embodiment 23 characterized in that the second antigen-binding moiety is capable of specifically binding to the same epitope on the target antigen as the first antigen-binding moiety.

26. The activatable fusion protein of any one of embodiments 1 to 23 characterized in that the first antigen-binding moiety is an antibody of the IgG type. 27. The activatable fusion protein of any one of embodiments 13 to 26 characterized in that the first antigen-binding moiety, the second antigen-binding moiety and the Fc region together form an antibody of the IgG type.

28. The activatable fusion protein of any one of embodiments 13 to 27 characterized in that the Fc domain is an IgG Fc domain, particularly an IgGl Fc domain or an IgG4 Fc domain.

29. The activatable fusion protein of any one of embodiments 26 to 28 characterized in that the activatable fusion protein comprises at least two full-length IgG antibody heavy chains and wherein the heavy chains of the antigen-binding moiety are of the y type (IgG), in particular of the yl type.

30. The activatable fusion protein of any one of embodiments 1 to 29 characterized in that the activatable fusion protein comprises at least two light chains and wherein the light chains are selected from the kappa (K) and/or lambda (A) subtype.

31. The activatable fusion protein of any one of embodiments 13 to 30 characterized in that the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular towards Fey receptor.

32. The activatable fusion protein of any one of embodiments 13 to 31 characterized in that the Fc domain is of the human IgGl subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

33. The activatable fusion protein of any one of embodiments 13 to 32 characterized in that the Fc domain comprises a modification promoting the association of the first and second Fc domain heavy chain polypeptide.

34. The activatable fusion protein of any one of embodiments 13 to 33 characterized in that the first Fc domain heavy chain polypeptide comprises knobs and the second Fc domain heavy chain polypeptide comprises holes according to the knobs into holes method.

35. The activatable fusion protein of any one of embodiments 13 to 34 characterized in that the first Fc domain heavy chain polypeptide comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second Fc domain heavy chain polypeptide comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

36. The activatable fusion protein of any one of embodiments 1 to 35 characterized in that the first and the second antigen-binding moieties are Fabs and that in one of the Fabs the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

37. The activatable fusion protein of embodiment 36 characterized that in the constant domain CL of one of the two Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

38. The activatable fusion protein of embodiment 36 or 37 characterized that in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CHI the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

39. The activatable fusion protein of any one of embodiments 1 to 38, characterized in that the target antigen is selected from the group consisting of alpha- synuclein, Amyloid beta, BCMA, BTLA, CD3e, CD4, CD8, CD 14, CD 16 (FcgRIIIa), CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD44, CD47, CD52, CD70, CD109, CD123, CD137, CEACAM5, c-MET, CTLA4, DLL3, CXCR4, EDB-FN, EpCAM, epidermal growth factor receptor (EGFR), EPO Receptor, FAPa, FGFR2, FGFR3, GD-2, GP100, GITR, GLP-1 receptor, GM-CSF, GPC3, Grp78, Hedgehog, HER2, HER3, HLA-G, ICAM (ICAM-1, -2, -3, -4, -5), IGF-1R, IL-1R1, IL-4Ra, Integrin av, b7 integrin subunit, a4b7 integrin, a4 integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notchl, Notch3, NRP1, NRP2, 0X40, PAR-2, PD-1, PD-L1, PDGFR, PSA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-Cl, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1, TGFp, TGF-Y, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2 or 5T4. 40. The activatable fusion protein of embodiment 39 characterized in that the target antigen is selected from the group consisting of PD1, IL-2, CD8 and CD19.

41. The activatable fusion protein of any one of embodiments 1 to 40 characterized in that the ligand is a cytokine selected from the group consisting of interferons, interleukins, chemokines, lymphokines, monokines, colony-stimulating factors, and tumour necrosis factors, in particular from the group consisting of interferons and interleukins.

42. The activatable fusion protein of embodiment 41 characterized in that the ligand is a cytokine selected from the group consisting of BMP, CSF-1, insulin, GLP-

I, HGH, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-

I I, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL- 23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL- 35, IL-36, GM-CSF, FGF, EGF, G-CSF, IFNa, IFNP, IFNy, PDGF, TGFp, TNFa, TNFP, VEGF, or EPO.

43. The activatable fusion protein of embodiment 41 or 42 characterized in that the cytokine is selected from the group consisting of IL-2, IL-7, IL-21 and IFNa.

44. The activatable fusion protein of any one of embodiments 1 to 40 characterized in that the ligand is an antigen-binding moiety.

45. The activatable fusion protein of embodiment 39 characterized in that the antigen-binding moiety is an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of to of BCMA, CD3, CD20, CD 19, CD27, CD28, CD40, CD47, CD 123, CD 137, CEA, CTLA4, DLL3, EpCAM, HLA-G, GP100, GITR, HER2, IL-7, kynureninase, MICA, MICB, 0X40, IL-2, PD-1, the extracellular domain of TGFBR2, TNF, or VEGF-C.

46. The activatable fusion protein of any one of embodiments 1 to 45 characterized in that the ligand is a non-cytokine ligand selected from the group consisting of a growth factor, a chemokine, an antibody, an antibody fragment, an enzyme, a receptor ligand, an affinity peptide ligand, a peptide hormone, a receptor agonist, a receptor antagonist, an enzyme, a soluble receptor, a protein toxin, a soluble ligand, an extracellular region of a cell surface receptor, an extracellular region of a cell surface ligand, a small molecule, or any combination thereof.

47. An isolated nucleic acid encoding the activatable fusion protein of any one of embodiments 1 to 46. 48. A host cell comprising the nucleic acid of embodiment 47.

49. An in vitro method of producing an activatable fusion protein of any one of embodiments 1 to 46 comprising culturing the host cell of embodiment 48 under conditions suitable for the expression of the activatable fusion protein.

50. The method of embodiment 49, further comprising recovering the activatable fusion protein from the host cell.

51. An activatable fusion protein produced by the method of embodiment 49 or embodiment 50.

52. A pharmaceutical composition comprising the activatable fusion protein of any any one of embodiments 1-46 or 51 and a pharmaceutically acceptable carrier.

53. The activatable fusion protein of any any one of embodiments 1-46 or 51 or the pharmaceutical composition of embodiment 52 for use as a medicament.

54. The activatable fusion protein of any one of embodiments 1-46 or 51 or the pharmaceutical composition of embodiment 52 for use in treating cancer, viral infection or autoimmune disease.

55. Use of the activatable fusion protein of any one of embodiments 1-46 or 51 or the pharmaceutical composition of embodiment 52 in the manufacture of a medicament for treatment of cancer, viral infection or autoimmune disease.

56. An activatable fusion protein comprising

(a) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(b) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide,

(c) a ligand capable of specifically binding to a ligand binding moiety, and

(d) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising (al) the ligand fused at its C-terminus via a first peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the masking moiety, fused at its C- terminus via a second peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigen-binding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site.

57. An activatable fusion protein comprising

(a) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(b) a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide,

(c) a ligand capable of specifically binding to a ligand binding moiety, and

(d) a masking moiety comprising a single-chain antigen-binding moiety capable of specifically binding to the ligand, characterized in that the activatable fusion protein comprises a) a first polypeptide, comprising the (al) masking moiety fused at its C- terminus via a second peptide linker to the N-terminus of the heavy chain polypeptide of the first antigen-binding moiety, (a2) the heavy chain polypeptide of the first antigen-binding moiety, fused at its C-terminus to the N-terminus of the first heavy chain polypeptide of the Fc domain, and (a3) the first heavy chain polypeptide of the Fc domain, b) a second polypeptide, comprising (bl) the ligand, fused at its C-terminus via a first peptide linker to the N-terminus of the light chain polypeptide of the first antigen-binding moiety, and (b2) the light chain polypeptide of the first antigenbinding moiety, c) a third polypeptide, comprising (cl) the heavy chain polypeptide of the second antigen-binding moiety, fused at its C-terminus to the N-terminus of the second heavy chain polypeptide of the Fc domain, and (c2) the second heavy chain polypeptide of the Fc domain, and d) a fourth polypeptide, comprising the light chain polypeptide of the second antigen-binding moiety, wherein the first and the second peptide linker do not comprise a protease cleavage site.

58. An antibody that binds to huIL-2 or a variant thereof, wherein the antibody binds to an epitope of huIL-2 within amino acid residues 8-17, amino acid residue 30 and/or amino acid residues 77-81 of SEQ ID NO:81; and thus inhibits the binding of huIL-2 to human IL-2RPy and to human IL-2Ra.

59. The antibody of embodiment 58, wherein the epitope comprises amino acid residues corresponding to K8, Q13, E15, H16, N30, N77, H79 and R81 of SEQ ID NO:81; and thus inhibits the binding of huIL-2 to human IL-2RPy and to human IL-2Ra.

60. An antibody that binds to huIL-2 or a variant thereof, wherein the antibody comprises

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

61. The antibody of embodiment 58 or 59, comprising

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NON, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

62. The antibody of any one of embodiments 58 to 61, which is a monoclonal antibody. 63. The antibody of any one of embodiments 58 to 62, which is a human, humanized or chimeric antibody.

64. The antibody of any of embodiments 60 to 63, which is an antibody fragment that binds huIL-2 or a variant thereof.

65. The antibody of any of embodiments 58 to 64, comprising

(A) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;

(B) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13;

(C) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(D) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(E) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:31;

(F) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(G) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39;

(H) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42;

(I) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or

(J) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

66. An antibody that specifically binds to huIL-2 or a variant thereof comprising

(A) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8;

(B) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13;

(C) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(D) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(E) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:31;

(F) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(G) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39; (H) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42;

(I) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or

(J) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

67. The antibody of any of embodiments 58 to 66, wherein the antibody is a full length IgGl antibody or a Fab.

68. The antibody of any of embodiments 58 to 67, wherein the antibody binds huIL-2 with an affinity of 125 nM or huIL-2v with an affinity of 15,4 nM as measured by SPR assay.

69. The antibody of embodiment 68, wherein the antibody binds huIL-2 with an affinity of 1,5 nM or huIL-2v with an affinity of 0,9 nM as measured by SPR assay.

70. The antibody of any of embodiments 58 to 69, wherein the antibody is a multispecific antibody.

71. The antibody of any of embodiments 58 to 70 wherein the antibody is a Fab and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 56; (E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

72. The antibody of any of embodiments 58 to 70 wherein the antibody length antibody and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54; (D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 56;

(E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68, and wherein the antibody further comprises a human Fc region which comprises two human Fc region polypeptides selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72. SEQ ID NO:73, SEQ ID NO74: and SEQ ID NO: 75.

73. An antibody, which competes for binding to huIL-2 or a variant thereof with the antibody of any one of embodiments 58 to 72.

74. An isolated nucleic acid encoding the antibody of any of embodiments 58 to 73. 75. A host cell comprising the nucleic acid of embodiment 74.

76. A method of producing an antibody that binds to huIL-2 or a variant thereof comprising culturing the host cell of embodiment 75 under conditions suitable for the expression of the antibody.

77. The method of embodiment 76, further comprising recovering the antibody from the host cell.

78. An antibody produced by the method of embodiment 76 or 77.

79. A pharmaceutical composition comprising the antibody of any of embodiments 76 to 77 and a pharmaceutically acceptable carrier.

80. The antibody of any one of embodiments 58 to 73 or the pharmaceutical composition of embodiment 79 for use as a medicament.

81. The antibody of any one of embodiments 58 to 73 or the pharmaceutical composition of embodiment 79 for use in treating cancer, viral infection or autoimmune disease.

82. Use of the antibody of any one of embodiments 58 to 73 or the pharmaceutical composition of embodiment 79 in the manufacture of a medicament for cancer, viral infection or autoimmune disease.

83. A method of treating an individual having cancer, viral infection or autoimmune disease comprising administering to the individual an effective amount of the antibody of any one of embodiments 58 to 73 or the pharmaceutical composition of embodiment 79.

84. The method of embodiment 83 further comprising administering an additional therapeutic agent to the individual.

85. The method of embodiment 84 wherein the additional therapeutic agent is selected from the group consisting of anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.

86. The antibody of any one of embodiments 58 to 73 for use as a masking antibody for huIL-2, huIL-2v or a variant thereof. 87. The antibody of any one of embodiments 58 to 73 for as a masking moiety in an activatable fusion protein of any one of embodiments 1-46, 51, 56 or 57.

III. EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1: Generation of CISS molecules, precursor molecules and control molecules used in the following examples

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions. To generate the targeted CISS molecules, the CrossMab technology described in WO2016/016299 was used, in which VH/VL have been exchanged in one antibody arm and the CHI /CL interface of the other antibody arm has been modified by charge modifications, in combination with the knobs-into-holes technology in the CH3/CH3 interface to foster heterodimerization, as described in Regula et al. (2018) Protein Engineering, Design and Selection, 31(7-8):289-299.

Plasmids used for the production of CISS molecules and control molecules used in the assays were in some cases synthesized and supplied by an external provider (Twist Bioscience, USA). In other cases, DNA fragments were synthesized (Twist Bioscience, USA) and cloned into vectors using either Gibson assembly (Gibson et al., (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature Methods, 6(5): 343-348 and U.S. Pat. No. 8,968,999) (NEBuilder® HiFi DNA Assembly Master Mix E2621X (New England BioLabs)) or Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix M1100L (New England BioLabs)). Standardized Roche in-house vectors under the control of the CMV-promoter were used. a) Small scale expressions

For the transient expression of our CISS proteins and controls, molecule plasmids were transfected into Expi293 cells (Thermo Fisher Scientific, USA) in 4 ml of culture media according to standard protocols. Following an expression interval, cells were harvested via centrifugation and protein-containing supernatant was filtered using a 0.22 pm vacuum filtration system (Millipore). Supernatants were purified using affinity chromatography with CaptureSelect™ CHI -XL Affinity Matrix (Thermo Fisher Scientific, USA), using PBS buffer (10 mM sodium phosphate, 1 mM potassium phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride, pH 7.4) as equilibration and washing buffer and 25 mM citric acid (pH 3.0, pH adjustment with NaOH) as elution buffer. Tris 1.5 M (pH 7.5) was used for the adjustment of sample pH, delivering a final formulation of approximately 23 mM Citrate, 151 mM TRIS, pH 6.0-6.5. b) Mid-scale Expressions

Mid-Scale expression of molecules was achieved through the transient transfection of Expi293 cells (Thermo Fisher Scientific, USA) grown in 30 ml of media according to standard protocols. Following an expression interval, cells were harvested via centrifugation and protein-containing supernatant was filtered using a 0.22 pm vacuum filtration system (Millipore). Supernatants were purified using affinity chromatography with CaptureSelect™ CHI -XL Affinity Matrix or Mab Select SuRe-protein A resin (Cytiva Life Sciences, USA) using PBS buffer (10 mM sodium phosphate, 1 mM potassium phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride, pH 7.4) as equilibration and washing buffer. 25 mM citric acid (pH 3.0, pH adjustment with NaOH) was used for elution and Tris 1.5 M (ph 7.5) was used for sample pH adjustment. If expression levels were sufficiently high, protein was further fractionated using size exclusion chromatography (Superdex® 200, Cytiva Life Sciences, USA) in 20 mM Histidine, 140 mM NaCl, pH 6.0. c) Overview of tool and control molecules used in the Examples

In the examples, different types of tool molecules (e.g. that are immobilized or flown during SPR assays for assaying capture of the CISS molecules; target binding anitbodies that are used to block receptor molecules in cell assays) and control molecules were used. The molecules are described in more detail in the respective examples. For ease of reference, the sequences of these molecules are listed in Table E. Table E: Sequences and short descriptions of tool and control molecules used in the Examples

Example 2: SPR assay In the subsequent examples, surface plasmon resonance (SPR) based assays were used to determine the binding characteristics of the CISS Fab units and their “precursor” molecules (including Fabs only, mask-Fab fusions and cytokine-Fab fusions). In some instances, Fc-fused molecules were also assessed in addition to some Fc-fused bispecific molecules. SPR assays were used to examine numerous aspects of the molecules used in the following examples, including; the binding of the mask to the cytokine, the tolerances of the Fabs for N-terminal fusions with respect to their target binding, the capacity of the cytokines to bind to their receptors when fused, whether the masking of the cytokines within the CISS Fab molecules is functional, and whether or not the CISS Fab molecules were exhibiting the desired binding mutual exclusivity (whether the mask and ligand interaction sterically blocks Fab and target antigen interactions).

