Activatable fusion proteins and methods of use

Abstract

The present invention provides activatable fusion proteins and methods of use thereof. The present invention further provides anti-huIL-2 antibodies and methods of use thereof.

Description

Technical Field

[0001] This invention relates to activatable fusion proteins and methods of using them. Background Technology

[0002] Therapeutic antibodies have revolutionized modern medicine. Thanks to their high specificity, they can target specific molecules in a subject with unprecedented precision. While initial monoclonal antibody therapeutics were primarily limited to blocking the interaction between target molecules and natural ligands, thus disrupting signaling pathways in certain disease mechanisms, the technology quickly evolved from these simpler modes of action to more complex forms, such as bispecific antibodies that can target two different epitopes or targeted cytokines designed to localize cytokine activity to target tissues to trigger a local immune response without simultaneously affecting healthy tissues.

[0003] However, for many therapeutically active substances, such as cytokines or agonist antibodies, targeting them to the desired tissue with targeted antibodies is insufficient to prevent nonspecific and undesirable activity of the active substance in the periphery. This is particularly true for potent cytokines, such as interferon-alpha, which may trigger unwanted side effects.

[0004] Different molecular forms have been proposed to address this. For example, various attempts have been made to mask therapeutic agents, such as cytokines or CD3-binding (i.e., T-cell activation) antigen-binding domains, where the molecular masking binds to these agents (usually antibodies or antibody-derived molecules such as scFv, but peptide masks are also common). These masks are typically linked to the therapeutic agent via peptide linkers that are readily cleaved by target tissue-specific proteases (e.g., tumor proteases). Thus, once the molecule reaches the target tissue and releases the therapeutic agent at the target site, the masking is cleaved. Dual-binding masks capable of specifically binding to both the therapeutic agent and the antigen in the target tissue in a mutually exclusive manner have also been proposed. These molecules have been described as masking the therapeutic 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 therapeutic agent.

[0005] The ability of proteases to activate molecules depends on the presence of highly tissue-specific proteases. If a protease is expressed not only in the target tissue but also in healthy tissue, this can lead to unwanted peripheral side effects. Mutually exclusive dual-binding antibodies overcome this problem, but they require significant effort to develop, and not every target combination is suitable for dual binding.

[0006] Therefore, there is a persistent need for a universal molecular form that allows therapeutic agents to target defined cell populations or tissues with high specificity, while preventing undesirable side effects of therapeutic agents in healthy tissues. Summary of the Invention

[0007] This invention provides an activatable fusion protein and a method of using the same. It has been found that the biological activity of a ligand (such as a cytokine) can be made dependent on the presence of the target antigen by fusing a masking portion (e.g., an antibody or antibody fragment) capable of binding to the ligand and preventing it from exercising its biological activity (by blocking the interaction between the ligand and the ligand-binding portion) to both chains of an antigen-binding portion that binds to the target antigen. This demasking is achieved when the antigen-binding portion binds to its target antigen, thereby spatially interrupting the inhibitory interaction between the ligand (e.g., a cytokine) and the masking portion without requiring a protease-cleavable linker.

[0008] Therefore, this activatable fusion protein platform does not depend on the presence of tissue-specific proteases or other cleavage mechanisms specific to certain tissues. While the latter lead to irreversible changes in bioactive molecules, the mechanisms relied upon by the activatable fusion protein according to the invention are reversible once the target antigen is absent from the environment. Therefore, if the activatable fusion protein is retained in circulation for a longer period, the risk of off-target activity is reduced compared to molecules that rely on mechanisms based on more destructive principles such as cleavage or digestion. The fusion protein of the invention is also independent of other activation mechanisms that depend on the presence of certain chemicals or physicochemical conditions in the environment, such as ATP activation or pH-dependent activation.

[0009] The activatable fusion proteins of the present invention achieve target antigen-dependent activity through a simple arrangement of readily available polypeptide domains, thereby allowing for easy realization and optimization of their intended purpose. By altering the affinity of different components, molecules can be tailored to distinguish specific surface expression levels of target antigens. The activatable fusion proteins exhibit excellent signal-to-noise ratio and sensitivity, for example, compared to other forms of targeting molecules that depend on ligand attenuation (such as cytokines). Therefore, the activatable fusion proteins described herein also possess very good side-effect characteristics, exhibiting very little target activation at the periphery. In some embodiments, the activatable fusion proteins shown herein can act not only on surface molecules but also on target molecules in solution. They also allow activation to depend on the presence of two different receptors at the target tissue / cell, thus potentially increasing specificity to the target tissue.

[0010] The present invention described herein provides an activatable fusion protein comprising:

[0011] (a) A first antigen-binding portion, the first antigen-binding portion being 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.

[0012] (b) Ligands capable of specifically binding to ligand-binding sites, and

[0013] (c) Masking portions that can specifically bind to ligands.

[0014] Its features are,

[0015] The ligand is covalently linked via a first peptide linker to the N-terminus of one of the two polypeptides at the first antigen-binding site.

[0016] The masking portion is covalently linked via a second peptide linker to the N-terminus of the other of the two polypeptides in the first antigen-binding portion.

[0017] and

[0018] The first and second peptide linkers do not contain protease cleavage sites.

[0019] The target-dependent binding of the ligand to the ligand-binding moiety contained in the activatable fusion protein described herein is particularly independent of any proteolytic activity of the activatable fusion protein, especially any proteolytic cleavage. In one aspect, the activatable fusion protein is functional and exhibits its intact form and / or lacks target antigen-dependent activity of the protease.

[0020] One embodiment of the present invention is an activatable fusion protein, characterized in that the first antigen-binding portion is an antibody or an antibody fragment.

[0021] One embodiment of the present invention is an activatable fusion protein, characterized in that the antigen-binding portion is selected from the group consisting of: Fab, DutaFab, DAF, Fv, Fab', Fab'-SH, F(ab')2, biantibody, linear antibody, and multispecific antibody formed from antibody fragments.

[0022] One embodiment of the present invention is an activatable fusion protein, characterized in that the ligand is selected from the group consisting of or any combination thereof: growth factors, cytokines, chemokines, antibodies, antibody fragments, enzymes, receptor ligands, affinity peptide ligands, peptide hormones, receptor agonists, receptor antagonists, enzymes, soluble receptors, protein toxins, soluble ligands, extracellular regions of cell surface receptors, extracellular regions of cell surface ligands, and small molecules. In a specific embodiment, the ligand included in the activatable fusion protein is a cytokine.

[0023] Essentially, in the activatable fusion proteins described herein, the ligand can be any molecule capable of specifically binding to another molecule that acts as the ligand-binding moiety, and the binding must 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: growth factor receptors, cytokine receptors, antigens, ligand receptors, enzyme substrates, fluorescent labels, radiolabels, or hormone receptors. In a particular embodiment, the ligand contained in the activatable fusion protein is a cytokine receptor.

[0024] One embodiment of the present invention is an activatable fusion protein, characterized in that the ligand is a cytokine selected from the group consisting of interferon, interleukin, chemokines, lymphokines, monokines, colony-stimulating factors, and tumor necrosis factor. In a specific embodiment, the activatable fusion protein is a cytokine selected from the group consisting of interferon and interleukin. 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-1α, IL-1β, 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, IFNα, IFNβ, IFNγ, PDGF, TGFβ, TNFα, TNFβ, VEGF or EPO. In another embodiment, the ligand is a cytokine selected from the group consisting of: IL-1, IL-1α, IL-1β, 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, IFNα, IFNβ, and IFNγ. In a specific embodiment, the cytokine is selected from the group consisting of: IL-2, IL-7, IL-21, and IFNα.

[0025] One embodiment of the present invention is an activatable fusion protein characterized in that the masking portion is selected from the group consisting of: antibodies, antibody fragments, single-chain antigen-binding portions, peptide masks, anti-idiotype antibodies or anti-idiotype antibody fragments, receptors, protein inhibitors, or binding proteins capable of specifically binding to a ligand. In a particular embodiment of the present invention, when the ligand is an antibody or antibody fragment, the masking portion is an anti-idiotype antibody or anti-idiotype antibody fragment (e.g., scFv, VHH) or an antigen of an antibody or antibody fragment used as a ligand. In another embodiment of the present invention, the activatable fusion protein is characterized in that the masking portion is an antigen-binding portion selected from the group consisting of: scFv, VHH, and single-domain antibodies (sdAb).

[0026] One embodiment of the present invention is an activatable fusion protein, characterized in that the masking portion reversibly binds to a ligand (e.g., a cytokine). Another embodiment of the present invention is an activatable fusion protein, characterized in that the binding of the masking portion to the ligand (e.g., a cytokine) spatially hinders the binding of the ligand to the ligand-binding portion (e.g., a cytokine receptor). Yet another embodiment of the present invention is an activatable fusion protein, characterized in that the binding of the masking portion to the ligand (e.g., a cytokine) spatially hinders the binding of the first antigen-binding portion to the antigen. In other words, the binding of the first antigen-binding portion to the target antigen spatially competes with the binding of the masking portion to the ligand.

[0027] One embodiment of the present invention is an activatable fusion protein, characterized in that the affinity of the first antigen-binding portion of the activatable fusion protein for the target antigen is reduced compared to the affinity of the individual first antigen-binding portion. Another embodiment of the present invention is an activatable fusion protein, characterized in that the binding of the first antigen-binding portion to the target antigen releases the ligand (e.g., cytokines) from the masking portion bound to the ligand. A third embodiment of the present invention is an activatable fusion protein, characterized in that the binding of the first antigen-binding portion to the target antigen prevents the masking portion from (re)binding to the ligand (e.g., cytokines).

[0028] One embodiment of the present invention is an activatable fusion protein, characterized in that it further comprises a second antigen-binding domain. In one embodiment, the second antigen-binding domain is an antibody capable of specifically binding to an antigen, an antibody fragment (such as Fab, DutaFab, DAF, Fv, Fab', Fab'-SH, F(ab')2, a biantibody, a linear antibody, a multispecific antibody formed from an antibody fragment, scFv, a nanobody, VHH, or a variable domain (VNAR) of a neoantigen receptor), an antibody mimic (such as DARPIN, affibody, monobody, or anticalin), an endogenous protein domain that interacts with a target, an engineered TCR, or a peptide. In a particular embodiment, the second antigen-binding domain 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 domain is Fab, DutaFab, DAF, or DBA. In another embodiment, the second antigen-binding portion contained in the activatable fusion protein described herein is a single-chain antigen-binding portion, preferably a single-chain antigen-binding portion selected from the group consisting of: scFv, scFab, VHH, VNAR, domain antibody (dAb), DARPin, affibody, monobody, anticalin, and single-domain antibody (sdAb).

[0029] One embodiment of the present invention is an activatable fusion protein, characterized in that it further comprises an Fc domain, the Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide.

[0030] One embodiment of the present invention is an activatable fusion protein characterized in that a first antigen-binding portion is covalently linked to the N-terminus or C-terminus of one of two Fc domain heavy chain polypeptides. In one embodiment, a masking portion is linked at its N-terminus to the N-terminus or C-terminus of one of the two Fc domain heavy chain polypeptides. The fusion between the first antigen-binding domain and the Fc domain can be via a peptide linker, which may also include or be composed of an immunoglobulin hinge region.

[0031] One embodiment of the present invention is an activatable fusion protein, characterized in that a first heavy chain polypeptide or a first light chain polypeptide of the first antigen-binding portion is covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide via its C-terminus a) or b) via covalently linked to the C-terminus of the first Fc domain heavy chain polypeptide.

[0032] One embodiment of the present invention is an activatable fusion protein characterized in that a second antigen-binding moiety is covalently linked to the N-terminus or C-terminus of one of two Fc domain heavy chain polypeptides. In one embodiment, the activatable fusion protein is characterized in that the second antigen-binding moiety is Fab, Dutafab, DAF, or DBA, and the second heavy chain polypeptide or the second light chain polypeptide of the second antigen-binding moiety is a) covalently linked at its C-terminus to the N-terminus of the second Fc domain heavy chain polypeptide, or b) covalently linked to the C-terminus of the first or 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: scFv, scFab, VNAR, domain antibody (dAb), DARPin, affibody, monobody, antiicalin, and single-domain antibody (sdAb), nanobody, and VHH. The fusion between the second antigen-binding domain and the Fc domain is via a peptide linker, which may also include or consist of an immunoglobulin hinge region.

[0033] One embodiment of the present invention is an activatable fusion protein, characterized in that...

[0034] a) The first antigen-binding moiety is covalently linked to the N-terminus of the heavy chain polypeptide with the first Fc domain, and the second antigen-binding moiety is covalently linked to the N-terminus of the heavy chain polypeptide with the second Fc domain, or

[0035] b) The first antigen-binding portion is covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide or the second Fc domain heavy chain polypeptide, and the second antigen-binding portion is covalently linked to the C-terminus of the first Fc domain heavy chain polypeptide or the second Fc domain heavy chain polypeptide.

[0036] One embodiment of the present invention is an activatable fusion protein characterized in that a second antigen-binding moiety is covalently linked to the N-terminus of a ligand (e.g., a cytokine) via a third peptide linker, and the third peptide linker does not contain a protease cleavage site.

[0037] One embodiment of the present invention is an activatable fusion protein characterized in that the N-terminus or C-terminus of the first heavy chain polypeptide of the Fc domain is covalently linked to the N-terminus of a ligand (e.g., a cytokine) via a third peptide linker, the second antigen-binding moiety is covalently linked 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 the third and fourth peptide linkers do not contain protease cleavage sites.

[0038] One embodiment of the present invention is an activatable fusion protein, wherein the masking portion includes a single-chain antigen-binding portion.

[0039] The characteristic feature is that the activatable fusion protein comprises:

[0040] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0041] b) A second polypeptide comprising (b1) a ligand (e.g., a cytokine) fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus and (b2) the light chain polypeptide of the first antigen-binding moiety.

[0042] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0043] d) The fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0044] One embodiment of the present invention is an activatable fusion protein, wherein the masking portion includes a single-chain antigen-binding portion.

[0045] The characteristic feature is that the activatable fusion protein comprises:

[0046] a) A first polypeptide comprising (a1) a ligand (e.g., a cytokine) fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0047] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding portion at its C-terminus and (b2) a light chain polypeptide of the first antigen-binding portion.

[0048] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0049] d) The fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0050] One embodiment of the present invention is an activatable fusion protein, wherein the masking portion includes a single-chain antigen-binding portion, and the second antigen-binding portion includes a single-chain antigen-binding portion.

[0051] The characteristic feature is that the activatable fusion protein comprises:

[0052] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0053] b) A second polypeptide comprising (b1) a ligand (e.g., a cytokine) fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus and (b2) the light chain polypeptide of the first antigen-binding moiety, and

[0054] c) A third polypeptide comprising (c1) a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0055] One embodiment of the present invention is an activatable fusion protein, wherein the masking portion includes a single-chain antigen-binding portion, and the second antigen-binding portion includes a single-chain antigen-binding portion.

[0056] The characteristic feature is that the activatable fusion protein comprises:

[0057] a) A first polypeptide comprising (a1) a ligand (e.g., a cytokine) fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0058] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding portion at its C-terminus and (b2) a light chain polypeptide of the first antigen-binding portion, and

[0059] c) A third polypeptide comprising (c1) a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0060] One embodiment of the invention is an activatable fusion protein characterized by a second antigen-binding moiety capable of binding specifically to an antigen that is the same as or different from the target antigen. Selecting a second antigen-binding moiety capable of binding specifically to the same antigen as the first antigen-binding moiety is particularly useful when the target antigen, although exhibiting specific expression in the desired target tissue, is not expected to be expressed in very high quantities. Selecting a second antigen-binding moiety binding to a different antigen as the first antigen-binding moiety can be useful, for example, when the target antigen is also expressed in tissues other than the target tissue. Combining two different target antigens can increase the specificity of the activatable fusion protein of the invention if only the desired target tissue exhibits expression of both antigens.

[0061] One embodiment of the present invention is an activatable fusion protein characterized in that a second antigen-binding moiety (a) binds to a target antigen and (b) binds to an epitope on the target antigen that is different from the epitope bound by the first antigen-binding moiety. Choosing the second antigen-binding moiety may be useful because it will not saturate the epitope on the target antigen bound by the first antigen-binding moiety, and thus the epitope remains accessible to the first antigen-binding moiety for demasking of ligands (e.g., cytokines). By selecting a second antigen-binding moiety that binds to a different epitope on the same target antigen as the first antigen-binding moiety, the activation mechanism of the activatable fusion protein can occur intramolecularly rather than intermolecularly, provided that the linkers and orientations in the activatable fusion protein allow simultaneous binding to two epitopes in the same antigen molecule. Thus, the activatable fusion protein of the present invention can be activated not only by membrane-bound target antigens but also by soluble target antigens, and can further exhibit enhanced demasking.

[0062] One embodiment of the invention is an activatable fusion protein characterized in that a second antigen-binding moiety binds to the same epitope on the target antigen as the first antigen-binding moiety. By selecting two antigen-binding moieties that bind to the same epitope, the activatable fusion protein can be made more dependent on the expression level of the target antigen molecule in the target tissue, because if another target antigen molecule is available that is sufficiently close to the target antigen molecule bound by the second antigen-binding moiety, the ligand (e.g., cytokine) can only be released by the first antigen-binding moiety.

[0063] One embodiment of the present invention is an activatable fusion protein, characterized in that the first antigen-binding portion is an IgG type antibody. In a specific embodiment of the present invention, the activatable fusion protein is characterized in that the first antigen-binding portion, the second antigen-binding portion, and the Fc region together form an IgG type antibody. In some embodiments, the activatable fusion protein is characterized in that the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain of the activatable fusion protein is an IgG1 Fc domain or an IgG4 Fc domain.

[0064] One embodiment of the present invention is an activatable fusion protein characterized in that it comprises at least two full-length IgG antibody heavy chains, wherein the heavy chain of the antigen-binding portion is γ-type (IgG), particularly γ1-type.

[0065] One embodiment of the present invention is an activatable fusion protein, characterized in that the activatable fusion protein comprises at least two light chains, wherein the light chain of the antigen-binding portion is selected from the Kappa (κ) and / or Lambda (λ) subtypes.

[0066] One embodiment of the present invention is an activatable fusion protein, characterized in that the target antigen is selected from the group consisting of: α-synuclein, amyloid-β, 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, FAPα, 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-4Rα, integrin αv, b7 integrin subunit, a4b7 integrin, a4 Integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notch1, Notch3, NRP1, NRP2, OX40, PAR-2, PD-1, PD-L1, PDGFR, P SA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-C1, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1. TGFβ, TGF-γ, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2, or 5T4. In a specific embodiment of the invention, the target antigen is selected from the group consisting of: PD1, PD-L1, CD8, and CD19.

[0067] One embodiment of the present invention is an activatable fusion protein, characterized in that the ligand is an antigen-binding portion.

[0068] One embodiment of the present invention is an activatable fusion protein, characterized in that the antigen-binding portion is an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of: α-synuclein, amyloid-β, 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, FAPα, 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-4Rα, integrin αv, b7 integrin subunit, a4b7 integrin, a4 Integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notch1, Notch3, NRP1, NRP2, OX40, PAR-2, PD-1, PD-L1, PDGFR, P SA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-C1, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1. TGFβ, TGF-γ, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2 or 5T4.

[0069] One embodiment of the present invention is an activatable fusion protein, characterized in that the antigen-binding portion is an antibody or antibody fragment capable of specifically binding to an antigen selected from the group consisting of: BCMA, CD3, CD20, CD19, CD27, CD28, CD40, CD47, CD123, CD137, CEA, CTLA4, DLL3, EpCAM, GP100, GITR, HER2, HLA-G, IL-7, kynurenase, MICA, MICB, OX40, PD-L1, PD-1, the extracellular domain of TGFBR2, TNF, or VEGF-C. In a specific embodiment of the present invention, the target antigen is selected from the group consisting of: PD1, PD-L1, CD8, and CD19.

