Mutually masked antibody-cytokine fusion proteins and methods of use thereof

Abstract

Described herein are compositions and methods for producing masked antibodies useful in a variety of therapeutic indications. TIFF2025528468000021.tif58170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 403,465, filed September 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates generally to masked antibody compositions and methods of use thereof.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NOVI_051_001WO_SeqList_ST26.xml, size: 18,584 bytes, and creation date: August 28, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Background of the Invention Antibodies are one of the most successful classes of drugs, benefiting from high target specificity and low inherent toxicity. Despite these favorable characteristics, toxicity can occur if the target antigen is also expressed at significant levels on non-diseased tissues (Hansel et al., 2010). This is particularly important for cancer treatment, where antibodies often mediate cell killing through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), direct killing of target cells, antibody-drug conjugates (ADCs), or T cell redirection using bispecific antibodies targeting CD3 on T cells and tumor-associated antigens on tumor cells.

[0005] Antibodies have been engineered in many ways to improve their efficacy. They have been humanized or isolated from human sequences to reduce the potential for immunogenicity. Fc domains have been engineered to tailor interactions with Fc receptors and downstream effector functions or pharmacokinetic properties. More recently, many approaches have been developed to create bispecific and multispecific antibody formats that enable novel modes of action.

[0006] To further increase specificity and limit on-target toxicity, novel approaches aim to efficiently activate antibodies when exposed to specific conditions, such as those found within the tumor microenvironment (TME). A variety of genetic engineering strategies have been applied to generate antibodies that are activatable under conditions found in the TME or in other diseased tissues.

[0007] Over the past decade, antibodies have been genetically engineered to be sensitive to various stimuli, including pH, light, temperature, ions, effector molecules, antigen combinations, and proteases (Lucchi et al., 2021). Therefore, these antibodies become activated under specific conditions, i.e., they are either capable of binding to their antigen or not. One of the main approaches utilized to activate antibodies relies on specific proteases preferentially expressed in the tumor membrane (TME). Protease-activated antibodies are based on the introduction of a masking domain, which blocks the antibody's binding to its cognate antigen and is connected via a cleavable linker. Once the antibody reaches the tumor site, the cleavable linker is cleaved by the protease, releasing the masking domain and restoring antibody binding activity at the tumor site. Because cleavage is mediated by proteases overexpressed in the tumor membrane (TME), in contrast to healthy tissues with low protease activity, the interaction between the antibody and its target is prevented by the masking domain, limiting on-target off-tumor toxicity.

[0008] Different masking strategies can be used to prevent paratope-epitope interactions. Affinity-based masks are specific to a given antibody and occupy the antibody paratope, preventing it from interacting with the epitope on the antigen. Generally, the interaction must be of weak or moderate affinity so that the mask is released from the antibody upon cleavage of the linker. Therefore, the affinity of the mask is an important parameter to adjust for each antibody-mask pair. The affinity mask can be a peptide of an anti-idiotypic antibody fragment. On the other hand, steric hindrance-based masks do not specifically interact with the antibody paratope but inhibit steric hindrance-mediated binding of the antibody (Bleuez et al., 2022).

[0009] Affinity-based masking was first introduced in 2009. A recombinant epidermal growth factor receptor (EGFR) fragment was fused to a single-chain Fv fragment (scFv) derived from an EGFR-targeting antibody via a linker cleavable by a protease expressed in the tumor necrosis factor (TME). The masked scFv poorly associated with EGFR, but protease treatment to produce an unmasked scFv restored association. Since this first example, various affinity-based masked antibodies have emerged. Among them, anti-CD166 (CX-2009), anti-PD-L1 (CX-072), and anti-CD71 (CX-2029) antibodies have reached clinical trials. Anti-CTLA-4 (XT101) demonstrated tumor-selective pharmacodynamic effects and efficacy in preclinical models. For affinity-based mask design, the selected peptide must have adequate affinity for the antibody paratope to mask its binding, but also a weak enough affinity to be released once cleaved. Furthermore, a specific peptide must be developed for each antibody.

[0010] Steric hindrance masking was introduced more recently by Chen et al. in 2017. They demonstrated that fusing a latency-associated peptide (LAP) to an anti-EGFR or anti-TNFα antibody reduced their binding activity, which could be restored after protease cleavage. Similarly, the addition of polyethylene glycol (PEG) chains to recombinant proteins and antibody fragments has been shown to interfere with protein-protein interactions and biological activity through the steric hindrance of the large PEG chains, which nonspecifically mask the protein-protein interaction surface.

[0011] In healthy tissues, extracellular protease levels are usually low, and their activity is tightly regulated by inhibitors present in the tissue. In tumor tissues, on the other hand, their expression levels and activity can be significantly upregulated. Altered protease expression and activity are hallmarks of cancer, which play a key role in cancer development at multiple stages, from tumorigenesis to metastasis (Vasiljeva et al., 2019). For example, proteases are involved in cancer cell invasion in healthy tissues by degrading basement membranes and extracellular matrix (ECM).

[0012] Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) are among the upregulated proteases in the TME. MMPs are a family of zinc endopeptidases involved in cancer initiation, progression, and angiogenesis. They are upregulated in many cancer types. 23 MMPs have been identified in humans, and among them, MMP-9 is involved in many cancer development processes. uPA is a serine-endopeptidase involved in regulating tumor progression and metastasis. More specifically, uPA cleaves plasminogen to produce active plasmin, which initiates the degradation of ECM components (Mahmood et al., 2011). MMP-9 and uPA are often used in the design of activatable antibodies.

[0013] Cytokines are key players in the immune response and mediate intercellular communication, making them interesting therapeutic agents (Berraondo et al., 2019). Interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-15, and IL-21, can be used to treat cancer and other diseases. However, their therapeutic use through systemic administration often results in undesirable side effects, such as hypotension, flu-like symptoms, nausea, diarrhea, and cardiac arrhythmias.

[0014] Among the above, interleukin-2 (IL-2) and interleukin-15 (IL-15) are related cytokines that can stimulate immune cells through interaction with their receptors. They bind to their respective alpha receptor subunits (IL-2Rα and IL-15Rα) and their shared receptor beta and gamma subunits (IL-2 / IL-15Rβγ). IL-2 is known as a mediator of T cell proliferation, differentiation, and survival. Meanwhile, IL-15 is known as a mediator of proliferation and differentiation of NK and CD8 memory T cells.