Mask and cytokine function could be assessed through simple flowing of the cytokine or cytokine receptor over captured Fab-mask or Fab-cytokine fusion molecules, respectively, and analysis of binding. Flowing target antigen over these captured Fab-mask and Fab-cytokine fusions can reveal whether the Fab is tolerant of fusions to either N-terminus. In each case, where binding is retained, the fusions are presumed to be correctly formed and functional. Subsequently, a strong reduction in or the absence of binding when flowing cytokine, cytokine receptor and target antigen as analytes over captured CISS molecules may indicate that the masking interaction is functional (which should prevent cytokines and cytokine receptors from binding) and that steric hindrance of the targeting Fab is occurring due to this interaction.

Each assay was performed using either a Series S CM4 (Cytiva 29104989) or a Series S CM5 (Cytiva 29104988) sensor chip onto which an anti-human Fab was immobilized using the “Human Fab Capture Kit” (Cytiva 28958325) via amine coupling. The surface coupling of anti-human Fab (10 pg/ml) (Cytiva 28958325) to the sensor chips typically resulted in around 1500-3000 response units (RU) onto each flow cell of the 8 channels of the sensor chip. Biacore 8k and 8k+ (GE Healthcare) machines were used. The analytes flown and the concentrations used are described in the respective examples.

During a run, samples were captured to the chip and the analytes were flown at multiple concentrations for assessment. The sensor chip surface was regenerated after each analysis cycle using 10 mM Glycine pH 2.0. In all experiments, the sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05 % v/v Surfactant P20, pH 7.4). Flow cell temperature was set to 25 °C and sample compartment temperature to 10 °C. The system was primed with running buffer. The samples were injected for 180 seconds with a concentration of 50-100 nM and bound to the second flow cell. Analytes were injected at a set of concentrations (225 nM, 75 nM, 25 nM, and 0 nM; or 250 nM, 100 nM, 20nM and 0 nM) over each sample for 120 seconds followed by a dissociation time of 240 s, 300 s or 480 s and a single 60 s regeneration steps with 10 mM Glycine pH 2. The equilibrium constant and kinetic rate constants were determined by fitting the data to a 1 : 1 Langmuir interaction model. A capture summary is included for capture evaluation. Through the comparison and assessment of many sensorgrams using proteins expressed and purified using the small scale method outlined in Example 1, a standardized capture level assessment was applied to assist in the interpretation of SPR results which factors to some degree both for the low capture of the desired product, which may diminish the possibility of an easily interpretable sensorgram, and also for the real or apparent capture of material other than the investigated product. For the summary, the following capture value cutoffs were selected: capture above 30 is characterized as “ok”, capture between 20 and 30 “low” and below 20 is considered to be “VL/N” (very low or no capture).

Binding was further assessed through the comparison of capture RU with the RUs in the presence of the highest concentration of flown analyte (abbreviated as ”Cap_Xxxnm” for a concentration of xx nm). Specifically, this response ratio (RR) was obtained by comparing the “analyte late binding” value (RU 5 seconds before analyte injection is complete) with the theoretical R_max (Capture RU/Molecular Weight of the captured molecule x molecular weight of the analyte x valency of the captured molecule).

Response ratio (%) = 100 / R_max theoretical x Analyte Late binding

For the purpose of screening the molecules, an additional subjective binding assessment (“SumBdg”) was performed using each of the sensorgrams which factored kinetic values, the response ratio in addition to manual assessment of the curves and fit in comparison to similar and control molecules. “Binding” (B) was assigned to sensorgrams that appeared to show significant amounts of binding to the analyte, “No / minimal binding” (N/MB) for sensorgrams that appeared did not clearly show appreciable binding to analyte, “low binding” (LB) was used where binding was deemed to be significantly perturbed relative to controls, but was likely not completely eliminated.

Example 3: HEK-Blue™ cell line generation

Reporter cell lines were generated for the testing of CISS molecules and precursors in a target dependent or independent manner. The cell lines were based on the HEK-Blue™ reporter cells (Invivogen). a) Modified HEK-Blue™ IL-2 reporter cell lines

HEK-Blue™ IL-2 cells (Invivogen hkb-il2) were used for assays. The HEK reporter cell recapitulates the human IL-2R signaling pathway via expression of the IL-2R chains (IL-2Ra, P and y subunits) and effectors of the downstream signaling cascade (JAK3 and STAT5) and a STAT5-inducible secreted alkaline phosphatase (SEAP) reporter system. Upon addition of Quanti-Blue™ substrate (Invivogen rep- qbs), absorbance at 650 nm is measured, correlating with SEAP levels and IL-2R activity. b) PD1 positive HEK-Blue™ IL-2 reporter cell lines (low / medium / high PD1 expression)

To assess activation of the IL-2 pathway in a PD1 -dependent manner, a HEK- Blue™ IL-2 reporter cell line (Invivogen hkb-il2) was genetically engineered to express PD1. For modification, a transposon vector system was used, which consists of two plasmids: transposon vector, in which the gene of interest is flanked by two inverted/direct repeats IR/DR, and a vector encoding the Sleeping Beauty transposase SBIOOx. Full-length cDNA encoding human PD1 was subcloned into the transposon vector, carrying a neomycin-resistance. In the final plasmid PD1 expression is under the control of a UBC promoter. The transposon and the transposase vectors were co-transfected into HEK-Blue IL-2 reporter cells (Invivogen, #hkb-IL2) using Lipofectamine 2000 Reagent (Invitrogen, #11668019) according to the manufacturer’s protocol. HEK-Blue IL-2 cells were maintained in DMEM media (PAN, #P04-03609) supplemented with 10% FCS (Gibco, #10500), 2 mM L-Glutamine (PAN, #P04-80100), lx HEK Blue CLR selection solution containing blasticidin, hygromycin, zeocin (Invivogen, #hb-csm) and Ipg/mL puromycin (Gibco, #A11138-03).

HEK-Blue IL-2 cells stably expressing human PD1 were isolated by single cell sorting with a BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Stable cell clones were screened for PD1 expression and expanded. The expression level and stability was confirmed by flow cytometry analysis using PE mouse anti-human PD1 (Clone EH12.2H7) (BioLegend, #329906) over a period of 3 weeks.

In addition, a QUANTI-Blue assay (Invivogen, #rep-qbs2) with human IL-2 stimulation (Miltenyi Biotec, #130-097-748) was performed according to the manufacturer's protocol in order to confirm that the reporter activation via the IL-2 pathway was not affected by stable transfection of PD1. The selected clone 4 (CL023813) with low human PD1 expression, clone 26 (CL023814) with medium human PD1 expression and clone 42 (CL023814) with high human PD1 expression showed comparable IL-2 stimulation to HEK-Blue IL-2 parental cell line. c) CD8 / PD1 positive HEK-Blue™ IL-2 reporter cell lines

To assess activation of the IL-2 pathway in a CD8 and PD1 dependent manner, a HEK Blue IL-2 reporter cell line was genetically engineered to express human CD8 and PD1. For modification, a transposon vector system was used, which consists of two plasmids: transposon vector in which the gene of interests is flanked by two inverted/direct repeats IR/DR, and a vector encoding the Sleeping Beauty transposase SBIOOx. Full-length cDNA encoding of either human PD1 or CD8 was subcloned into the transposon vector, carrying a neomycin-resistance. In the final plasmids, PD1 expression is under the control of a human PGK promoter and CD8 upstream of a CMV promoter in order to drive different expression levels of the transgenes. All three plasmids (the two transposon vectors and the transposase vector) were co-transfected into HEK-Blue IL-2 reporter cells (Invivogen, #hkb-IL2) using Lipofectamine 2000 Reagent (Invitrogen, #11668019) according to the manufacturer’s protocol. HEK-Blue IL-2 cells were maintained in DMEM media (PAN, #P04-03609) supplemented with 10% FCS (Gibco, #10500), 2 mM L- Glutamine (PAN, #P04-80100), lx HEK Blue CLR selection solution containing blasticidin, hygromycin, zeocin (Invivogen, #hb-csm) and Ipg/mL puromycin (Gibco, #A11138-03).

After an antibiotic selection phase with geneticin (Gibco, #44890) to enrich the stable transfected cell population, HEK Blue IL-2 cells stably expressing human PD1 and CD8 were isolated by single cell sorting with a BD FACS Aria III cell sorter (BD Biosciences). Stable cell clones were screened for PD1 and CD8 co-expression and expanded. The expression level and stability was confirmed by flow cytometry using PE mouse anti-human PD1 (Clone EH12.2H7, BioLegend, #329906) and APC anti human CD8a (clone RPA-T8, BioLegend, #301014), over a period of 3 weeks.

In addition, a QUANTI-Blue assay (Invivogen, #-qbs2) with human IL-2 stimulation (Miltenyi Biotec, #130-097-748) was performed according to the manufacturer's protocol in order to ensure that the reporter activation via the IL-2 pathway was not affected by stable transfection of PD1 and CD8. The selected clone 11 (DBR Concept ID CL023901) with low human PD1 expression and high CD8 expression level showed effective but slightly lower IL-2 stimulation compared to HEK Blue IL-2 parental cell line. d) Modified HEK-Blue™ IFNa/p reporter cell lines

HEK-Blue™ IFNa/p cells (Invivogen hkb-ifnab) were used to assess activation of the IFNAR1/2 pathway by CISS molecules comprising an Interferon alpha molecule. The HEK reporter cell allows for the detection of bioactive type 1 human interferons. Upon binding of IFN-a2a to IFNAR1/2 receptors, the JAK/STAT/ISGF3 pathway is triggered, leading finally to the expression of the reporter gene which is under the control of the ISG54 promoter containing an IFN- stimulated response element (ISRE). The reporter gene SEAP (secreted alkaline phosphatase) is produced by the cell and secreted into the medium. Its amount correlates with the extent of IFNAR1/2 receptor activation. The SEAP levels in the medium can be measured by using a SEAP detection reagent like QUANTI-Blue™ and the color change of the detection reagent by the SEAP activity can be measured with a spectrophotometer. e) PD-L1 positive HEK-Blue™ IFNa/ reporter cell lines

In order to assess PD-L1 dependent activation of IFNARs HEK-Blue™ IFNa/p reporter cell were modified to express high amounts of PD-L1. Full-length cDNA encoding human PD-L1 was subcloned into a mammalian expression vector. The plasmid was transfected into HEK-Blue IFNa/p (Invivogen, #hkb-IFNab) cells using Lipofectamine 3000 Reagent (Invitrogen, #L3000015) according to the manufacturer’s protocol. HEK-Blue IFNa/p cells were maintained in DMEM media (PAN, #P04-03596) supplemented with 10% FCS (Gibco, #10500), 2 rnM L- Glutamine (PAN, #P04-80100), 30 pg/mL Blasticidin (Gibco, #A1113903) and 100 pg/mL Zeocin (Gibco, #R25001). Two days after transfection, Hygromycin (PAN, #P06-08020) was added to 200 pg/mL.

After initial selection, the cells with the highest cell surface expression of PD-L1 were sorted by BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Surface expression and stability was confirmed by flow cytometry analysis using PE mouse anti-human CD274 (Clone MIH1) (BD Biosciences, #557924) over a period of 4 weeks.

In addition, a QUANTI-Blue assay (Invivogen, #rep-qbs2) was performed according to the manufacturer's protocol in order to confirm that the reporter activation via the IFNa pathway was not affected by stable transfection of PD-L1. The selected clone 45 (DBR Concept ID CL022702) showed comparable IFNa stimulation to HEK-Blue IFNa/p parental cells. Quantification of the cell surface PD-L1 using a bead based quantification kit (BD, #340495) revealed low PD-L1 surface expression level for HEK-Blue IFNa/p parental cells (~300 PD-L1 molecules/cell on cell surface; herein also referred to as “parental cells”) and high PD-L1 surface expression levels of clone 45 (-25000 PD-L1 molecules/cell on cell surface).

Example 4: Generation and testing of molecules to identify suitable cytokine linker lengths

In order to assess whether the Fab and cytokine subunits within a CISS molecule can simultaneously bind to the target antigen and cytokine receptors in the intended context, generated Fab-cytokine fusions with an attenuated IL-2v molecule were generated for testing in PD1 positive and negative IL-2 reporter cell assays. The cell lines used for these assays were made as described in Example 3.

An overview of the molecules that were created for this example is shown in Table 1. Various Fab fusion proteins were generated by fusing the ligand IL-2v via a linker to the N-termini of different Fabs comprising the binding regions of the three anti-PDl antibodies 1040int, oncL, and Pembrolizumab. Since IL-2v leads to quite potent receptor activation, an attenuated variant of IL-2v, IL-2v Q126T (having decreased IL-2RP affinity), was used as ligand in the Fab-fusions for this assay in order to better distinguish the PD1 -dependent receptor activation from IL-2v binding directly to its receptor without binding of PD1.

In order to investigate whether the selection of the N-terminus that the IL-2v was fused to had an impact on the target binding of the Fab, two corresponding sets of molecules were generated, one where IL-2v_Q126T was fused to the N-terminus of the light (kappa) chain of the Fab molecule and another one where it was fused to the N-terminus of the heavy chain (HC) of the Fab molecule.

Moreover, different molecules with three different linker lengths (6x, lOx, and 14x) attached to the anti-PDl Fab 1040 were generated to assess whether the linker is long enough to reach between the target and the cytokine receptor on the cell surface. For the N-terminal fusions of IL-2v_Q126T to the pembrolizumab- derived, only linker lengths lOx and 6x were used, resulting in 17 molecules overall. Table 1: Molecules generated to test target-binding of anti-PDl Fab IL-2 fusions

To perform the assay, our fusion molecules and controls (controls prepared in duplicate) were prepared as titration series and applied to HEK-Blue IL-2 reporter cells. The PD1 negative (parental) IL-2 reporter cell line (“HEK-Blue IL-2”) and the PD1 positive (CL1AA1486) IL-2 reporter cell line (“HEK-Blue IL-2 PD1”) were used. An additional condition was used where the PD1 positive reporter cells were preblocked with antibodies (250 nM each of P1AH4157 and P1AG3741 for 1 h by 37°C) known to be competitive with those of the molecules being assayed (“HEK- Blue IL-2 PD1 block”). First, 1x10⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat- inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant- nr-1) in a 384 well plate, followed by 25 pl of the blocking antibodies or media for 1 h 37°C incubation. Subsequently, 3x titration series (12 steps) of the various test molecules and controls were prepared, starting at 15 nM (final concentration in assay well between 5 nM and 0.00003 nM). 25 pL of each test molecule at each concentration was transferred to 384-well flat bottom plates. As positive control, recombinant IL-2v (P1AD6412) was used in the same concentration range. Incubation was performed overnight at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) was prepared and 20 pL/well was distributed among wells of new 384- well flat bottom plates (#781098), followed by addition of 20 pL of supernatants of the treated cells. Substrate turnover by the expressed SEAP ran for 2 to 6 h before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

The results of the HEK-Blue assay are shown in Figure 4 A-D. A comparison of the response to the IL-2v-only control P1AF6412 between the three conditions confirm that all conditions all had a similar IL-2 reporter response, while the two anti-PDl-only controls P1AH4157 and P1AG3741 did not elicit any IL-2 reporter response at all (Figure 4 D). Where the linker between the cytokine and the Fab was of sufficient length to cover the distance between the target and the cytokine receptor at the cell surface, the observed signal should be enhanced in the presence of the target. Where the observed signal was not enhanced, this may signify that binding to the cell surface is not occurring effectively, that the cytokine does not permit fusion to the selected terminus, that there is a protein quality issue or that the molecule localizes to the cell surface but that the cytokine is sequestered at the target in a conformation that is not amenable to cytokine receptor binding. The results confirmed that for all targeting anti-PDl Fabs that were tested, at least one fusion site and linker length could be identified for which the cytokine and the PD1 Fab was tolerant of fusions. This is clear from the heightened activation of IL-2 pathways in the “HEK-Blue IL-2 PD1” (= PD1 positive) condition relative to the “HEK-Blue IL-2” and “HEK-Blue IL-2 PD1 block” conditions. The Fab Cytokine fusions generated with the anti-PDl Fab 1040int (Figure 4 A) showed generally good tolerance for the fusions. All variants except for the shortest fusion (6x) to the Fab light chain showed differentiated receptor activation, potentially this linker may be too short in this orientation for good function. For the anti-PD 1 -Fabs Pembrolizumab (Figure 4 B) and 376 (Figure 4 C) some variants also were clearly functional, as PD1 dependent IL-2 activation could be seen with the molecules P1AI4351, P 1 AI4360, P 1 AI4359 and P 1 AI4361.

Example 5: Generation and testing of CISS Fab molecules a) Generation of the molecules

In order to identify the optimal parameters for the CISS mode of action, a pool of 37 different PD1 IL-2v CISS molecules was designed that were based on a Fab variant of the anti-PDl antibody 1040int that functioned as the first antigenbinding moiety here. IL-2v (the ligand) was attached to the heavy chain (HC) of the anti-PDl Fab, while the anti-IL-2v mask (the masking moiety) was attached to the light chain (LC). The format of the generated molecules corresponds to the schematic depiction shown in Figures 1 A and 2 C.