[0070] One embodiment of the present invention is an activatable fusion protein, characterized in that the ligand is a non-cytokine ligand selected from the group consisting of or any combination thereof: growth factors, chemokines, antibodies, antibody fragments, enzymes, receptor ligands, affinity peptide ligands, peptide hormones, receptor agonists, receptor antagonists, enzymes, soluble receptors, protein toxins, soluble ligands, extracellular regions of cell surface receptors, extracellular regions of cell surface ligands, and small molecules.

[0071] This invention provides an isolated nucleic acid that encodes an activated fusion protein as described herein.

[0072] The present invention provides a host cell containing such nucleic acids.

[0073] This invention provides an in vitro method for producing an activatable fusion protein as described herein, the in vitro method comprising culturing host cells under conditions suitable for expressing the activatable fusion protein as described herein. In a particular embodiment, the method further comprises recovering antibodies from the host cells.

[0074] This invention provides an activatable fusion protein produced by such a method.

[0075] The present invention provides a pharmaceutical composition comprising an activatable fusion protein as described herein and a pharmaceutically acceptable carrier.

[0076] This invention provides an activatable fusion protein or pharmaceutical composition as described herein, which is used as a drug.

[0077] This invention provides an activatable fusion protein or a pharmaceutical composition as described herein for the treatment of cancer, viral infection, or autoimmune disease.

[0078] This invention provides the use of an activatable fusion protein as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament. In one embodiment, the medicament is used to treat cancer, viral infection, or autoimmune disease.

[0079] One embodiment of the present invention is an activatable fusion protein comprising (A) a first antigen-binding portion 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 portion 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 the ligand-binding portion (e.g., a cytokine receptor); and (D) a masking portion comprising a single-chain antigen-binding portion capable of specifically binding to the ligand.

[0080] The characteristic feature is that the activatable fusion protein comprises:

[0081] a) A first polypeptide comprising (a1) a ligand fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding moiety at its C-terminus via a first peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding moiety fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0082] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a second peptide linker and (b2) a light chain polypeptide of the first antigen-binding moiety.

[0083] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0084] d) A fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0085] Neither the first nor the second peptide linker contains a protease cleavage site.

[0086] One embodiment of the present invention is an activatable fusion protein comprising (A) a first antigen-binding portion 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 portion 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 the ligand-binding portion (e.g., a cytokine receptor); and (D) a masking portion comprising a single-chain antigen-binding portion capable of specifically binding to the ligand.

[0087] The characteristic feature is that the activatable fusion protein comprises:

[0088] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0089] b) A second polypeptide comprising (b1) a ligand fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a first peptide linker and (b2) the light chain polypeptide of the first antigen-binding moiety.

[0090] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0091] d) A fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0092] Neither the first nor the second peptide linker contains a protease cleavage site.

[0093] One embodiment of the present 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 the 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.

[0094] The characteristic feature is that the activatable fusion protein comprises:

[0095] a) A first polypeptide comprising (a1) a ligand fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding moiety at its C-terminus via a first peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding moiety fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0096] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a second peptide linker, and (b2) a light chain polypeptide of the first antigen-binding moiety, and

[0097] c) A third polypeptide comprising (c1) a second antigen-binding moiety fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0098] Neither the first nor the second peptide linker contains a protease cleavage site.

[0099] One embodiment of the present 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 the 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.

[0100] The characteristic feature is that the activatable fusion protein comprises:

[0101] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0102] b) A second polypeptide comprising (b1) a ligand fused at the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a first peptide linker, and (b2) the light chain polypeptide of the first antigen-binding moiety, and

[0103] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0104] Neither the first nor the second peptide linker contains a protease cleavage site.

[0105] This invention further provides antibodies that bind to IL-2 or its variants. It has been found that the antibodies of this invention bind not only to human IL-2 but also to IL-2v, an attenuated variant of human IL-2 with eliminated CD25 (IL-2Rα) binding. The anti-IL-2 antibodies disclosed herein compete with IL-2Rβ for IL-2 / IL-2v binding. By masking IL-2 through disruption of interaction with the high-affinity receptor IL-2Rβ (rather than the low-affinity receptor IL-2Rγ), they are able to reduce target-mediated drug disposal (TMDD) by preventing significant binding to the cell surface, and—because they do not aggregate via IL-2Rβ, thus not increasing local concentrations—also increase IL-2 masking efficiency. In some embodiments of the invention, the anti-IL-2 antibodies disclosed herein not only block the binding of IL-2 to IL-2Rβγ by competing with IL-2Rβγ for IL-2 binding, but also surprisingly simultaneously prevent the binding of IL-2 to IL-2Rα (CD25). Such anti-IL-2 antibodies exhibiting effective competitive binding to IL-2Rα are very useful as maskers of the IL-2 molecule because they completely eliminate IL-2 activity, making wild-type IL-2 available for treatment, rather than attenuated variants such as IL-2v. This is an advantage because introducing amino acid substitutions to attenuate cytokines carries the risk of increasing immunogenicity in patients. In the context of the activatable fusion proteins described herein, the anti-IL-2 antibodies disclosed herein, when used as a masking portion of IL-2, possess the ability to bind to IL-2Rα on the cell surface once IL-2 is released, thus facilitating overall tumor targeting of the activatable fusion protein. In the context of cancer treatment, anti-IL2 antibodies that bind to IL-2Rα can prove helpful because they can prevent IL-2Rα from binding to Treg and Breg. Furthermore, since IL-2Rα is a high-affinity receptor, masking it by reducing TMDD (the mechanism by which a drug binds to its pharmacological target to a certain extent, thus affecting its pharmacokinetic characteristics) may have a positive impact.

[0106] One embodiment of the invention is an antibody that binds to IL-2 or a variant thereof, wherein the antibody binds to an IL-2 epitope within amino acid residues 8-17, 30, and / or 77-81 of SEQ ID NO:81; and thus inhibits the binding of IL-2 to IL-2Rβγ and IL-2Rα. In one embodiment, the epitope comprises amino acid residues corresponding to K8, Q13, E15, H16, N30, N77, H79, and R81 of SEQ ID NO:81; and thus the binding of the anti-IL2 antibody to IL-2 inhibits the binding of IL-2 to IL-2Rβγ and IL-2Rα.

[0107] One embodiment of the present invention is an antibody that binds to IL-2 or a variant thereof, wherein the antibody comprises

[0108] (A) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0109] (B) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0110] (C) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0111] (D) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0112] (E) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0113] (F) 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 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:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0114] (G) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0115] (H) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0116] (I) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; or

[0117] (J) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17.

[0118] One embodiment of the present invention is an antibody comprising...

[0119] (A) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0120] (B) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0121] (C) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0122] (D) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0123] (E) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0124] (F) 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 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:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0125] (G) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0126] (H) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0127] (I) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; or

[0128] (J) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17.

[0129] In a particular embodiment of the invention, the antibody comprises

[0130] (A) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0131] (B) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0132] (C) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0133] (D) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:33; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0134] (E) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0135] (F) 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 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;

[0136] (G) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; or

[0137] (F) 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 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.

[0138] In another specific embodiment of the invention, the antibody comprises: a heavy chain variable domain (VH) comprising: (a) CDR-H1 containing the amino acid sequence of SEQ ID NO:20, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:46, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:23, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO:17.

[0139] In one embodiment of the invention, the antibody is a monoclonal antibody. In another embodiment, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In a particular embodiment, the antibody is an antibody fragment that binds to IL-2 or a variant thereof.

[0140] One embodiment of the present invention is an antibody comprising...

[0141] (A) The VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:8;

[0142] (B) The VH sequence of SEQ ID NO:12 and the VL sequence of SEQ ID NO:13;

[0143] (C) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19;

[0144] (D) The VH sequence of SEQ ID NO:24 and the VL sequence of SEQ ID NO:25;

[0145] (E) The VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO:31;

[0146] (F) The VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:36;

[0147] (G) The VH sequence of SEQ ID NO:38 and the VL sequence of SEQ ID NO:39;

[0148] (H) The VH sequence of SEQ ID NO:41 and the VL sequence of SEQ ID NO:42;

[0149] (I) The VH sequence of SEQ ID NO:44 and the VL sequence of SEQ ID NO:45; or

[0150] (J) The VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0151] In one embodiment of the invention, the antibody that specifically binds to IL-2 or a variant thereof comprises:

[0152] (A) The VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:8;

[0153] (B) The VH sequence of SEQ ID NO:12 and the VL sequence of SEQ ID NO:13;

[0154] (C) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19;

[0155] (D) The VH sequence of SEQ ID NO:24 and the VL sequence of SEQ ID NO:25;

[0156] (E) The VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO:31;

[0157] (F) The VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:36;

[0158] (G) The VH sequence of SEQ ID NO:38 and the VL sequence of SEQ ID NO:39;

[0159] (H) The VH sequence of SEQ ID NO:41 and the VL sequence of SEQ ID NO:42;

[0160] (I) The VH sequence of SEQ ID NO:44 and the VL sequence of SEQ ID NO:45; or

[0161] (J) The VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0162] In a specific embodiment of the present invention, the antibody comprises:

[0163] (A) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19;

[0164] (B) The VH sequence of SEQ ID NO:24 and the VL sequence of SEQ ID NO:25;

[0165] (C) The VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO:31;

[0166] (D) The VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:36;

[0167] (E) The VH sequence of SEQ ID NO:38 and the VL sequence of SEQ ID NO:39;

[0168] (F) The VH sequence of SEQ ID NO:41 and the VL sequence of SEQ ID NO:42;

[0169] (G) The VH sequence of SEQ ID NO:44 and the VL sequence of SEQ ID NO:45; or

[0170] (H) The VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0171] In another specific embodiment of the invention, the antibody comprises the VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:48.

[0172] In one embodiment of the present invention, the antibody is a full-length IgG1 antibody or Fab.

[0173] In one embodiment, the antibody binds to IL-2 with an affinity of ≤ 125 nM or to IL-2v with an affinity of ≤ 15.4 nM, as measured by an SPR assay.

[0174] In one embodiment, the antibody binds to IL-2 with an affinity of ≤ 1.5 nM or to IL-2v with an affinity of ≤ 0.9 nM, as measured by an SPR assay.

[0175] In one embodiment, the antibody is a multispecific antibody.

[0176] In one embodiment, the antibody is Fab and comprises:

[0177] (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:50;

[0178] (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:52;

[0179] (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54;

[0180] (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56;

[0181] (E) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58;

[0182] (F) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:59 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:60;

[0183] (G) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:61 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:62;

[0184] (H) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing the amino acid sequence of SEQ ID NO:64;

[0185] (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

[0186] (J) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:67 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:68.

[0187] In one specific embodiment, the antibody is Fab and comprises:

[0188] (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54;

[0189] (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56;

[0190] (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58;

[0191] (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:60;

[0192] (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;

[0193] (F) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing the amino acid sequence of SEQ ID NO:64;

[0194] (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

[0195] (H) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:67 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:68.

[0196] In another specific embodiment, the antibody is 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.

[0197] In one embodiment, the antibody is a full-length antibody and comprises:

[0198] (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:50;

[0199] (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:52;

[0200] (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54;

[0201] (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56;

[0202] (E) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58;

[0203] (F) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:59 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:60;

[0204] (G) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:61 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:62;

[0205] (H) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing the amino acid sequence of SEQ ID NO:64;

[0206] (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

[0207] (J) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:67 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:68.

[0208] Furthermore, the antibody further comprises a human Fc region containing 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 NO:74 and SEQ ID NO:75.

[0209] In one particular embodiment, the antibody is a full-length antibody and comprises:

[0210] (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54;

[0211] (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56;

[0212] (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58;

[0213] (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:59 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:60;

[0214] (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;

[0215] (F) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing the amino acid sequence of SEQ ID NO:64;

[0216] (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

[0217] (H) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:67 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:68.

[0218] Furthermore, it includes a human Fc region comprising a human Fc region polypeptide 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 NO:74 and SEQ ID NO:75.

[0219] In another specific embodiment, the antibody as 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 comprising 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 NO:74 and SEQ ID NO:75.

[0220] One embodiment of the present invention is an antibody that competes with the antibodies of any one of Examples 58 to 72 for binding to IL-2 or a variant thereof.

[0221] One embodiment of the invention is an isolated nucleic acid encoding the antibody described herein. Another embodiment of the invention is a host cell containing a nucleic acid encoding the antibody described herein. One embodiment of the invention is a method for producing an antibody that binds to IL-2 or a variant thereof, the method comprising culturing the host cell under conditions suitable for antibody expression. In one embodiment, the method further comprises recovering the antibody from the host cell. Another embodiment of the invention is an antibody produced by the method described herein.

[0222] One embodiment of the present invention is a pharmaceutical composition comprising an antibody as described herein and a pharmaceutically acceptable carrier.

[0223] One embodiment of the present invention is an anti-IL-2 antibody as described herein or a pharmaceutical composition as described herein, which is used as a medicine.

[0224] One embodiment of the present invention is an anti-IL-2 antibody as described herein or a pharmaceutical composition as described herein, for the treatment of cancer, viral infection or autoimmune disease.

[0225] One embodiment of the present invention is the use of an antibody or pharmaceutical composition as described herein in the manufacture of a medicament for cancer, viral infection, or autoimmune disease.

[0226] One embodiment of the present invention is a method for treating an individual suffering from cancer, viral infection, or autoimmune disease, the method comprising administering to the individual an effective amount of an antibody or pharmaceutical composition as described herein. In one embodiment, the method further comprises administering to the individual an adjunct therapeutic agent. In one embodiment, the adjunct therapeutic agent is selected from the group consisting of: anticancer agents (e.g., microtubule disruptors), antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapy, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, or antiangiogenic agents.

[0227] One embodiment of the present invention uses the anti-IL-2 antibody described herein as a masking antibody for IL-2, IL-2v, or variants thereof. Another embodiment of the present invention uses the anti-IL-2 antibody described herein as a masking portion in the activatable fusion protein described herein. Attached Figure Description

[0228] Figure 1 A: Schematic representation of an exemplary Fab-based, activatable fusion protein (“CISS Fab molecule”). This figure illustrates the reversible binding between a ligand (here, a cytokine) and a masking moiety (here, an anti-cytokine masker, “α-cytokine”) and how it spatially blocks the antigen-binding moiety (“α-target”) from approaching the target antigen (not depicted).

[0229] Figure 1B: Schematic illustration of an exemplary activatable fusion protein comprising a “CISSFab molecule” and a second antigen-binding moiety as an additional targeting arm for fusion with the Fc domain, thereby generating an IgG-based molecule. The molecule shown here consists of four polypeptide chains.

[0230] Figure 1C: Schematic illustration of an exemplary activatable fusion protein comprising a “CISSFab molecule” and a second antigen-binding moiety as an additional targeting arm for fusion with the Fc domain, wherein the second antigen-binding moiety is a single-chain antigen-binding moiety (specifically VHH). The molecule shown here consists of three polypeptide chains.

[0231] Figure 2 A: Schematic illustration of ligand Fab fusion used as a CISS Fab precursor molecule in an example.

[0232] Figure 2B: Schematic illustration of masking Fab fusion used as a CISS Fab precursor molecule in an example.

[0233] Figure 2C: Schematic representation of an exemplary complete CISS Fab molecule.

[0234] Figure 3A: Schematic illustration of the mechanism of action of the CISS Fab molecule. In solution, in the absence of the target antigen, the masking portion of the CISS Fab molecule binds to the ligand, preventing the ligand from binding to the ligand-binding portion. In the presence of the target antigen, Fab binds to it, while simultaneously preventing the masking portion from binding to the ligand; the ligand now freely binds to the ligand-binding portion.

[0235] Figure 3B: Schematic illustration of an activatable fusion protein of the “targeting CISS molecule” type. (I) In solution, the ligand (dot-like ellipses) is masked by the masking portion (dark dot-like rectangles) and cannot bind to the ligand-binding portion expressed on the cell surface. When the interaction between the ligand and the masking portion oscillates open, the ligand-binding portion is typically at a very low concentration relative to the ligand compared to the masking portion, thus the masking interaction is usually re-established. (II) In the presence of the target antigen, the activatable fusion protein binds to the cell surface via the second antigen-binding portion. (III) After the second antigen-binding portion binds to the target antigen on the cell surface, the effective antigen concentration of the first antigen-binding portion increases. When the interaction between the ligand and the masking portion now oscillates between open and closed, it opens the binding surface of the first antigen-binding portion to bind to the target antigen. (IV) The first antigen-binding portion binds to the second cell surface target antigen, keeping the ligand mask open. (V) The ligand interacts with the ligand-binding portion on the cell surface.

[0236] Figures 4A through 4D: Results of HEK-Blue assays (Fab cytokine fusions) of CISS precursor molecules used to identify appropriate cytokine linker lengths. The X-axis shows the concentration of molecules in contact with cells during the assay (in [nM]) on a logarithmic chemical scale. The Y-axis shows the signal intensity (OD). 620nm ).

[0237] Figures 5A through 5E: Results of HEK-Blue assays of CISS Fab molecules used to assess the masking ability of CISS Fab molecules. The X-axis shows the concentration of molecules in contact with cells during the assay (in [nM]) on a logarithmic chemical scale. The Y-axis shows the signal intensity (OD). 620nm ).

[0238] Figures 6A to 6B: Exemplary datasets of PD1-IL-2v CISS Fab and precursors of each desired property among the desired properties of CISS molecules.

[0239] Figure 6 A: Confirms that the length of the cytokine linker is sufficient to reach the HEK-Blue assay of the cytokine receptor.

[0240] Figure 6B: Results of SPR assays used to select potentially functional CISS Fab molecules.

[0241] Figure 7: Results of HEK-Blue IL-2 reporter cell assays assessing the activity of two CISS-targeting IL-2 molecules in PD1-dependent and independent environments.

[0242] Figure 8: Comparison of HEK-Blue IL-2 reporter gene assay results for PD1-IL-2v CISS with and without the PD1-binding target subunit.

[0243] Figure 9: Results of HEK-Blue IL-2 reporter gene assay showing that mutually exclusive CISS switching enhances function compared to simple cytokine masking.

[0244] Figures 10A to 10B: Showing the results of HEK-Blue IL-2 reporter gene assays using CD8 IL-2v CISS Fab molecules (and precursor molecules) from HEK-Blue IL-2 reporter gene cells expressing CD8, compared to HEK-Blue IL-2 reporter gene cells that do not express CD8 on their surface.

[0245] Figures 11A to 11B: Schematic illustration of different exemplary T cell conjugate molecular forms utilizing the CISS Fab unit.

[0246] Figure 11A: The antigen-binding moiety of this CISS T-cell conjugate form (“CD3 outer”) targets the target antigen. The ligand is an anti-CD3 single-chain Fv masked by anti-idiotype VHH. The second antigen-binding moiety (“targeting arm”) also targets the target antigen.

[0247] Figure 11B: The antigen-binding moiety of this CISS T-cell conjugate form (“CD3 interior”) is the anti-CD3 Fab arm. The ligand is the anti-target antigen scFv masked by the idiotype VHH. The second antigen-binding moiety (“target arm”) binds to the target antigen.

[0248] Figure 12: Schematic illustration of the action mode of the "CD3 external" CISS T cell binding agent molecule.

[0249] Figure 13: A schematic illustration of an exemplary CISS molecular form activated via an intramolecular mechanism and its mode of action. This form comprises a first antigen-binding moiety, a second antigen-binding moiety, a ligand, and a masking moiety. In this example, both the second antigen-binding moiety and the masking moiety are single-chain antigen-binding moieties. The ligand is covalently linked via a peptide linker to the N-terminus of either the heavy-chain or light-chain polypeptide of the first antigen-binding moiety. The masking moiety is covalently linked via a peptide linker at its C-terminus to the N-terminus of another polypeptide of the first antigen-binding moiety. The second antigen-binding moiety binds to the masking moiety via a peptide linker at its C-terminus or its N-terminus.

[0250] Figures 14A to 14C: Schematic illustration of exemplary alternative targeted CISS molecular forms. In addition to the CISS module, these forms also contain an Fc domain and a second antigen-binding moiety.