[0015] IL-2 is FDA-approved for the treatment of advanced and metastatic melanoma. However, systemic administration of IL-2 is associated with severe side effects, limiting its clinical use. Furthermore, IL-2 is involved in the development of regulatory T cells (Tregs), which prevent the development of effective antitumor immunity.

[0016] Therefore, there remains a need to develop cytokine therapeutics that mediate immune activation specifically at tumor sites without systemic side effects. One way to limit the side effects of IL-2 is to make it specifically active at tumor sites (Puskas et al., 2011). Furthermore, by combining IL-2 with the extracellular domain (ECD) of IL-2Rα, it is possible to preferentially activate IL-2Rβ and γ, which are expressed by CD8 T cells and NK cells, while limiting the activation of Tregs, which express IL-2Rα.

[0017] The combination of cytokines and their receptors as steric hindrance masks in activatable antibodies not only keeps the cytokines inactive, but also keeps the linked antibodies inactive outside the tumor. Upon reaching the TME, cleavage of the linker by proteases overexpressed in the TME releases both the active cytokine and the functional antibody. This strategy allows for a dual therapeutic effect: enabling activation of both the antibody and cytokine at the tumor site; while limiting unwanted activation in healthy tissues and the systemic circulation. Summary of the Invention

[0018] In various aspects, the invention provides antibody fusion proteins having the following structure: a first antigen-binding domain having a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second antigen-binding domain comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2). A cytokine is linked via a first protease-cleavable linker (i) to the N-terminus of L1 and / or L2; (ii) to the N-terminus of H1 and / or H2; or (iii) to the N-terminus of L1, L2, H1, and / or H2.

[0019] The antibody fusion proteins described above further comprise at least a portion of a cytokine's cognate receptor linked to the N-terminus of H1 and / or H2 or the N-terminus of L1 and / or L2 via a second protease linker. Different cytokines (e.g., IL-2 or IL-15) can be incorporated into the constructs of the invention. The cytokine's partial receptor can be the extracellular portion of their respective cognate receptor, such as IL-2Rα, IL-2Rβ, or IL-2Rγ, IL-2, or a combination thereof.

[0020] The antibody fusion proteins described above further have the cytokine and its cognate receptor sequentially linked to the N-terminus of the same light or heavy chain of the antibody in different orders using the same or different linkers.

[0021] The components of the invention can be combined in various ways to achieve different levels of masking of (i) the antigen-binding domain and (ii) the cytokine, depending on the expected mode of action of the fusion protein and the potential toxicity of the unmasked antigen-binding domain or cytokine.

[0022] In some aspects of the invention, cytokine sequences can be modified to alter their interactions with various receptors, thereby modulating their biological activity when masked or unmasked. Similarly, receptor sequences can also be modified to alter their interactions with cognate cytokines. In addition, affinity or antigen-binding domains can be modified to modulate their binding capacity when masked or unmasked.

[0023] If the antigen-binding domain contains an Fc domain (e.g., in an antibody), the Fc can be selected for its ability to bind to an Fc receptor and drive effector functions such as ADCC or CDC. The Fc portion can also be silenced or enhanced by introducing mutations to further modulate activity. [Brief explanation of the drawings]

[0024] [Figure 1]

[0023] Figure 1 is an illustration of a mutual masking and activation approach by fusing a cytokine receptor complex to the N-terminus of an antibody. Masking can be removed by proteolytic cleavage. The antibody has no binding specificity for the cytokine or cytokine receptor complex. [Figure 2] 1 shows different possible configurations of antibody-cytokine / receptor fusions. [Figure 3] The different constructs that were generated are shown. The "n°" designation represents the different Novimmune construct configurations. [Figure 4] Strategies and combinations of elements to achieve the desired mode of action are presented. [Figure 5] Illustration of a reciprocal masking and activation approach using CD47 antibodies and IL2-IL2Rα as an example. In this case, unmasking of the antibody-cytokine / receptor fusion in the tumor microenvironment restores CD47-SIRPα blockade, resulting in increased tumor cell phagocytic activity and IL-2 signaling that mediates T cell activation and proliferation. [Figure 6]Schematic representation of examples of vector maps generated for expression of different construct configurations. [Figure 7A] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 7B] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 7C] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 7D] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 7E] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 7F] SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 is shown. [Figure 8] 1 shows an example of the binding profile of an antibody-cytokine / receptor fusion before and after cleavage using Bio-Layer Interferometry (BLI) technology. [Figure 9A] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9B] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9C] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9D]Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9E] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9F] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9G] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9H] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9I] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9J] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9K] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9L] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9M] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9N]Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9O] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9P] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9Q] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9R] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9S] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9T] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9U] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9V] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9W] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9X]Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9Y] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9Z] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 9AB] Binding profiles of antibody-cytokine / receptor fusions on CD47 at the surface of peak cells before and after proteolytic cleavage by MMP-9 are shown. [Figure 10A] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10B] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10C] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10D] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10E] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10F]IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10G] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10H] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10I] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10J] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10K] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10L] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10M] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10N] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10O]IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10P] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10Q] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10R] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10S] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10T] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10U] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10V] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10W] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10X]IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10Y] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 10Z] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ IL-2 reporter system is shown. [Figure 11A] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11B] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11C] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11D] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11E] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11F]IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11G] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11H] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11I] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11J] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11K] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11L] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. [Figure 11M] IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9 using the HEK-Blue™ CD122 / CD132 reporter system is shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention The present disclosure provides for the generation of protease-activatable antibody and cytokine or cytokine / receptor fusions in a single construct. The cytokine and / or cytokine / receptor mask the antibody binding site, and conversely, the antibody masks the cytokine or cytokine / receptor. Mutual masking simultaneously reduces the biological activity of the antibody and cytokine or cytokine / receptor. Mutual masking activity is mediated by steric hindrance, as the antibody has no affinity for the cytokine / receptor. The antibody is linked to the cytokine and / or cytokine / receptor via one or more protease-cleavable linkers. Upon cleavage by a protease upregulated in the TME, both the antibody and cytokine / receptor are released into the TME in forms with fully or partially restored biological activity.