The sequences of the anti-IL-2v masks are listed in Table 2. Different linker lengths were used for attaching both the cytokine (6x and 14x) and the anti-IL-2 v mask (2x and 6x) to the first-antigen binding moiety. For the scFv masks P029.221.AM2, P017.333, P017.093 and MT204, two different orientations were tested, VH-VL (i.e. VH C-terminally linked to N-terminus of VL via peptide linker) and VL-VH (i.e. VL C-terminally linked to N-terminus of VH via peptide linker). In instances where the mask was derived from a standard antibody (221.AM2, 093 and 333), corresponding scFvs were generated using a 21 amino acid Gly-Ser linker in both orientations (VH-VL and VL-VH). An overview of the molecules generated for this assay is shown in Table 3.

Table 2 - IL-2v binding masks (scFv in KH (heavy chain-light chain) orientation) and sequences

The plasmids for the CISS Fab molecules of Table 3 were generated using gene fragment synthesis (Twist Bioscience, USA) and Gibson assembly® as described in Example 1. Expressions and purifications were performed according to the small-scale expression method outlined in Example 1. Some of the molecules shown in Table 3 were additionally also generated according to the mid-scale protocol as described in Example 1 using CH1-XL resin.

37 molecules were thus generated. These molecules presented potential complete CISS Fab molecules and were to be screened for mutually exclusive (or altered) binding between the target antigen PD 1 and the masking interaction between the mask and the cytokine IL-2v, via SPR and - for selected molecules - also in a HEK-Blue assay. For each mask, a Fab mask-only fusion was made as control molecule. The attenuated IL-2v molecule, IL-2 Q126T, and the anti-PDl Fab only (1040int) were used as additional controls. Table 3 - PD1 IL-2 CISS Fab molecules generates for proof-of-concept

b) SPR testing of the CISS Fab PD1-IL-2 molecules

First, the CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify molecules with properties that were suitable for potential CISS function. SPR assays were performed using the Fab capture setup described in Example 2. Analyte was flown at 20 nM, 100 nM and 250 nM. 50 nM - 100 nM of the CISS molecules or precursors were captured. Dissociation times were 240 to 480 seconds.

Three analytes were flown to assess the function of the PD 1 -IL-2v molecules: a) Human PDl-Fc protein was flown in order to assess for anti-PDl -Fab-to- PD1 or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-PDl Fab to PD1 interaction. Use of PD1 as an analyte is also of interest for mask (Figure 2 A) or IL-2v (Figure 2 B) individual fusions to an N-terminus of the anti-PDl Fab. Fusion PD1 kinetics can be compared to those of the Fab alone in order to assess for the alteration ofPDl binding. b) IL-2Rbg-Fc was flown as analyte to assess for function of the mask or to confirm correct folding of a free IL-2v component. Where a mask is concerned, if IL-2Rbg did not bind or binding was greatly diminished it was assumed that at least the IL-2Rb binding surface of IL-2v was masked or otherwise altered. Depending on the kinetic, binding of the IL-2Rbg may suggest either binding to IL-2rbg or to IL-2rb alone, the former suggesting an absence of effective masking and the latter that the mask may obscure the IL-2Rg interface alone. c) Lastly, IL-2v was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding for complete CISS Fabs.

In Table 4, the results of the SPR assays demonstrating target binding are summarized. The Table shows the capture level of the molecules in the cycle in which 250 nM of analyte was subsequently flown, response ratio (in [%]) and a summary of the observed binding to the target for each analyte flown. “B” indicates “binding”, “LB” indicates “low binding” and “N/MB” indicates “no/minimal binding”. Due to the high-throughput methods used in producing the molecules for the assay, not all proteins could be produced with satisfactory quality, leading in some instances to SPR sensorgrams that were inconclusive. Quality of the capture is indicated in the table as “Ok” for satisfactory capture, “Low” for low capture and VL/N for “very low/no capture”.

Many sensorgrams were imperfect due to protein quality issues, however, for many of the generated molecules, the desired CISS Fab mutual exclusivity (no target binding whilst the masking interaction is in place) could be demonstrated. For example, with mask 093 in the VL-VH orientation with a 2 amino acid linker on the 1040 VLN-terminus and the IL-2v on the VH N terminus with a 14 amino acid linker CISS mutual exclusivity seemed to be present (molecule Pl AI4382). The precursors of this molecule, including the mask-Fab fusion (P1AI4461) and IL-2v Q126T-Fab fusions (P1AI4346) retained binding to PD1 and also had binding to IL-2v and IL-2Rbg respectively. Similarly, mask 333 also exhibited dramatically lower PD1 binding with a similarly oriented CISS Fab (molecule P1AI4381, precursors P1AI4460 and P1AI4346) and mask 221. AM2 too, although with the scFv oriented VH-VL (P1AI4385, precursors P1AI4464 and P1AI4346).

Table 4: Results of the SPR assays for the CISS Fab PD1 IL-2 molecules

CISS molecules made with the MT204 mask appeared to retain some IL-2v binding, this is likely because the MT204 prevents IL-2v binding to the IL-2R gamma but not IL-2R beta and thus binding is present to IL-2Rbg although reduced. IL-2Rb mask molecules were difficult to interpret potentially due to protein quality issues. c) Functional characterization of the CISS Fab molecules using HEK blue assay

To evaluate the masking capacity of the CISS Fab molecules, a selection of the molecules of Table 3 were used for a HEK-blue IL-2 reporter cell assay which was performed as described in Example 4 using the “HEK-Blue IL-2”, “HEK-Blue IL-2 PDl” and HEK-Blue IL-2 PD1 Block” conditions. The results of the experiment are shown in Figures 5 A - E. In comparison, Figure 4 D shows the effect of the IL-2v molecule alone on the cells. The tested molecules were of the format shown in Figure 1 A, i.e. they were based on Fabs and did not comprise an additional targeting arm or an Fc domain. The primary aim of this investigation was to determine whether the CISS molecules effectively masked the IL-2v molecule. For the majority of CISS molecules tested, a very substantial reduction in IL-2R activity is seen in all conditions relative to IL-2v only. A large reduction of signal may be indicative a functional masking interaction but could also be indicative of dysfunctional protein in some cases. In some instances, IL-2R activity appears to be much higher in the “HEK-Blue IL-2 PD1” condition, this may be indicative of a PD1 dependent activation due either to PD 1 binding when the mask oscillates open or to PD 1 binding and localization to the cell surface with the mask closed. PD1 binding can occur with the mask closed in the event that mutual exclusivity is not present or due to multimer formation (masks and cytokines interacting with the masks and cytokines of other molecules) which may in some cases permit antigen binding.

Example 6: Selection of suitable CISS Fab molecules based on SPR and cell assay data

Selections for potentially functional CISS Fabs can be made based on the data obtained in the cell assays and SPR experiments outlined in the Examples 4 and 5. Figure 6 displays an example dataset of a PDl-IL-2v CISS Fab and precursors that have each of the desired properties for a CISS molecule. Such a molecule could be selected for the addition of a targeting arm and further testing. Table 5 summarizes the molecules discussed.

Table 5 - Exemplary data set to demonstrate CISS Fab selection

Figure 6 B shows the results of SPR assays, essentially as performed in Example 2 (analyte concentrations 20 nM, 100 nM and 250 nM with dissociation time of 240 s for sensorgrams a, b, c and d, analyte concentrations 25 nM, 75 nM and 225 nM with dissociation time of 480 s for sensorgrams e, f, g, and h) show that the PD1 binder functions in isolation to bind PD1 (Figure 6 B, sensorgram a) and continues to bind when fused to either the mask or cytokine at each N-terminus (Figures 6, sensorgrams e and g). The mask functions to bind IL-2v (Figure 6 B, sensorgram f) and the IL-2v Q126T binds to its receptors (Figure 6 B, sensor gram h) when fused to the Fab in isolation. The complete CISS molecule (P1AI4382, Figure 6 B, sensorgrams b, c and d) displays apparent mutual exclusivity and does not bind to PD1 since the mask and IL-2v are presumably interacting and sterically blocking the paratope, furthermore external IL-2v and IL-2Rbg cannot bind to the molecules since the masking interaction occurring within the molecule is occupying the requisite interaction surfaces. HEK-Blue assays with the relevant molecules in Table 5, performed essentially as in Examples 4 and 5, confirm that the cytokine linker is of sufficient length to reach the cytokine receptors (Figure 6 A, P1AI4346) when bound to PD1 and also that the CISS Fab effectively masks the IL-2v cytokine (P1AI4382).

Where it is not possible to obtain all of these selection data due to the potential absence of some control or CISS molecules or experimentation components such as SPR analytes or assay cell lines, selections can nevertheless be made for further investigation based on the findings of data that is available.

Example 7: Generation of targeted PD1 IL-2 CISS antibody molecules

Two CISS Fab molecules of interest were used to generate PDl-targeted IL- 2v CISS molecules that comprise an additional Fab arm for targeting the CISS molecule to the cell surface (for a schematical representation of the molecule format see Figure 1 B). The proposed mechanism of action for such a targeted CISS molecule is shown in Figure 3 B. The second antigen-binding moiety binds to the target molecule on the cell surface, which generally increases the local concentration of the target molecule relative to the CISS molecule and facilitates the binding of the CISS targeting binder which releases the ligand to bind to the ligand-binding moiety. The presence of the second antigen-binding moiety thus increases not only the efficiency, but also the specificity of target-dependent binding to the ligand-binding moiety.

Two CISS molecules were made using a lower affinity variant of the 221.AM2 mask previously used. The IL-2 variant IL-2v (with abolished CD25 binding) was used in these molecules, since the targeting to CD25 positive cells was not desired. The Fab part of the anti-PDl (376) antibody was always used as a targeting arm. The Fab part of the anti-PDl (1040int) antibody was used for generating the CISS arm. The linker for the masks had a length of 2 AA, the cytokine linkers had lengths of 14 or 18 AA. The tested targeted CISS molecules are shown in Table 6. The SEQ ID NO:s of the generated molecules are shown in Table 7.

Plasmids were created using a combination of Gene Synthesis (Twist Bioscience, USA) and Gibson Assembly (NEBuilder® HiFi DNA Assembly Master Mix E2621X (New England BioLabs)) according to example 1. Expressions and purifications were performed according to the Mid-scale expression procedure in example 1 using MabSelect SuRe-protein A resin (Cytiva Life Sciences, USA) for affinity purification. Mass spectrometry as well as reducing and non-reducing CE- SDS was used to confirm whether molecules were intact and of the expected size.

Table 6: Targeted CISS molecules

Table 7: Sequences of targeted CISS molecules and controls

HEK-Blue IL-2 reporter cell assays were used to assess the IL-2 activity of the targeted CISS molecules in a PD1 dependent and independent context. To perform the assay, our fusion molecules and controls were prepared as a titration series and applied to HEK-Blue IL-2 reporter cells. The PD1 negative (parental) HEK-Blue IL-2 reporter cell line (“HEK-Blue IL-2” condition) and the PD1 positive (CL1AA1486) IL-2 reporter cell line (“HEK-Blue IL-2 PD1”) were used. Additionally, the PD1 positive reporter cells (CL1AA1486) were preblocked with antibodies (500 nM each of P1AH4157 and P1AG3741 and Ih by 37°C) known to be competitive with those of the molecules being assayed (“HEK-Blue IL-2 PD1 block”). First, 1x10⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat- inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/mlNormocin (InvivoGen Catalog number ant-nr-1 ) in a 384 well plate, followed by 25 pl of the blocking antibody mixture or media for Ih 37°C incubation. Subsequently, 3x titration series (12 steps) of the various test molecules and controls were prepared, starting at 15 nM (final concentration in assay well between 5 nM and 0.00003 nM). 25 pL per fusion protein of each concentration were transferred to 384-well flat bottom plates. As positive control, recombinant IL- 2v (P1AD6412) was used in the same concentration range. Incubation of the test molecules and cells ran overnight at 37°C. QUANTI-Blue solution (Invivogen, #rep- qbs) was prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (#781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by the expressed SEAP ran for 2 to 6 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

The results are shown in Figures 7 A, B and C. As expected, huIL-2v alone (Figure 7 C, control) led to high IL-2 receptor activation in all three conditions, independent of the presence of free PD1 on the cell surface. The targeted CISS molecules Pl AJ4205 and Pl AJ4207 showed good masking efficiency as observable through the comparison of these molecules (Figure 7 A and B) with the huIL-2v control (Figure 7 C) in the “HEK-Blue IL-2” and “HEK-Blue IL-2 PD1 block” conditions. Through the comparison of the “HEK-Blue IL-2 PD1” with the “HEK- Blue IL-2” and “HEK-Blue IL-2 PD1 block” conditions it is clear that these molecules exhibit strong PD1 dependent IL-2 activity (Figures 7A and B).

Example 8: Targeting units increase CISS function

A targeting domain can be used as a means of localizing the CISS molecule to a region of relatively high concentration of the target epitope such that the CISS binder is more likely to bind to its target once it enters an open conformation and thereby leave the cytokine unmasked and able to activate its receptors. To test the influence of the targeting domain on the CISS functionality and to compare targeted and untargeted molecules directly, the molecules shown in Table 8 were generated. A schematic depiction of these molecules is shown in Figure 1 C. All molecules contained the anti-PDl Fab 1040int as first antigen-binding moiety and the cytokine variant IL-2v as ligand. Instead of a second Fab, an anti-PDl VHH (G05) was used as additional anti-PDl targeting arm in this example. The 093 mask was attached to the antigen-binding moiety in the orientation VK VH with a 2x linker. Corresponding molecules wherein the CISS Fab is fused to the N-terminus of an Fc domain, but having no targeting arm were generated and tested against the corresponding molecules having a targeting arm.

Table 8: PDl-targeted IL-2v CISS molecules with second targeting arm

Plasmids were generated by gene synthesis and Golden gate assembly as described in Example 1. Molecules were expressed and purified according to the mid-scale purification protocol described in Example 1 using CaptureSelect™ CH1- XL Affinity Matrix (Thermo Fisher Scientific, USA).

Figure 8 shows representative data using HEK-Blue IL-2 reporter assays comparing PDl-IL-2v CISS with and without targeting subunits binding to PD1, using molecules P1AJ7869 and P1AJ7915.

To perform the assay, test molecules and controls were prepared as a titration series onto either the PD1 negative (parental) IL-2 reporter cell line (“HEK-Blue IL- 2”) or the PD1 positive (CL1AA1486) IL-2 reporter cell line (“HEK-Blue IL-2 PD1”). First, 3x titration series (12 steps) of the various fusion proteins and controls were prepared, starting at a concentration of up to 460 nM, the range differs for each protein (final concentrations in the assay wells were between 230 nM and 0.0039 nM). 25 pL of each test molecule at each concentration was transferred to 384-well flat bottom plates. Subsequently, 1x10⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat-inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant-nr-1) into the 384 well plate. As a positive control, recombinant IL-2v (P1AD6412) was used in the same concentration range. Incubation of cells and test molecules ran overnight at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) was prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (#781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by expressed SEAP ran for 2 to 6 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2). As can be seen in Figure 8, a targeting arm significantly increased the PD1 specific activity for the CISS molecule as can be seen by comparing molecules P1AJ7869 (targeted) and P1AJ7915 (untargeted). The results confirm that the targeting arm can greatly enhance function in some scenarios. These results also demonstrate that the additional targeting arm may consist of standard antibody domains (e.g. a Fab or scFv) or may also be any number of other types of protein binders, such as VHHs, endogenous protein domains, or antibody mimetics (e.g. DARPin, Anticalin, Affibodies, etc.). Potentially other targeting moieties could also be conjugated for use.

Example 9: Mutually Exclusive CISS switching enhances function relative to simple cytokine masking

When a CISS molecule is localized to the cell surface via a second antigenbinding domain as targeting arm, it relies upon the mask being held open when it enters the “Off’ state by either CISS binding to the target antigen and/or cytokine binding to the receptor. Since the cytokine can activate receptors by direct binding to its receptors whilst the mask is in the “off’ state, not all activity is dependent upon first binding to the target of the CISS Fab via the additional targeting arm. To test this, targeted IL-2v CISS molecules having the same second targeting arm, but different first antigen-binding moieties were made.

The plasmids for the CISS Fab molecules of Table 9 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix Ml 100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA).