[0251] Figure 14A: The first antigen-binding domain of the CISS module is not directly linked to the Fc domain. Instead, it is covalently linked to the C-terminus of a masking portion at the N-terminus of one of the heavy-chain polypeptides via a peptide linker. This masking portion itself is covalently linked to the C-terminus of one of the two Fc domain heavy-chain polypeptides at its N-terminus via another peptide linker. The second antigen-binding domain is covalently linked to the C-terminus of the other of the two Fc domain heavy-chain polypeptides via another peptide linker via one of its N-termini. In this example, the second antigen-binding portion is CrossFab (indicated by different coloring of the variable domain) to facilitate correct heavy-chain / light-chain pairing during manufacturing.

[0252] Figure 14 B: The first antigen-binding portion of the CISS module is covalently linked to the N-terminus of the Fc domain at the C-terminus of its heavy chain polypeptide via a peptide linker, while the second antigen-binding portion is covalently linked to the C-terminus of one of the Fc domain heavy chain polypeptides at the N-terminus via another peptide linker.

[0253] Figure 14C: Schematic illustration of the mechanism of action of the molecule shown in Figure 14A. The molecule binds to the target antigen on the cell surface via a second antigen-binding moiety (“targeting arm”). When the masking agent temporarily releases a 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 masking agent-cytokine interaction and releasing the cytokine to bind to a cytokine receptor (not shown) on the cell surface.

[0254] Figure 15: SPR sensor diagrams of molecules P1AJ7955, P1AI4373 (1040int Fab), P1AJ7982 (a bispecific molecule containing Fc, with a nonfunctional Fab (DP47) fused to one Fc peptide and a G05VHH fused to another Fc peptide), and P1AJ6652. P1AJ7955 exhibits significantly higher affinity than either PD1 conjugate alone. This indicates the ability of P1AJ7955 to bind to PD1 twice, resulting in improved binding. The 221 scFv contained within molecule P1AJ7955 maintains good binding with IL-2v, indicating no fusion intolerance induced by G05VHH. SPR results for P1AJ6652 confirm that the masking interaction appears to be functional (as indicated by the absence of IL-2v or IL-2Rbg binding).

[0255] Figure 16: Comparison of results from HEK-Blue IL-2 reporter gene assays using attenuated IL-2 variant Q126T targeting the PD1 IL-2 CISS molecule with those using IL-2v (which has full IL-2R signaling) as a cytokine.

[0256] Figure 17: Comparison of HEK-Blue IL-2 reporter gene assays using masking portions of the PD1 IL-2 CISS molecule with different affinities for cytokines.

[0257] Figure 18: Schematic illustration of the SPR assay setup used to determine the competition between generated anti-IL-2 Fab and IL-2Rβγ for IL-2 binding.

[0258] Figures 19A to 19C: Schematic illustration of SPR-based competitive binding assays used to determine whether the binding of generated anti-IL-2 Fab to IL-2 competes with the binding of IL-2 to IL-2Rbg.

[0259] Figures 20A to 20B: Schematic illustration of SPR-based competitive binding assays used to determine whether the binding of generated anti-IL-2 / IL-2v specific Fab competes with the binding of IL-2 to IL-2Ra (CD25).

[0260] Figure 21: Results of SPR analysis used to determine the binding affinity of generated anti-IL-2 / IL-2v Fab using a Biacore 8K or 8K+ instrument (Cytiva). Binding kinetics with human IL-2v (right column), human IL-2 (middle column), and CD79B dimer (control, left column) were analyzed in this assay.

[0261] Figure 22: Results of SPR-based competitive binding assays used to determine whether the binding of generated anti-IL-2 Fab to IL-2 competes with the binding of IL-2 to IL-2Rbg.

[0262] Figures 23A to 23B: Results of SPR-based competitive binding assays used to determine whether the binding of the generated anti-IL-2 Fab to IL-2 competes with the binding of IL-2 to IL-2Ra (CD25).

[0263] Figures 24A to 24F: Results of SPR analysis used to determine the affinity of anti-IL-2 Fab for affinity maturation.

[0264] Figures 25A to 25B: Results of SPR analysis for evaluating IL-2Rbg and IL-2Ra competition against IL-2 Fab with 221-derived point mutations.

[0265] Figures 26A to 26C: Results of X-ray diffraction analysis of IL-2 binding to the affinity-mature anti-IL-2 Fab P029-221.AM2 and its effect on binding to IL-2Rαβγ.

[0266] Figure 26A: Structure of the P029.221.AM2 IL-2 complex.

[0267] Figure 26B: IL-2 bound to P029-221.AM2 superimposed on the IL-2 / IL-2Rαβγ complex (PDB 2erj).

[0268] Figure 26 C: P029-221.AM2 superimposed on the IL-2 / IL-2αβγ complex.

[0269] Figures 27A to 27F: Structural comparison of IL-2 bound to 221.AM2 and IL-2 bound to the trimer receptor complex (PDB2erj) to elucidate the competition between P029.221.AM2 and IL-2Rα for IL-2 binding.

[0270] Figure 27 A: IL-2 / P029.221.AM2 complex superimposed on unbound IL-2 (PDB 2erj, front).

[0271] Figure 27 B: IL-2 / P029.221.AM2 complex superimposed on unbound IL-2 (PDB erj, back side).

[0272] Figure 27 C: Superposition of IL-2 bound to P021.221.AM2 and unbound IL-2 (PDB 2erj, front).

[0273] Figure 27 D: Superposition of IL-2 bound to P021.221.AM2 and unbound IL-2 (PDB 2erj, details).

[0274] Figure 27 E: IL-2 (PDB 2erj) / IL-2Rα complex superimposed on IL-2 bound to P021.221.AM2.

[0275] Figure 27 F: IL-2 (PDB 2erj) / IL-2Rα complex superimposed on IL-2 bound to P021.221.AM2, details.

[0276] Figures 28A to 28B: Comparison of results from activated HEK-Blue IFNα reporter gene assays performed on PD-L1-IFNα CISS molecules with and without the second antigen-binding moiety that targets PD-L1 on the cell surface.

[0277] Figure 28 A: Chart illustrating IFNα activation in parental HEK-Blue™ IFNα / β reporter gene cell lines with relatively low PD-L1 expression (“HEK-Blue IFNα PD-L1 low”).

[0278] Figure 28 B: Graph illustrating IFNα activation in cells with high expression of PD-L1 positive IFNα reporter gene (clone 45, CL022702, "HEK-Blue IFNa PD-L1 high").

[0279] Figure 29: Comparison of IL-2 activation assays of CD8 IL-2 CISS molecules (CISS Fab and targeting CISS molecules) in CD8a-negative (parental) HEK-Blue IL-2 reporter cells (labeled "HEK-BlueIL-2", solid lines and circles) and PD1 and CD8a-positive IL-2 reporter cell lines (CL023901; labeled "HEK-Blue IL-2 CD8a", dashed lines and triangles). Additionally, the condition where CD8a molecules on PD1 and CD8a-positive IL-2 reporter cells (CL023901) were pre-blocked by the OKT8 variant (P1AF4823) (labeled "HEK-Blue IL-2 CD8a Blocked", dashed lines and squares).

[0280] Figure 30: Schematic illustration of the STAT-5P assay used to determine IL-2R signaling in cis (activation of the IL-2 receptor on the same cell) and trans (activation of the IL-2 receptor on another cell). For this assay, CD4 T cells from healthy donor PBMCs were sorted and activated for 3 days with anti-CD3 and anti-CD28 antibodies to induce PD-1 expression. After 3 days, cells were labeled with Cell Trace Violet (CTV) or unlabeled cells, and then unlabeled cells were blocked with anti-PD-1 antibody. These PD-1 pre-blocked cells were co-cultured with PD-1 + CTV-labeled cells and treated with different concentrations of immunoconjugates. STAT-5 phosphorylation was assessed using flow cytometry, and data analysis provided insights into the molecular power of IL-2R signaling, both dependent on and independent of PD-1 expression.

[0281] Figures 31A to 31C: Results of IL-2R signal transduction measurements.

[0282] Figure 32: Schematic illustration of the STAT-5-P assay used to determine IL-7R activation. CD4 T cells from healthy donor PBMCs were sorted and activated for 3 days with anti-CD3 and anti-CD28 antibodies to induce PD-1 expression. After 3 days, cells were labeled with Cell Trace Violet (CTV) or unlabeled cells, and then unlabeled cells were blocked with anti-PD-1 antibody. These PD-1 pre-blocked cells were co-cultured with PD-1 + CTV-labeled cells and treated with different concentrations of immunoconjugates. STAT-5 phosphorylation was assessed using flow cytometry, and data analysis provided insights into the molecular efficacy of IL-7R signaling, both dependent on and independent of PD-1 expression. Detailed Implementation

[0283] I. Definition

[0284] Unless otherwise defined herein, the scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. The methods and techniques of this disclosure are generally performed according to conventional methods known in the art. Unless otherwise defined herein, the terms and techniques related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are generally known and commonly used in the art.

[0285] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of".

[0286] When the term “about” is used in conjunction with specific values ​​(such as temperature, concentration, time, etc.) in this article, it should refer to a change of + / - 1% in the specific value referred to by the term “about”.

[0287] 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 to a ligand-binding moiety (e.g., a cytokine receptor) in the presence of a target antigen immediately adjacent to the fusion protein (particularly the first antigen-binding moiety that binds to the target antigen).

[0288] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody, ligand) and its binding partner (e.g., antigen, ligand-binding moiety). Unless otherwise specified, 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 molecule X for its partner Y can generally be determined by the dissociation constant (K). D The term ) indicates binding affinity. Affinity can be measured using conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0289] As used herein, the term "antigen" refers to a polypeptide macromolecule that binds to an antigen-binding moiety to form an antigen-binding moiety-antigen complex. Useful antigens can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, the proteins referred to as antigens in this document (e.g., α-synuclein, amyloid-β, 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, FAPα, 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-4Rα, integrin αv, b7 integrin subunit, a4b7 integrin, a4 Integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notch1, Notch3, NRP1, NRP2, OX40, PAR-2, PD-1, PD-L1, PDGFR, P SA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-C1, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1. TGFβ, TGF-γ, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2, or 5T4) can be any naturally occurring protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The antigen can also be a variant of a naturally occurring polypeptide with one or more amino acid substitutions. In one particular embodiment, the antigen is a human protein.

[0290] As used herein, the terms "antigen-binding moiety" or "antigen-binding domain" refer 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, an antigen-binding moiety comprises a heavy chain and a light chain. In some embodiments, the heavy chain and light chain are fused via a peptide linker. In some embodiments, an antigen-binding moiety comprises two polypeptide chains. Specific antigen-binding moieties of the activatable fusion proteins described herein include an antigen-binding domain of an antibody comprising a variable region of the antibody heavy chain and a variable region of the antibody light chain. In some embodiments, an antigen-binding moiety may comprise all or a portion of at least one antibody constant region as further defined herein and known in the art. Available heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Available light chain constant regions include any of two isotypes: κ and λ. In some embodiments, the antigen-binding moiety is a single-chain antigen-binding moiety, such as scFv, scFab, VHH, VNAR, domain antibody (dAb), or single-domain antibody (sdAb). In some embodiments, the single-chain antigen-binding portion is an antibody mimic, such as DARPin, monobody, affibody, or anticalin.

[0291] As used herein, the terms “first” and “second” for antigen-binding moieties, peptide linkers, Fab fragments, etc., are used for ease of distinction when there is more than one of each type of moieties (e.g., within a single fusion protein). Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific sequence or orientation to the bispecific antigen-binding molecule.

[0292] The terms "anti-target antigen (TA) antigen-binding moiety" and "antigen-binding moiety binding to target antigen (TA)" refer to antigen-binding moieties capable of specifically binding to a target antigen (TA) with sufficient affinity, such that the antigen-binding moiety can be used as a diagnostic and / or therapeutic agent targeting TA. In one aspect, as measured, for example, by surface plasmon resonance (SPR), the degree of binding of the anti-TA antigen-binding moiety to unrelated, non-TA proteins is less than about 10% of the binding of the antigen-binding moiety to TA. In some aspects, the dissociation constant (Ki) of the antigen-binding moiety binding to TA is... D ) is ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10-13 M). When the antigen-binding site K D When the concentration is 1 μM or less, the antigen-binding moiety is said to be "specifically bound" to the TA. In some respects, the anti-TA antigen-binding moiety binds to an epitope of the target antigen that is conserved across target antigens from different species.

[0293] The term “antibody” is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0294] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0295] The term "epitope" refers to a site on an antigen (whether protein- or non-protein-based) that binds to an antigen-binding moiety or any other antigen-binding moiety. Epitopes can be formed from continuous amino acid extensions (linear epitopes) or contain discontinuous amino acids (conformational epitopes), for example, due to the folding of antigens, i.e., spatial proximity through tertiary folding of protein antigens. Linear epitopes typically remain bound to the antigen-binding moiety after exposure to a denaturing agent, while conformational epitopes are typically destroyed upon treatment with a denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique stereoconformation.

[0296] Competitive binding can be used to readily determine whether an antigen-binding moiety binds to the same target antigen (TA) epitope as the reference anti-TA antigen-binding moiety or competes with the reference anti-TA antigen-binding moiety for binding. For example, "antigen-binding moiety binding to the same epitope as the reference anti-TA antigen-binding moiety" means an antigen-binding moiety that blocks 50% or more of the binding of the reference anti-TA antigen-binding moiety to its antigen in a competitive assay, and conversely, the reference antigen-binding moiety blocks 50% or more of the binding of the antigen-binding moiety to its antigen in a competitive assay. Similarly, for example, to determine whether an antigen-binding moiety binds to the same epitope as the reference anti-TA antigen-binding moiety, the reference antigen-binding moiety is saturated with TA. After removing excess reference anti-TA antigen-binding moiety, the ability of the anti-TA antigen-binding moiety in question to bind to TA is evaluated. 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 antigen-binding moiety in question binds to a different epitope than the reference anti-TA antigen-binding moiety. However, if the anti-TA antigen-binding moiety in question fails to bind to TA after saturation binding of the reference anti-TA antigen-binding moiety, the anti-TA antigen-binding moiety in question may bind to the same epitope as the reference anti-TA antigen-binding moiety. To confirm whether the antigen-binding moiety in question binds to the same epitope or is blocked due to steric hindrance, conventional experiments can be used (e.g., peptide mutation and binding assays 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 performed in two settings, i.e., both antibodies are saturated antibodies. If, in both settings, only the first (saturated) antibody is able to specifically bind to TA, it can be concluded that the anti-TA antibody in question and the reference anti-TA antibody compete for binding to TA.

[0297] In some respects, such as those measured in competitive binding assays, if one antibody inhibits the binding of another antibody by 1, 5, 10, 20, or 100 times at least 50%, at least 75%, at least 90%, or even 99% or higher, the two antibodies are considered to bind to the same or overlapping epitopes (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0298] In some respects, if virtually all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of another antibody, then the two antibodies are considered to have “overlapping epitopes.”

[0299] As used herein, the term “CISS” refers to “Competitive Interaction Spatial Switching,” which is the mode of action of a conditionally activatable molecule described herein that depends on a specific arrangement within a polypeptide complex of at least a ligand (e.g., a cytokine), a masking moiety, and an antigen-binding moiety, such that the state of the ligand is masked or unmasked depending on the presence of a target antigen. The term “CISS molecule” is used herein as an abbreviation for “activatable fusion protein” and is interchangeable with it. It refers to any molecule comprising an arrangement as described herein of at least a ligand, a masking moiety, and an antigen-binding moiety, such that the masking and demasking of the ligand depends on the presence of a target antigen. “CISS Fab” or “CISS Fab molecule” refers to a CISS molecule consisting essentially of Fab as the antigen-binding moiety, with both the ligand and the masking moiety covalently linked to the antigen-binding moiety via a peptide linker. The linker and the masking moiety are preferably covalently linked to the N-terminus of the Fab heavy and light chains in such CISS molecules. As used herein, the term "CISS molecule" refers to any molecule that contains a CISS Fab to deliver CISS functionality throughout the molecule. CISS molecules may contain further polypeptide domains, such as Fc domains, further Fab domains, antibody fragments, or other polypeptides (such as cytokines).

[0300] A “targeted CISS molecule” is a CISS molecule that comprises a ligand (e.g., a cytokine), a masking portion, and an antigen-binding portion, as well as at least one additional antigen-binding portion that binds to an antigen that is the same as or different from the antigen-binding portion of the CISS module, thus facilitating the targeting of the CISS molecule to a desired target tissue / molecule. Targeted CISS molecules may contain additional polypeptide domains, such as Fc domains.

[0301] An antibody's "class" 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 some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some respects, an antibody is an IgG1 isotype. In some respects, an antibody is an IgG1 isotype with P329G, L234A, and L235A mutations to reduce the function of the Fc region effector. In other respects, an antibody is an IgG2 isotype. In some respects, an antibody is an IgG4 isotype with an S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody, based on the amino acid sequence of its constant structural domain, can be classified into one of two types, known as Kappa (κ) and Lambda (λ).

[0302] The terms “covalent link” or “fusion” are used interchangeably herein. As used herein, “(covalent) link to the N-terminus of a polypeptide” means that a portion is linked to the polypeptide via a covalent bond at or near the N-terminus of the polypeptide. In one particular aspect, portions (e.g., Fab and Fc domains) are linked by a peptide bond between the C-terminus of one portion (i.e., the free carboxyl group at the C-terminus of the polypeptide chain) and the N-terminus of another portion (i.e., the free amino group at the N-terminus of the polypeptide) (either directly via an amide bond between the carboxyl and amino groups or via one or more peptide linkers). In another aspect, a portion is linked to the polypeptide via an amino acid side chain of the polypeptide. Fusion between various domains of an activatable fusion molecule can be via a peptide linker, which may also include or consist of an immunoglobulin hinge region, particularly when the Fab, as a (first and / or second) antigen-binding domain, is fused to the Fc domain.

[0303] As used herein, the term "cytokine" refers to small, secreted regulatory proteins ranging from approximately 5 kDa to 20 kDa that are essential for cell signaling, particularly within the immune system. Cytokines specifically include molecules such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monocytes, and colony-stimulating factors, and are produced by a range of cell types, including (but not limited to) immune cells (macrophages, B and T lymphocytes, mast cells, monocytes) and non-immune cells (endothelial cells, fibroblasts, mesenchymal cells). Cytokines regulate immune responses to inflammation and infection and modulate various cellular functions, such as survival, growth, and gene expression, and can be classified as pro-inflammatory and anti-inflammatory cytokines. Some cytokines can also mobilize the immune system to fight cancer. Cytokines function through specific receptors and play a crucial role in balancing humoral and cellular immune responses and regulating the maturation, growth, and response of cell populations. The cytokines discussed herein may be naturally occurring (e.g., possessing wild-type sequences) or carry specific mutations to alter their function, activity, or specificity. Cell surface receptors that match cytokines are also referred to herein as "cytokine receptors". The binding of a cytokine to a cytokine receptor can trigger a cascade of intracellular signaling that can then alter cellular function, including upregulation and / or downregulation of genes or transcription factors, thereby producing other cytokines or increasing the number of surface receptors for other molecules. Examples of the cytokine family include bone morphogenetic protein (BMP), chemokine ligand (CCL), CXC motif ligand (CXCL), growth / differentiation factor (GDF), growth hormone, interferon (IFN), interleukin (IL), and tumor necrosis factor (TNF). In particular, interleukins 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 hematopoietic cells. In certain aspects of the invention disclosed herein, the cytokine may be an interleukin or an interferon. In one aspect, cytokines can be members of the IL-1 family or IL-2 subfamily, interferon (IFN) subfamily or IL-10 subfamily.

[0304] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0305] The “effective amount” of a pharmaceutical agent (e.g., a pharmaceutical composition) refers to the amount that is sufficient to effectively achieve the desired therapeutic or preventative outcome at the necessary dose for the necessary period of time.