[0026] Thus, the present disclosure enables: (i) mutual dual masking of antibody-cytokine / receptor fusions in the circulation and in healthy tissues; (ii) release of the active molecule (i.e., antibody, cytokine, cytokine receptor) upon proteolytic cleavage; (iii) an unmasked antibody capable of binding to its target (i.e., specifically binding to its cognate antigen); (iv) a biologically active cytokine / receptor capable of signaling through its cognate receptor; and (v) obtaining increased therapeutic activity of antibody-cytokine / receptor cytokine fusions because two distinct modes are released compared to previous masking strategies in which the mask has no function upon release. (See Table 1.)

[0027] Different molecular designs and architectures can be used to generate the antibody-cytokine / receptor fusions of the present invention.

[0028] In the first configuration, the cytokine is fused to the N-terminus of an antibody light chain, and the extracellular portion of the cytokine receptor is fused to the N-terminus of an antibody heavy chain. This configuration allows the cytokine to interact with the extracellular portion of the cytokine receptor. Because the N-terminus of the heavy chain and the N-terminus of the light chain are close to the antibody binding site, steric hindrance mediated by the cytokine / receptor fusion and mutual inhibition of cytokine activity are facilitated by this configuration.

[0029] In the second configuration, the cytokine is fused to the N-terminus of the antibody heavy chain and the extracellular portion of the cytokine receptor is fused to the N-terminus of the antibody light chain. This swapped configuration also promotes steric hindrance as described for the first configuration.

[0030] In other configurations, only the cytokine is fused to either the N-terminus of the light chain or the N-terminus of the heavy chain.

[0031] In other configurations, the cytokine is fused to both the N-terminus of the light chain and the N-terminus of the heavy chain.

[0032] In another configuration, the cytokine and the first extracellular portion of the cytokine receptor are fused to the N-terminus of the light chain, and the second extracellular portion of the cytokine receptor is fused to the N-terminus of the antibody heavy chain. Such a configuration can increase cytokine masking.

[0033] In another configuration, the cytokine and the first extracellular portion of the cytokine receptor are fused to the N-terminus of the heavy chain, and the second extracellular portion of the cytokine receptor is fused to the N-terminus of the antibody light chain. Similarly, such a configuration can increase cytokine masking.

[0034] In another configuration, the cytokine is fused to the N-terminus of the light chain and two extracellular portions, i.e., the first and second extracellular portions of the cytokine receptor, are fused to the N-terminus of the antibody heavy chain to further block cytokine activity.

[0035] In other configurations, the cytokine is fused to the N-terminus of the heavy chain and two extracellular portions, i.e., the first and second extracellular portions of the cytokine receptor, are fused to the N-terminus of the antibody light chain to further block cytokine activity.

[0036] In each of the above possible configurations, the linker and protease-cleavable sequence can be varied to simultaneously optimize both masking efficiency and proteolytic cleavage efficiency. In some embodiments, the antibody fusion protein is comprised of a first protease-cleavable linker. In some embodiments, the antibody fusion protein is comprised of a first protease-cleavable linker and a second protease-cleavable linker. In some embodiments, the antibody fusion protein is comprised of a first protease-cleavable linker, a second protease-cleavable linker, and a third protease-cleavable linker. In some embodiments, the antibody fusion protein is comprised of three or more cleavable linkers. In some aspects, the cleavable linker is one or more of SEQ ID NO:7 (CM1), SEQ ID NO:8 (CM2), or SEQ ID NO:9 (CM3).

[0037] In some embodiments, the cleavable linker is cleaved by a protease or peptidase that is upregulated or present in higher amounts in the TME compared to healthy peripheral tissues, hi some embodiments, the cleavable linker is cleaved by MMP-9.

[0038] In the above possible configurations, the cytokine can be modified by mutagenesis to alter its interaction with one or several of its cognate receptors and to modify its biological activity.

[0039] In some embodiments, the antibody fusion protein comprises a first portion of a cognate receptor for a cytokine. In some embodiments, the antibody fusion protein comprises a second portion of a cognate receptor for a cytokine. In some embodiments, the first portion of the cognate receptor is any one of the extracellular portions of IL-2Rα, IL-2Rβ, IL-2Rγ, or IL-15Rα Sushi 1. In some embodiments, the second portion of the cognate receptor is any one of the extracellular portions of IL-2Rα, IL-2Rβ, IL-2Rγ, or IL-15Rα Sushi 1. In some embodiments, the first portion of the cognate receptor is IL-2Rα and the second portion of the cognate receptor is IL-2Rβ. In some embodiments, the first portion of the cognate receptor is IL-2Rβ and the second portion of the cognate receptor is IL-2Rα. In some embodiments, the first portion of the cognate receptor is IL-2Rα and the second portion of the cognate receptor is IL-2Rγ. In some embodiments, the first portion of the cognate receptor is IL-2Rγ and the second portion of the cognate receptor is IL-2Rα.

[0040] Other configurations combining a cytokine with one or several extracellular portions of its receptor can be linked to the N-terminus of the antibody heavy and / or light chain using protease-cleavable or non-cleavable linkers to achieve various degrees of masking of the cytokine and antibody. A non-exhaustive representation of possible configurations is shown in Figures 2 and 3.

[0041] In some of the constructs, the cytokine used for N-terminal fusion may be, but is not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, and domain receptors derived from their respective receptors, including, when the cytokine used is IL-2, IL-2Rα, IL-2Rβ, or IL-2Rγ, or any combination thereof. In some embodiments, the mutant cytokine is mutated IL-2 or mutated IL-15. In some embodiments, the mutated IL-2 includes one or more of the following mutations: C125S, F42A, D20T, or Q126T.

[0042] Any antibody can be masked according to the present invention, including, but not limited to, antibodies targeting CD47, CD3, CD28, PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD40, CD40L, OX40, OX40L, ICOS, ICOSL, CD70, CD27, CD28, GITR, GITRL, TIGIT, TIM3, LAG3, CEACAM5, EGFR, SIRPα, CD20, CD19, BCMA, FcRH5, CD38, PSMA, CD73, HER2, HER3, cMet, GPC3, EpCAM, GPRC5D, and MUC-16. In some embodiments, the antibody fusion protein comprises an antibody specific for CD47, such as the K91 or K33 antibodies.

[0043] Different sequences can be used as linkers and protease-sensitive linkers to connect the antibody and cytokine or cytokine receptor domains.