HEK Blue IL-2 reporter cell assays were performed as described in Example 8, data is shown in Figure 9. Two CISS molecules of the structure shown in Figure 1 C were tested that were identical aside from their first antigen-binding moieties (Table 9). Both molecules contained the cytokine variant IL-2v as ligand which was attached to the N-terminus of the light chain (LC) of the first antigen-binding moiety via a 14x linker. As in Example 8, an anti-PDl VHH (G05) was used as second anti- PD1 targeting arm. The 093 mask was attached to the heavy chain (HC) N-Terminus of the antigen-binding moiety in the orientation VK VH with a 2x linker. The antigen-binding moieties bind to either PD1 (1040int Fab) or have no known specificity (DP47 Fab).

Since the DP47 clone is considered to be essentially inactive for binding, any observed receptor activity from this CISS variant is assumed to be predominantly due to direct IL-2v to IL-2R binding whilst the mask is in the “off’ state. Some PD1 dependent activation appears to be present but the effect is relatively low (Figure 9, upper left panel). By contrast, where a PD1 dependent mutually exclusive switch is included, greatly increased potency is observed (Figure 9, upper right panel). Untargeted (Figure 9, lower left panel) or PD-1 targeted huIL-2v without masking or CISS functionality (Figure 9, lower right panel) are shown for comparison and show no signfificant difference between target-expressing and target-non-expressing reporter cells.

Table 9: PD1- and DP47 targeted IL-2v CISS molecules with second targeting arm

Example 10: Unmasking in an intramolecular as opposed to intermolecular fashion

The molecules shown in Table 10 are created to show the molecules unmasking in an intramolecular as opposed to intermolecular fashion. The proposed mechanism for this intramolecular unmasking is shown in Figure 13. In the absence of the target antigen, the masking moiety binds to the ligand, here a receptor binder (e.g. a cytokine). In the presence of the target antigen, the second antigen-binding moiety will bind to the target antigen first. When the masking moiety opens, the first antigen-binding moiety (here a Fab directed to the target antigen) will also bind to the target at a separate epitope. The two-fold binding to the target antigen by the first and the second antigen-binding moieties results in efficient release of the ligand (i.e. the receptor binder) which is now free to bind to the ligand-binding moiety (i.e. the ligand’s receptor) and trigger receptor activation. a) SPR screening and generation of molecules

In order to find molecules with the capacity to bind to a single PD1 twice in the context of the desired conformation we made precursor molecules to test for binding to PD1 which excluded the IL-2v cytokine. These precursors were constructed with the ultimate aim of taking some of those CISS Fabs candidates with known good screening properties based on cell assay and SPR results from examples 2-4 and adding a second PD1 binder (VHH G05) to the N-terminus of the mask (VHH G05 binds non-competitively with the 1040int binder to PD1). For example, fusion of IL-2v to the N terminus and of the heavy chain of 1040int Fab with a 14x linker, with the 221.AM2 mask (VH-VL) fused to the light chain N terminus with a 2 amino acid linker looked promising in screening SPR and cell assays (examples 2- 4).

We therefore constructed a precursor intramolecular molecule based on this orientation which excluded the IL-2v but included the 221.AM2 mask on the N- terminus of the light chain (via a 2x linker), we further added the G05 targeting VHH N-terminally to this via a 20x Gly-Ser linker. An Fc was also added to this molecule (molecule P1AJ7955) in order to facilitate potential further testing. Finally we also made complete molecules featuring CISS Fabs with N-terminal G05 VHH fusions. One example (P1AJ6652) is similar in construction to molecule P1AJ7955 but features a VH N-terminal IL-2v on a 14x linker, a lower affinity variant of the 221 mask (either V3 which is the non-matured parental mask P029.221 and has the lowest affinity, or V2 which differs from V3 by the amino acid substitution IhlO2F and has an affinity that is between V3 and the 221.AM2 mask) and no Fc. Intramolecular unmasking is potentially more efficient than many intermolecular scenarios partly because when the mask opens the PD1 epitope of the 1040int anti- PD1 is theoretically at rather high effective concentration relative to the 1040int paratope independent of cellular surface concentrations.

The plasmids for the CISS Fab molecules of Table 10 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix Ml 100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA). Pl AJ6652 was expressed twice, once with the small scale purification and expression protocol in example 1 and once with the aforementioned mid-scale protocol. Table 10: PDl-targeted IL-2v CISS molecules for intramolecular unmasking

We then used SPR to compare the potential tandem binding precursor molecules with molecules that featured either of the PD1 binders (1040 or G05) in isolation. SPR was performed as described in example 2 using 50 nM capture and analyte concentrations of 25 nM, 75 nM and 225 nM. Dissociation time was 480 s. Figure 15 shows example sensorgrams using molecules P1AJ7955, P1AI4373 (1040int Fab) and P1AJ7982 (Fc containing bispecific molecule featuring a nonfunctional Fab (DP47) fused to one Fc polypeptide and the G05 VHH to the other Fc polypeptide). When comparing the PD1 analyte sensorgrams of P1AJ7955 with the two individual PD1 binders it is clear that the affinity of P1AJ7955 is significantly higher than either of PD1 binders alone. This is indicative of a capacity for Pl AJ7955 to bind to the PD1 twice resulting in improved binding. When IL-2v is used as an analyte we can also see that the 221.AM2 scFv contained within molecule Pl AJ7955 retains good binding indicating no fusion intolerance induced by the G05 VHH. P1AJ6652 (small scale purified material) was also examined in an identical SPR setup but with a shorter dissociation time of 30 s. SPR results for P1AJ6652 confirm that the masking interaction appears to be functional (as seen by the absence of IL- 2v or IL-2Rbg binding). The kinetic of PD1 binding for molecule P1AJ6652 also appears to be quite close to molecule P1AJ7982 (G05 VHH bispecific molecule) which can be explained by the G05 VHH being the dominant driver of PD1 binding in both cases. b) HEK-Blue assays

HEK-Blue IL-2 reporter cell assays are used to examine the P1AJ6652 molecule with potential intramolecular unmasking (CISS Fab and targeting VHH binding two different epitopes of the same PD1). To perform the assay, our test molecule and control (P1AD6412) are prepared as a titration series and applied to reporter cells. Three HEK-blue cell linesare used; the PD1 negative (parental) IL-2 reporter cell line (“HEK-Blue IL-2”); the high PD1 expression IL-2 reporter cell line (CL023814: “HEK-Blue IL-2 PD1 high”); and the low PD1 expression IL-2 reporter cell line (CL023813: “HEK-Blue IL-2 PD1 low”). In addition, each of these cell lines is used in an additional preblocked condition with PD1 antibodies (500 nM each of P1AH4157 and P1AG3741 and Ih by 37°C) known to be competitive with the PD1 binders of the molecule being assayed (“HEK-Blue IL-2 block”, “HEK-Blue IL-2 PD1 High block”, “HEK-Blue IL-2 PD1 Low block”).

First, 1x10⁴ reporter cells/well are seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat- inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/mlNormocin (InvivoGen Catalog number ant-nr-1 ) in a 384 well plate. Thereafter, 25 pl of the blocking antibody mix or cell media only is distributed to each well, cells are then incubated for 1 h at 37°C. Subsequently, 3x titration series (12 steps) of the various fusion proteins and controls are prepared in triplicate, concentrations vary by protein starting at 100 nM (final concentration in assay well between 50 nM and 0.0003 nM). As a positive control, recombinant IL- 2v (Pl AD6412) is used in the same concentration range. 25 pL of the test molecules of each concentration are transferred to 384-well flat bottom plates. Cells and test molecules are incubated for 16-20 hrs at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) is prepared and 20 pL/well is distributed among wells of new 384-well flat bottom plates (#781098), followed by addition of 20 pL of supernatants of the treated cells. Substrate turnover by produced SEAP runs for 2.5 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

Example 11: Targeted PDl-IL-2v CISS p-Stat5 Assay

Upon induction of the IL-2 signaling pathway a protein called STAT5 is phosphorylated, phospho-STAT5 (p-STAT5) can be fluorescently stained using antibodies and populations of cells can thereby be assessed for IL-2 activation. Such a p-STAT5 assay is used to analyze the PD1 dependent IL-2 activity when applying CISS molecules to activated donor T cells. The molecules shown in Table 11 are tested. Table 11 - PDl-targeted IL-2v CISS molecules for p-Stat5 assay

The plasmids for the CISS Fab molecules of Table 11 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix Ml 100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA).

IL-2R Signaling Assay

The following assay was performed to determine the potency and cis/trans-signaling of a PD-l-IL-2v immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide with additional mutations) was provided.

For this purpose, CD4 T cells from healthy donor PBMCs were sorted with CD4 beads (Miltenyi, #130-045-101) and activated for 3 days in presence of 1 pg/ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and half of the cells were labeled with Cell Trace Violet (CTV) (5 pM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were left unlabeled.

Then, the unlabelled cells were incubated with a saturating concentration of a competing anti-PD-1 antibody (in-house molecule, 10 pg/ml) for 30 minutes at RT followed by several washing steps to remove the excess unbound anti-PD-1 antibody (“PD-1 blocking”). Thereafter, the PD-1 pre-blocked cells (25 pl, 6*10⁶ cells/ml) were co-cultured 1 : 1 with the PD-1+ CTV-labeled cells (25 pl, 6xl0⁶ cells/ml) in a V-bottom plate before being treated for 12 minutes at 37 °C with increasing concentrations of treatment immunoconjugates (50 pl, 1 : 10 dilution steps). To preserve the phosphorylation state, an equal amount of Phosphoflow Fix Buffer I (100 pl, 557870, BD Bioscience) was added after 12 minutes incubation with the various constructs. The cells were then incubated for an additional 30 minutes at 37 °C before being permeabilized overnight at -80 °C with Phosphoflow PermBuffer III (558050, BD Bioscience). On the next day, STAT-5 in its phosphorylated form was stained for 30 minutes at 4 °C by using an anti- STAT- 5P antibody (47/Stat5(pY694) clone, 562076, BD Bioscience).

The cells were acquired at the fluorescence-activated cell sorting (FACS) BD- Symphony A3/A5 (BD Bioscience) instrument. The frequency of STAT-5P was determined with Flow Jo (VI 0) and plotted with GraphPad Prism (v8).

The dose-response curves on PD-1+ T cells provided information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 mediated delivery, showed the potency of the molecules in providing IL-2R signaling independently from PD- 1 expression.

Table E: Results of the IL-2R Signaling Assay after 15 min

Table F: Results of the IL-2R Signaling Assay after 1 h

When examined using this STAT5-p assay, “093” variant masked IL2v CISS molecules (P1AL7925, top row, and P1AL7926, bottom row) produced a clear window of PDl-dependent function (Table E and F; Figure 31A). This can be observed through comparison of PD1 blocked and non-blocked conditions. FAP- IL2v (P1AA5355 FAP-IL2v) is present as a control and represents untargeted IL2v since FAP is not expressed on the cells present (see Table E and F).

Through comparison of molecules Pl AJ7935 (no targeting arm, middle row) and P1AL7893 (PD1 -targeting VHH, top row), we can see a clear added benefit of the PD1 targeting arm in this setup (Table E and F; Figure 31 B).

In Figure 31 B, bottom row, we further observe the potency of the “intramolecular” molecule P1AJ6652, which is capable of binding to a single PD1 molecule twice. Here a 143x increase in potency is seen in the PDl-positive scenario relative to the PD1 -blocked (comparison of EC50 values, Table F).

In Figure 31 C the capacity of the “221” mask to inhibit IL2Ra in addition to IL2Rb is leveraged to make three CISS molecules that use IL2wt. Once again, we see a strong increase in IL2R signaling in the PD1 non-blocked scenario relative to the blocked indicating that these CISS molecules deliver targeted cytokine function.

Example 12: Modification of the cytokine can enhance CISS specificity

The target specificity of CISS molecules is to some extent dependent upon the affinity of the cytokine for its receptors. Where the affinity for the receptors is higher there is greater risk of direct cytokine to cytokine receptor binding when the mask interaction is off without first binding to the target. Therefore, mutations of the cytokine that reduce cytokine receptor binding but do not abolish signaling could be useful in improving specificity since the CISS Fab should more preferentially bind to the target when open, It can also be used as a means to adjust the cytokine potency of the molecule without modifying binding to the target and thereby potential affinity and dose dependent secondary functions of the molecule such as for example receptor antagonism may be unaffected.

Table 12 shows the targeted CISS example molecules with variations of the IL-2 cytokine. The plasmids for the CISS molecules of Table 12 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix M1100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA)

Table 12 - PDl-targeted IL-2v CISS molecules with different cytokine variants

In order to assess these molecules HEK-Blue™ IL-2 reporter cell assays were performed as described in Example 8. Figure 16 shows HEK-blue™ IL-2 assay data using the same targeted CISS molecule but for a single amino acid substitution in the IL-2v, Q126T. The Q126T mutation lowers affinity for IL-2Rb without completely abolishing IL-2R signaling when the cytokine is localized to the cell surface. When compared to the analogous IL-2v molecule (P1AJ7893; Figure 16, left image) the PD1- IL-2v Q126T targeted CISS variant (Pl AJ7904; Figure 16, right image) shows lower activity on PD1 positive cells “HEK-Blue IL-2 PDF’, the Q126T variant also shows a decrease in IL-2R activity in the PD1 negative “HEK-Blue IL-2” condition thereby demonstrating that the CISS molecule can be further finetuned by mutation of the cytokine. Example 13: CD8 IL-2v CISS Fab molecules

In order to demonstrate that the CISS mode of action also works for target antigens other than PD1, the set of CD8-targeted IL-2v CISS molecules shown in Table 13 was designed based on a Fab variant of the anti-CD8 antibody 0KT8vl 1 which functioned as the first antigen-binding moiety in these molecules. Plasmids were generated by gene synthesis and Golden gate assembly as described in Example 1. Molecules were expressed and purified according to the small-scale purification protocol described in Example 1. IL-2v (the ligand) was attached either to the heavy or the light chain of the anti-CD8 Fab, while the anti-IL-2v mask (the masking moiety) was attached to the N-terminus of the other chain. The format of the generated molecules corresponds to the schematic depiction shown in Figures 1 A and 2 C. Different linker lengths were used for attaching both the cytokine (14x and 18x) and the anti-IL-2v mask (2x and 4x) to the first-antigen binding moiety. For the scFv mask P017.093, the orientation VL-VH (VH attached to the Fab light chain C- terminus) was used. The SEQ ID NO:s of the molecules are listed in Table 14.

Molecules without either the masking moiety or the IL-2v moiety were used as controls where available. As an additional control IL-2 CD8 fusion proteins and IL-2v were used. The CD8 IL-2v CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify clones that suggested satisfactory masking properties and the ability for mutually exclusive binding. The SPR based assays were further used to determine the binding kinetics of the CISS Fab units. Their respective “precursor” molecules (the Fabs alone, mask-Fab fusions without IL-2v and IL-2v- Fab fusions without mask) were used as control. An Fc fused bispecific mAb molecules with a single 0KT8vl 1 targeting arm was used for the assessment of 0KT8vl 1 binding (P1AJ7944).

Table 13: CD8 IL-2 CISS Fab molecules tested

Each assay was performed essentially as described in Example 4. Three analytes were flown to assess the function of the CD8 IL-2v molecules: a) Human hCD8a-Fc protein was flown in order to assess for anti-CD8a- Fab-to-CD8a or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-PDl Fab to PD1 interaction. Use of PD1 as an analyte is also of interest for mask or IL-2v individual fusions to an N-terminus of the anti-PD 1

Table 14 - Sequences of tested molecules

Fab. Fusion PD1 kinetics can be compared to those of the Fab alone in order to assess for the alteration of PD1 binding. b) IL-2Rbg-Fc was flown as analyte to assess for function of the mask or to confirm correct folding of a free IL-2v component. Where a mask is concerned, if IL-2Rbg did not bind or binding was greatly diminished it was assumed that at least the IL-2Rb binding surface of IL-2v was masked or otherwise altered. Depending on the kinetic, binding of the IL-2Rbg may suggest either binding to IL-2rbg or to IL-2rb alone, the former suggesting an absence of effective masking and the latter that the mask may obscure the IL-2Rg interface alone. c) Lastly, IL-2v was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions and may also deliver an indication of correct folding for complete CISS Fabs.

In Table 15, the results of the SPR assays demonstrating target binding are summarized. The Table shows the capture level of the molecules in the cycle in which 225 nM of analyte was subsequently flown, Response ratio (in [%]) and a summary of the observed binding to the target. “B” indicated “binding”, “LB” indicates “low binding” and “N/MB” indicates “no/minimal binding”.