[0306] The terms “Fc region,” “Fc,” “Fc fragment,” and “Fc domain” are used interchangeably herein and refer to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or the antibody can comprise a cleaved variant of the full-length heavy chain. This could be the case where the last 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 C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may or may not be present. Typically, the Fc region contains two heavy chain polypeptides. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc region is represented herein as lacking a C-terminal glycine-lysine dipeptide. In one aspect, a heavy chain comprising the Fc region as specified herein is included in an antibody according to the invention, the heavy chain comprising additional C-terminal glycine-lysine dipeptides (G446 and K447, EU numbering system). In another aspect, a heavy chain comprising the Fc region as specified herein is included in an antibody according to the invention, the heavy chain comprising additional C-terminal glycine residues (G446, according to EU index number). Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as 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).

[0307] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0308] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more (particularly three or more) moieties, wherein the components of the fusion protein are linked to each other directly or via peptide linkers. One or more moieties of the fusion proteins disclosed herein may be antibodies or antibody fragments comprising two or more polypeptide chains. It should be clarified that "fusion protein" as described herein also includes fusion protein complexes comprising more than one polypeptide chain, wherein at least one polypeptide chain is a fusion polypeptide comprising two or more moieties linked to each other directly or via peptide linkers. For example, a "fusion protein" as described herein may also be an IgG antibody, wherein a ligand (e.g., a cytokine) and a masking moiety are covalently linked via peptide linkers to the N-terminus of the heavy and light chains of one of the two Fab arms of the IgG antibody, respectively. In this example of a fusion protein as used herein, only two of the four polypeptide chains contain the fusion moiety. For clarity, individual polypeptide chains of an antibody or antibody fragment portions of a fusion protein may be non-covalently linked to each other, for example, via disulfide bonds.

[0309] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells.

[0310] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0311] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human CDR and amino acid residues from a human FR. In some respects, humanized antibodies will substantially contain at least one, typically two, variable domains, wherein all or substantially all CDRs correspond to the CDRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0312] As used herein, the term "hypervariant region" or "HVR" refers to the regions within the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, such as the "complementarity-determining region" ("CDR").

[0313] Typically, an antibody contains six CDRs; three in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs in this document include:

[0314] (a) Hypercyclic rings appearing at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987));

[0315] (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0316] (c) Antigen contact sites appearing at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0317] Unless otherwise stated, the CDR designation is based on Kabat et al. (ibid.). Those skilled in the art will understand that the CDR designation may also be based on Chothia (ibid.), McCallum (ibid.), or any other scientifically accepted naming system.

[0318] Unless otherwise specified, as used herein, the term "interleukin-2" or "IL-2" refers to any naturally occurring IL-2 from any vertebrate source, including mammals (such as primates, e.g., humans) and rodents (e.g., mice and rats). The term includes unprocessed IL-2 as well as any form of IL-2 produced through cellular processing. The term also covers naturally occurring IL-2 variants, such as splice variants or allelic variants. An exemplary amino acid sequence of 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 amino acid sequence shown in SEQ ID NO: 83, which is absent in mature IL-2 molecules. When used with respect to IL-2, "full-length" is intended to refer to a mature, naturally occurring IL-2 molecule of length. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, for example, SEQ ID NO: 81).

[0319] As used herein, the “wild-type” form of IL-2 is a form of IL-2 that is otherwise identical to the mutant IL-2 polypeptide, except that the wild-type form contains wild-type amino acids at every amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is full-length IL-2 (i.e., IL-2 is not fused or conjugated with any other molecule), then the wild-type form of the mutant is full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide (e.g., an antibody chain) downstream of IL-2, then the wild-type form of the IL-2 mutant is IL-2 fused with the same downstream polypeptide and having a wild-type amino acid sequence. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (a mutated or modified sequence within the non-truncated portion of IL-2), then the wild-type form of the IL-2 mutant is a similarly truncated IL-2 with a wild-type sequence. For the purpose of comparing the IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants with the corresponding wild-type forms of IL-2, the term wild-type covers a form of IL-2 that contains one or more amino acid mutations that do not affect IL-2 receptor binding compared to naturally occurring IL-2, such as, for example, replacing alanine with cysteine ​​at position 125 of human IL-2.

[0320] As used herein, the terms “IL-2 mutant” or “mutant IL-2 polypeptide” are intended to encompass any mutated form of the IL-2 molecule, including full-length IL-2, truncated IL-2, and IL-2 linked to another molecule, such as by fusion or chemical conjugation. Various forms of IL-2 mutants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation, or modification of a wild-type amino acid residue normally located at that position. Mutants obtained through amino acid substitution are preferred. Unless otherwise specified, IL-2 mutants may be referred to herein as mutant IL-2 peptide sequences, mutant IL-2 polypeptides, mutant IL-2 proteins, or mutant IL-2 analogs. Various forms of IL-2 have been named in connection with the sequence shown in SEQ ID NO: 81. Various names may be used herein to indicate the same mutation. For example, a mutation from phenylalanine to alanine at position 42 can be represented as 42A, A42, A42, L42A, or Phe42Ala.

[0321] In some embodiments, the wild-type IL-2 used for the purposes of this invention comprises an amino acid substitution C125A.

[0322] As used herein, the terms “IL-2 variant,” “IL-2v,” or “interleukin-2v” refer to an interleukin-2 variant with eliminated CD25 binding. While different IL-2 variants with eliminated CD25 binding are available, the variant labeled IL-2v herein is SEQ ID NO: 82, which has amino acid exchanges T3A, F42A, Y45A, L72G, and C125A, and is frequently used during the examples herein.

[0323] Unless otherwise specified, the terms “CD25,” “alpha-subunit / α-subunit of the IL-2 receptor,” “IL-2Rα,” or “IL-2Ra” are used interchangeably herein and refer to any natural CD25 from any vertebrate source, including mammals (such as primates, e.g., humans) and rodents (e.g., mice and rats). The term covers “full-length” unprocessed CD25, as well as any form of CD25 produced through cellular processing. The term also covers naturally occurring CD25 variants, such as splice variants or allelic variants. In some embodiments, CD25 is human CD25. An exemplary amino acid sequence of human CD25 (with a signal sequence, an Avi-tag, and a His-tag) is shown in SEQ ID NO: 85 and found, for example, in UniProt entry number P01589 (version 185).

[0324] As used herein, the terms “IL-2Rβγ” or “IL-2Rbg” refer to the heterodimeric form of the IL-2 receptor, which consists of the receptor γ-subunit (also known as the common cytokine receptor γ-subunit, γc, or CD132, and referred to herein as “IL-2Rγ”) and the receptor β-subunit (also known as CD122 or p70, and referred to herein as “IL-2Rβ”) (see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)). IL-2 acts as a lymphocyte growth and stimulating factor, signaling via activation of the heterodimeric IL-2 receptor complex or, further, the heterotrimeric IL-2 receptor complex including IL-2Rα (CD25).

[0325] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In some respects, the individual or subject is a human.

[0326] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some respects, antibodies are purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0327] The term "masking" refers to the spatial obstruction of binding of a polypeptide domain (e.g., the antigen-binding domain of an antibody) to a target sequence, or the spatial obstruction of binding of a ligand (e.g., its corresponding cytokine receptor) to its ligand-binding portion.

[0328] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists 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 ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct in vitro and / or in vivo (e.g., in a host or patient) expression of antibodies used in this invention. 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 the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate in vivo antibodies (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101823 B1).

[0329] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0330] "Isolated nucleic acid encoding anti-TA antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of anti-TA antibody, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0331] As used herein, the term "ligand" 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 the "ligand-binding moiety". In one aspect, the ligand may be a growth factor, cytokine, chemokine, antibody, antibody fragment, enzyme, receptor ligand, affinity peptide ligand, peptide hormone, receptor agonist, receptor antagonist, enzyme, soluble receptor, protein toxin, soluble ligand, extracellular region of a cell surface receptor, extracellular region of a cell surface ligand, small molecule, or any combination thereof, preferably a cytokine.

[0332] For example, in ligand binding, the ligand is typically a signal-triggered molecule that binds to a site on a target protein. This binding can lead to changes in the protein's conformation (or shape) and / or function, thereby affecting biological processes.

[0333] As used herein, the term "ligand-binding moiety" refers to a molecule, particularly a polypeptide molecule, containing 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: growth factor receptors, cytokine receptors, antigens, ligand receptors, enzyme substrates, fluorescently labeled, radiolabeled, or hormone receptors, preferably cytokine receptors.

[0334] As used herein, a “masking portion” (sometimes also simply referred to as a “masker,” “protein mask,” or “antibody mask”) is a polypeptide capable of binding to another polypeptide (e.g., a ligand) and reducing or completely eliminating the ability of a ligand (e.g., a cytokine) to specifically bind to a ligand-binding portion (e.g., a cytokine receptor). In some aspects, the masking portion comprises an antibody or an antibody fragment complementary to itself. In other aspects, the masking portion is a polypeptide that is a natural binding partner of a ligand (e.g., a ligand receptor). In one aspect, the masking portion comprises an antibody, an antibody fragment single-chain antigen-binding portion, a peptide mask (anti-idiotype antibody, anti-idiotype antibody fragment (e.g., scFv, VHH), or a receptor, protein inhibitor, or binding protein capable of specifically binding to a ligand.

[0335] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. Each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, from the N-terminus to the C-terminus, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain.

[0336] The term "peptide linker" refers to a peptide containing one or more amino acids (typically about 2 to 20, but sometimes up to 40). Peptide linkers are known in the art or described herein. Suitable non-immunogenic peptide linkers are, for example, (GG). n (G3S) n (SEQ ID NO: 91) or (G4S) n (SEQ ID NO: 92) Peptide linkers, where "n" is typically a number between 1 and 10 (usually between 2 and 4, especially 2). Peptide linkers of particular interest are GG, GGGG (SEQ ID NO: 86), SGSGSG (SEQ ID NO: 87), and (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 that is at least 1, preferably 2 to 6. Table A shows different peptide linkers that can be used to covalently link different parts of the CISS molecule.

[0337] Table A – Peptide linkers for CISS molecules

[0338]

[0339] In one aspect of the invention, the length of the first peptide linker connecting the ligand (e.g., a cytokine) to the first antigen-binding site is sufficient to allow interaction with the masking site without causing stress that would result in poor interaction and the formation of polymers. The length of the first peptide linker further ensures that the interaction between the ligand and the masking site is sufficiently close to the complementary site of the first antigen-binding site to exhibit near or complete repulsion in the binding of the ligand to the ligand-binding site. In another aspect, when the first antigen-binding site is simultaneously bound to a target antigen, the length of the first peptide linker is sufficient to reach the ligand-binding site (e.g., on the surface of the target cell).

[0340] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and for alignment purposes without considering any conserved substitutions as part of sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, registered under US Copyright Registry No. TXU510087 and described in WO 2001 / 007611.

[0341] Unless otherwise stated, for the purposes of this article, the ggsearch program of FASTA package version 36.3.8c or later was used to generate the percentage values ​​of amino acid sequence identity using the BLOSUM50 comparison matrix. The FASTA package was created by WR Pearson and DJ Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; WR Pearson (1996) “Effective proteins sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a public server accessible via fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global rather than local alignment. The percentage of amino acid identity is given in the output alignment header.

[0342] The terms “pharmaceutical composition” or “pharmaceutical formulation” refer to a formulation in which the active ingredient contained therein is in a biologically effective form and does not contain any additional components that would have unacceptable toxicity to a subject to whom the pharmaceutical composition will be administered.

[0343] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical composition or formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0344] As used herein, the term "polypeptide" (sometimes also referred to as "polypeptide chain") refers to...

[0345] As used, the term "target antigen" (sometimes also referred to herein as TA for readability, particularly in the context of the antigen-binding portion of an anti-target antigen) refers to an antigen or a portion thereof present in or on a target cell or tissue. For example, target antigens may be selected for the purpose of target-dependent activation or blocking of ligand-binding portions (e.g., cytokine receptors or cell surface receptors) because they can be used to target ligands (such as, for example, cytokines or receptor-binding ligands) to desired target cells or tissues. Target antigens may be bound by an antigen-binding domain of an antigen-binding portion (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 above. In some embodiments, the target antigen may be located on a functionalized surface that can be introduced into a diseased tissue or organ (e.g., by injection) to activate a systemically administered activatable fusion protein active in that region.

[0346] As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of a disease in the treated individual and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the antibodies of the present invention are used to delay the development of disease or slow its progression.

[0347] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, p. 6) (WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be separated using either the VH or VL domain from the antibody binding that antigen, to screen libraries containing complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0348] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0349] II. Compositions and Methods

[0350] In one aspect, the invention is based in part on the discovery that conditionally active fusion proteins exhibiting mutually exclusive binding to the target antigen and ligand can be obtained by arranging an antigen-binding portion, a ligand (e.g., a cytokine), and a masking portion that binds the ligand in a manner disclosed herein, and further demonstrates conditional binding of the ligand to the ligand-binding portion (e.g., a cytokine receptor), i.e., the binding of the ligand to the ligand-binding portion depends on the presence of the target antigen near the ligand-binding portion, without resorting to the use of mutually exclusive bispecific binders (“switch binders” or “flip binders”), which are difficult to generate and may not be able to prepare each target antigen / ligand combination with the desired quality.

[0351] The activatable fusion protein described herein is based on a form that allows for great versatility and broad applicability, as the only building blocks required to prepare such an activatable fusion protein are: a) an antigen-binding moiety specific to the desired target antigen and b) a masking moiety of the desired ligand that prevents the ligand from binding to the ligand-binding moiety and thus exerting its biological function; these are molecules readily available using existing techniques. The effect achieved by the activatable fusion protein reported herein depends on the first antigen-binding moiety binding to the target antigen in such a manner that the distance between the masking moiety and the ligand is significantly increased. Therefore, the masking moiety and the ligand, fused to the first antigen-binding moiety via a peptide linker, can no longer contact each other, effectively blocking the binding of the masking moiety to the ligand. This allows the ligand to freely bind to the ligand-binding moiety. The activatable fusion protein described herein does not depend on specific requirements for the target tissue, such as the presence of tissue-specific proteases. Moreover, the mechanism for activating the activatable fusion protein described herein is reversible and non-destructive. In the absence of the target antigen, the masking moiety re-links to the ligand and restores the masking effect. Therefore, if the fusion protein remains in circulation for an extended period, off-target activity is significantly reduced.

[0352] Therefore, the fusion protein of the present invention differs from known molecular platforms. The ligand (e.g., cytokine) fused to the first antigen-binding moiety is the (primary) bioactive entity of the activatable fusion protein described herein, and the binding of the first antigen-binding moiety to the target antigen releases the ligand to exert its biological function by spatially hindering the interaction between the ligand and the masking moiety, without relying on mechanisms such as cleavage or digestion of the masking moiety, such as proteases.

[0353] Excellent target-dependent binding and activation of target cells expressing the ligand-binding moiety can be achieved using the activatable fusion protein of the present invention, particularly if the activatable fusion protein of the present invention comprises a second antigen-binding moiety, which acts as a targeting arm to localize the molecule to the surface of the target cell. The antigen bound by the second antigen-binding moiety may be the same as or different from the antigen bound by the first antigen-binding moiety.

[0354] In one aspect, the ligand-binding activity of the activatable fusion protein is reduced; for example, if the ligand is a cytokine, it has reduced cytokine receptor binding activity. In one embodiment, if the ligand is a cytokine, the cytokine receptor activation activity of the activatable fusion protein is at least about 10 times lower than that of a fusion protein containing a cytokine peptide but without a masking portion.

[0355] The activatable fusion protein of the present invention can be used, for example, to diagnose or treat diseases such as cancer, viral infections, or autoimmune diseases.

[0356] In some respects, activatable fusion proteins as described herein

[0357] This illustrates target-dependent binding of ligands to their binding sites, and particularly target-dependent activation of cytokine receptors (if the ligand is a cytokine) or target-dependent enzyme activity (if the ligand is an enzyme) (e.g., protease activity, phosphorylation, etc.).

[0358] If the level of the target antigen is below a certain threshold or the target antigen is not present at all, it indicates reduced or inhibited binding of the ligand to the ligand-binding moiety, and / or

[0359] The ligand is released from the masking portion independently of the presence of (target-specific) protease so that the ligand binds to the ligand-binding portion.

[0360] 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 linked via a first peptide linker to the N-terminus of one of the two polypeptides of the first antigen-binding moiety, the masking moiety is covalently linked via a second peptide linker to the N-terminus of the other of the two polypeptides of the first antigen-binding moiety, and neither the first nor the second peptide linker contains a protease cleavage site. The target-dependent binding of the ligand to the ligand-binding moiety in the activatable fusion protein described herein is particularly independent of any proteolytic activity of the activatable fusion protein, especially any proteolytic cleavage, for example, in a target tissue. In one aspect, the activatable fusion protein is functional and exhibits its intact form and / or lacks target antigen-dependent protease activity.

[0361] A. Exemplary CISS molecular form

[0362] In some aspects of activatable fusion proteins, the first antigen-binding portion is an antibody or antibody fragment.

[0363] For a functional CISS molecule to become an antigen-binding moiety, the ligand and masking moiety need to be arranged in the manner described herein, i.e., the ligand is covalently linked via a first peptide linker to the N-terminus of one of the two polypeptides of the first antigen-binding moiety, and the masking moiety is covalently linked via a second peptide linker to the N-terminus of the other of the two polypeptides of the first antigen-binding moiety. Neither the first nor the second peptide linker is sensitive to protease cleavage; i.e., they do not contain protease cleavage sites. To achieve activatable binding of the ligand (e.g., a cytokine) to the ligand-binding moiety (e.g., a cytokine receptor), the antigen-binding moiety is selected as the first antigen-binding moiety, wherein the N-termini of the heavy and light chains are closely adjacent, for example, as in Fab. Therefore, in another aspect of the activatable fusion protein described herein, the first antigen-binding moiety is an antibody fragment selected from the group consisting of: Fab, DutaFab, DAF, Fv, Fab', Fab'-SH, F(ab')2, biantibodies, linear antibodies, and multispecific antibodies formed from antibody fragments. In another embodiment, the first antigen-binding portion is an engineered T-cell receptor (TCR), such as a soluble TCR (which lacks a transmembrane domain) or a single-chain TCR, wherein the variable α region and the variable β region of the TCR are linked by a peptide linker.

[0364] It should be understood that CISS molecules can also be generated based on a first antigen-binding moiety containing only one polypeptide chain (such as DARPin). In such embodiments, the ligand and masking moiety are attached to the N-terminus and C-terminus of the first antigen-binding moiety via peptide linkers.

[0365] In one aspect of the activatable fusion protein described herein, the ligand is selected from the group consisting of or any combination thereof: growth factors, cytokines, chemokines, antibodies, antibody fragments, enzymes, receptor ligands, affinity peptide ligands, peptide hormones, receptor agonists, receptor antagonists, enzymes, soluble receptors, protein toxins, soluble ligands, extracellular regions of cell surface receptors, extracellular regions of cell surface ligands, small molecules, preferably cytokines.

[0366] In one aspect of the activatable fusion protein described herein, the ligand-binding portion to which a ligand can specifically bind is selected from the group consisting of: growth factor receptors, cytokine receptors, antigens, ligand receptors, enzyme substrates, fluorescent labels, radioactive reagents, radiolabeled or hormone receptors, preferably cytokine receptors.

[0367] In one aspect, the masking portion included in the activatable fusion protein described herein is selected from the group consisting of: antibodies, antibody fragments, single-chain antigen-binding portions (which may also be antibody mimics, such as DARPin, monobody, affibody, or anticalin), peptide maskers, anti-idiotype antibodies, anti-idiotype antibody fragments (e.g., scFv, VHH, dAb, VNAR) (only when the ligand is an antibody / antibody fragment), or receptors that bind specifically to the ligand.

[0368] In some aspects of activatable fusion proteins, the masking portion is a single-chain antigen-binding portion selected from the group consisting of: scFv, scFab, VHH, VNAR, domain antibody (dAb), DARPin, affibody, monobody, anticalin, and single-domain antibody (sdAb).