[0044] The affinity of the antibody or antigen-binding moiety can be engineered to optimize the difference in biological activity between the masked and unmasked forms, thereby minimizing potential peripheral toxicity while maintaining anti-tumor activity.

[0045] Similarly, the activity of cytokines can be modified to optimize the difference in biological activity between masked and unmasked forms, thereby minimizing potential peripheral toxicity while maintaining antitumor activity.

[0046] Antibody Fc domains can be selected for their ability to bind to Fc receptors and drive effector functions such as ADCC, ADCP, or CDC. The Fc portion can also be silenced or enhanced by introducing mutations to further modulate activity. Different Fc selections combined with different configurations can result in antibody-cytokine receptors with different safety and activity profiles that can be exploited in the context of the present invention.

[0047] The above different components, i.e., the location of cytokine fusion, the presence and number of extracellular portions of cytokine receptors, antibody affinity, modification of cytokine activity, and selection of Fc portion, can be combined to find the optimal configuration depending on the antibody and cytokine used, resulting in the desired mode of action and optimal safety and efficacy. Mutations that enhance or reduce Fc gamma receptor or complement interaction can be pursued herein. Mutations that modulate FcRn interaction to alter antibody half-life can be pursued herein. A list of possible mutations is described in Antibodies 2020, 9(4), 64. See https: / / doi.org / 10.3390 / antib9040064, which is incorporated herein by reference.

[0048] In some embodiments, the Fc comprises at least one L234A, or L235A, or P329A mutation. In some embodiments, the Fc comprises L234A, L235A, and P329A mutations.

[0049] In an ideal scenario, the construct would be effective at blocking both cytokines and antibodies, and thus could incorporate high affinity antibodies and cytokines that retain full activity. High affinity antibodies and fully active cytokines could also be used if the antibodies have limited peripheral toxicity and cytokine blockade is effective.

[0050] If this is not achievable, or if the desired mode of action is more antibody-driven, then one can focus on efficient antibody masking, allowing the use of potent antibodies with high peripheral toxicity or other drawbacks. In that case, if optimal cytokine masking in the context of optimal antibody blockade is difficult to achieve, then a cytokine of lower potency can be purposely incorporated into the fusion construct.

[0051] Conversely, if cytokine function is the primary driver of the intended mode of action, the focus can be on effective cytokine blockade and the incorporation of low affinity antibodies, thus limiting its undesired effects in the periphery.

[0052] Furthermore, the selection of an active, less active, or inactive Fc portion provides another layer of optimization of activity in the tumor and limiting peripheral toxicity. For example, if the focus of the construct is on the cytokine component and the antibody is not completely blocked, a high affinity antibody can still be used if the Fc is silent to limit non-tumor side effects.

[0053] The combination of elements and strategies enabled by the present invention to achieve the desired mode of action is shown in FIG.

[0054] In one embodiment, the antibody used is a high-affinity anti-human CD47 antibody, and the cytokine receptor complex is IL-2 with IL-2Rα and / or IL-2Rβ and / or IL-2Rγ. CD47 is overexpressed in a wide range of cancers but is also ubiquitously expressed in healthy tissues, including red blood cells (RBCs). Interaction of CD47 with transmembrane signal-regulatory protein-α (SIRPα), which is expressed on the surface of macrophages, inhibits phagocytosis. Therefore, blocking the interaction of CD47 with SIRPα activates phagocytes and mediates phagocytosis. However, anti-CD47 antibodies bind to and block CD47 on all cells, resulting in the undesirable toxicity and poor pharmacokinetic properties observed with anti-CD47 monoclonal antibodies administered to patients. Thus, according to the present invention, a CD47 antibody masked with a cytokine / receptor complex effectively prevents CD47 binding in the periphery, limiting toxicity and improving pharmacokinetic properties, and can effectively block the CD47-SIRPα interaction upon activation by proteolytic cleavage in the TME. This blockade enhances phagocyte activity and activates the innate immune system. At the same time, the released IL-2 can activate immune cells, including T cells, within the TME while avoiding peripheral toxicity. As an example, a mutual masking and activation approach using a CD47 antibody and IL-2 / IL-2Rα is shown in Figure 5.

[0055] The components of the invention are not limited to monoclonal antibodies of any isotype or monoclonal antibodies containing mutations that modulate Fc-mediated activity, but are also applicable to other antibody formats including, but not limited to, antibody fragments, bispecific antibodies, antibody-drug conjugates, antibody fusion proteins, and other binding protein scaffolds such as single domain antibodies (e.g., camelid VHHs).

[0056] In some embodiments, the antibody fusion protein is human IgG1, or human IgG2, or human IgG3, or human IgG4, or human IgA, or human IgE, or human IgM.

[0057] In some embodiments, the antibody fusion protein is a bispecific antibody, wherein the first antigen-binding domain binds to a first antigen and the second antigen-binding domain binds to a second antigen, and the first antigen and the second antigen are not the same antigen.

[0058] While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. [Example]

[0059] Example 1. Design and molecular cloning of antibody-cytokine / receptor fusions The anti-CD47 antibodies K91 and K33 (see U.S. Patent Application No. 17 / 701,573 (NOVI-048 / 001US), the contents of which are incorporated herein by reference in their entirety) were used to design several antibody-cytokine / receptor fusions (see Table 1). Either IL-2 (SEQ ID NO: 1), IL-15 (SEQ ID NO: 5), IL-2Rα (SEQ ID NO: 2), IL-2Rβ (SEQ ID NO: 3), and / or IL-2Rγ (SEQ ID NO: 4), or the sushi domain of IL-15Rα (SEQ ID NO: 6), were fused to the N-terminus of either the antibody light or heavy chain using different sets of connecting linkers (linkers 1, 2, 3, 4, 5, and 6) and linkers cleavable by selected proteases. Cleavable moiety 1 (CM1) refers to the linker sequence cleavable by the tumor protease MMP-9 (VHMPLGFLGP; SEQ ID NO: 7). Cleavable moiety 2 (CM2) refers to a linker cleavable by uPA (TSTSGRSANPRG; SEQ ID NO: 8). Cleavable moiety 3 (CM3) refers to a linker cleavable by uPA, matriptase, legumain, and MMP-2 / 7 / 9 / 14 (EAGRSANHTPAGLTGP; SEQ ID NO: 9). The cleavable linker was followed by a flexible glycine serine (GS) linker, kept short to favor steric hindrance. In some of the constructs containing IL-2 as the cytokine component, mutations were introduced into the IL-2 sequence to stabilize IL-2 or alter its interaction with different components of the IL-2R. Several control constructs were also designed that did not contain the cleavable linker (n860 and n900).