SPR results show that precursor molecules behaved as desired, OKT8vl l Fab binds to hCD8a and all mask-Fab and IL-2v Q126T-Fab fusions also bind to hCD8a but also to IL-2v and IL-2Rbg respectively. For numerous CISS Fab generated molecules, apparent mutual exclusivity could be demonstrated. P1AJ6285 is an example of interest. P1AJ6285 has the 093 mask (VL-VH) fused to the OKT8vl 1 N-terminus of the OKT8vl 1 Fab VH with the IL-2v fused to the VL via a 14 amino acid linker. As discussed precursors for P1 J6285 behave as expected (P1AJ7944, P1AJ6281, P1AJ6295) in SPR whilst P1AJ6285 has abolished binding for all 3 analytes (hCD8a, IL-2v, IL-2rbg) indicating that the masking interaction is likely occurring and sterically inhibiting the binding of hCD8a. As such all mechanistic criteria that we assess via SPR are met for Pl AJ6285 to be a potentially functional CISS molecule once targeted.

Table 15 - Results of the SPR assays

HEK-blue IL-2 assays with CD8 positive and negative cells were used in order to assess whether the Fab and cytokine subunits within the CD8 IL-2 CISS molecule can simultaneously bind to the target and cytokine receptors in the intended context, we generated Fab-cytokine fusions with an attenuated IL-2v molecule for testing in IL-2 reporter cell assays. We also sought to assess whether the complete CD8a CISS molecule successfully masks the IL-2v.

To perform the assay, our test molecules and controls were prepared as a titration series and applied to IL-2 reporter cells. The PD1 negative (parental) IL-2 reporter cell line (“HEK-Blue IL-2”) or the PD1 and CD8A positive reporter cells (CL023901 : “HEK-Blue IL-2 CD8”). First, 3x titration series (12 steps) of the various test molecules and controls were prepared in triplicate. All molecules were diluted to a starting concentration of 100 nM (final concentration in well between 50 nM and 0.0003 nM) except for controls P1AD6412 and P1AE4422 which were diluted to a starting concentration of 10 nM (final concentration in well between 5 nM and 0.00003 nM). 25 pL of each test molecule at each concentration was transferred to 384-well flat bottom plates. Subsequently, IxlO⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat-inactivated FBS (Anprotec, #AC-SM- 0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant-nr-1) into a 384 well plate. Test molecules and Cells were incubated for 16-20 hrs at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) was prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by expressed SEAP ran for 2 to 6 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

In Figure 10, HEK-blue data is shown using CISS Fab P1AJ6285 and its precursors as an example. The results show that, despite a higher sensitivity of the “HEK-Blue IL-2” cell line to IL-2v when compared to the “HEK-Blue IL-2 CD8a” cell line (Figure 10 B), the CD8a targeting of IL-2v Q126T in the Cytokine Fab fusion molecule P1AJ6295 leads to higher IL-2 signaling activity from the “HEK- Blue IL-2 CD8” cell line than the “HEK-Blue IL-2” cell line (Figure 10 A). This suggests that the IL-2Rs can be effectively reached and bound by IL-2v whilst CD8a is also bound. When comparing IL-2v with the CISS Fab Pl AJ6285, it is clear that in both conditions the IL-2v is much more potent and this suggests that the CISS molecule is effectively masking its internal IL-2v. Therefore P1AJ6285 appears to satisfy all of our screening criteria in terms of for a potential targeted CISS molecule.

Example 14: CD8 IL-2 CISS molecules (CD8-targeted) The addition of a targeting arm to a CISS Fab can confer enhanced specificity and potency to the molecule. In order to make a targeted CISS CD8 IL-2v molecule an Fc and VHH targeting arm (VHH5v2 targeting CD8a) were attached to the 0KT8vl l Fab. 0KT8vl l is further fused to the 093 mask and IL-2v using the orientation and linker lengths of the screened Pl AJ6285 candidate. The plasmids for the CISS Fab molecules of Table 16 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix Ml 100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA). Mass Spectrometry was used to confirm the correct formation of the protein.

Table 16 - Generated CD8-targeted CD8 IL-2 CISS molecules

Table 17 - Sequences of the CD8-targeted CD8 IL-2 CISS molecules

HEK-Blue IL-2 reporter Assays

This experiment details the potency of molecules displayed in Table 16 in HEK-Blue IL-2 reporter assay and their dependence upon CD8a. To perform the assay, our fusion molecules and controls were applied as a titration series onto IL-2 reporter cells. The CD8a negative (parental) HEK-Blue IL-2 reporter cell line (“HEK-Blue IL-2”) and the PD1 and CD8A positive IL-2 reporter cell line (CL023901 : “HEK-Blue IL-2 CD8a”) were used. Additionally, a condition in which the CD8a molecules on the PD1 and CD8A positive IL-2 reporter cells (CL023901) are pre-blocked with an OKT8 variant (P1AF4823) was used (“HEK-Blue IL-2 CD8a block”). Importantly, whilst this antibody is capable of blocking OKT8 it is not competitive with the VHH5v2 binder.

Reporter cells were incubated for 1 hour in a 15ml falcon with either blocking antibody (OKT8 variant monoclonal antibody (P1AF4823 at 1 pM) plus media (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat- inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant- nr-1) or media plus Normocin alone. 25 pL of these cells (containing IxlO⁴ reporter cells/well) were then dispensed into wells of a 384 well plate. Thereafter, 25 pl of the blocking antibody mix or cell media only was distributed to each well, cells were then incubated for 1 h at 37°C. Subsequently, First, 3x titration series (12 steps) of the various test molecules and controls are prepared in triplicate. 25 pL of the test molecules of each concentration are transferred to 384-well flat bottom plates. Cells and test molecules were incubated for 16-20 hrs at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) was prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (#781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by produced SEAP ran for 2.5 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

Results of the assays are shown in Figure 29. Figure 29 shows the dose dependent IL2 activity stimulated by the CD8a-IL2v CISS Fab (Pl AL7886, middle), targeted CISS fab (P1AL7927, bottom) and IL2v control (P1AD6412, top) molecules using the IL2 parent cell line. Through the comparison of the three graphs of Figure 29, it is clear that the mask contained within the two CISS molecules is capable of strongly inhibiting IL2 activity. Since the CISS Fab unit is the same in both the targeted and untargeted CISS Fab, we unsurpisingly find that the level of IL2 inhibition is also very similar. Through comparison of IL2v function between cell lines in the top graph it is clear that the CD8 positive variant of the IL2 reporter cell line is much less sensitive to IL2 activity (dotted line with triangles). The IL2 blocking function of the anti-IL2 mask is once again clear through the comparison of the graphs on the top (IL2v control) and the middle. IL2 activation in HEKblue cells not expressing CD8 is greatly reduced for Pl AL7927 and Pl AL7886. It is clear that a significant amount of CD8 selectivity is possible via the CISS Fab alone, as indicated by the decrease in IL2 activity when the CD8 is blocked (dashed line and squares). Furthermore, when comparing the two graphs it is clear that the second antigen-binding moiety significantly boosts this CD8-dependent CISS function. Blocking of the targeted construct is not possible owing to the non-availibility of a competitor with the VHH5v2 targeting arm. However, since the CISS Fab and targeted CISS Fab act highly similar on the IL2 reporter cells (solid line and circles), it is highly likely that successful blocking of the targeted CISS Fab would be somewhat similar to the blocking of the CISS Fab seen in the graph in the middle of Figure 29.

Example 15: IL18R IL-2v CISS molecules

To test whether CISS type mutual exclusivity could also be transferred to combinations of CISS binders wherein the antigen-binding moiety was an anti- IL18R antibody, a screen was performed to find functional anti-IL18R IL-2v CISS molecules (see Table 18). The anti-IL18R Fab 1G12 AB11.6 was combined with IL-2v. All molecules used the anti-IL-2v mask 093. Two different linker lengths for both IL-2v molecule (14x, 18x) and mask (2x, 4x) as well as alternating fusions of IL-2v and mask to heavy and light chain of the anti-IL18R binder were tested. The molecules had the format shown in Figure 2 C and did not contain an Fc domain or a targeting arm. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecules were expressed and purified according to the small-scale purification protocol described in example 1.

Table 18: IL18R1 IL-2v CISS molecules

Table 19 - SEQ ID NO:s of the molecules of Table 18

The IL18R1 IL-2v CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify clones that suggested satisfactory masking properties and the ability for mutually exclusive binding. The SPR based assays were further used to determine the binding kinetics of the CISS Fab units. Their respective “precursor” molecules (mask-Fab fusions without IL-2v and IL-2v-Fab fusions without mask) were used as control.

Each assay was performed essentially as described in Example 4. Three analytes were flown to assess the function of the IL18Rl-IL-2v molecules: a) Human IL18R1-Fc dimer protein was flown in order to assess for anti- IL18Rl-Fab-to-IL18Rl or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-IL18Rl Fab to IL18R1 interaction. Use of IL18R1 as an analyte is also of interest for mask (Figure 2 A) or IL-2v (Figure 2 B) individual fusions to an N-terminus of the anti-IL18Rl Fab. b) IL-2Rbg-Fc was flown as analyte to assess for function of the mask or to confirm correct folding of a free IL-2v component. Where a mask is concerned, if IL-2Rbg did not bind or binding was greatly diminished it was assumed that at least the IL-2Rb binding surface of IL-2v was masked or otherwise altered. Depending on the kinetic, binding of the IL-2Rbg may suggest either binding to IL-2rbg or to IL-2rb alone, the former suggesting an absence of effective masking and the latter that the mask may obscure the IL-2Rg interface alone. c) Lastly, IL-2v was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding for complete CISS Fabs.

In Table 20, the results of the SPR assays demonstrating target binding are summarized. The Table shows capture at 225 nM, response ratio (in [%]) and a summary of the observed binding to the target. “B” indicated “binding”, “LB” indicates “low binding” and “N/MB” indicates “no/minimal binding”.

Where capture levels were sufficient SPR result show that the IL18R1 binder retains the capacity to bind when fusions are present on either N terminus (P1AJ6484, P1AJ6485, P1AJ6487, P1AJ6486 and P1AJ6500). In addition all precursor mask-Fab fusions bound to IL-2v and the single IL-2v Q126T-Fab fusion to capture well also bound to IL-2Rbg. For numerous CISS Fab generated molecules, apparent mutual exclusivity could be demonstrated (between the mask-cytokine interaction and the Fab-target interaction as indicated by the inability of complete CISS molecules to bind to IL18R1). The mask-cytokine fusion orientation appeared to be key to mutual exclusivity. For example, whilst molecule Pl AJ6488 appeared to display incomplete mutual exclusivity with the 093 mask fused to the VH and the cytokine fused to the VL, Pl AJ6492 appears to be mutually exclusive with the same linker lengths but with the fusion orientations switched.

Table 20: SPR-Results

Example 16: CD19 IFNalpha constructs

To test whether the CISS type mutual exclusivity can also be transferred to other combinations of antigen-binding moieties, masking moieties and cytokines, a screen was performed to find functional CD 19 IFNalpha CISS molecules (see Table 21). A CD19 Fab (based on anti-CD19 antibody Blinatumumab, “Blina”) was combined with either wildtype IFNalpha or IFNalpha_L30A (attenuated). All molecules used the anti-IFNalpha mask P7. Two different linker lengths for both IFNalpha molecule (14x, 18x) and mask (2x, 4x) were used. The molecules had formats as shown in Figure 2 and did not contain an Fc domain or a targeting arm. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecules were expressed and purified according to the small-scale purification protocol described in example 1. Table 21: Tested CD19 IFNalpha constructs with Blinatumumab anti- CD19

Table 22: SEQ ID NO: for molecules shown in Table 21

The CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify clones that suggested satisfactory masking properties and the ability for mutually exclusive binding. The SPR based assays were further used to determine the binding kinetics of the CISS Fab units. Their respective “precursor” molecules (mask-Fab fusions without IFNa and IFNa-Fab fusions without mask) were used as control.

Each assay was performed essentially as described in Example 4. Three analytes were flown to assess the function of the CD19 IFNalpha molecules: a) Human CD 19-Fc protein was flown in order to assess for anti-CD 19- Fab-to-CD19 or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-CD19 Fab to CD19 interaction. Use of CD19 as an analyte is also of interest for mask (Figure 2 A) or IFNa (Figure 2 B) individual fusions to an N-terminus of the anti-CD19 Fab. Fusion CD19 kinetics can be compared to those of the Fab alone in order to assess for the alteration of CD 19 binding. b) IFNAR2-his was flown as analyte to assess for function of the mask or to confirm correct folding of a free IFNa component. Where a mask is concerned, if IFNAR2 did not bind or binding was greatly diminished it was assumed that the IFNa was masked or otherwise altered. IFNAR-2 may or may not bind clearly to IFNa L30A due to low the low affinity present for this attenuated variant of IFNa. c) Lastly, IFNa was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding for complete CISS Fabs.

In Table 23, the results of the SPR assays demonstrating target binding are summarized. The Table shows the capture level of the molecules in the cycle in which 225 nM of analyte was subsequently flown, response ratio (in [%]) and a summary of the observed binding to the target. “B” indicated “binding”, “LB” indicates “low binding” and “N/MB” indicates “no/minimal binding”.

SPR results show that precursor molecules behaved largely as desired. CD 19 binding was present for the mask-Fab and IFNa L30A-Fab fusions. The P7 mask bound to IFNa when fused to the Fab in isolation. Binding of the IFNAR-2 receptor ECD (extracellular domain) was not clearly observable for the IFNa L30A-Fab fusions, likely because the capture is relatively low and the affinity is also rather low when the L30A mutation is introduced. A selection of CISS Fab CD19-IFNa molecules showed apparent mutual exclusivity with target binding not possible whilst the masking interaction is ongoing. CISS Fab P1AJ6705 is an example of a CISS molecule of potential interest based on this SPR screening. Table 23: SPR results for CD19 IFNalpha constructs with Blinatumumab

Example 17: PD-L1 IFNa constructs

To test whether the CISS functionality can also be transferred to other combinations of CISS binders, masks and cytokines, a screen was performed to find functional PD-L1 IFNalpha CISS molecules (see Table 24). A PD-L1 binder (Atezolizumab Fab) was combined with either wildtype IFNalpha or IFNalpha_L30A (attenuated). All molecules used the anti-IFNalpha mask P7 (VL- VH). Two different linker lengths for both IFNalpha molecule (14x, 18x) and mask (2x, 4x) were used. The molecules had formats as shown in Figure 2 and did not contain an Fc domain or a targeting arm. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecules shown in Table 24 were expressed and purified according to the small-scale purification protocol described in example 1. The SEQ ID NO: for the molecules are listed in Table 25.

The CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify clones that suggested satisfactory masking properties and the ability for mutually exclusive binding. The SPR based assays were further used to determine the binding kinetics of the CISS Fab units. Their respective “precursor” molecules (mask-Fab fusions without IFNa and IFNa-Fab fusions without mask) were used as control. Table 24: Tested PD-L1 IFNalpha constructs with anti-PD-Ll

Table 25 - SEQ ID NO: of molecules shown in Table 24

Each assay was performed essentially as described in Example 4. Three analytes were flown to assess the function of the PD-L1 IFNalpha molecules: a) Human PD-Ll-Fc protein was flown in order to assess for anti-PD-Ll-Fab-to-PD-Ll or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-PD-Ll Fab to PD-L1 interaction. Use of PD-L1 as an analyte is also of interest for mask (Figure 2 A) or IFNa (Figure 2 B) individual fusions to an N-terminus of the anti-PD- Ll Fab. Fusion PD-L1 kinetics can be compared to those of the Fab alone in order to assess for the alteration of PD-L1 binding. b) IFNAR2-his was flown as analyte to assess for function of the mask or to confirm correct folding of a free IFNa component. Where a mask is concerned, if IFNAR2 did not bind or binding was greatly diminished it was assumed that the IFNa was masked or otherwise altered. IFNAR-2 may or may not bind clearly to IFNa L30A due to low the low affinity present for this attenuated variant of IFNa. c) Lastly, IFNa was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding for complete CISS Fabs.

In Table 26, the results of the SPR assays demonstrating target binding are summarized. The Table shows the capture level of the molecules in the cycle in which 225 nM of analyte was subsequently flown, response ratio (in [%]) and a summary of the observed binding to the target. “B” indicated “binding”, “LB” indicates “low binding” and “N/MB” indicates “no/minimal binding”.