[0369] In one aspect of the activatable fusion protein provided herein, the masking portion reversibly binds to a ligand. In some aspects, the binding of the masking portion to the ligand spatially hinders the binding of the first antigen-binding portion to the antigen. In another aspect, the affinity of the first antigen-binding portion in the activatable fusion protein is reduced compared to the affinity of the first antigen-binding portion itself. In yet another aspect, when the masking portion binds to the ligand, the binding of the first antigen-binding portion of the activatable fusion protein to the antigen is blocked. In a further aspect, when the first antigen-binding portion binds to the target antigen, the interaction between the ligand and the masking portion is interrupted and the masking portion is prevented from binding the ligand. In yet another aspect, when the antigen-binding portion binds to the target antigen, the ligand is released from the ligand-binding portion to bind to the ligand-binding portion.

[0370] The ligand and masking moiety are covalently linked (preferably at their C-terminus) to the N-terminus of the heavy-chain and light-chain polypeptides of the first antigen-binding moiety via a peptide linker. The peptide linker is typically a short peptide containing one or more amino acids (usually about 2 to 20, but sometimes up to 40 or more). The peptide linker of the activatable fusion protein described herein does not contain a protease cleavage site; that is, neither the ligand nor the masking moiety will detach when the activatable fusion protein exerts its therapeutic effect in the target tissue. Even after reaching its physiological target, the molecule described herein will retain its conditionally therapeutic activity. This allows for the use of molecules with long half-lives. Even if these molecules continue to circulate in vivo after reaching their target tissue, they will exhibit very low undesirable activity in the periphery because the masking moiety will rebind to the ligand once the target antigen is no longer present.

[0371] 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 Fab, DutaFab, scFab, DAF, Fv, Fab', Fab'-SH, or F(ab')2 fragments, particularly the Fab fragment. In another embodiment, the second antigen-binding moiety is a single-chain antigen-binding moiety, such as scFv, scFab, VHH, VNAR, domain antibody (dAb), DARPin, affibody, monobody, anticalin, and single-domain antibody (sdAb). In some aspects, the second antigen-binding moiety is covalently linked to the first antigen-binding moiety directly or via a peptide linker. The first antigen-binding moiety and the second antigen-binding moiety may also form an F(ab')2 fragment.

[0372] In 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 should be understood that including an Fc domain in the activatable fusion protein described herein can be useful in many ways, for example, for providing a linking site for additional domains (e.g., antigen-binding moieties), but also for delivering desired properties of the molecule, such as customized half-life or Fcγ-dependent cytotoxicity.

[0373] In one aspect, the first antigen-binding moiety in the activatable fusion protein described herein is covalently linked to the N-terminus or C-terminus of one of the two Fc domain heavy chain polypeptides. In a particular aspect, the first antigen-binding moiety is covalently linked to the N-terminus or C-terminus of one of the two Fc domain heavy chain polypeptides. In a particular aspect, the first antigen-binding moiety is covalently linked 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 Fab, Dutafab, DAF, or DBA, and the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding moiety is (a) covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide via its C-terminus, or (b) covalently linked to the C-terminus of the first Fc domain heavy chain polypeptide. In another aspect, the first antigen-binding portion of the activatable fusion protein is Fab, Dutafab, DAF, or DBA, and the first heavy chain polypeptide or the first light chain polypeptide of the first antigen-binding portion is (a) covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide, or (b) covalently linked to the C-terminus of the first Fc domain heavy chain polypeptide, particularly the C-terminus of the first Fc domain heavy chain polypeptide.

[0374] In some aspects, the second antigen-binding moiety included in the activatable fusion protein is covalently linked to the N-terminus or C-terminus of one of the two Fc domain heavy chain polypeptides. In some aspects, the second antigen-binding moiety is Fab, Dutafab, DAF, or DBA, and the second heavy chain polypeptide or the second light chain polypeptide of the second antigen-binding moiety is covalently linked to the N-terminus of the second Fc domain heavy chain polypeptide via its C-terminus (a) or (b) via its C-terminus. In another embodiment, the second antigen-binding moiety is a single-chain antigen-binding moiety, such as scFv, scFab, VHH, or a single-domain antibody.

[0375] In one aspect, the activatable fusion protein described herein includes a first antigen-binding moiety covalently linked to (a) the N-terminus of a first Fc domain heavy chain polypeptide and a second antigen-binding moiety covalently linked to the N-terminus of a second Fc domain heavy chain polypeptide, or (b) the first antigen-binding moiety covalently linked to the N-terminus of either the first or second Fc domain heavy chain polypeptide and the second antigen-binding moiety covalently linked to the C-terminus of either the first or second Fc domain heavy chain polypeptide.

[0376] As an illustration, Figure 1 shows two examples of the activatable fusion proteins described herein: an exemplary CISSFab (Figure 1A) and an exemplary targeted CISS molecule (Figure 1B). The molecule shown in Figure 1A is based on an anti-TA Fab, in which a ligand (here: a cytokine) and a masking moiety (here: an scFv capable of specifically binding to a cytokine) are fused to the N-termini of the heavy and light chains of the Fab, respectively. The molecule shown in Figure 1B has a structure similar to IgG. One Fab arm of the IgG molecule forms the basis of the CISS module, in which the ligand and masking moiety are covalently linked to the N-termini of the heavy and light chains of the Fab, respectively. The other Fab arm serves as a second antigen-binding moiety, providing the molecule with additional targeting to the target tissue (by binding to TA or another antigen located in the target tissue).

[0377] An exemplary form of an activated fusion protein that can be activated via an intramolecular mechanism is shown in Figure 13, comprising a first antigen-binding 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. In the context of targeting CISS molecules, the term “intramolecular” means that the first and second antigen-binding moieties bind to different epitopes on the same target antigen in such a manner that the two antigen-binding moieties binding to the same target antigen molecule result in the release of a ligand (e.g., a cytokine) to bind to the ligand-binding moieties (e.g., a cytokine receptor). (If the first and second antigen-binding moieties are chosen such that they cannot simultaneously bind specifically to the same target antigen molecule, they depend on the simultaneous binding of two different target antigen molecules; this mechanism is referred to herein as “intermolecular.”) The ligand is covalently linked at its C-terminus to the N-terminus of the heavy-chain or light-chain polypeptide of the first antigen-binding moiety via a peptide linker. The masking moieties are covalently linked at their C-terminus to the N-terminus of another polypeptide chain of the first antigen-binding moiety via a peptide linker. The second antigen-binding moiety binds to the masking moiety via a peptide linker at its C-terminus or its N-terminus.

[0378] Figure 14 illustrates a further exemplary form of the activatable fusion protein according to the invention. Figures 14A and 14B illustrate alternative targeting CISS molecules. These have alternative forms that, in addition to the CISS module, contain an Fc domain and a second antigen-binding moiety. In the form shown in Figure 14A, the first antigen-binding moiety of the CISS module is not directly linked to the Fc domain, but is covalently linked via a peptide linker at the N-terminus of one of its polypeptide chains to the C-terminus of a masking portion, which itself is covalently linked via another peptide linker at its N-terminus to the C-terminus of one of the two Fc domain heavy chain polypeptides, while the second antigen-binding moiety is covalently linked via yet another peptide linker via its N-terminus to the C-terminus of the other of the two Fc domain heavy chain polypeptides. Figure 14 In the form shown in B, the first antigen-binding portion of the CISS module is covalently linked to the N-terminus of the Fc domain at the C-terminus of its heavy-chain polypeptide via a peptide linker, while the second antigen-binding portion is covalently linked to the C-terminus of one of the Fc domain heavy-chain polypeptides at its N-terminus via another peptide linker. It should be understood that the linker sites can vary between different polypeptide chains of Fab and the Fc domains used to assemble the CISS molecule.

[0379] Figure 14C exemplarily illustrates the mode of action of the molecule shown in Figure 14A. The molecule binds to the target antigen on the cell surface via a second antigen-binding moiety (“targeting arm”). When the masking agent temporarily releases a 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 masking agent-cytokine interaction and releasing the cytokine to bind to a cytokine receptor on the cell surface (not shown here).

[0380] In another aspect, the second antigen-binding moiety included in the activatable fusion protein described herein is covalently linked to the N-terminus of a ligand (e.g., a cytokine) via a third peptide linker that does not contain a protease cleavage site. In a particular aspect, the first and second antigen-binding moieties are capable of simultaneously and specifically binding to two different epitopes on the same target antigen molecule. The proximity of the second antigen-binding moieties to the ligand, the masking moieties, and the first antigen-binding moieties allows for intramolecular activation of the ligand, which can lead to the release of the ligand even if the target antigen is soluble. In some aspects, the second antigen-binding moieties covalently linked to the N-terminus of the masking moieties are single-chain antigen-binding moieties selected from the group consisting of: scFv, scFab, VHH, VNAR, domain antibodies (dAb), DARPin, affibody, monobody, anticalin, and single-domain antibodies (sdAb).

[0381] When the second antigen-binding moiety binds to the target antigen, the effective concentration of the target antigen near the activatable fusion protein increases, promoting the binding of the first antigen-binding moiety to the target antigen. This keeps the masking moiety open, allowing ligands (e.g., cytokines) to freely bind to the ligand-binding moiety (e.g., cytokine receptors). Intramolecular demasking generally increases demasking efficiency because it is independent of the cell surface concentration of the receptor and should utilize the higher effective concentration achieved through intramolecular binding. This can be advantageous where an entire population with a given receptor is targeted or where a target in solution or a fixed target is desired.

[0382] In some exemplary forms of the aforementioned activatable fusion proteins, the N-terminus or C-terminus of the first heavy chain polypeptide of the Fc domain is covalently linked to the N-terminus of the masking portion via a third peptide linker (Figure 14A), and in some exemplary forms, the second antigen-binding portion is covalently linked to the N-terminus or C-terminus of the second heavy chain polypeptide of the Fc domain via a fourth peptide linker, via the N-terminus of its heavy chain polypeptide or light chain polypeptide (Figures 14A and 14B). It should be noted that the third and fourth peptide linkers do not contain protease cleavage sites.

[0383] The activatable fusion proteins containing antibody-derived domains (such as Fc domains or Fab domains) described herein typically comprise more than one polypeptide chain. Some forms described herein comprise three or four polypeptide chains. In one aspect of the activatable fusion protein described herein, the masking portion comprises a single-chain antigen-binding portion, and the activatable fusion protein comprises:

[0384] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus via a peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0385] b) A second polypeptide comprising (b1) a ligand (e.g., a cytokine) fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety via a peptide linker at its C-terminus and (b2) the light chain polypeptide of the first antigen-binding moiety.

[0386] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0387] d) The fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0388] In one aspect of the activatable fusion protein described herein, the masking portion comprises a single-chain antigen-binding portion, and the activatable fusion protein comprises:

[0389] a) A first polypeptide comprising (a1) a ligand (e.g., a cytokine) fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of the first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​the first heavy chain polypeptide having an Fc domain.

[0390] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a peptide linker and (b2) a light chain polypeptide of the first antigen-binding moiety.

[0391] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0392] d) The fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0393] In one aspect of the activatable fusion protein described herein, the masking portion comprises a single-chain antigen-binding portion, and the activatable fusion protein comprises:

[0394] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion at its C-terminus via a peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0395] b) A second polypeptide comprising (b1) a ligand (e.g., a cytokine) fused at the N-terminus of a light chain polypeptide of the first antigen-binding moiety via a peptide linker at its C-terminus and (b2) the light chain polypeptide of the first antigen-binding moiety, and

[0396] c) A third polypeptide comprising (c1) a second antigen-binding portion fused at the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus as a single-chain antigen-binding portion and (c2) a second heavy chain polypeptide with an Fc domain.

[0397] In one aspect of the activatable fusion protein described herein, the masking portion comprises a single-chain antigen-binding portion, and the activatable fusion protein comprises:

[0398] a) A first polypeptide comprising (a1) a ligand (e.g., a cytokine) fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of the first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​the first heavy chain polypeptide having an Fc domain.

[0399] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a peptide linker and (b2) a light chain polypeptide of the first antigen-binding moiety, and

[0400] c) A third polypeptide comprising (c1) a second antigen-binding portion fused at the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus as a single-chain antigen-binding portion and (c2) a second heavy chain polypeptide with an Fc domain.

[0401] In another aspect, the activatable fusion protein includes a second antigen-binding moiety capable of binding specifically to an antigen that is the same as or different from the target antigen. In some aspects, the second antigen-binding 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 bound by the first antigen-binding moiety. In some aspects, the second antigen-binding moiety binds specifically to an epitope on the target antigen that is the same as the first antigen-binding moiety.

[0402] Often, the second antigen-binding moiety is selected to specifically bind to the same target antigen as the first antigen-binding moiety, or even to the same epitope on the target antigen as the first antigen-binding moiety. These embodiments have been found particularly useful if the target antigen exhibits high specificity for the desired target tissue (e.g., tumor tissue or specific organ tissue). Using a first antigen-binding moiety and a second antigen-binding moiety that specifically bind to the same epitope on the target antigen, or even using the same antigen-binding moiety as the first antigen-binding moiety and the second antigen-binding moiety, can result in the activated fusion protein having higher specificity for target cells with particularly high concentrations of the target antigen on its surface, because the fusion protein is only activated when there are at least two target antigens on the cell surface that are sufficiently close so that the first and second antigen-binding moieties can bind to them simultaneously (because they cannot bind to the same target antigen simultaneously, as one would block the binding of the other).

[0403] For activatable fusion proteins, the inclusion of a second antigen-binding moiety is particularly useful. This moiety can bind specifically to an antigen other than the target antigen, especially when the concentration of the desired target antigen is insufficient, or if the expression of the target antigen is not specific enough for the target tissue or cell of the desired mode of action. In such cases, the second antigen-binding moiety can be used to target the second antigen in order to improve specificity for a particular target cell / tissue.

[0404] In one aspect of the activatable fusion protein described herein, the first antigen-binding moiety is an IgG antibody or a portion thereof. In some aspects, one arm of the IgG antibody contains the first antigen-binding moiety, and the other arm of the IgG antibody contains 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 IgG antibody. In some aspects, the Fc domain is an IgG Fc domain, particularly an IgG1 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 chain of the antigen-binding moiety is γ-type (IgG), particularly γ1 type. In another aspect, the activatable fusion protein comprises at least two light chains, and the light chain of the antigen-binding moiety is selected from the Kappa (κ) and / or Lambda (λ) subtypes.

[0405] In one aspect, the activatable fusion protein described herein comprises an Fc domain containing one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In another aspect, the Fc domain belongs to the human IgG1 subclass and contains amino acid mutations L234A, L235A, and P329G (according to Kabat EU index numbers).

[0406] In one aspect, the activatable fusion protein described herein comprises an Fc domain containing modifications that promote association between the first Fc domain heavy chain polypeptide and the second Fc domain heavy chain polypeptide. In some aspects, according to the mortar and pestle method, the first Fc domain heavy chain polypeptide comprises a pestle, and the second Fc domain heavy chain polypeptide comprises a pore. In some aspects, the first Fc domain heavy chain polypeptide comprises amino acid substitutions S354C and T366W (according to Kabat EU index numbers), and the second Fc domain heavy chain polypeptide comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index numbers).

[0407] Molecular complexes containing more than one different heavy / light chain pairing are at risk of forming undesirable heavy / light chain pairings during manufacturing. To prevent such mispairing, the CrossMab technique can be used for the activatable fusion protein described herein. Thus, in one aspect, the first and second antigen-binding portions of the activatable fusion protein described herein are Fabs, and in one of these Fabs, the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain; that is, one of these 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 independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). In a particular aspect, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to the Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number).

[0408] In one aspect of the activatable fusion protein described herein, the target antigen to which the first antigen-binding moiety specifically binds is selected from the group consisting of: α-synuclein, amyloid-β, 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, FAPα, 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-4Rα, integrin αv, b7 integrin subunit, a4b7 integrin, a4 Integrin, LAG3, LIGHT, LRP1, MAdCAM, MHC, MUC1, MICA, MICB, NKG2D, NKp30, nKp46, Notch1, Notch3, NRP1, NRP2, OX40, PAR-2, PD-1, PD-L1, PDGFR, P SA, PSMA, SLAMF6, SR-A1, SR-A3, SR-A4, SR-A5, SR-A6, SR-B, dSR-C1, SR-D1, SR-E1, SR-F1, SR-F2, SR-G, SR-H1, SR-H2, SR-11, SR-J1, Syndecan 1. TGFβ, TGF-γ, TCR, gdTCR, TGFBR1, TGFBR2, TIM-3, TLR2, TLR3, Trap, Trop2, VAP-1, VCAM, VEGF, VEGFR1, VEGFR2, or 5T4. In certain aspects, the target antigen may be selected from the group consisting of: PD1, PD-L1, CD8, and CD19.

[0409] In one aspect of the activatable fusion protein described herein, the ligand contained in the activatable fusion protein is a cytokine selected from the group consisting of interferon, interleukin, chemokine, lymphokine, monokinase, colony-stimulating factor, and tumor necrosis factor. 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 interferon and interleukin. In some aspects, the ligand is a cytokine selected from the group consisting of BMP, CSF-1, insulin, GLP-1, HGH, IL-1, IL-1α, IL-1β, 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, IFNα, IFNβ, IFNγ, PDGF, TGFβ, TNFα, TNFβ, VEGF or EPO. In one such aspect, the ligands are cytokines selected from the group consisting of: IL-1, IL-1α, IL-1β, 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, IFNα, IFNβ, and IFNγ. In some aspects, the ligands are cytokines selected from the group consisting of: IL-2, IL-7, IL-21, and IFNα. In one aspect, cytokines can be mutant proteins, variants, active subunits, active fragments, and / or attenuated variants of naturally occurring cytokines.

[0410] In one particular aspect, the activatable fusion protein comprises:

[0411] (a) A first antigen-binding portion, wherein the first antigen-binding portion is a Fab capable of specifically binding to a target antigen.

[0412] (b) Cytokines that can specifically bind to cytokine receptors, and

[0413] (c) The masking component, which is a VHH or scFv that can specifically bind to cytokines.

[0414] The invention is characterized in that the cytokine is covalently linked to the N-terminus of one of the two polypeptides of the first antigen-binding site via a first peptide linker, the masking portion is covalently linked to the N-terminus of the other of the two polypeptides of the first antigen-binding site via a second peptide linker, and the first and second peptide linkers do not contain protease cleavage sites.

[0415] In another aspect, the activatable fusion protein includes:

[0416] (a) A first antigen-binding portion, wherein the first antigen-binding portion is a Fab capable of specifically binding to a first target antigen.

[0417] (b) A second antigen-binding portion, which is capable of specifically binding to the first or second target antigen, preferably Fab.

[0418] (c) An Fc domain comprising a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide.

[0419] (d) Cytokines that can specifically bind to cytokine receptors, and

[0420] (e) A masking component, which is a VHH or scFv that can specifically bind to cytokines.

[0421] The fusion is characterized in that a cytokine is covalently linked to the N-terminus of one of the two polypeptides of the first antigen-binding moiety via a first peptide linker, and a masking portion is covalently linked to the N-terminus of the other 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 linked to the N-terminus of the heavy chain polypeptide of the first Fc domain, and the second antigen-binding moiety is covalently linked to the N-terminus of the heavy chain polypeptide of the second Fc domain. Neither the first nor the second peptide linker contains a protease cleavage site. When the second antigen-binding moiety is Fab, it is linked to the Fc domain at the C-terminus of its heavy chain or its light chain, particularly its heavy chain. Fusion between various domains of the activatable fusion molecule can be via a peptide linker, which may also include or consist of an immunoglobulin hinge region, particularly when Fab, as the (first and / or second) antigen-binding domain, is fused to the Fc domain.

[0422] In some aspects of the activatable fusion proteins disclosed herein, the response that has therapeutic activity and / or activates the patient’s intended mode of action is not (only) the ligand but the first antigen-binding part.