[0060] The different constructs generated are listed in Table 1 and shown in Figure 3.

[0061] Table 1: Structure and composition of masked antibody constructs. Detailed structure of masked antibodies with single or multiple masking domains, with different components listed. Constructs are numbered for ease of understanding. TIFF2025528468000002.tif210170TIFF2025528468000003.tif220170TIFF2025528468000004.tif207170TIFF2025528468000005.tif203170TIFF2025528468000006.tif223170TIFF2025528468000007.tif215170

[0062] To generate antibody-cytokine / receptor fusions, expression plasmids encoding these different constructs were generated. The expression vectors contained an origin of replication, a kanamycin resistance gene, and two expression cassettes under the transcriptional control of the human cytomegalovirus promoter (hCMV) for expression in mammalian cells of HC and LC with or without a cleavable masking domain. An SV40 promoter and glutamine synthetase gene were also present for expression in CHO cells. An example of an expression vector and a description of the corresponding structure are shown in Figure 6. Synthetic sequences consisting of a masking domain, a flexible linker, a cleavable linker, and another flexible linker were purchased from Eurofins and flanked by appropriate restriction enzyme sites for molecular cloning into the expression vector. Ten micrograms of the expression vector pNOVI K91 (allowing expression of the high-affinity anti-CD47 antibody K91) or pNOVI K33 (allowing expression of the low-affinity anti-CD47 antibody K33) and 10 micrograms of synthetic DNA inserts encoding the different constructs listed in Table 1 were digested with 10 units of the appropriate restriction enzyme for 1 hour at 37°C. The digested vector was dephosphorylated by incubating with 40 units of alkaline phosphatase for 15 minutes at 37°C. The vector and insert were then loaded onto E-Gel Agarose containing 1.2% SYBR Safe DNA Gel Stain (Invitrogen) and purified twice using the MiniElute Gel Extraction Kit (Qiagen) according to the Qiagen protocol. After purification, 15–30 ng of insert DNA was ligated to 40–50 ng of vector using the Rapid DNA Ligation Kit (Roche). Ligation controls were performed with DNA vector alone and DNA insert alone. 30 μL of E. coli XL1 competent cells were slowly thawed on ice, added to the ligation reaction, and kept on ice for 30 minutes. The cells were then heat shocked by incubating at 42°C for 1 minute, then on ice for 2 minutes.The cells were recovered in 500 μL of SOC medium (Invitrogen) for 1 hour at 37°C with agitation at 1250 rpm. The cells were then spread onto LB agar plates containing kanamycin and incubated at 37°C, ON. Several colonies were randomly picked and plated onto a master plate to isolate individual clones, which were then cultured in 5 mL of LB medium containing kanamycin at 37°C, ON. DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen). Analytical DNA digestion was performed to confirm the presence of inserts, followed by sequencing of clones with the expected insert size. A mixture containing approximately 200 ng of DNA and 1 μM of primers in a final volume of 5 μL was used for Sanger sequencing. The sequence was aligned with a theoretical reference sequence using Sequencher software. Clones with the correct sequence were inoculated at 37°C, ON. DNA was extracted and purified using the PureLink HiPure Plasmid Filter DNA Purification Kit (Invitrogen) according to the Invitrogen Maxiprep protocol, and purified DNA was sequenced as described above.

[0063] Example 2. Expression and purification of antibody-cytokine / receptor fusions The expression vectors of Example 1 were transiently transfected into Expi293 cells, and the corresponding antibody-cytokine / receptor fusions were purified and characterized. Expi293 cells were cultured in Expi293 expression medium (ThermoFisher) containing 25 mg / L gentamicin (Gibco) at 37°C, >80% relative humidity, 8% CO2, and with agitation at 120 rpm. On the day of transfection, cells were cultured at 3 x 10 6The cells were diluted to 1000 cells / mL. 50 mL of cells were transfected using polyethyleneimine (PEI) (Polysciences) transfection reagent. A DNA mixture was prepared using 1.3 mL of NaCl and 62.5 μg of DNA. The DNA mixture was added dropwise to a PEI mixture prepared with 1.3 mL of NaCl and 250 μL of PEI and incubated at room temperature for 10 minutes. The DNA / PEI mixture was then transferred dropwise to Expi293 cells. The transfected cells were incubated at 37°C, with a relative humidity of >80%, 8% CO2, and stirring at 120 rpm. After 6 days of culture, the supernatant was collected and filtered through a 0.22 μm membrane using a Sartoclear Dynamics Lab V kit (Sartorius). The antibody was purified by affinity chromatography using an FcXL affinity matrix (ThermoFisher). An appropriate amount of FcXL resin, prewashed three times with phosphate-buffered saline (PBS), was added to the supernatant and incubated at 4°C, 15 rpm, and ON. The sample was then centrifuged at 2000 rpm and 4°C for 10 min to recover the resin, and the flow-through was discarded. The resin was washed twice with PBS, transferred to an Amicon Pro device (Merck), washed again with PBS, and centrifuged at 200 g for 5 min. Elution was then performed with an elution buffer of 50 mM glycine pH 3.5 neutralized 1 / 10 (v:v) with 1 M Tris-HCl, pH 7.5. Three 3 mL elution fractions were applied, collected, and transferred onto an Amicon 50 kDa membrane (Merck) pre-equilibrated with formulation buffer (25 mM histidine, 125 mM NaCl, pH 6.0). Three stages of dilution / concentration with formulation buffer were performed, with centrifugation at 3500 rpm between each stage. The desalted and concentrated samples were then collected and transferred to LoBind tubes (Eppendorf). The antibody concentration was measured by Nanodrop.

[0064] Example 3. Characterization of antibody-cytokine / receptor fusions The purity, molecular size, and integrity of the antibody-cytokine / receptor fusions were assessed by SDS-PAGE. Purified antibody-cytokine / receptor fusions were loaded onto a NuPAGE gel (Invitrogen) under denaturing and reducing conditions. Five micrograms of protein diluted in PBS was incubated with NuPAGE LDS 4X buffer (Invitrogen) containing 4% β-mercaptoethanol at 95°C for 5 minutes. Migration was performed at 150V for 45 minutes in 1X MES NuPAGE running buffer (Invitrogen). For antibody integrity, the gel was stained with Coomassie blue, and aggregation status was assessed by SEC-UPLC using an Acquity UPLC BEH SEC column (Waters) with a 0.2 M sodium phosphate (pH 6.8) mobile phase.