SPR results show that precursor molecules behaved largely as desired. PD- L1 binding was present for the mask-Fab and IFNa L30A-Fab fusions. The P7 mask bound to IFNa when fused to the Fab in isolation. Binding of the IFNAR-2 receptor ECD (extracellular domain) was not clearly observable for the IFNa L30A-Fab fusions, likely because the capture is relatively low and the affinity is also rather low when the L30A mutation is introduced. All tested CISS Fabs with the P7 mask fused to the VH N terminus and the IFNa to the VL N terminus did not show complete mutual exclusivity (as indicated by the capacity of the CISS Fabs to bind PD-L1 whilst the masking interaction was in place). In addition, CISS Fabs in the opposite orientation did show apparent mutual exclusivity where the mask linker was 2 amino acids (P1AJ6812 and P1AJ6813) but PD-L1 binding was incompletely blocked where a 4 amino acid linker was used (Pl AJ7259, Pl AJ6814). Table 26: SPR results for PD-L1 IFNalpha constructs with Atezolizumab

Example 18: PD-L1 IFNa targeted constructs In order to produce targeted PD-L1 IFNa CISS molecules we used linker lengths and component orientations from the molecule Pl AJ6812 which appeared to demonstrate mutual exclusivity in our screening assay. We essentially fused this CISS Fab to an Fc domain and added a VHH (KN035) as second antigen-bindin moiety (“targeting arm”) (molecule P1AL7929). Figure 1 C shows a schematic representation of this molecule’s format. Molecules tested are displayed in Table 27.

In all molecules tested, IFNa was linked to the first antigen-bindin moiety via a 18x peptide linker. The masking moiety was linked to the first antigen-binding moiety via a 2x linker. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecule P1AL7929 was expressed and purified according to the mid-scale purification protocol described in example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA). All other molecules were expressed and purified according to the small-scale purification protocol described in example 1. Table 27: Tested molecules for targetd PD-L1 IFNa CISS constructs

Table 28: SEQ ID Nos of the molecules shown in Table 27

The molecules displayed in Table 27 are tested in the HEK-Blue assay system. For that, the fusion molecules and controls are applied as a titration series onto IFNalpha reporter cells, the parental HEK-Blue™ IFNa/p reporter cell line which has relatively low PD-L1 expression (“HEK-Blue IFNa PD-L1 low”), or the high expressing PD-L1 positive IFNalpha reporter cells (clone 45, CL022702, “HEK-Blue IFNa PD-L1 high”), that have been generated as described in Example 3 d) and e). Pre-blocked conditions are also included for both cell lines with Atezolizumab (P1AE0282) which is competitive with the Atezo Fab of the CISS Fab and also with KN035 (“HEK-Blue IFNa PD-L1 low blocked”, “HEK-Blue IFNa PD- L1 high blocked”).

First, 1x10⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat- inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant-nr-1) into a 384 well plate. Thereafter, 25 pl of the blocking antibody mix (Atezolizumab monoclonal antibody P1AE0282, 750 nM) or cell media only were distributed to each well, cells were then incubated for 1 h at 37°C. Subsequently, 3x titration series (12 steps) of the various test molecules were prepared in triplicate (300 nM starting concentration, final concentration in assay well between 100 nM and 0.0006 nM). 25 pL of the test molecules of each concentration were transferred to 384-well flat bottom plates. Cells and test molecules were incubated for 16-20 hrs at 37°C. QUANTI-Blue solution (Invivogen, #rep-qbs) is prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (#781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by produced SEAP ran for 2.5 hours before measuring the optical density (OD) at 620 nm (Tecan Safire 2).

HEK-Blue Cell Assay results for the parental IFNa reporter cell line with and without PD-L1 blocking are shown in Figure 28 A. Importantly, whilst these cells have not been modified to express PD-L1, PD-L1 is expressed at a relatively low level (see Example 3). CISS Fab molecule P1AJ6812 (Figure 28 A, upper left corner) can be seen to effectively inhibit IFNa when compared to the IFNa N-terminal Fc fusion control (P1AF6940, Figure 28 A lower right corner). Through examination of the attenuated IFNa molecule P1AJ6816 (Figures 28 A, lower left corner), it is clear that when fused N-terminally onto the heavy chain of the Atezo Fab, IFNa is capable of reaching its receptors when the molecule is bound to PD-L1. This can be observed through the decreased function of the molecules when PD-L1 is blocked, which suggests that the high level of IFNAR activation in the non-blocked scenario is dependent upon PD-L1 binding. The KN035 VHH targeted CISS Fab (P1AL7929, Figure 28 A upper right corner) shows strong PD- L1 dependent activation.

HEK-Blue IFNa cell assay results for the high expressing PD-L1 cell line (see example 3) are shown in Figure 28 B. CISS Fab molecule P1AJ6812 (Figure 28 B upper left corner) can be seen to have PD-L1 dependent activity in this high expression scenario, this can be observed by comparing the blocked and non blocked scenarios. The targeted CISS Fab (Pl AL7929, Figure 28 B upper right corner) has comparable activity to its untargeted counterpart (Pl AJ6812, Figure 28 B upper left corner) in the blocked scenario but the targeting arm can be seen to greatly enhance activation where PD-L1 can be freely bound.

Example 19: PD1 IL-21 constructs

To test whether the CISS functionality can also be transferred to other combinations of CISS binders, masks and cytokines, a screen was performed to find functional PD1 IL-21 CISS molecules (see Table 29). The anti-PDl Fab 1040 was combined with IL-21 and the anti-IL-21 mask “Aviza” (a Fab based on the antibody avizakimab). Two different linker lengths for both IL-21 molecule (14x, 18x) and mask (2x, 4x). IL-21 was fused to the heavy chain and masks were fused to the light chain of the 1040 anti-PDl Fab. The molecules had the format shown in Figure 2 C and did not contain an Fc domain or a targeting arm. Plasmids were generated by gene synthesis and Golden gate assembly as described in Example 1. Molecules were expressed and purified according to the small-scale purification protocol described in example 1 with the exception of plasmid P1AJ7944 which was produced using the mid-scale expression and purification protocol from example 1 with CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA).

Table 29: PD1 IL-21 CISS molecules

Table 30: SEQ ID Nos of the molecules shown in Table 29 and 31

The CISS Fab molecules were screened using Surface plasmon resonance (SPR) to identify molecules with properties that are suitable for potential CISS function. SPR assays were performed using the Fab capture setup described in Example 2. 50nM of molecule was captured, analyte concentrations were 25 nM, 75 nM, 225 nM and the dissociation time was 240 s.

Three analytes were flown to assess the function of the PD1 -IL-21 molecules: a) Human PDl-Fc protein was flown in order to assess for anti- PDl-Fab-to-PDl or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the maskcytokine interaction sterically inhibits the anti-PDl Fab to PD1 interaction. Use of PD1 as an analyte is also of interest for mask (Figure 2 A) or IL-21 (Figure 2 B) individual fusions to an N-terminus of the anti-PDl Fab. Fusion PD1 kinetics can be compared to those of the Fab alone in order to assess for the alteration of PD1 binding. b) IL-21R-Fc was flown as analyte to assess for function of the mask or to confirm correct folding of a free IL-21 component. Where a mask is concerned, if IL-21R-Fc did not bind or binding was greatly diminished it was assumed that the IL-21 was masked or otherwise altered. c) Lastly, IL-21-Fc was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding and function of the masks when fused in mask-Fab only fusions.

The results are shown in Table 31. Due to the high-throughput methods used in producing the molecules for the assay, not all proteins could be produced with satisfactory quality, leading in some instances to SPR sensorgrams that were inconclusive.

The SPR results showed that Avizakimab functions as an scFv both orientations (P1AJ7200 and P1AJ7201). In addition, as previously seen, fusion to the N-termini of 1040 does not prevent binding to PD1 (e.g. Pl AJ7192, Pl AJ7201). All CISS Fab molecules appeared to prevent PD1 binding to the 1040 and therefore mutual exclusivity is thought to occur. A very low amount of IL-21R-Fc binding may be present for complete CISS molecules which may indicate a protein quality issue resulting in some free IL-21 that is available for IL-21R binding. Table 31: SPR results for PD1 IL-21 CISS molecules

Example 20: PD1 IL7 molecules

To test whether the CISS functionality can also be transferred to other combinations of CISS binders, masks and cytokines, a screen was performed to find functional PD1 IL7 CISS molecules (see Table 33). The anti-PDl Fab 1040 was combined with IL7 and the anti-IL7 mask DRSPAI L7B (a Fab based on the antibody DRSPAI L7B). Two different linker lengths were used for the mask (2x, 4x) and a single linker length for IL7 (18x). IL7 was fused to the heavy chain and masks were fused to the light chain of the 1040 anti-PDl Fab. The molecules had the format shown in Figure 2 C and did not contain an Fc domain or a targeting arm. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecules of Table 32 were expressed and purified according to the small-scale purification protocol described in example 1 with the exception of plasmid P1AJ7944 which was produced using the mid-scale expression and purification protocol from example 1 with CaptureSelect™ CHI -XL Affinity Matrix (Thermo Fisher Scientific, USA). Table 32: PD1 IL7 CISS molecules

Table 33: SEQ ID Nos of the molecules shown in Table 32

The CISS Fab molecules were screened using Surface plasmon resonance

(SPR) to identify molecules with properties that are suitable for potential CISS function. SPR assays were performed using the Fab capture setup described in Example 2. 50 nM of molecule was captured, analyte concentrations were 25 nM, 75 nM, 225 nM and the dissociation time was 240 s. Three analytes were flown to assess the function of the PD1-IL-7 molecules: a) Human PDl-Fc protein was flown in order to assess for anti-PDl -Fab-to- PD1 or mask-to-cytokine mutual exclusivity of the full CISS Fab molecules, whereby the reduction of binding may suggest that the mask-cytokine interaction sterically inhibits the anti-PDl Fab to PD1 interaction. Use of PD1 as an analyte is also of interest for mask (Figure 2 A) or IL7 (Figure 2 B) individual fusions to an N-terminus of the anti-PDl Fab. Fusion PD1 kinetics can be compared to those of the Fab alone in order to assess for the alteration of PD1 binding. b) IL7Ra-IL-2Rg-Fc was flown as analyte to assess for function of the mask or to confirm correct folding of a free IL7 component. Where a mask is concerned, if IL7Ra-IL-2Rg-Fc did not bind or binding was greatly diminished it was assumed that the IL7 was masked or otherwise altered. c) Lastly, IL7-Fusion was flown in order to assess the function of the masking moieties for Mask-to-Fab fusions (Figure 2 B) and may also deliver an indication of correct folding and function of the masks when fused in mask-Fab only fusions.

The results are shown in Table 34. Due to the high-throughput methods used in producing the molecules for the assay, not all proteins could be produced with satisfactory quality, leading in some instances to SPR sensorgrams that were inconclusive.

Table 34: SPR results for PD1 IL7 CISS molecules

The SPR results showed that DRSPAI-L7B functions as an scFv in both orientations. In addition, as previously seen, fusion to the N-termini of 1040 does not prevent binding to PD1. All CISS Fab molecules appeared to prevent PD1 binding to the 1040 and therefore mutual exclusivity is thought to occur. A low amount of IL7Ra-IL-2Rg-Fc binding appears to be present for at least some complete CISS molecules though with a rather different kinetic to IL7-Fab to IL7Ra-IL-2Rg-Fc binding. This may indicate that the DRSPAI-L7B is not blocking all elements of the IL7 receptor complex to IL7 binding interaction with some minimal modified binding retained.

Example 21: PD1-IL7 targeted CISS

In order to produce targeted PD1 IL7 CISS molecules we used linker lengths and component orientations from the molecule P1AJ7168 which appeared to demonstrate mutual exclusivity in our screening assay. For each targeted molecule we essentially fused variants of this CISS Fab to an Fc domain and added a VHH as a targeting arm (G05). Lower affinity masks were generated for these molecules from the DRSPAI-L7B parent mask. Variants 3 and 4 (DRSPAI-L7B V3 and V4) of the mask were alternately used in the targeted CISS molecules. Both masks are of much lower affinity than the parent, DRSPALL7B V3 has a higher affinity than DRSPAL L7B V4. In addition, an attenuation of IL7 was also used in some of the molecules to modulate the potency of the CISS molecules. Molecules tested are display in Table 35. Plasmids were generated by gene synthesis and Golden gate assembly as described in example 1. Molecules were expressed and purified according to the midscale purification protocol described in example 1 using CaptureSelect™ CHI -XL Affinity Matrix (Thermo Fisher Scientific, USA).

Table 35: PD1-IL7 targeted CISS generated for this experiment

PDl-IL7wt was used as a control for the pStat5 assay. Table 36: SEQ ID Nos of the molecules shown in Table 35

The molecules shown in Table 35 were tested for CISS functionality in a in a p-Stat5 Assay (see Figure 33 for the principle of the assay).

IL-7R Signaling Assay

The following assay was performed to determine the potency and cis/trans- signaling ofaPD-l-IL-7 CISS molecule or immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-7 polypeptide that is masked or contains additional mutations.

For this purpose, CD4 T cells from healthy donor PBMCs were sorted with CD4 beads (Miltenyi, #130-045-101) and activated for 3 days in presence of 1 pg/ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and half of the cells were labeled with Cell Trace Violet (CTV) (5 pM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were left unlabeled.

Then, the unlabelled cells were incubated with a saturating concentration of a competing anti-PD-1 antibody (in-house molecule, 10 pg/ml) for 30 minutes at RT followed by several washing steps to remove the excess unbound anti-PD-1 antibody. Thereafter, the PD-1 pre-blocked cells (25 pl, 6*10⁶ cells/ml) were cocultured 1 : 1 with the PD-1⁺ CTV-labeled cells (25 pl, 6xl0⁶ cells/ml) in a V-bottom plate before being treated for 12 minutes at 37 °C with increasing concentrations of treatment immunoconjugates (50 pl, 1 : 10 dilution steps). To preserve the phosphorylation state, an equal amount of Phosphoflow Fix Buffer I (100 pl, 557870, BD Bioscience) was added after 12 minutes incubation with the various constructs. The cells were then incubated for an additional 30 minutes at 37 °C before being permeabilized overnight at -80 °C with Phosphoflow PermBuffer III (558050, BD Bioscience). On the next day, STAT-5 in its phosphorylated form was stained for 30 minutes at 4 °C by using an anti-STAT-5P antibody (47/Stat5(pY694) clone, 562076, BD Bioscience).

The cells were acquired at the fluorescence-activated cell sorting (FACS) BD-Symphony A3/A5 (BD Bioscience) instrument. The frequency of STAT-5P was determined with Flow Jo (VI 0) and plotted with GraphPad Prism (v8).

The dose-response curves on PD-1⁺ T cells provided information on the potency of the assessed molecules in signaling through the IL-7R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 mediated delivery, showed the potency of the molecules in providing IL-7R signaling independently from PD-1 expression.

Table G: Results of IL-7R Signaling Assay after 15 minutes

Table H: Results of IL-7R Signaling Assay after 1 hour

Both complete CISS molecules using wildtype IL7 molecule exhibited decent target dependent IL7R signaling. A lower affinity IL7 mask was associated with a larger window of PD1 specificity (P1AL7932) when compared to a higher affinity (Pl AL7931) variant. Both molecules were more selective than the Fab fused with IL7wt alone (P1AJ7159) as well as the FAP-IL7wt molecule which is untargeted in this context. Similary, when an attenuated variant of IL7 (K81E, which has reduced affinity to IL7R complex) was used in complete CISS molecules, PD1 enhanced IL7R signaling was observed (P1AL7933, P1AL7934). When the lower affinity mask was used the CISS molecule was more potent and selective (P1AL7934 compared to P1AL7933). These CISS molecules were superior in selectivity to the PDl-targeted IL7 K81E alone (P1AL7935) all the frequency of STAT5-P positive cells was lower.

Example 22: CISS molecules with wildtype IL-2

Wildtype IL-2 contains three receptor interaction surfaces which bind to CD25 (IL-2Ra) with high affinity (KD ~10'⁸), CD 122 (IL-2Rb) with lower affinity (KD ~10'⁶'⁷) and CD132 (IL-2Rg) (when IL-2 is already in complex with CD25 and CD 122 or CD 122 alone). Using mask 221, which directly masks the IL-2Rb interaction surface and allosterically inhibits the interaction with CD25 (see example 25), we generated targeted PD1 CISS molecules containing the wildtype IL-2. Table 37 shows example molecules of PD1-IL-2 CISS variants

Table 37: IL-2 wildtype targeted PD1 CISS variants

The plasmids for the CISS Fab molecules of Table 37 were generated using either external gene synthesis and vector cloning (Twist Bioscience, USA) or gene fragment synthesis (Twist Bioscience, USA) and Golden Gate Assembly (PaqCI R0745L and NEBridge® Ligase Master Mix Ml 100L (New England BioLabs)) as described in Example 1. Expressions and purifications were performed according to the mid-scale scale expression method outlined in Example 1 using CaptureSelect™ CH1-XL Affinity Matrix (Thermo Fisher Scientific, USA).

Table 38: SEQ ID Nos of the molecules shown in Table 37

HEK-Blue IL-2 reporter assays were used to assess the potency of the PD1- IL-2 CISS molecules and their PD1 dependency. To perform the assay, our test molecules and controls were prepared as a titration series and applied to reporter cells. The PD1 negative (parental) IL-2 reporter cell line (“HEK-Blue IL-2”) and the PD1 positive (CL1AA1486: “HEK-Blue IL-2 PD1”) IL-2 reporter cell line were used.