[0423] In one aspect of the activatable fusion protein 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-1 / 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 should be understood that if the antigen-binding moiety is used as a ligand included in the activatable fusion protein described herein, the masking portion will generally be an anti-idiotype antigen-binding moiety capable of specifically binding to the antigen-binding moiety used as a ligand.

[0424] The CISS molecular forms described herein can be used to achieve different types of modes of action. In one aspect, an activatable T-cell or NK-cell conjugate is disclosed. The activatable T-cell or NK-cell conjugate comprises a first antigen-binding moiety capable of specifically binding to a target antigen, a ligand as an antigen-binding moiety capable of specifically binding to T-cell or NK-cell antigens (such as CD3, TCR, or CD28), and a masking moiety capable of specifically binding to the antigen-binding moiety as the ligand (i.e., an anti-idiotype antibody or antibody fragment capable of specifically binding to the anti-T-cell or anti-NK-cell antigen-binding moiety). One advantage of this form is that different target antigens can be used without having to generate new anti-idiotype masks for the antigen-binding moiety that binds to the target antigen. Figure 11A shows a schematic depiction of such an exemplary activatable T-cell conjugate, which is essentially composed of an IgG-type antibody capable of specifically binding to a target antigen, wherein the ligand, anti-CD3ε scFv, and masking moiety, anti-CD3 VHH, are bound to one of the two Fab arms of the IgG antibody. In this example, the other arm serves as a second antigen-binding moiety. In the absence of target-expressing cells, anti-CD3ε scFv is bound by the anti-CD3 VHH masking moiety (Figure 12). Once the activatable T cell conjugate and both antigen-binding moieties are bound to the target antigen, anti-CD3ε scFv is released from the mask and freely binds to its antigen CD3ε on T cells, thus recruiting T cells to the tumor and inducing T cell activation and tumor cell elimination.

[0425] In another aspect, the activatable T-cell or NK-cell conjugates disclosed herein may further comprise a first antigen-binding moiety, such as CD3, TCR, CD28, CD16a, or NKG2D, capable of specifically binding to a T-cell or NK-cell antigen, while a ligand is capable of specifically binding to a second target antigen, and a masking agent is an anti-idiotypic antibody or antibody fragment (e.g., VHH) capable of specifically binding to the antigen-binding moiety of the ligand herein. In a particular aspect, the ligand is an antigen-binding moiety that binds to the target antigen. Figure 11B illustrates a schematic depiction of such an exemplary activatable T-cell conjugate consisting essentially of a bispecific IgG-type antibody comprising two Fab arms, one Fab arm capable of specifically binding to CD3ε and the other Fab arm capable of specifically binding to the target antigen. In this example, the ligand, anti-TA scFv, and masking moiety, and the anti-TA idiotypic VHH are covalently linked to one Fab arm of an IgG antibody capable of specifically binding to CD3ε. In the absence of target-expressing cells, anti-target scFv is partially bound by the anti-TA VHH masking component. Once the activatable T cell conjugate binds to the target antigen via the anti-TA scFv and the anti-TA Fab arm, the spatial binding of anti-CD3ε scFv to its antigen CD3ε on T cells is no longer blocked, thus recruiting T cells, γδ T cells, or other innate immune cells (e.g., NK cells, macrophages / monocytes, neutrophils) and inducing T cell activation and tumor cell elimination.

[0426] The activatable T cell conjugates described herein are particularly capable of modulating affinity and cohesion to select between cells expressing targets at low and high densities, especially when combined with two antigen-binding moieties that bind to two different target antigens. They further strongly reduce the binding of circulating T cells to CD3ε by “masking” CD3 and reduce potential mode-of-action-driven cytokine release syndrome by “masking” the anti-CD3 antigen-binding domain. Like other activatable fusion proteins described herein, the second antigen-binding moieties can bind to the same or different antigens as the first antigen-binding moieties or ligands. In cases where the activatable T cell or NK cell conjugate contains a second antigen-binding moiety that binds to an antigen different from the first antigen-binding moieties, this may result in increased specificity for target cell subsets expressing both antigens. In some embodiments, the activatable T cell conjugates described herein are CD3 bispecific or CD3 trispecific T cell conjugates.

[0427] In another aspect, this disclosure provides an activatable fusion protein as an activatable antagonist, wherein the first antigen-binding portion is an antagonistic conjugate capable of binding specifically to, for example, essential membrane transport proteins or channels.

[0428] In another respect, the ligand can be a non-cytokine ligand selected from the group consisting of or any combination thereof (i.e., polypeptides other than cytokines): growth factors, chemokines, antibodies, antibody fragments, enzymes, receptor ligands, affinity peptide ligands, peptide hormones, receptor agonists, receptor antagonists, enzymes, soluble receptors, protein toxins, soluble ligands, extracellular regions of cell surface receptors, extracellular regions of cell surface ligands, and small molecules.

[0429] In one aspect, an activatable fusion protein is provided, comprising:

[0430] (A) A first antigen-binding portion, which is capable of specifically binding to a target antigen and comprises at least a first heavy chain polypeptide and at least a first light chain polypeptide.

[0431] (B) A second antigen-binding portion comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide.

[0432] (C) A ligand (e.g., a cytokine) capable of specifically binding to a ligand-binding moiety (e.g., a cytokine receptor), and

[0433] (D) The masking portion, which includes a single-chain antigen-binding portion capable of specifically binding to a ligand.

[0434] Furthermore, it is characterized in that the activatable fusion protein comprises:

[0435] a) A first polypeptide comprising (a1) a ligand fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding moiety at its C-terminus via a first peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding moiety fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0436] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a second peptide linker and (b2) a light chain polypeptide of the first antigen-binding moiety.

[0437] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0438] d) A fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0439] Neither the first nor the second peptide linker contains a protease cleavage site.

[0440] In one aspect, an activatable fusion protein is provided, comprising:

[0441] (A) A first antigen-binding portion, which is capable of specifically binding to a target antigen and comprises at least a first heavy chain polypeptide and at least a first light chain polypeptide.

[0442] (B) A second antigen-binding portion comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide.

[0443] (C) A ligand (e.g., a cytokine) capable of specifically binding to a ligand-binding moiety (e.g., a cytokine receptor), and

[0444] (D) The masking portion, which includes a single-chain antigen-binding portion capable of specifically binding to a ligand.

[0445] Furthermore, it is characterized in that the activatable fusion protein comprises:

[0446] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0447] b) A second polypeptide comprising (b1) a ligand fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a first peptide linker and (b2) the light chain polypeptide of the first antigen-binding moiety.

[0448] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain, and

[0449] d) A fourth polypeptide, which is a light chain polypeptide containing the second antigen-binding moiety.

[0450] Neither the first nor the second peptide linker contains a protease cleavage site.

[0451] One embodiment of the present invention is an activatable fusion protein comprising:

[0452] (A) A first antigen-binding portion, which is capable of specifically binding to a target antigen and comprises at least a first heavy chain polypeptide and at least a first light chain polypeptide.

[0453] (B) The second antigen-binding region,

[0454] (C) A ligand (e.g., a cytokine) capable of specifically binding to a ligand-binding moiety (e.g., a cytokine receptor), and

[0455] (D) The masking portion, which includes a single-chain antigen-binding portion capable of specifically binding to a ligand.

[0456] The characteristic feature is that the activatable fusion protein comprises:

[0457] a) A first polypeptide comprising (a1) a ligand fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding moiety at its C-terminus via a first peptide linker, (a2) a heavy chain polypeptide having a first antigen-binding moiety fused to the N-terminus of a first heavy chain polypeptide having an Fc domain at its C-terminus, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0458] b) A second polypeptide comprising (b1) a masking portion fused to the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a second peptide linker, and (b2) a light chain polypeptide of the first antigen-binding moiety, and

[0459] c) A third polypeptide comprising (c1) a second antigen-binding moiety fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0460] Neither the first nor the second peptide linker contains a protease cleavage site.

[0461] One embodiment of the present invention is an activatable fusion protein comprising:

[0462] (A) A first antigen-binding portion, which is capable of specifically binding to a target antigen and comprises at least a first heavy chain polypeptide and at least a first light chain polypeptide.

[0463] (B) A second antigen-binding moiety comprising a single-chain antigen-binding portion (or composed of a single-chain antigen-binding portion).

[0464] (C) A ligand (e.g., a cytokine) capable of specifically binding to a ligand-binding moiety (e.g., a cytokine receptor), and

[0465] (D) The masking portion, which includes a single-chain antigen-binding portion capable of specifically binding to a ligand.

[0466] The characteristic feature is that the activatable fusion protein comprises:

[0467] a) A first polypeptide comprising (a1) a masking portion fused to the N-terminus of a heavy chain polypeptide having a first antigen-binding portion fused to the N-terminus of a first heavy chain polypeptide having an Fc domain fused to the C-terminus of the first heavy chain polypeptide, and (a3) ​​a first heavy chain polypeptide having an Fc domain.

[0468] b) A second polypeptide comprising (b1) a ligand fused at the N-terminus of a light chain polypeptide of the first antigen-binding moiety at its C-terminus via a first peptide linker, and (b2) the light chain polypeptide of the first antigen-binding moiety, and

[0469] c) A third polypeptide comprising (c1) a heavy chain polypeptide with a second antigen-binding portion fused to the N-terminus of a second heavy chain polypeptide with an Fc domain at its C-terminus and (c2) a second heavy chain polypeptide with an Fc domain.

[0470] Neither the first nor the second peptide linker contains a protease cleavage site.

[0471] In a further aspect, the activatable fusion protein according to any of the foregoing aspects can be combined, alone or in combination, with any of the features described in Sections 1 through 8 below:

[0472] 1. Antibody fragments

[0473] In some respects, the activatable fusion proteins described herein contain antibody fragments. These antibody fragments may be specifically contained within antigen-binding moieties or ligands.

[0474] In one aspect, the antibody fragment is a Fab, DutaFab, scFab, DAF, Fv, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digests an intact antibody to produce two identical antigen-binding fragments called "Fab" fragments. Each "Fab" fragment contains a heavy chain variable domain and a light chain variable domain (VH and VL, respectively), as well as a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, the term "Fab" or "Fab fragment" refers to an antibody fragment comprising a light chain polypeptide containing the VL and CL domains and a heavy chain polypeptide containing the VH and CH1 domains. A "Fab' fragment" differs from a Fab fragment in that a residue is added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain are accompanied by a free thiol group. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0475] In another respect, antibody fragments are biantibodies, trisomic antibodies, or tetrasomic antibodies. A “bisomic antibody” is an antibody fragment having two antigen-binding sites, which can 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). Trisomic and tetrasomic antibodies are also described by Hudson et al. in Nat. Med. 9:129-134 (2003).

[0476] In a further aspect, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. Specifically, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab fragments can be further stabilized by generating interchain disulfide bonds via the 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).

[0477] On the other hand, antibody fragments are single-chain variable fragments (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the antibody's heavy chain variable domain (VH) and light chain variable domain (VL), linked by a linker. Specifically, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. For reviews of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (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.

[0478] On the other hand, antibody fragments are single-domain antibodies. A "single-domain antibody" is an antibody fragment containing all or part of the antibody's heavy chain variable domain or all or part of the antibody's light chain variable domain. In some respects, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, for example, U.S. Patent No. 6,248,516 B1).

[0479] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and recombinant production from recombinant host cells (e.g., E. coli), as described herein.

[0480] 2. Multispecific antibodies

[0481] In some respects, the activatable fusion proteins provided herein comprise multispecific antibodies, such as bispecific antibodies. A “multispecific antibody” is a monoclonal antibody that has binding specificity to at least two distinct sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some respects, multispecific antibodies have three or more binding specificities. In some respects, one of the binding specificities is against the target antigen, and another specificity is against any other antigen. In some respects, bispecific antibodies can bind to two (or more) different epitopes of the same target antigen. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0482] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and engineered “mortar and pestle structures” (see, for example, U.S. Patent 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects for the preparation of antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain techniques to avoid light chain mismatch problems (see, for example, WO 98 / 50431); using “dual antibody” techniques for the preparation of bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fc-dimers. (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

[0483] This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having 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. Bispecific antibodies or their antigen-binding fragments also include “dual-acting FAbs” or “DAFs” that contain antigen-binding sites that bind to the target antigen and another different antigen or to two different epitopes of the target antigen (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0484] Multispecific antibodies can also be provided in an asymmetric form, wherein there is domain interchange in one or more binding arms having the same antigen specificity, i.e., by exchanging the VH / VL domain (see, for example, WO 2009 / 080252 and WO2015 / 150447), the CH1 / CL domain (see, for example, WO 2009 / 080253), or the complete Fab arm (see, for example, WO 2009 / 080251, WO 2016 / 016299, and also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, multispecific antibodies contain cross-Fab fragments. The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Cross-Fab fragments comprise polypeptide chains consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), as well as polypeptide chains consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0485] Various further molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0486] This article also includes a specific type of multispecific antibody, which is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell (e.g., tumor cells) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3), for retargeting T cells or NK cells to kill the target cell. Therefore, in some respects, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one binding specificity is against the target antigen and the other against CD3.

[0487] Examples of bispecific antibody forms that can be used for this purpose include, but are not limited to, so-called “BiTE” (bispecific T-cell conjugate) molecules, in which two scFv molecules are fused via a flexible linker (see, for example, WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Bäuerle, ExpCell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem bispecific antibodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56). (1999)); “DART” (dual affinity retargeting) molecules, which are based on a dual antibody form but characterized by a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomab antibodies, which are fully hybridized mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0488] 3. Molecular variants

[0489] In some respects, amino acid sequence variants of the activatable fusion proteins provided herein are envisioned. For example, it may be desirable to alter the binding affinity and / or other biological properties of antigen binding. Amino acid sequence variants of the antigen-binding moiety can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0490] 4. Fc region variant

[0491] In some respects, one or more amino acid modifications may be introduced into the Fc region of the activatable fusion protein provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0492] In some aspects, the present invention contemplates activatable fusion proteins possessing some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the activatable fusion protein is important, but certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the activatable fusion protein lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells mediating ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. 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 for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, 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, non-radioactive assays may be used (see, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Mountain View, California); and CytoTox 96). ®Non-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the activatable fusion protein does not bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0493] Fc regions with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acids at positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0494] Certain Fc regions where binding with FcR is improved or weakened are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).)

[0495] In some respects, the activatable fusion protein contains an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbers of the residues).

[0496] In some aspects, the activatable fusion protein comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues). In one aspect, the substitutions are L234A and L235A (LALA). In some aspects, the activatable fusion protein further comprises D265A and / or P329G in an Fc region derived from the human IgG1 Fc region. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and P329G (LALA-PG). (See, for example, WO 2012 / 130831). In another aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and D265A (LALA-DA).

[0497] In some respects, alterations are made in the Fc region that result in changes (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0498] Antibodies with prolonged half-life and improved neonatal Fc receptor (FcRn) binding, responsible for transferring maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117:587 (1976), and Kim, JK et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following 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, for example, a substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF et al. J. Biol. Chem. 281 (2006) 23514-23524), and can be used in the activatable fusion proteins described herein.

[0499] Fc region residues crucial for the mouse Fc-mouse FcRn interaction have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF et al., J. Immunol 169 (2002) 5171-5180). The interaction involves residues I253, H310, H433, N434, and H435 (EU numbers of the residues) (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, JK et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be crucial for the interaction between human Fc and mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819). Studies on the human Fc-human FcRn complex showed that residues I253, S254, H435, and Y436 were crucial for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). 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 Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0500] In some aspects, the activatable fusion protein comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310, and / or 435 of the Fc region (EU numbers of the residues). In some aspects, the activatable fusion protein comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are I253A, H310A, and H435A. See, for example, Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0501] In some aspects, the activatable fusion protein comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, and / or 433, and / or 436 of the Fc region (EU numbers of the residues). In some aspects, the activatable fusion protein comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are H310A, H433A, and Y436A. (See, for example, WO 2014 / 177460 A1).

[0502] In some aspects, the activatable fusion protein comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, for example, substitutions at positions 252, and / or 254, and / or 256 of the Fc region (EU numbers of the residues). In some aspects, the activatable fusion protein comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are M252Y, S254T, and T256E. For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0503] When an activatable fusion protein as reported herein includes an Fc region, the C-terminus of the heavy chain in the Fc region may be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, an activatable fusion protein comprising an Fc domain heavy chain polypeptide including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbers of amino acid positions). In one aspect of all aspects 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 number of amino acid position).

[0504] 5. Exemplary anti-IL-2 antibody

[0505] In one aspect, the present invention provides an antibody that binds to human IL-2 (huIL-2). In one aspect, an isolated antibody that binds to human IL-2 having SEQ ID NO:81 is provided. In one aspect, an isolated antibody that binds to an attenuated variant IL-2v (SEQ ID NO:82) is provided. In one aspect, an isolated antibody that binds to both human IL-2 having SEQ ID NO:81 and the attenuated variant IL-2v (SEQ ID NO:82) is provided. In one aspect, the present invention provides an antibody that specifically binds to human IL-2. In one aspect, the present invention provides antibodies that specifically bind to both human IL-2 and human IL-2v. In some aspects, the anti-huIL-2 antibody binds to the surface of human IL-2 and / or IL-2v containing helices A and C and to the loop between helices B and C (Stauber DJ et al., PNAS 2005). In some respects, anti-huIL-2 antibodies block the interaction between IL-2 and / or IL-2v and IL-2Rβγ. In another respect, anti-huIL-2 antibodies also block the binding of IL-2 and / or IL-2v to IL-2Rα. In yet another respect, anti-huIL-2 antibodies bind to human IL-2 and / or IL-2v with an affinity of ≤ 1.1 nM (particularly ≤ 0.8 nM).

[0506] In one aspect, the present invention provides an anti-huIL-2 antibody comprising:

[0507] (A) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0508] (B) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;

[0509] (C) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0510] (D) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0511] (E) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0512] (F) 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 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:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0513] (G) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0514] (H) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17;

[0515] (I) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; or

[0516] (J) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17.

[0517] In any of the aspects provided herein, the anti-huIL-2 antibody is humanized. In one aspect, the anti-huIL-2 antibody further comprises a recipient human framework, such as a human immunoglobulin framework or a human common framework.

[0518] In a further aspect, the anti-huIL-2 antibody comprises the following CDR-H1, CDR-H2, and CDR-H3 amino acid sequences:

[0519] (A) The VH domain of SEQ ID NO:7 and the VL domain of SEQ ID NO:8;

[0520] (B) The VH domain of SEQ ID NO:12 and the VL domain of SEQ ID NO:13;

[0521] (C) The VH domain of SEQ ID NO:18 and the VL domain of SEQ ID NO:19;

[0522] (D) The VH domain of SEQ ID NO:24 and the VL domain of SEQ ID NO:25;

[0523] (E) The VH domain of SEQ ID NO:30 and the VL domain of SEQ ID NO:31;

[0524] (F) The VH domain of SEQ ID NO:35 and the VL domain of SEQ ID NO:36;

[0525] (G) The VH domain of SEQ ID NO:38 and the VL domain of SEQ ID NO:39;

[0526] (H) The VH domain of SEQ ID NO:41 and the VL domain of SEQ ID NO:42;

[0527] (I) The VH domain of SEQ ID NO:44 and the VL domain of SEQ ID NO:45; or

[0528] (J) VH domain of SEQ ID NO:47 and VL domain of SEQ ID NO:48.