[0065] The characterization of the different antibody-cytokine / receptor fusions is summarized in Table 2.

[0066] Table 2. Yields and aggregation levels of antibody-cytokine / receptor fusions TIFF2025528468000008.tif236170TIFF2025528468000009.tif70170

[0067] Overall constructs with only IL-15 on the LC or on the LC and HC showed lower expression levels. Better expression levels were observed with the combined masking of IL-15 on the LC and IL-15Rα on the HC, suggesting better stability due to a possible interaction between IL-15 and IL-15Rα. Constructs masked with IL-2 or IL-2Rα showed good expression levels. Constructs with IL-2 fused to both the LC and HC showed high aggregation levels (42%, 53%). These constructs also showed unexpected patterns in SDS-PAGE analysis. Constructs with IL-2 on the LC and IL-2Rα on the HC showed good expression and low aggregation levels (less than 1%), suggesting good stability due to a possible interaction between IL-2 and IL-2Rα. Similar observations can be made for other constructs containing several extracellular domains of cytokine receptors that can be expressed and purified (Table 2). For some constructs, aggregation levels could not be determined as they showed abnormal profiles on SEC-UPLC due to their molecular weights being significantly different from IgG (denoted as ND in Table 2).

[0068] Based on this initial characterization, antibody-cytokine / receptor fusions were selected for further biological characterization.

[0069] Example 4. Proteolytic cleavage of the masking domain Proteolytic cleavage of antibody-cytokine / receptor fusions was assessed. 3 μg of antibody was treated with 10 units of hMMP-9 (Abcam) in a reaction buffer containing 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, and 20 μM ZnCl2 (pH 7.5) in a final volume of 20 μL. The reaction was carried out at 37°C for 4 to 5 hours. Cleavage of the masking domain was assessed by SDS-PAGE analysis under reducing and denaturing conditions, as previously described.

[0070] The antibody-cytokine / receptor fusions were incubated with recombinant MMP-9, and the cleavage efficiency was visualized by SDS-PAGE under denaturing and reducing conditions (Figure 7). mAbs K91 (n46) and K33 (n22) were used as controls. As shown, before cleavage, mAbs K91 and K33 exhibited two bands corresponding to the HC (approximately 48 kDa) and LC (approximately 23 kDa) (Figures 7A, 7C-7E, and 7E, respectively). After MMP-9 treatment (approximately 60 kDa), n361 was not cleaved efficiently, and strong bands corresponding to the masked HC and LC were still present. In contrast, n41, n281, and n291 were cleaved more efficiently, revealing bands corresponding to the unmasked HC and LC, as well as one band likely corresponding to IL-2 (approximately 15 kDa). The IL-2Rα, not visible on the gel, likely comigrated with the LC, as their molecular weights are similar (approximately 24 kDa for IL-2Rα and 23 kDa for LC) (Figure 7A). Overall, all constructs could be cleaved by proteases with the expected bands appearing on SDS-PAGE gels (Figures 7B-7D and 7F). As expected, the uncleavable constructs n860 and n900 remained intact after protease cleavage (Figure 7E).

[0071] Example 5. Antibody binding profiles before and after proteolytic cleavage of antibody-cytokine / receptor fusions The ability of the masking domain to inhibit antibody binding activity was assessed by biolayer interferometry (BLI). Wild-type mAbs K91(n46) and K33(n22) were used as positive controls. Binding experiments were performed to assess masking efficiency and binding recovery after cleavage of the masking domain. BLI was performed using an Octet RED96 system (Sartorius). HIS-tagged human CD47 was diluted to 2.5 μg / mL in Kinetic Buffer (KB) (Sartorius) and loaded onto a HIS1K biosensor (anti-HIS tag antibody biosensor, Sartorius) for 300 seconds. The loaded biosensor was immersed in antibody diluted to 15 μg / mL in KB for 300 seconds to monitor association. The biosensor was then transferred to the KB and allowed to dissociate for 60 seconds. The binding profiles were then analyzed using ForteBio Data Analysis software.

[0072] Examples of binding profiles obtained with cleaved and uncleaved antibody-cytokine / receptor fusions are shown in Figure 8. Binding capacities for all constructs are shown in Table 3. Some constructs were not characterized after cleavage (denoted as ND).

[0073] All constructs showed lower association to the target antigen than the control antibody. Furthermore, masking efficacy generally correlated with the molecular weight of the masking domain, consistent with steric hindrance of the masking moiety.

[0074] The binding activity of the antibody-cytokine / receptor fusions before and after cleavage was also assessed by flow cytometry. Peak cells derived from HEK 293, a human cell line expressing CD47, were used to assess masking efficacy and CD47 binding recovery by flow cytometry. Peak cells were diluted to 1.2 × 10 in cold FACS buffer (PBS, 2% BSA). 6 Diluted to 3 x 10 cells / mL 5Cells per well were added to a 96-well V-bottom plate. The plate was then centrifuged at 1300 rpm for 5 minutes at 4°C. The supernatant was removed, and the cells were washed twice with FACS buffer. Serial dilutions of antibody-cytokine / receptor fusions predigested with MMP-9 or undigested antibody-cytokine / receptor fusions as described above were prepared in FACS buffer. An irrelevant antibody was included. 150 μL of each diluted antibody was then added to the corresponding well and incubated for 30 minutes at 4°C. The cells were washed twice with FACS buffer, and 100 μL of mouse anti-human Fc-PE conjugated secondary antibody was added. The plate was incubated for 20 minutes at 4°C. The cells were then washed twice with FACS buffer. Finally, 150 μL of SYTOX Blue Dead cell dye (ThermoFisher) diluted 1 / 5000 in FACS buffer was added before flow cytometric detection using a Cytoflex (Beckman Coulter). Acquisition was performed for 10,000 events per well. Data were analyzed using FlowJo.

[0075] Binding of antibody-cytokine / receptor fusions corresponding to the different constructs before and after proteolytic cleavage to cell surface CD47 is shown in FIG.