First, 3x titration series (12 steps) of the test molecules were prepared in triplicates, starting at 100 nM (final concentration in assay well between 50 nM and 0.0003 nM). 25 pL per fusion protein of each concentration was transferred to 384- well flat bottom plates. As positive control, recombinant IL-2v (P1AD6412) was used in the same concentration range. Subsequently, IxlO⁴ reporter cells/well were seeded in 25 pL medium (DMEM high glucose (4.5 g/L glucose) (PAN, Catalog number P04-03609) + 10 % heat-inactivated FBS (Anprotec, #AC-SM-0014Hi) + 2 mM L-glutamine (PAN, Catalog number P04-80100) + lOOpg/ml Normocin (InvivoGen Catalog number ant-nr-1) into the 384 well plate. Incubation of the test molecules and cells ran overnight at 37°C. QUANTI-Blue solution (Invivogen, #rep- qbs) was prepared and 20 pL/well was distributed among wells of new 384-well flat bottom plates (#781098), followed by the addition of 20 pL of supernatants of the treated cells. Substrate turnover by the expressed SEAP ran for 2 to 6 hours before measuring of the optical density (OD) at 620 nm (Tecan Safire 2).

Results are shown in Figure 17 A-D. Using this assay setup the lower affinity masks 221_V2 (Figure 17, middle image) and 221_V3 (Figure 17, right image) appeared to be ineffective at masking the wildtype IL-2. Targeted CISS molecules using these masks (P1AJ7910 and P1AJ7913) had a similar potency in the “HEK- Blue IL-2” and “HEK-Blue IL-2 PD1” cell lines. By contrast, in Figure 17, left image, the higher affinity “221” can be seen to have successfully masked the IL-2 and also delivered PD1 dependent IL-2 activity within the context of the targeted CISS molecule (Molecule P1AJ7907). This dataset demonstrates the necessity of using the correct affinity mask to avoid target independent activation and also demonstrates the utility of the 221 mask for the inhibition of wildtype IL-2.

Example 23: Constructs used for selection and characterization of binders for huIL-2 / huIL-2v masking moieties Table 39 lists the constructs that were used for generating and characterizing the anti-IL-2/IL-2v antigen binding domains that were made for use as huIL-2/huIL-2v masking moieties in the CISS molecules described herein.

Table 39: List of constructs that were used for generating and characterizing IL-2/IL-2v antigen-binding moieties

Example 24: Generation of recombinant human IL-2 / IL-2v protein for binding assays a) huIL-2 /huIL-2v expression and purification

The human IL-2v cytokine (internal batch number Pl AD6412-013) has been transiently expressed in CHO and purified via the C-terminal his-tag at WuXi Biologies. Purification was carried out by applying the expressed molecules to the following chromatographic steps:

1. Ni Excel column for affinity chromatography of the C-terminal his-tag (buffer for column equilibration and wash: 20 mM PB, 150 rnM NaCl, 20 mM imidazole, pH 7.4; elution buffer: 20 rnM PB, 150 mM NaCl, 500 mM imidazole, pH 7.4).

2. Superdex 200 size-exclusion chromatography (equilibration buffer, running buffer and formulation buffer: 20 mM Histidine-HCl, 140 mM NaCl, pH 6.0).

Protein purity was determined by SEC-HPLC (monomer peak 100%), nonreducing CE-SDS (main peak 98.7%), reducing CE-SDS (main peak 98.5%) and the protein identity was confirmed by LC-MS. The endotoxin level was determined to be <0.05 EU/mg and the final concentration was 3.4 mg/mL. Recombinant human IL-2 was purchased from Peprotech, catalog 200-02. b) Biotinylation of tool proteins

Recombinant huIL-2 /huIL-2v protein were chemically biotinylated by using a 4 and 3 molar excess of EZ-Link™ Sulfo-NHS-SS-Biotin (ThermoFischer Scientific, #A39258), respectively. The reaction was carried out for 1 hour at room temperature and the excess of biotin was removed by two steps of dialysis to PBS IX, pH 7.4. Biotinylation was assessed via pull-down assay, showing that in both cases more than 90% of the protein was biotinylated. The quality of the biotinylated protein was assessed by SEC and SPR. Example 25: Selection and characterization of huIL-2 /huIL-2v binders a) Generation of Fabs binding specifically to huIL-2 / huIL-2v

Generation of Fabs binding specifically to huIL-2 / huIL-2v was carried out by phage display. For this, recombinant biotinylated huIL-2 / huIL-2v was used (biotinylation was performed as described in Example 24 b above).

Diverse Roche in-house phage display libraries of synthetic human Fab fragments were utilized. In all libraries, the CHI domain of the Fab fragments was fused via a linker to a truncated gene-III protein to facilitate phage display.

Phage library panning was performed in four rounds, wherein the first round was performed with lOOOnM of biotinylated huIL-2 / huIL-2v pre-immobilized on Dynabeads™ M-280 Streptavidin magnetic beads (Thermo Fisher catalog number 11206D), and rounds 2 -4 were performed with 500 nM of biotinylated huIL-2 and 500 nM, 250 nM, 250 nM of biotinylated huIL-2v, followed by capture of Fab-on- phage/target complexes on the beads. In round 2 and 4 Neutravidin-coupled magnetic beads (Sera-Mag SpeedBeads Neutravidin-Coated Magnetic Particles, Cytiva: 78152104010350) were used. In round 1, captured phage clones bearing target-specific Fabs in combination with the beads were directly infected into logphase TGI E. coli cells, and rescued using M13KO7 helper phage, according to standard protocols. In rounds 2-4, the Fabs bearing phages were eluted from the magnetic beads using 100 mM DTT prior to being used for infection.

For the screening of phage display outputs, a polyclonal plasmid miniprep of the respective selection round was prepared from the infected TGI E. coli cells. Plasmids were digested using BamHI restriction endonuclease, which cuts the phagemid pDuta4 upstream and downstream of the phage Fd gene 3 domain. Plasmids were recircularized by ligation, generating an in-frame fusion of a T7 tag at the C-terminus of the DutaFab CHI domain. The ligated polyclonal plasmids encoding T7-tagged DutaFabs were transformed into TGI E. coli cells, and single colonies were picked into microtiter plates. Soluble Fabs were expressed in microtiter plates and supernatants were clarified by centrifugation.

The Fab culture supernatants were screened and specific binders were identified by ELISA as follows: 20 pL mixture of biotinylated antigen huIL-2 / huIL- 2v (0.1 pg/mL final concentration in assay) and detection antibody anti human Ig kappa chain specific POD (Millipore AP502P, 1:2000 final in assay) and anti T7- Tag POD (Bethyl A190-116P, 1 : 10000 final in assay) were mixed with 5 pL Fab containing bacterial supernatant and added to streptavidin coated microtiter plates (MicroCoat 384 SA, 11974998001). After an incubation for 60 min at room temperature, the plates were washed 6 times with PBST (PBS, 0.1% Tween20). The binding of Fabs to huIL-2 and huIL-2v, respectively, was detected by adding 30 pL TMB (Sera Care, #5120-0087) substrate to the wells. After an incubation of 5 min at RT the reaction was stopped by adding 30 pl IM HC1. The absorbance was then measured at OD 450 nm with a Microplate Reader (Biotek Powerwave). Clones expressing huIL-2 / huIL-2v specific Fabs were identified and the corresponding phagemids were sequenced.

365 unique sequences were discovered and subsequently grouped into families. 121 clones were chosen according to the ELISA results, sequence frequency and sequence similarity and expressed in E. coli. Fabs were purified from the E. coli supernatant via a one-step affinity chromatography purification on CaptureSelect™ IgG-CHl resin (Thermo Fisher; Catalog number: 1943200 IL). The quality of the purified Fabs was analyzed via size exclusion chromatography. b) SPR characterization of Fabs binding specifically to huIL-2 / huIL- 2v

The binding affinities of the Fabs were assessed by SPR-analysis using a Biacore 8K or 8K+ instrument (Cytiva). Binding kinetics of binding to human huIL- 2v, human huIL-2 and to CD79B dimer (control) were analyzed (results shown in Table 40)

Table 40: Binding affinity of selected huIL-2 / huIL-2v binding clones

Briefly, anti human-Fab antibody (Cytiva; Catalog number 28958325) was immobilized to a Series S CM5 Sensor Chip (Cytiva, Catalog number 29149603) according to the manufacturer's instructions. 100 nM Fabs were captured 10 pl/min, 60 sec) and 0 nM, 10 nM, 50 nM and 150 nM of antigen was flown at 30 pl/min for 120 sec followed by a 240 second dissociation window at a flow rate of 30 pl/min. The surface was regenerated by injecting 10 mM Glycine pH 2 for 60s at a flow rate of 30 pl/min. The sensograms are shown in Figure 21.

Competitive binding of the huIL-2 / huIL-2v specific Fabs to huIL-2Rbg heterodimer was investigated as follows. A schematic depiction of the experimental set-up is shown in Figure 18. A Human Antibody capture kit (Cytiva, Catalog number BR100839) was used to immobilize lOpg/ml of IgG capture antibody (“Anti-Fc”) (applied for 500 seconds at a flow rate of 10 pl/min) to a Series S CM5 Sensor Chip (Cytiva, Catalog number 29149603) using standard amine coupling chemistry. To assess competitive binding, 100 nM IL-2Rbg-Fc heterodimer was captured to the surface of the chip by flowing it at 10 ul/min for 120 seconds. 100 nM huIL-2 / huIL-2v was then injected for 120 seconds at a rate of 10 pl/min. 100 nM Fabs were flown subsequently for 120 seconds at a rate of 30 pl/min, followed by a 10 seconds dissociation window. Finally the chip surface was regenerated by injecting 3M MgCh for 60 seconds at a flow rate of 30 pl/min. Competitive binding was assessed by individual evaluation of the sensorgrams (see Figure 22 A - B). IL- 2Rbg noncompetitive Fabs are expected to bind IL-2Rbg-bound huIL-2 / huIL-2v, resulting in a SPR signal increase. IL-2Rbg competitive Fabs are expected to be unable to bind IL-2Rbg-bound huIL-2 / huIL-2v, resulting in no SPR signal increase.

Competitive binding of the huIL-2 / huIL-2v specific Fabs to IL-2Rbg heterodimer was further confirmed as follows (a schematic depiction of the experimental set-up is shown in Figure 19). a Human Antibody capture kit (Cytiva, Catalog number BR100839) was used to immobilize lOpg/ml of IgG capture antibody (applied for 500 seconds at a flow rate of 10 pl/min) to a Series S CM5 Sensor Chip (Cytiva, Catalog number 29149603) using standard amine coupling chemistry. To assess competitive binding, 100 nM IL-2Rbg heterodimer (Pl AE2657) was captured to the surface of the chip by flowing it at 10 ul/min for 60 seconds. 50 nM huIL-2 / huIL-2v was pre-mixed with 0 nM (Figure 19 A), 100 nM (Figure 19 B) and 500 nM (Figure 19 C) Fab and subsequently flown for 120 seconds at 30 pl/min, followed by a 240 seconds dissociation window. Finally, the chip surface was regenerated by injecting 3M MgCh for 60 seconds at a flow rate of 30 pl/min. Competitive binding was assessed by individual evaluation of the sensorgrams (see Figure 22). IL-2Rbg competitive binders are expected to have a decreased SPR signal compared to the control (huIL-2 / huIL-2v + 0 nM Fab). The extent of such decrease depends both on the Fab binding affinity and on the molar excess of Fab used. As shown by Figure 22, all three tested binders show competetive binding to huIL-2/huIL-2v with IL2Rbg. Competitive binding of the huIL-2 / huIL-2v specific Fabs to IL-2Ra (CD25) was investigated as follows (a schematic depiction of the experimental set-up is shown in Figure 20). A Series S Sensor Chip NTA (Cytiva, BRI 0053) was used to immobilize 100 nM of IL-2Ra-His (P1AE7552, applied for 120 seconds at a flow rate of 10 pl/min). To assess competitive binding, 100 nM huIL-2 was injected at 30 ul/min for 120 seconds. Running buffer (Figure 20 A) or 150 nM Fab (Figure 20 B) were subsequently flown for 120 seconds at 30 pl/min, followed by a 120 seconds dissociation window. The surface was regenerated by injecting 350 mM EDTA for 60s at a flow rate of 30 pl/min. Sensograms are shown in Figure 23. Competitive binding was assessed by comparing the “Fab” sensorgram (Figure 23 A, dotted line) with the “buffer” sensorgram (negative control; Figure 23 A, solid line). IL-2Ra non-competitive binders are expected to have increased SPR signal compared to the buffer control. IL-2Ra competitive binders are expected to have an SPR trace comparable to the buffer control. The competition experiment shows that 093 and 333 are not IL-2Ra competitive (the SPR signal increases at tO, i.e. upon injection of 100 nM Fab, which indicates that the Fab is able to bind to huIL-2 that is bound to IL-2Ra) while 221 is IL-2Ra competitive (no SPR signal increase upon 100 nM Fab injection).

Competitive binding to IL-2Ra (CD25) was further investigated as follows. lOpg/ml of THE™ His Tag Antibody, mAb, mouse (Genescript, catalog A00186S, applied for 500 seconds at a flow rate of 10 pl/min) were immobilized to a Series S CM5 Sensor Chip (Cytiva, Catalog number 29149603) using standard amine coupling chemistry. To assess competitive binding, 100 nM IL-2Ra-His (Pl AE7552) was captured to the surface of the chip by flowing it at 10 pl/min for 60 seconds. 50 nM huIL-2 was pre-mixed with 0 nM, 100 nM and 500 nM Fab (Figure 24 B) and subsequently flown for 120 seconds at 30 pl/min, followed by a 240 seconds dissociation window. The surface was regenerated by injecting 10 mM Glycine pH 2 for 60 seconds at a flow rate of 30 pl/min. Competitive binding was assessed by individual evaluation of the sensorgrams. IL-2Ra competitive binders are expected to have a decreased SPR signal compared to the control (huIL-2 + 0 nM Fab). The extent of such decrease is dependent both on the Fab binding affinity and on the excess of Fab used. IL-2Ra non-competitive binders are expected to have an SPR signal (RU) higher than the control. This effect is due to the higher mass of the complex huIL-2-Fab compared to the mass of huIL-2 alone. This experiment confirmed that 093 and 333 are not IL-2Ra competitive while 221 is IL-2Ra competitive. In summary, approximately 20 huIL-2 / huIL-2v binders showing nM / pM binding affinity as determined via SPR were identified. Most of the binders had comparable binding affinity for both huIL-2 and huIL-2v and were found to be IL- 2Rbg competitive. Only one binder, P029.221, was found to be IL-2Ra competitive as well. Binders P029.221 (“221”), P017.093 (“093”) and P017.333 (“333”) were selected as building blocks for the huIL-2v CISS constructs. Additional binders with lower binding affinity (high nM or pM) were identified but not further characterized.

Example 26: Affinity maturation of clone 221

Binder 221 was of special interest due to its ability to block both the IL-2Rbg interaction and the IL-2Ra (CD25) interaction, as shown by SPR (see Example 25, Figure 22 and Figure 23). Binding affinity was found to be 15.4 nM for huIL-2v and 125 nM for huIL-2. We speculated that given the high binding affinity of the IL-2 / IL-2RaPy complex, to have a functional huIL-2 mask the binding affinity needed to be improved. Towards this end, we affinity-matured the binder as described hereafter. a) Site saturation mutagenesis (SSM)

In order to identify point mutations bearing a positive effect in terms of binding affinity, a Site Saturation Mutagenesis (SSM) approach was used. 22 positions known to be relevant for binding according to the Fab library design (hl, h2, h28, h29, h30, h31, h32, h33, h53, h96, hlOO, hl02, k29, k31, k32, k51, k53, k54, k55, k56, k57, k92 according to Kabat numbering; the letters “h” and “k” indicate whether the position is located in the heavy “h” or the kappa light “k” chain) were chosen for randomization. NNK degenerated overlapping primers were designed for each position, and mutants were generated using the QuickChange Site- Directed Mutagenesis Kit (Agilent, catalog 200519). The mutated plasmid was transformed into TGI E. coli cells and plated into agar plates. b) Supernatant screening, expression and SPR characterization of point mutant hits

Single colonies (170 - 180 per mutated position, in order to cover the amino acid diversity introduced by using the NNK primers) were picked into microtiter plates, soluble Fabs were expressed in microtiter plates and supernatants were clarified by centrifugation.