[0529] In one aspect, the anti-huIL-2 antibody comprises:

[0530] (A)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:1; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:3; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:7; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:8 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0531] (B)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:9; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:10; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:11; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:12; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:13 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0532] (C)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:14; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:16; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:18; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:19 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0533] (D)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:20; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:21; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) containing the following: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:23; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:24; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:25 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0534] (E)(a) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29; and (f) a 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 with the amino acid sequence of SEQ ID NO:30; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:31 ... The amino acid sequence has a VH domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0535] (F)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:14; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:32; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:33; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:34; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:35; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:36 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0536] (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 comprising 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 with the amino acid sequence of SEQ ID NO:38; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:39 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0537] (H)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:40; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:37; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:23; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:41; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:42 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0538] (I)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:26; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:37; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:29; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:44; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:45 ... The amino acid sequence has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or

[0539] (J)(a) CDR-H1 containing the amino acid sequence of SEQ ID NO:20; (b) CDR-H2 containing the amino acid sequence of SEQ ID NO:46; and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) CDR-L1 containing the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO:23; and (f) CDR-L3 containing 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 with the amino acid sequence of SEQ ID NO:47; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:48 ... The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0540] In one aspect, the anti-huIL-2 antibody comprises:

[0541] (A) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:7 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:8;

[0542] (B) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:12 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:13;

[0543] (C) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:18 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:19;

[0544] (D) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:24 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:25;

[0545] (E) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:30 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:31;

[0546] (F) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:35 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:36;

[0547] (G) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:38 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:39;

[0548] (H) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:41 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:42;

[0549] (I) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:44 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:45; or

[0550] (J) A VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:47 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:48.

[0551] In one aspect, the anti-huIL-2 antibody comprises:

[0552] (A) A VH domain containing the amino acid sequence of SEQ ID NO:7; and a VL domain containing the amino acid sequence of SEQ ID NO:8;

[0553] (B) A VH domain containing the amino acid sequence of SEQ ID NO:12; and a VL domain containing the amino acid sequence of SEQ ID NO:13;

[0554] (C) A VH domain containing the amino acid sequence of SEQ ID NO:18; and a VL domain containing the amino acid sequence of SEQ ID NO:19;

[0555] (D) A VH domain containing the amino acid sequence of SEQ ID NO:24; and a VL domain containing the amino acid sequence of SEQ ID NO:25;

[0556] (E) A VH domain containing the amino acid sequence of SEQ ID NO:30; and a VL domain containing the amino acid sequence of SEQ ID NO:31;

[0557] (F) VH domain containing the amino acid sequence of SEQ ID NO:35; and VL domain containing the amino acid sequence of SEQ ID NO:36;

[0558] (G) VH domain containing the amino acid sequence of SEQ ID NO:38; and VL domain containing the amino acid sequence of SEQ ID NO:39;

[0559] (H) VH domain containing the amino acid sequence of SEQ ID NO:41; and VL domain containing the amino acid sequence of SEQ ID NO:42;

[0560] (I) A VH domain containing the amino acid sequence of SEQ ID NO:44; and a VL domain containing the amino acid sequence of SEQ ID NO:45; or

[0561] (J) A VH domain containing the amino acid sequence of SEQ ID NO:47; and a VL domain containing the amino acid sequence of SEQ ID NO:48.

[0562] In one aspect, the anti-huIL-2 antibody comprises:

[0563] (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:50;

[0564] (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:52;

[0565] (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54;

[0566] (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56;

[0567] (E) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58;

[0568] (F) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:59 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:60;

[0569] (G) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:61 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:62;

[0570] (H) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing the amino acid sequence of SEQ ID NO:64;

[0571] (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

[0572] (J) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:67 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:68.

[0573] In one aspect, an anti-huIL-2 antibody is provided, wherein the antibody is Fab and comprises a VH sequence as described in any of the aspects provided above and a VL sequence as described in any of the aspects provided above. In one aspect, the antibody comprises VH and VL sequences as described 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, including post-translational modifications of those sequences. In one aspect, the antibody comprises heavy and light chain sequences from 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, including post-translational modifications of those sequences.

[0574] In a further aspect of the invention, the anti-huIL-2 antibody according to any of the foregoing aspects is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one aspect, the anti-huIL-2 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment. In one aspect, the anti-huIL-2 antibody is Fab.

[0575] In one aspect, the anti-huIL-2 antibody according to any of the above aspects is DutaFab. In one aspect, the anti-huIL-2 antibody includes a human IL-2 complementary site and a non-binding complementary site (i.e., a complementary site that does not bind to any epitope) within a homologous pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the non-binding complementary site contains amino acid residues from the antigen-binding moiety CDR-H2, CDR-L1, and CDR-L3, and wherein the IL-2 complementary site contains amino acid residues from the antigen-binding moiety CDR-H1, CDR-H3, and CDR-L2.

[0576] On the other hand, the antibody is a full-length antibody, such as a complete IgG1 antibody or other antibody class or isotype as defined herein.

[0577] In a further aspect, the antibody 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 a 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 described herein is an IgG1 isotype / subtype 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 a human heavy chain constant region of SEQ ID NO:78 or SEQ ID NO:79. In one aspect, an additional C-terminal glycine (Gly446) is present. In another aspect, both C-terminal glycine (Gly446) and C-terminal lysine (Lys447) are present.

[0578] In one aspect, the antibody described herein comprises at least one Fab fused directly or via a peptide linker to the N-terminus of an Fc region polypeptide at the C-terminus of its heavy chain polypeptide, wherein the Fab has the amino acid sequence disclosed above, and wherein the Fc region polypeptide has the amino acid sequence disclosed above. In a particular aspect, the antibody described herein comprises two Fabs, each Fab being covalently linked directly or via a peptide linker to one of the two N-termini of two Fc region polypeptides at the C-terminus of its heavy chain polypeptide, wherein the Fab has the amino acid sequence disclosed above, and wherein the Fc region polypeptide has the amino acid sequence disclosed above.

[0579] In one aspect, an anti-huIL-2 antibody is provided, comprising 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.

[0580] Table B: SEQ ID NO of anti-IL-2 Fab CDR:

[0581]

[0582] Table C: Description of the amino acid sequence of anti-IL-2 antibody

[0583]

[0584] In a further aspect, the anti-huIL-2 antibody according to any of the foregoing aspects can be combined, alone or in combination, with any of the features described in Sections 3 through 8 below:

[0585] 6. Antibody affinity

[0586] In some respects, the antibodies presented herein have dissociation constants (Ki) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. D (e.g., 10) -8 M or lower, for example from 10 -8 M to 10 -10 M, for example, from 10 -9 M to 10 -10 M).

[0587] In one aspect, Biacore is used. ® Surface plasmon resonance method for measuring K D For example, using Biacore ® Assays were performed using an 8K or 8K+ instrument (Cytiva) at 25°C with an anti-Fab capture setting. In one aspect, the CM5 carboxymethyl dextran biosensor chip (Cytiva; catalog number 29149604) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-human Fab antibody (Cytiva; catalog number 28958325) was diluted to 10 μg / ml with 10 mM sodium acetate at pH 5 and then injected at a flow rate of 10 μl / min for 500 s to obtain approximately 5000 response units (RU) of conjugate protein. Following injection of the anti-human Fab antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, 100 nM Fab was injected at a flow rate of 10 μl / min into HBS-EP+ buffer 1x (Cytiva, catalog number BR100669) at 25°C for 60 seconds. 0 nM, 10 nM, 50 nM, and 150 nM antigens (IL-2, IL-2v, or CD79B) diluted in HBS-EP+ buffer 1x were injected at 30 µl / min for 120 seconds, followed by a 240-second dissociation window at 30 µl / min. Surface regeneration was achieved by injecting 10 mM glycine (pH 2) at 30 µl / min for 60 seconds. The association rate (kJ / L) was calculated by simultaneously fitting association and dissociation sensor maps using a simple one-to-one Langmuir binding model (Biacore 8K Control Software version 4.0.8.20368). on) and dissociation rate (k off Equilibrium dissociation constant (K) D ) Calculated as ratio k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0588] 7. Antibody fragments

[0589] In some respects, the fusion proteins provided herein comprise one or more antibody fragments. In other respects, the antibodies provided herein are antibody fragments.

[0590] In one respect, antibody fragments are Fab, Fab', Fab'-SH, or F(ab')2 fragments, particularly Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments called "Fab" fragments, each containing a heavy chain variable domain and a light chain variable domain (VH and VL, respectively), as well as a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing the VL and CL domains and a heavy chain containing the VH and CH1 domains. A "Fab' fragment" differs from a Fab fragment in that a residue is added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain have a free thiol group. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0591] In some respects, the antibody fragment is DutaFab. DutaFab is a Fab in which a pair of VH and VL domains specifically binds to two distinct epitopes, one complementary site containing amino acid residues from CDR-H2, CDR-L1, and CDR-L3, and the other complementary site containing amino acid residues from CDR-H1, CDR-H3, and CDR-L2. In one respect, DutaFab contains two non-overlapping complementary sites within a homologous VH / VL pair and binds to two distinct epitopes in a mutually exclusive manner (“Dutaflip”).

[0592] In another respect, antibody fragments are biantibodies, trisomic antibodies, or tetrasomic antibodies. A “bisomic antibody” is an antibody fragment having two antigen-binding sites, which can 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). Trisomic and tetrasomic antibodies are also described by Hudson et al. in Nat. Med. 9:129-134 (2003).

[0593] In a further aspect, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. Specifically, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab fragments can be further stabilized by generating interchain disulfide bonds via the 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).

[0594] On the other hand, antibody fragments are single-chain variable fragments (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the antibody's heavy chain variable domain (VH) and light chain variable domain (VL), linked by a linker. Specifically, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. For reviews of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (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.

[0595] On the other hand, antibody fragments are single-domain antibodies. A "single-domain antibody" is an antibody fragment containing all or part of the antibody's heavy chain variable domain or all or part of the antibody's light chain variable domain. In some respects, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, for example, U.S. Patent No. 6,248,516 B1).

[0596] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and recombinant production from recombinant host cells (e.g., E. coli), as described herein.

[0597] 8. Chimeric antibodies and humanized antibodies

[0598] In some respects, the antibodies provided herein are chimeric antibodies. In some respects, the fusion proteins provided herein comprise chimeric antibodies or fragments of chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, 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 instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0599] In some respects, chimeric antibodies are humanized antibodies. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent nonhuman antibody. Humanized antibodies typically contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of the human constant region. In some respects, some FR residues in the humanized antibody are replaced by corresponding residues from the nonhuman antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0600] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “surface reshaping”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach for FR reorganization).

[0601] Human architectural regions that can be used for humanization include, but are not limited to: architectural regions selected using a “best-fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); architectural regions derived from the common sequences of human antibodies from specific subgroups of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatic mutant) architectural regions or human germline architectural regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and architectural regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok ... Biol. Chem. 271:22611-22618 (1996)).

[0602] 9. Human antibodies

[0603] In some respects, the antibodies provided herein are human antibodies. In other respects, the fusion proteins provided herein contain human antibodies or fragments of human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0604] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a complete antibody with a human variable region in response to antigen stimulation. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, a description of XENOMOUSE. TMThe technologies described are described in U.S. Patent Nos. 6,075,181 and 6,150,584; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication No. US 2007 / 0061900 describing VelociMouse® technology. The human variable region derived from intact antibodies produced by such animals can be further modified, for example, by combining it with different human constant regions.

[0605] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, 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 produced via human B-cell hybridoma technology are also described by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridoma). 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).

[0606] Human antibodies can also be generated by isolating variable domain sequences selected from human phage display libraries. These variable domain sequences can then be bound to the desired human constant domain. The technique for selecting human antibodies from antibody libraries is described below.

[0607] 10. Antibodies derived from a library

[0608] In some respects, the antibody or antigen-binding moiety provided herein is derived from a library. In other respects, the fusion protein provided herein comprises an antibody, particularly an antibody fragment, such as Fab or DutaFab derived from a library. The antibodies of the present invention can be isolated by screening a combinatorial library for antibodies having one or more desired activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al., Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with desired binding properties. Such methods are reviewed in, for example, Frenzel et al. mAbs 8:1177-1194 (2016); Bazan et al. Human Vaccines and Immunotherapeutics 8:1817-1828 (2012); and Zhao et al. Critical Reviews in Biotechnology 36:276-289 (2016), as well as Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., editors, Human Press, Totowa, NJ, 2001) and Marks and Bradbury Methods in Molecular Biology 248:161-175 (Lo, editors, Human Press, Totowa, NJ, 2003).

[0609] In some phage display methods, the entire set of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library. Antigen-binding phages can then be screened from this library, as described by Winter et al. in *Annual Review of Immunology* 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described by Griffiths et al. in *EMBO Journal* 12: 725-734 (1993). In addition, natural libraries are synthesized by cloning an unrearranged V gene segment from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 region and perform in vitro rearrangement, 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, U.S. Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 and U.S. Patent Publications Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0610] Further examples of methods known in the art for screening combinatorial libraries of antibodies having one or more desired activities include ribosome and mRNA display, and methods for displaying and selecting antibodies on bacterial, mammalian, insect, or yeast cells. Methods for yeast surface display are reviewed, for example, in Scholler et al. Methods in Molecular Biology 503:135-56 (2012), Cherf et al. Methods in Molecular Biology 1319:155-175 (2015), and Zhao et al. Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al. Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al. PNAS 94:4937-4942 (1997).

[0611] In this paper, antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

[0612] 11. Multispecific antibodies

[0613] In some respects, the antibodies presented herein are multispecific antibodies, particularly bispecific antibodies. A “multispecific antibody” is a monoclonal antibody that has binding specificity to at least two distinct sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some respects, multispecific antibodies have three or more binding specificities. In some respects, one of the binding specificities is against human IL-2, while the others are against any other antigen. In some respects, bispecific antibodies can bind to two (or more) distinct epitopes of human IL-2. Multispecific (e.g., bispecific) antibodies can also be used to target cytotoxic agents or cells to cells expressing human IL-2. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0614] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and engineered “mortar and pestle structures” (see, for example, U.S. Patent 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic guiding effects to prepare antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using universal light chain technology to avoid light chain mismatch problems (see, for example, WO 98 / 50431); using “dual antibody” technology to prepare bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fc-heterodimers. (scFv) dimer (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al., J. Immunol. 147: 60 (1991).

[0615] This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having 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. Bispecific antibodies or their antigen-binding fragments also include “dual-acting Fab” or “DAF”, which contain antigen-binding sites that bind to human IL-2 and another different antigen or two different epitopes of human IL-2 (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0616] Multispecific antibodies can also be provided in an asymmetric form, wherein there is domain interchange in one or more binding arms having the same antigen specificity, i.e., by exchanging the VH / VL domain (see, for example, WO 2009 / 080252 and WO2015 / 150447), the CH1 / CL domain (see, for example, WO 2009 / 080253), or the complete Fab arm (see, for example, WO 2009 / 080251, WO 2016 / 016299, and also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises CrossFab. The terms “CrossFab,” “xFab,” or “cross-Fab” refer to a Fab in which the variable or constant regions of the heavy and light chains are exchanged. CrossFab comprises a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0617] Various further molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0618] This article also includes a specific type of multispecific antibody, which is a bispecific antibody designed to simultaneously bind to surface antigens and activation-invariant components (such as CD3) of the T cell receptor (TCR) complex on target cells (e.g., tumor cells and / or immune cells in the tumor microenvironment) for retargeting T cells to kill the target cells. Therefore, in some respects, the antibodies presented herein are multispecific antibodies, particularly bispecific antibodies, where one binding specificity is against human IL-2 and the other against CD3.

[0619] Examples of bispecific antibody forms that can be used for this purpose include, but are not limited to, so-called “BiTE” (bispecific T-cell conjugate) molecules, in which two scFv molecules are fused via a flexible linker (see, for example, WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Bäuerle, ExpCell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem bispecific antibodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56). (1999)); “DART” (dual affinity retargeting) molecules, which are based on a dual antibody form but characterized by a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomab antibodies, which are fully hybridized mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0620] 12. Antibodies and activatable fusion protein variants

[0621] In some respects, amino acid sequence variants of the activatable fusion proteins and antibodies provided herein are envisioned. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antigen-binding moiety contained in the antibody or activatable fusion protein. Amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or activatable fusion protein or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody or activatable fusion protein. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0622] a) Substitution, insertion, and deletion variants

[0623] In some respects, antibody variants (antigen-binding domains) with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table D. More substantial variations are provided under the heading “Exemplary Substitutions” in Table D, as further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities (e.g., preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0624] Amino acids can be grouped based on common side-chain characteristics:

[0625] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0626] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0627] (3) Acidic: Asp, Glu;

[0628] (4) Alkaline: His, Lys, Arg;

[0629] (5) Residues affecting chain orientation: Gly, Pro;

[0630] (6) Fang ethnic group: Trp, Tyr, Phe.

[0631] Non-conservative substitution would require swapping members of one of these categories for members of another category.

[0632] One type of substitution variant involves replacing one or more highly variable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more resulting variants selected for further research will alter (e.g., improve) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody, relative to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0633] For example, changes (e.g., substitutions) can be made in the CDR to improve antibody affinity. Such alterations can occur in CDR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues in contact with the antigen (detecting the binding affinity of the resulting variant VH or VL). Affinity maturation achieved by constructing and reselecting from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., editors, Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is subsequently screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, in which several CDRs are... Residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutations or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0634] Table D

[0635]

[0636] In some respects, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as described herein) can be made within the CDR. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some variant VH and VL sequences provided above, each CDR either remains unchanged or contains more than one, two, or three amino acid substitutions.

[0637] A useful method for identifying residues or regions of antibody or antigen-binding moieties that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) in Science 244:1081-1085. In this method, residues or target groups of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

[0638] Amino acid sequence insertions include the fusion of amino and / or carboxyl terms of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or peptide that increases the antibody's serum half-life.

[0639] b) Glycosylation variants

[0640] In some respects, the activatable fusion proteins or antibodies provided herein can be modified to increase or decrease the degree of antibody glycosylation, particularly in cases where the activatable fusion protein contains an Fc domain. The addition or deletion of glycosylation sites in antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0641] When an activatable fusion protein or antibody contains an Fc region, the oligosaccharide linked thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0642] In one aspect, antibody and activatable fusion protein variants with non-fucosylated oligosaccharides are provided, i.e., oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. Such non-fucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides lacking the fucose residues that link the first GlcNAc in the stem of the biantennary oligosaccharide structure. In another aspect, antibody variants with an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to natural or parental antibodies are provided. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO 2006 / 082515, and measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking fucosylated residues relative to the sum of all oligosaccharides (e.g., complex, heterozygous, and high-mannose structures) linked to Asn 297. Asn 297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn 297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US 2003 / 0157108 and US 2004 / 0093621.

[0643] Examples of cell lines capable of producing antibodies with reduced fucosylation or activatable fusion proteins include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, especially in Example 11), and knockout cell lines such as α-1,6-fucosylation transferase 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 eliminated GDP-fucose synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0644] In a further aspect, antibody variants or variants of activatable fusion proteins provide bipartite oligosaccharides, for example, wherein biantennary oligosaccharides linked to the Fc region of the antibody are bipartitely GlcNAc. As described above, such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, 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.

[0645] Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0646] c) Fc region variant

[0647] In some respects, one or more amino acid modifications may be introduced into the Fc region of the fusion protein or antibody provided herein to generate Fc region variants. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0648] In some aspects, the present invention covers fusion protein or antibody variants having some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody is important, but certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. 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 for assessing ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, 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, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and CytoTox 96). ®Non-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0649] Antibodies or fusion proteins with reduced effector function include antibodies in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acids 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0650] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0651] In some respects, fusion proteins or antibody variants contain an Fc region with one or more amino acid substitutions that can improve ADCC, such as substitutions at Fc region positions 298, 333, and / or 334 (EU numbers of residues).

[0652] In some aspects, the fusion protein or antibody variant includes an Fc region with one or more amino acid substitutions that weaken FcγR binding, such as substitutions at Fc region positions 234 and 235 (EU numbers of residues). In one aspect, the substitutions are L234A and L235A (LALA). In some aspects, the antibody variant further includes D265A and / or P329G in an Fc region derived from the human IgG1 Fc region. On one hand, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and P329G (LALA-PG). (See, for example, WO 2012 / 130831). On the other hand, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and D265A (LALA-DA).