[0076] As expected, wild-type antibody K91 n46 showed strong binding to Peak cells before and after proteolytic cleavage (Figures 9A-9R and 9T-9AB). In contrast, the lower-affinity anti-CD47 wild-type antibody K33 n22 showed much lower binding levels (Figure 9S). All antibody-cytokine / receptor fusions showed strongly reduced binding on Peak cells before cleavage, accompanied by a decreased EC50 for binding, compared to mAb K91 n46 (Figures 9B-9AB). Masked n2 showed a weaker binding reduction (Figure 9A). After cleavage, unmasked antibodies showed different levels of binding recovery. Furthermore, constructs not containing the cleavable linker (n860 and n900) showed reduced binding profiles that were not altered by proteolytic cleavage (Figures 9U and 9X).

[0077] Peak cell binding confirmed the masking efficacy provided by the masking domain fused to the N-terminus of the mAb via steric hindrance. The GS linker is required to allow efficient cleavage and effective recovery of binding.

[0078] Table 3: Binding profile of masked cytokine-antibody constructs. Masking of antibody binding was assessed by biolayer interferometry (BLI) technology and flow cytometry. ECs before and after cleavage were measured by flow cytometry. 50 Binding recovery was assessed by dividing the TIFF2025528468000010.tif216170TIFF2025528468000011.tif225170TIFF2025528468000012.tif245170

[0079] Example 6. IL-2 signaling activity of antibody-cytokine / receptor fusions before and after protease treatment The HEK-Blue™ IL-2 reporter system, which expresses high-affinity trimeric IL-2R (IL-2Rα, IL-2Rβ, and IL-2Rγ), was used to assess the IL-2 signaling capacity of antibody-cytokine / receptor fusions before and after proteolytic cleavage. The cell lines express human JAK3 / STAT5 and STAT5-inducible SEAP reporter genes. Binding of IL-2 to the IL-2R results in the secretion of SEAP, which can be monitored using QUANTI-BLUE™ solution. 20 μL of serially diluted antibody was added per well of a flat-bottom 96-well plate, to which 180 μL of HEK-Blue™ IL-2 cells (approximately 100,000 cells) were added and incubated at 37°C in a CO2 incubator for 24 hours. 20 μL of supernatant was transferred to a flat-bottom 96-well plate containing 180 μL of QUANTI-BLUE™ solution. Control wells containing uninduced HEK-Blue™ IL-2 cells were also included. After 1-3 hours of incubation at 37°C, SEAP levels were determined by reading the absorbance at 620-655 nm using a spectrophotometer.

[0080] Activity measured using a dose response of antibody-cytokine / receptor fusions before and after protease treatment is shown in Figure 10. The results, summarized in Table 4, show reduced IL-2 signaling activity in uncleaved antibody-cytokine / receptor fusions compared to cleaved antibody-cytokine / receptor fusions for most of the listed constructs. IL-2 mutant candidates with lower affinity for subunits of the IL-2R show reduced signaling before and after cleavage (Figures 10N-10Q and 10Z). Constructs that do not contain cleavable linkers (n860 and n900) showed only minimal differences before and after protease cleavage (Figures 10T and 10W).

[0081] Table 4: IL-2 activity profile of cytokine-antibody constructs before and after proteolytic cleavage. Masking and restoration of IL-2 activity was assessed using HEK Blue IL-2 cells (Invivogen). IL-2 EC obtained before and after cleavage. 50 IL-2 activity recovery was evaluated by dividing the IL-2 activity by the mean IL-2 activity. TIFF2025528468000013.tif214170TIFF2025528468000014.tif146170TIFF2025528468000015.tif11128

[0082] Example 7. IL-2R B and IL-2R G IL-2 signaling activity of antibody-cytokine / receptor fusions before and after protease treatment on cells expressing only The HEK-Blue™ CD122 / CD132 reporter system was used to assess the IL-2 signaling capacity of antibody-cytokine / receptor fusions before and after proteolytic cleavage in the context of low- to medium-affinity dimeric IL-2Rs (IL-2Rβ and IL-2Rγ). 20 μL of serially diluted antibody was added per well of a flat-bottom 96-well plate, followed by 180 μL of HEK-Blue™ CD122 / CD132 cells (approximately 100,000 cells) and incubation for 24 hours at 37°C in a CO2 incubator. 20 μL of supernatant was transferred to a flat-bottom 96-well plate containing 180 μL of QUANTI-BLUE™ solution. Control wells containing uninduced HEK-Blue™ CD122 / CD132 cells were also included. After incubation at 37°C for 1 to 3 hours, SEAP levels were determined by reading the absorbance at 620 to 655 nm using a spectrophotometer.

[0083] Activity measured using a dose response of antibody-cytokine / receptor fusions before and after protease treatment is shown in Figure 11. The results, summarized in Table 5, show that mutations to IL-2 reduce interaction with IL-2Rγ and decrease IL-2 signaling activity when compared to the trimeric IL-2R complex.

[0084] Table 5: IL-2 activity profile of cytokine-antibody constructs before and after proteolytic cleavage. Masking and restoration of IL-2 activity was assessed using HEK Blue CD122 / CD132 cells (Invivogen). IL-2 EC obtained before and after cleavage. 50 IL-2 activity recovery was evaluated by dividing the IL-2 activity by the mean IL-2 activity. TIFF2025528468000016.tif171170TIFF2025528468000017.tif11128

[0085] These examples demonstrate that in antibody-cytokine / receptor fusions, both antibody binding and cytokine activity are mutually impaired: proteolytic cleavage and release of the antibody and cytokine can restore full binding and signaling activity.

[0086] Example 8. Sequences used to design masking domains TIFF2025528468000018.tif126161TIFF2025528468000019.tif210161TIFF2025528468000020.tif74161The sequences in italics indicate the leader sequence.