The Fab culture supernatants were screened for improved affinity versus the parental version by ELISA as follows: 500 ng/ml a-F(ab’)2 (108 pl per well, JIR #109-006-006) was coated onto 384w Maxisorp plates (Nunc, 464718) for Ih at room temperature. After washing three times with 90 pl PBST (lx PBS, 0.1% Tween20), the surface was then blocked by adding 90 pl of blocking buffer (lx PBS, Roche # 11666789001; 2% BSA, Roche #10735086001; 0,05% Tween-20, usb #20605) with overnight incubation at 4°C. Next the surface was washed three times with 90 pl PBST. Subsequently 25 pl of Fab containing supernatants were added and incubated for 1 hour at room temperature. Unbound sample was washed off by washing three times with 90 pl PBST. Next, 25 pl of biotinylated huIL-2 (produced in house) was added in different concentrations (20, 200, 2000 ng/ml) and incubated for Ih at room temperature. The plate was then washed six times with 90 pl followed by a Ih incubation step with Streptavidin-POD (Roche #11089143001, 1 :4000) at room temperature. After washing three times with 90 pl PBST, 25 pl TMB (Sera Care, #5120-0087) were added. After 10 min incubation, the absorbance was measured at OD 370 nm with a Microplate Reader (Biotek Powerwave).

The identity of selected clones expressing Fabs showing increased ELISA signal relative to the parent molecule was revealed by Sanger sequencing. The identified hits were expressed in E.coli and binding to huIL-2 / huIL-2v was assessed by SPR as described in Example 25. c) Mutants design, expression and characterization

Beneficial point mutations were combined in silico in multiple fashions and the desired gene segments were prepared by chemical synthesis. The synthesized gene fragments were cloned into a suitable vector for expression in E.Coli by Twist Bioscience (San Francisco, US). d) Expression and SPR characterization of improved clones

Fabs were expressed in E.coli as described in Example 24. Binding to huIL- 2 / huIL-2v, IL-2Rbg competition and IL-2Ra competition were assessed as described in Example 25.

In summary, 71 unique hits (point mutants) were expressed and characterized. 15 mutants were found to have improved k_on, k_off, or both compared to the parent molecule (huIL-2 was chosen as antigen of reference). The following mutations were therefore combined in different fashions into 6 variants (numbering according to Kabat): VH: Ell; M29L; Q32S; Q32A; A33R; S53T; S53R; S53Q; F100V; I102F, I102Y; Vk: V51I; R54Q; E56S; N57P (for results see Figures 24 A - F and Table 41). Table 41: Binding affinity of 221-derived point mutants.

The improved variant P029.221.AM2 (“221. AM2”) showed a 70-fold improved dissociation constant for huIL-2 (1.5 nM vs 103 nM, when measured side- by-side in SPR, see Figures 25 A and B, and Table 41) and all the other variants showed at least a lOx improvement compared to the parent binder. IL-2Rbg and IL- 2Ra competition was also tested (Figure 25) and the affinity-matured clone showed a significantly improved potency in competing with the receptors, showing that an improved binding affinity translates into an improved potency in vitro. In addition, these competition experiments confirmed that the affinity maturation did not change the targeted huIL-2 epitope. Such outcome could not be assumed, taking into account the peculiar binding mode of 221.AM2 (see Example 27). Example 27: Elucidation of 221.AM2 huIL-2 binding mode via X-ray crystallography a) Expression and purification of 221.AM2_tagfree for X-ray crystallography

The fragment antigen-binding region (Fab region) of antibody 221.AM2 (internal concept number P1AI2583) has been generated by plasmin digestion from the one-armed human IgG OA P029.221.AM2 PG LALA AAA (internal concept number P1AH9927). The one-armed human IgG OA P029.221.AM2 PG LALA AAA was produced at Evitria. Suspension-adapted CHO KI cells (originally received from ATCC and adapted to serum-free growth in suspension culture at Evitria) were used. The seed was grown in eviGrow medium, a chemically defined, animal-component free, serum-free medium. Cells were transfected with eviFect, Evitria’ s custom-made, proprietary transfection reagent and grown after transfection in eviMake2, an animal-component free, serum-free medium. Supernatant was harvested by centrifugation and subsequent filtration (0.2 pm filter).

The supernatant was subsequently purified at Roche by Protein A MabSelectSure affinity chromatography (equilibration and wash buffer: 20 mMNa- Citrate, 20 mM Na-Phosphate, pH 7.5; elution buffer: 20 mM NaCitrate, pH 3.0; neutralization buffer: 0.5 M Na2HPO4, pH 8.0) and subsequent preparative sizeexclusion chromatography. The one-armed human IgG OA P029.221.AM2 PG LALA AAA was formulated into 20 mM Histidine, 140 mM NaCl, pH 6.0 with a monomer content of 97.2% (analytical size-exclusion chromatography), a CE-SDS main peak of 100% and an endotoxin concentration of < 0.061 EU/mg.

In a next step, 20 mg one-armed human IgG OA P029.221.AM2 PG LALA AAA were digested using 1.05U plasmin/mg IgG. The Fab region of antibody P029.221.AM2 was purified by CaptureSelect Pre-Packed IgG-CHl column (Thermo Scientific, Ref 494320005) and size-exclusion chromatography (HiLoad Superdex PG). The final product was formulated into 20mM Tris, 150 mM NaCl, pH 7.4 with a monomer content of 95.2% (analytical size-exclusion chromatography) and a CE-SDS main peak of 100%. b) 221.AM2-huIL-2 crystallization and structure determination

IL-2 lyophilisate (Peprotech, #200-02, Lot.: 071412) was dissolved in 100 mM acetic acid, pH 5.0. For complex formation huIL-2 was mixed in a 2.2 molar excess with Fab 221.AM2. After incubation on ice for 1 hour the complex was concentrated and buffer was exchanged against 20 mM MES, 150 mM NaCl, pH 5.5. Crystallization screening for complex crystals was performed at a concentration of 13.2 mg/ml. Crystallization droplets with 0.2 pl volume were set up at 21° C by mixing protein solution with reservoir solution in different ratios of 0.7/0.3 and 0.5/0.5 in vapor diffusion sitting drop experiments. The crystal used to determine the structure appeared within one day out of 100 mM Tris, 20 % (v/v) PEG Smear Broad (Molecular Dimensions, #MD2- 100-26), 200 mM (NH^SCU, pH 8.0 in a protein/precipitant ratio of 0.5/0.5. The crystal was mounted after two days and flash- cooled in liquid N2 with paraffin oil as cryoprotectant.

Data were collected from a single crystal at 100 K at the Swiss Light Source beamline PX-II with an EIGER2-16M detector at an wavelength of 1 A. Intensities were integrated with XDS [Kabsch, Wolfgang, "xds." Acta Crystallographica Section D: Biological Crystallography 66.2 (2010): 125-132.], scaled with AIMLESS and treated for anisotropy using STARANISO (Tickle, I. J., et al. "Staraniso." Global Phasing Ltd., Cambridge, /W (2O I 8)). Data from one crystal were merged to yield a 1.50 A resolution data set in space group C121 (see Table 42).

Table 42: Data collection and refinement statistics for P029.221.AM2

*Values in parentheses are for the highest-resolution shell.

Phases were generated by molecular replacement with PHASER (McCoy, Airlie J., et al. "Phaser crystallographic software." Journal of applied crystallography 40.4 (2007): 658-674.) using AlphaFold2 (Jumper, John, et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596.7873 (2021): 583-589.) search models.

Models were rebuilt in Coot (Casanal, Ana, Bernhard Lohkamp, and Paul Emsley. "Current developments in Coot for macromolecular model building of Electron Cryo-microscopy and Crystallographic Data." Protein Science 29.4 (2020): 1055-1064.) and refined with the CCP4 program REFMAC (Winn, Martyn D., et al.

"Overview of the CCP4 suite and current developments." Acta Crystallographica Section D: Biological Crystallography 67.4 (2011): 235-242.). The final refinement steps were performed with the program BUSTER (Bricogne, G., et al. "BUSTER, version 2.11. 2; Global Phasing Ltd." Cambridge, United Kingdom (2011)) using TLS protocols for B-values. The final structures contain two molecules per asymmetric unit (see Table 42). Table 43: List of main interactions between huIL-2 and 221. AM2

c) 221.AM2 competes for the same epitope of IL-2Rb on huIL-2

The binding mode of 221. AM2 was elucidated by X-ray diffraction (Figure 26 A). The structural analysis reveals that 221.AM2 binds to a surface of huIL-2 comprising helices A and C, and to the loop between helices B' and C (Stauber D. J., et al., PNAS 2005). The main interactions between huIL-2 and 221. AM2 were found to be as shown in Table 43.

With the help of the software PyMol, the co-crystal structure of 221.AM2 - huIL-2 was aligned to huIL-2 - IL-2Rabg complex (PDB 2erj), using huIL-2 as reference (Figure 26 B and C). The structural alignment revealed that 221. AM2 is sterically competitive with IL-2Rb, which also binds to the huIL-2 helices A and C. The structural analysis results were in agreement with the SPR competition experiments (Example 25 and Example 26). Unexpectedly, 221.AM2 is not a steric competitor of IL-2Ra (Figure 26 C). d) IL-2Ra competition of P029.221.AM2 is due to a binding-induced IL- 2 conformational change

The structural alignment of huIL-2 bound to 221.AM2 and huIL-2 bound to the trimeric receptor complex (PDB 2erj) revealed no major differences between the two structures, apart from the loop between helix A and A'and the helix A' (amino acids 29 - 43) and the region spanning between the C-ter of helix B' and the N-ter of helix C (amino acids 67 - 84) (Figure 27 A and B). Both regions of the Fab-bound huIL-2 results kinked and symmetrically shifted, keeping their relative distance, compared to the receptor bound cytokine (Figure 27 C and D). Such an effect can be explained by the different binding mode of IL-2Rb vs 221. AM2. IL-2Rb does not form any interaction with the aforementioned huIL-2 regions, while the Fab engages binding contacts via CDR LI and CDR L2. Such binding-induced huIL-2 conformational change is responsible for the IL-2Ra competition of 221. AM2. In detail, the structural alignment shows that within helix A', Lys36, Arg39 and Phe43 of the Fab-bound huIL-2 would sterically clash with Leu42, Tyr43 and Leu45 of IL- 2Ra. With regard to the helix B', the shift probably leads to missing interactions, e.g. huIL-2 Leu72 with IL-2Ra Tyr43 (Figure 27 E and F). Altogether, these combined effects determine the binding-induced IL-2Ra competition of 221.AM2, further confirming the SPR competition results.

Claims

PATENT CLAIMS

1. An activatable fusion protein comprising

(a) a first antigen-binding moiety capable of specifically binding to a target antigen and comprising at least a first heavy chain polypeptide and at least a first light chain polypeptide,

(b) a cytokine capable of specifically binding to a cytokine receptor, and

(c) a masking moiety capable of specifically binding to the cytokine, characterized in that the cytokine is covalently attached to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, the masking moiety is covalently attached to the N-terminus of the other one of the two polypeptides of the first antigen-binding moiety via a second peptide linker, and the first and the second peptide linker do not comprise a protease cleavage site.

2. The activatable fusion protein of claim 1 characterized in that the first antigen-binding moiety is an antibody or an antibody fragment.

3. The activatable fusion protein of claim 2 characterized in that the antibody fragment is selected from the group consisting of a Fab, a DutaFab, a DAF, an Fv, a Fab', a Fab’-SH, a F(ab')2, a diabody, a linear antibody, and a multispecific antibody formed from antibody fragments.

4. The activatable fusion protein of any one of claims 1 to 3, characterized in that the cytokine is selected from the group consisting of interferons, interleukins, chemokines, lymphokines, monokines, colony- stimulating factors, and tumour necrosis factors.

5. The activatable fusion protein of any one of claims 1 to 4 characterized in that the cytokine is selected from the group consisting of BMP, CSF-1, insulin, GLP- 1, HGH, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL- 23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL- 35, IL-36, GM-CSF, FGF, EGF, G-CSF, IFNa, IFNP, IFNy, PDGF, TGFp, TNFa, TNFP, VEGF, and EPO.

6. The activatable fusion protein of any one of claims 1 to 5, characterized in that the masking moiety is selected from the group consisting of an antibody, an antibody fragment, a single-chain antigen-binding moiety, a peptide mask (anti- idiotypic antibody, an anti-idiotypic antibody fragment (e.g. scFv, VHH), and a receptor, protein inhibitor or binding protein capable of binding specifically to the cytokine.

7. The activatable fusion protein of any one of claims 1 to 6, characterized in that the masking moiety is reversibly bound to the cytokine.

8. The activatable fusion protein of any one of claims 1 to 7, characterized in that the cytokine is hindered from binding to the cytokine receptor when the masking moiety is bound to the cytokine.

9. The activatable fusion protein of any one of claims 1 to 8, characterized in that the first antigen-binding moiety is hindered from binding to the antigen when the masking moiety is bound to the cytokine.

10. The activatable fusion protein of any one of claims 1 to 9, characterized in that it further comprises a second antigen-binding moiety comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide.

11. The activatable fusion protein of any one of claims 1 to 10, characterized in that it further comprises an Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc Domain heavy chain polypeptide.

12. The activatable fusion protein of claim 11, characterized in that a) the first antigen-binding moiety is covalently attached to the N-terminus of the first Fc domain heavy chain polypeptide and the second antigen-binding moiety is covalently attached to the N-terminus of the second Fc domain heavy chain polypeptide, or b) the first antigen-binding moiety is covalently attached to the N-terminus of the first or the second Fc domain heavy chain polypeptide and the second antigen- binding moiety is covalently attached to the C-terminus of the first or the second Fc domain heavy chain polypeptide.

13. The activatable fusion protein of any one of claims 10 to 12 characterized in that the second antigen-binding moiety is capable of specifically binding to an antigen that is the same as or a different antigen from the target antigen.

14. The activatable fusion protein of any one of claims 11 to 13 characterized in that the first antigen-binding moiety, the second antigen-binding moiety and the Fc region together form an antibody of the IgG type.

15. An antibody that binds specifically to huIL-2 or a variant thereof, wherein the antibody binds to an epitope of huIL-2 within amino acid residues 8-17, amino acid residue 30 and/or amino acid residues 77-81 of SEQ ID NO:81; and inhibits the binding of huIL-2 to human IL-2RPy and to human IL-2Ra.

16. The antibody of claim 15, wherein the epitope comprises amino acid residues corresponding to K8, Q13, E15, H16, N30, N77, H79 and R81 of SEQ ID NO:81; and inhibits the binding of huIL-2 to human IL-2RPy and to human IL-2Ra.

17. An antibody that binds specifically to huIL-2 or a variant thereof, wherein the antibody comprises

(A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

(B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:28, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:33, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(G) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (H) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;

(I) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:43, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or

(J) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

18. The antibody of any one of claims 15 - 17 comprising

(A) a VH sequence of SEQ ID NON and a VL sequence of SEQ ID NO: 8;

(B) a VH sequence of SEQ ID NO: 12 and a VL sequence of SEQ ID NO: 13;

(C) a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19;

(D) a VH sequence of SEQ ID NO:24 and a VL sequence of SEQ ID NO:25;

(E) a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID NON 1;

(F) a VH sequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36;

(G) a VH sequence of SEQ ID NO:38 and a VL sequence of SEQ ID NO: 39;

(H) a VH sequence of SEQ ID NO:41 and a VL sequence of SEQ ID NO:42; (I) a VH sequence of SEQ ID NO:44 and a VL sequence of SEQ ID NO:45; or

(J) a VH sequence of SEQ ID NO:47 and a VL sequence of SEQ ID NO:48.

19. The antibody of any one of claims 15 - 18 wherein the antibody is a Fab and comprises

(A) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 50;

(B) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 52;

(C) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 54;

(D) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 55 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 56;

(E) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(F) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(G) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:61 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(H) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 63 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:64; (I) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 66; or

(J) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 67 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:68.

20. An isolated nucleic acid encoding the activatable fusion protein of any of claims 1 - 14 or the antibody of any one of claims 15 - 19.

21. A host cell comprising the nucleic acid of claim 20.

22. A method of producing the activatable fusion protein of any of claims 1 - 14 or the antibody of any one of claims 15 - 19 comprising culturing the host cell of claim 20 under conditions suitable for the expression of the activatable fusion protein or the antibody, optionally further comprising the step of recovering the activatable fusion protein or the antibody from the host cell.

23. An activatable fusion protein or an antibody produced by the method of claim 22.

24. A pharmaceutical composition comprising the activatable fusion protein of any one of claims 1 - 14 or 23 or the antibody of any one of claims 15 - 19 or 23 and a pharmaceutically acceptable carrier.

25. The activatable fusion protein of any one of claims 1 - 14 or 23, the antibody of any one of claims 15 - 19 or 23 or the pharmaceutical composition of claim 24 for use as a medicament.