[0653] In some respects, alterations are made in the Fc region that result in changes (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0654] Antibodies with extended half-lives and improved neonatal Fc receptor (FcRn) binding, responsible for transferring maternal IgG 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.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following 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, for example, a substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0655] Fc region residues crucial for the mouse Fc-mouse FcRn interaction have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF et al., J. Immunol 169 (2002) 5171-5180). The interaction involves residues I253, H310, H433, N434, and H435 (EU numbers of the residues) (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, JK et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be crucial for the interaction between human Fc and mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819). Studies on the human Fc-human FcRn complex showed that residues I253, S254, H435, and Y436 were crucial for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). 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 Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0656] In some aspects, fusion proteins or antibody variants include Fc regions with one or more amino acid substitutions that reduce FcRn binding, such as substitutions at Fc region positions 253, and / or 310, and / or 435 (EU numbers of residues). In some aspects, fusion proteins or antibody variants include Fc regions with amino acid substitutions at positions 253, 310, and 435. In one instance, in Fc regions derived from the human IgG1 Fc region, the substitutions are I253A, H310A, and H435A. See, for example, Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0657] In some aspects, fusion proteins or antibody variants include an Fc region with one or more amino acid substitutions that reduce FcRn binding, such as substitutions at Fc region positions 310, and / or 433, and / or 436 (EU numbers of residues). In some aspects, antibody variants include Fc regions with amino acid substitutions at positions 310, 433, and 436. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are H310A, H433A, and Y436A. (See, for example, WO 2014 / 177460 A1).

[0658] In some aspects, fusion proteins or antibody variants include an Fc region with one or more amino acid substitutions that can enhance FcRn binding, such as substitutions at Fc region positions 252, and / or 254, and / or 256 (EU numbers of residues). In some aspects, fusion proteins or antibody variants include an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, in an Fc region derived from the human IgG1 Fc region, the substitutions are M252Y, S254T, and T256E. For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0659] The C-terminus of the heavy chain of the antibody reported herein may be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbers of amino acid positions). In one aspect of all aspects reported herein, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, EU index number of amino acid position).

[0660] d) Cysteine-engineered antibody variants

[0661] In some respects, it may be desirable to create cysteine-engineered antibodies or activatable fusion proteins, such as THIOMAB.TM An antibody, wherein one or more residues of the antibody or an activatable fusion protein are replaced by cysteine ​​residues. In certain aspects, the substituted residues are present at an accessible site of the antibody. As further described herein, by replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody or an activatable fusion protein and can be used to conjugate the antibody or activatable fusion protein to other parts (such as a pharmaceutical part or a linker-pharmaceutical part) to produce an immunoconjugate. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.

[0662] e) Antibody derivatives

[0663] In some respects, the fusion proteins or antibodies provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, and whether the antibody derivative will be used for a specific therapeutic purpose.

[0664] B. Recombination methods and compositions

[0665] Recombinant methods and compositions can be used to generate activatable fusion proteins or antibodies as described herein, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the activatable fusion protein are provided.

[0666] In cases where the activatable fusion protein is based solely on a first antigen-binding moiety comprising a heavy-chain polypeptide and a light-chain polypeptide, one covalently linked to a ligand and the other covalently linked to a masking moiety, two nucleic acids are required: one for the light-chain polypeptide and one for the heavy-chain polypeptide. Similarly, in the case of a natural antibody or a fragment of a natural antibody, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. These nucleic acids encode the amino acid sequence comprising the VL of the activatable fusion protein and / or the amino acid sequence comprising the VH of the activatable fusion protein (e.g., the light chain and / or heavy chain of the activatable fusion protein). These nucleic acids can be expressed on the same expression vector or on different expression vectors.

[0667] In cases where the activatable fusion protein further comprises a second antigen-binding moiety and an Fc domain of a heterodimeric heavy chain, four nucleic acids may be required: one for the first light chain, one for the first heavy chain containing the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain containing the second heteromonomer Fc region polypeptide. Similarly, in the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required: one for the first light chain, one for the first heavy chain containing the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain containing the second heteromonomer Fc region polypeptide. These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. These nucleic acids encode the amino acid sequence of the first VL constituting an activated fusion protein and / or the amino acid sequence of the first VH constituting the activated fusion protein containing the first heteromonomer Fc region and / or the amino acid sequence of the second VL constituting the activated fusion protein and / or the amino acid sequence of the second VH constituting the activated fusion protein containing the second heteromonomer Fc region (e.g., the first light chain and / or the second light chain and / or the first heavy chain and / or the second heavy chain of the activated fusion protein). These nucleic acids can be on the same expression vector or on different expression vectors, typically on two or three expression vectors, i.e., a single vector can contain more than one of these nucleic acids. An example of such bispecific antibodies is CrossMab (see, for example, Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, one of the heterologous singleton heavy chains contains a so-called "palm-like mutation" (T366W, and optionally one of S354C or Y349C), and the other contains a so-called "mortar-like mutation" (T366S, L368A, and Y407V, and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73), according to EU index number.

[0668] In one aspect of the invention, the activatable fusion protein or antibody according to the invention comprises an Fc domain comprising modifications that promote association between a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide. In one aspect, according to the mortar and pestle method, the first Fc domain heavy chain polypeptide comprises a pestle, and the second Fc domain heavy chain polypeptide comprises a pore. In a particular aspect of the invention, the activatable fusion protein is characterized in that the first Fc domain heavy chain polypeptide comprises amino acid substitutions S354C and T366W (according to Kabat EU index numbers), and the second Fc domain heavy chain polypeptide comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index numbers).

[0669] In one aspect of the invention, the activatable fusion protein or 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 antigen-binding moieties and the second antigen-binding moieties are Fabs, and in one of the Fabs, variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In one aspect, in the constant domain CL of one of the two Fab fragments, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). In a particular aspect of the invention, in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

[0670] In one aspect, an isolated nucleic acid is provided encoding an activatable fusion protein or antibody as reported herein. In another aspect, an isolated nucleic acid is provided encoding an activatable fusion protein as described in the methods reported herein. In yet another aspect, a host cell is provided comprising a nucleic acid encoding an activatable fusion protein as reported herein.

[0671] In one aspect, a method for preparing an activatable fusion protein or antibody is provided, wherein the method comprises: culturing a host cell containing nucleic acid encoding an activatable fusion protein or antibody as provided above under conditions suitable for the expression of the activatable fusion protein or antibody; and optionally recovering the activatable fusion protein or antibody from the host cell (and / or from a host cell culture medium). In another aspect, an activatable fusion protein or antibody produced by the method reported herein is provided.

[0672] To recombinantly produce an activatable fusion protein or antibody, a nucleic acid encoding the activatable fusion protein or antibody, such as as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the activatable fusion protein), or obtained through recombinant methods or chemical synthesis.

[0673] Suitable host cells for vectors used to clone or express peptides encoding activatable fusion proteins or antibodies include prokaryotic or eukaryotic cells as described herein. For example, activatable fusion proteins or antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in *E. coli*.) After expression, the activatable fusion protein or antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0674] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding peptides that can activate fusion proteins or antibodies. These eukaryotic microorganisms include fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in fusion proteins with partial or complete human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0675] Suitable host cells for expressing (glycosylated) activated fusion proteins or antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0676] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0677] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 or 293T cells as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (such as those described, for example, in Mather, JP et al., Annals NYAcad). (As described in 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, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody or other protein production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0678] On the one hand, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells).

[0679] C. Measurement

[0680] The physical / chemical properties and / or biological activities of the activatable fusion proteins and antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

[0681] 1. Combining measurements with other measurements

[0682] In one aspect, the activatable fusion protein of the present invention is tested for its antigen-binding activity by known methods such as ELISA, Western blotting, target and ligand binding affinity.

[0683] In some respects, this paper provides the dissociation constant (K0) of the antigen-binding domain of the activatable fusion protein. D ) is ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0684] In one aspect, BIACORE is used. ® Surface plasmon resonance method for measuring K D For example, BIACORE was used at 25°C with a fixed antigen CM5 chip at approximately 10 response units (RU). ® -2000 or BIACORE ® The assay was performed using a 3000-type (BIAcore, Inc., Piscavenge, NJ) biosensor chip (CM5, BIACORE, Inc.) activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), according to the supplier's instructions. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8 and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of conjugate protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, the antigen was injected into a solution containing 0.05% polysorbate 20 (TWEEN-20) at a flow rate of approximately 25 μl / min. TM The surfactant (PBST) in PBS was serially diluted twice (0.78 nM to 500 nM). A simple one-to-one Langmuir binding model (BIACORE) was used. ® Evaluation Software version 3.2 calculates the association rate (k) by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (koff Equilibrium dissociation constant (K) D ) Calculated as ratio k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate obtained by the above surface plasmon resonance determination exceeds 10... 6 M -1 s -1 The association rate can be determined using fluorescence quenching technology. This technique measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS at pH 7.2 at 25°C in the presence of gradually increasing antigen concentration. This can be achieved using a spectrometer such as an Aviv Instruments spectrophotometer equipped with a flow stop or an 8000 series SLM-AMINCO spectrophotometer. TM The readings were obtained using a stirred cuvette in a ThermoSpectronic spectrophotometer.

[0685] In an alternative method, K is measured by radiolabeled antigen binding assay (RIA). D In one aspect, RIA is performed using the Fab form of the target antibody and its antigen. For example, by titrating a series of unlabeled antigens in the presence of the minimum concentration ( 125 I) The labeled antigen was equilibrated with Fab, and the bound antigen was then captured using a plate coated with anti-Fab antibody to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen et al., J. Mol. Biol. 293:865-881(1999)). To determine the conditions used for the assay, MICROTITER was coated with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6). ® Multi-well plates (ThermoScientific) were incubated overnight, followed by blockade with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] The antigen was mixed with serial dilutions of the target Fab (e.g., consistent with the assessment of anti-VEGF antibody Fab-12 in Presta et al., (Cancer Res. 57:4593-4599(1997))). The target Fab was then incubated overnight; however, incubation may be prolonged (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution was then removed and mixed with 0.1% polysorbate 20 (TWEEN-20) in PBS. ® Wash the plate eight times. When the plate is dry, add 150 μl / well of scintillator (MICROSCINT-20). TM Packard), and in TOPCOUNT TM Use a γ counter (Packard) to count the filaments for several tens of minutes. Select the concentration of each Fab that gives a maximum binding of less than or equal to 20% for use in competitive binding assays.

[0686] 2. Activity Assay

[0687] In one aspect, an assay is provided for identifying bioactive, activatable fusion proteins. Bioactivity may include cytokine activity or enzyme activity. Antibodies exhibiting such bioactivity in vivo and / or in vitro are also provided.

[0688] In some aspects, such bioactivity of the activatable fusion protein of the present invention is tested, for example in cell-based assays such as the HEK-Blue™ reporter gene cell assay (Invivogen). Reporter gene cell lines can be generated for testing the activatable fusion protein in a target-dependent or independent manner. HEK-Blue™ IL-2 cells reconstruct the human IL-2R signaling pathway via expression of the IL-2R chain (IL-2Rα, β, and γ subunits) and downstream signaling cascade effectors (JAK3 and STAT5), as well as the STAT5-induced secretory alkaline phosphatase (SEAP) reporter gene system. Absorbance at 650 nm was measured after adding Quanti-Blue™ substrate (Invivogen rep-qbs) to correlate with SEAP levels and IL-2R activity. The HEK-Blue™ IL-2 reporter gene cell line can be modified by genetically engineering the HEK-Blue™ IL-2 reporter gene cell line (Invivogen hkb-il2) to express a desired target antigen (e.g., PD1) to assess target-dependent activation of the IL-2 pathway. For modification, a transposon vector system is used, consisting of two plasmids: a transposon vector containing two inverted / direct repeats (IR / DR) flanking the gene of interest for the target antigen, and a vector encoding the Sleeping Beauty transposase SB100x. The full-length cDNA encoding the target antigen is subcloned into the transposon vector, carrying neomycin resistance. In the final plasmid, target antigen expression is controlled by the UBC promoter. Following the manufacturer's protocol, the transposon and transposase vector were co-transfected into HEK-Blue IL-2 reporter cells (Invivogen, #hkb-IL2) using Lipofectamine 2000 reagent (Invitrogen, #11668019). HEK-BlueIL-2 cells were maintained in DMEM medium (PAN, #P04-03609) supplemented with 10% FCS (Gibco, #10500), 2 mM L-glutamine (PAN, #P04-80100), 1x HEKBlue CLR selectable solution containing styromycin, hygromycin, and bleomycin (Invivogen, #hb-csm), and 1 µg / mL puromycin (Gibco, #A11138-03).

[0689] HEK-Blue IL-2 cells stably expressing the target antigen were isolated by single-cell sorting using a BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Stable cell clones were screened for target antigen expression and amplification. Expression levels and stability over 3 weeks were confirmed by flow cytometry analysis using an anti-human TA antibody.

[0690] Additionally, a QUANTI-Blue assay with human IL-2 stimulation (Invivogen, #rep-qbs2) (Miltenyi Biotec, #130-097-748) can be performed according to the manufacturer's protocol to confirm that the reporter gene activation of the IL-2 pathway is not affected by stable transfection with the transgenic target antigen.

[0691] In some aspects, such biological activity of the activatable fusion protein of the present invention is tested, for example, in cell-based assays such as p-Stat5 assays. Following induction of cytokine (e.g., IL-2) signaling pathways, a protein called STAT5 is phosphorylated in activated donor T cells. Phospho-STAT5 (p-STAT5) can be fluorescently stained using antibodies, and thus the activation of cytokines (e.g., IL-2) in a cell population can be assessed.

[0692] D. Pharmaceutical Composition

[0693] In a further aspect, a pharmaceutical composition comprising any of the activatable fusion proteins or antibodies provided herein is provided, for example, in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the activatable fusion proteins or antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0694] Pharmaceutical compositions of activatable fusion proteins or antibodies as described herein are prepared by mixing such activatable fusion proteins or a...

Claims

1. An activatable fusion protein comprising (a) A first antigen-binding portion, the first antigen-binding portion being 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) Cytokines, said cytokines being capable of specifically binding to cytokine receptors, and (c) A masking portion, said masking portion being capable of specifically binding to the cytokine. Its features are, The cytokine is covalently linked via a first peptide linker to the N-terminus of one of the two polypeptides at the first antigen-binding site. The masking portion is covalently linked via a second peptide linker to the N-terminus of the other of the two polypeptides in the first antigen-binding portion. and The first peptide linker and the second peptide linker do not contain protease cleavage sites.

2. The activatable fusion protein according to claim 1, characterized in that, The first antigen-binding portion is an antibody or an antibody fragment.

3. The activatable fusion protein according to claim 2, characterized in that, The antibody fragments are selected from the group consisting of: Fab, DutaFab, DAF, Fv, Fab', Fab'-SH, F(ab')2, biantibody, linear antibody, and multispecific antibody formed from antibody fragments.

4. The activatable fusion protein according to any one of claims 1 to 3, characterized in that, The cytokines are selected from the group consisting of: interferon, interleukin, chemokine, lymphokine, monokinetic factor, colony-stimulating factor, and tumor necrosis factor.

5. The activatable fusion protein according to 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-1α, IL-1β, 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-3 3. IL-34, IL-35, IL-36, GM-CSF, FGF, EGF, G-CSF, IFNα, IFNβ, IFNγ, PDGF, TGFβ, TNFα, TNFβ, VEGF and EPO.

6. The activatable fusion protein according to any one of claims 1 to 5, characterized in that, The masking portion is selected from the group consisting of: antibodies, antibody fragments, single-chain antigen-binding portions, peptide maskers (anti-idiotype antibodies, anti-idiotype antibody fragments (e.g., scFv, VHH), and receptors, protein inhibitors, or binding proteins that can specifically bind to the cytokine.

7. The activatable fusion protein according to any one of claims 1 to 6, characterized in that, The masking portion reversibly binds to the cytokine.

8. The activatable fusion protein according to any one of claims 1 to 7, characterized in that, When the masking portion binds to the cytokine, the binding of the cytokine to the cytokine receptor is blocked.

9. The activatable fusion protein according to any one of claims 1 to 8, characterized in that, When the masking portion binds to the cytokine, the binding of the first antigen-binding portion to the antigen is blocked.

10. The activatable fusion protein according to any one of claims 1 to 9, characterized in that, The activatable fusion protein further comprises a second antigen-binding portion, the second antigen-binding portion comprising at least a second heavy chain polypeptide and at least a second light chain polypeptide.

11. The activatable fusion protein according to any one of claims 1 to 10, characterized in that, The activatable fusion protein further comprises an Fc domain, wherein the Fc domain comprises a first Fc domain heavy chain polypeptide and a second Fc domain heavy chain polypeptide.

12. The activatable fusion protein according to claim 11, characterized in that, a) The first antigen-binding portion is covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide, and the second antigen-binding portion is covalently linked to the N-terminus of the second Fc domain heavy chain polypeptide, or b) The first antigen-binding portion is covalently linked to the N-terminus of the first Fc domain heavy chain polypeptide or the second Fc domain heavy chain polypeptide, and the second antigen-binding portion is covalently linked to the C-terminus of the first Fc domain heavy chain polypeptide or the second Fc domain heavy chain polypeptide.

13. The activatable fusion protein according to any one of claims 10 to 12, characterized in that, The second antigen-binding portion can specifically bind to antigens that are the same as or different from the target antigen.

14. The activatable fusion protein according to any one of claims 11 to 13, characterized in that, The first antigen-binding portion, the second antigen-binding portion, and the Fc region together form an IgG antibody.

15. An antibody that specifically 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 inhibits the binding of huIL-2 to human IL-2Rβγ and to human IL-2Rα.

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-2Rβγ and to human IL-2Rα.

17. An antibody that specifically binds to huIL-2 or a variant thereof, wherein the antibody comprises (A) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (B) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (C) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:14, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (D) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (E) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:27, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:28; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (F) 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 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:34, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (G) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (H) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; (I) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:26, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17; or (J) A heavy chain variable domain (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:20, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:22; and a light chain variable domain (VL) comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

17.

18. The antibody according to any one of claims 15 to 17, comprising: (A) The VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:8; (B) The VH sequence of SEQ ID NO:12 and the VL sequence of SEQ ID NO:13; (C) The VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:19; (D) The VH sequence of SEQ ID NO:24 and the VL sequence of SEQ ID NO:25; (E) The VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO:31; (F) The VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:36; (G) The VH sequence of SEQ ID NO:38 and the VL sequence of SEQ ID NO:39; (H) The VH sequence of SEQ ID NO:41 and the VL sequence of SEQ ID NO:42; (I) The VH sequence of SEQ ID NO:44 and the VL sequence of SEQ ID NO:45; or (J) The VH sequence of SEQ ID NO:47 and the VL sequence of SEQ ID NO:

48.

19. The antibody according to any one of claims 15 to 18, wherein the antibody is Fab and comprises (A) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:49 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:50; (B) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:51 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:52; (C) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:53 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:54; (D) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:55 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:56; (E) A heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:57 and a light chain polypeptide containing the amino acid sequence of SEQ ID NO:58; (F) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:59 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:60; (G) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:61 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:62; (H) Heavy chain polypeptide containing the amino acid sequence of SEQ ID NO:63 and light chain polypeptide containing 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) Heavy chain polypeptides containing the amino acid sequence of SEQ ID NO:67 and light chain polypeptides containing the amino acid sequence of SEQ ID NO:

68.

20. An isolated nucleic acid encoding an activatable fusion protein according to any one of claims 1 to 14 or an antibody according to any one of claims 15 to 19.

21. A host cell comprising the nucleic acid according to claim 20.

22. A method for producing an activatable fusion protein according to any one of claims 1 to 14 or an antibody according to any one of claims 15 to 19, the method comprising culturing a host cell according to claim 20 under conditions suitable for expressing 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 antibody, generated by the method according to claim 22.

24. A pharmaceutical composition comprising: an activatable fusion protein according to any one of claims 1 to 14 or 23 or an antibody according to any one of claims 15 to 19 or 23; and a pharmaceutically acceptable carrier.

25. The activatable fusion protein according to any one of claims 1 to 14 or 23, the antibody according to any one of claims 15 to 19 or 23, or the pharmaceutical composition according to claim 24, used as a pharmaceutical.

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