[0087] References Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug;18(8):585-608. Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJT. The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov. 2010 Apr;9(4):325-38. Lucchi R, Bentanachs J, Oller-Salvia B. The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control.ACS. Bleuez C, Koch WF, Urbach C, Hollfelder F, Jermutus L. Exploiting protease activation for therapy. Drug Discov Today. 2022 Jun;27(6):1743-54. Chen IJ,Chuang CH,Hsieh YC,Lu YC,Lin WW,Huang CC,et al.Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains.Sci Rep.2017 Sep 14;7:11587。 Vasiljeva O,Hostetter DR,Moore SJ,Winter MB.The multifaceted roles of tumor-associated proteases and harnessing their activity for prodrug activation.Biol Chem.2019。 Mahmood N,Mihalcioiu C,Rabbani SA.Multifaceted Role of the Urokinase-Type Plasminogen Activator(uPA)and Its Receptor(uPAR):Diagnostic,Prognostic,and Therapeutic Applications.Front Oncol.2018 Feb 12;8:24。 Berraondo P,Sanmamed MF,Ochoa MC,Etxeberria I,Aznar MA,Perez-Gracia JL,et al.,Cytokines in clinical cancer immunotherapy.Br J Cancer.2019 Jan;120(1):6-15。 Puskas J,Skrombolas D,Sedlacek A,Lord E,Sullivan M,Frelinger J.Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases.Immunology.2011 Jun;133(2):206-20。

Claims

1. The following structure: (a) a first antigen-binding domain comprising a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and (b) a second antigen-binding domain comprising a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2). an antibody fusion protein having the following structure: the cytokine is linked via a first protease-cleavable linker to (i) at the N-terminus of L1 and / or L2; (ii) at the N-terminus of the H1 and / or H2; or (iii) at the N-terminus of the L1, L2, H1 and / or H2 It is connected, the first antigen-binding domain and the second antigen-binding domain are not specific for the cytokine; The antibody fusion protein.

2. The antibody fusion protein of claim 1 , further comprising a second protease linker.

3. 3. The antibody fusion protein of claim 2, further comprising at least a first portion of a cognate receptor of said cytokine linked via said second protease linker.

4. 4. The antibody fusion protein of claim 3, wherein the second protease linker is linked to the N-terminus of the H1 and / or H2.

5. The antibody fusion protein of claim 3 , wherein the second protease linker is linked to the N-terminus of L1 and / or L2.

6. The antibody fusion protein of claim 3 , wherein the second protease linker is linked to the cytokine.

7. The antibody fusion protein of any one of claims 1 to 6, further comprising at least a second portion of said cognate receptor of said cytokine.

8. The antibody fusion protein of any one of claims 1 to 7, further comprising a third protease linker.

9. 9. The antibody fusion protein of any one of claims 1 to 8, wherein the at least one second portion of the cognate receptor of the cytokine is linked to the antibody fusion via a third protease linker.

10. 10. The antibody fusion of claim 9, wherein the third protease linker is linked to any one of the N-terminus of H1, H2, L1, or L2, the first portion of the cognate receptor of the cytokine, or the cytokine.

11. The antibody fusion protein of any one of claims 1 to 10, further comprising a non-cleavable linker.

12. The antibody fusion protein of any one of the preceding claims, wherein the cytokine is IL-2, IL-15, mutated IL-2, or mutated IL-15.

13. 10. The antibody fusion protein of claim 9, wherein said at least one first portion of said cognate receptor is any one of the extracellular portions of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15Rα Sushi 1.

14. 10. The antibody fusion protein of claim 9, wherein said at least one second portion of said cognate receptor is any one of the extracellular portions of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15Rα Sushi 1.

15. 10. The antibody fusion protein of any one of the preceding claims, which is specific for CD47.

16. 16. The antibody fusion protein of claim 15, comprising a K91 or K33 antibody.

17. 10. The antibody fusion protein of any one of the preceding claims, wherein the IL-2 cytokine comprises one or more of the following mutations: C125S, F42A, D20T, or Q126T.

18. 10. The antibody fusion protein of claim 1, further comprising a modified Fc domain.

19. 19. The antibody fusion protein of claim 18, which is human IgG1, or human IgG2, or human IgG3, or human IgG4, or human IgA, or human IgE, or human IgM.

20. 10. The antibody fusion protein of any one of the preceding claims, wherein the antibody fusion protein is a bispecific antibody, wherein the first antigen-binding domain binds to a first antigen and the second antigen-binding domain binds to a second antigen, and wherein the first antigen and the second antigen are not the same antigen.

21. 21. A method for masking the binding activity of an antibody fusion protein according to any one of claims 1 to 20, wherein the binding activity of said antibody fusion protein to its cognate target is reduced.

22. 22. The method of masking of claim 21, wherein the cognate target binding activity of the antibody fusion protein is reduced by steric hindrance of the first antigen-binding domain and / or the second antigen-binding domain.

23. 23. The method of masking according to claim 22, wherein the steric hindrance is removed by the cleavage activity of a matrix metalloproteinase.

24. The masking method according to any one of claims 21 to 23, wherein the antibody fusion protein binding activity before and after cleavage by matrix metalloproteinases is determined by surface plasmon resonance or by biolayer interferometry.

25. 25. The method of masking of claim 24, wherein the binding activity of the antibody protein fusion is determined before cleavage (BC) and after cleavage (AC), and binding recovery is determined by the ratio of BC to AC.

26. 26. The method of masking according to claim 25, wherein the ratio of BC to AC is at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 150.

27. A method of masking according to any one of claims 21 to 23, wherein cytokine activity before and after cleavage by matrix metalloproteinases is determined by measuring said cytokine activity using a cytokine signalling cellular reporter system.

28. 28. The method of masking of claim 27, wherein the EC50 cytokine signaling activity of the antibody protein fusion is determined by the EC50 before cleavage (BC) and the EC50 after cleavage (AC), and the recovery of cytokine signaling activity is determined by the ratio of BC to AC.

29. 29. The method of masking according to claim 28, wherein the ratio of BC to AC is at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 120.

30. 21. A method of treating a human disease in a subject by administering a therapeutically effective amount of the antibody fusion protein of any one of claims 1 to 20.

31. 31. The method of treating human disease of claim 30, wherein the antibody fusion protein is activated by matrix metalloproteinase cleavage in or near tumor tissue.

32. 32. The method of treating a human disease according to any one of claims 30 or 31, wherein the human disease is cancer.

33. 33. The method of treating human disease according to claim 32, wherein the cancer is one of bladder cancer, breast cancer, colon and rectal cancer, lung cancer, melanoma cancer, endometrial cancer, kidney cancer, leukemia, lymphoma, pancreatic cancer, prostate cancer, brain cancer, central nervous system cancer, stomach cancer, esophageal cancer, thyroid cancer, head and neck cancer, ovarian cancer, or oral cancer.

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