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

Abstract

Described herein are compositions and methods for generating masked antibodies useful for a variety of therapeutic indications.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 403,465, filed on September 2, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to compositions of masked antibodies and methods of use thereof.

[0004] References to Electronic Sequence Listings

[0005] 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 Art

[0006] Antibodies are one of the most successful drug classes, with the advantages of high targeting specificity and low intrinsic toxicity. Despite such favorable features, toxicity may occur if the targeted antigen is also expressed at significant levels on non-diseased tissues (Hansel et al., 2010). This is particularly important for the treatment of cancer, where antibodies typically mediate cell killing through different mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), direct killing of target cells, antibody-drug conjugates (ADC), or T cell redirection using bispecific antibodies targeting CD3 on T cells and tumor-associated antigens on tumor cells.

[0007] Antibodies have been engineered in many ways to improve their efficacy. They have been humanized or isolated from human sequences to reduce immunogenicity potential. Fc domains have been engineered to modulate their interactions with Fc receptors and downstream effector functions or pharmacokinetic properties. Recently, many methods have been developed to generate bispecific and multispecific antibody formats that can achieve new modes of action.

[0008] Furthermore, to increase specificity and limit on-target toxicity, new approaches aim to efficiently activate antibodies when exposed to specific conditions, such as those found within the tumor microenvironment (TME). A variety of engineering strategies have been applied to generate antibodies that can be activated under conditions found in the TME or in other diseased tissues.

[0009] Over the past decade, antibodies have been engineered to become sensitive to a variety of stimuli, including pH, light, temperature, ions, effector molecules, antigen combinations, and proteases (Lucchi et al., 2021). Therefore, under specific conditions, these antibodies are activated, that is, they can bind or not bind to their antigens. One of the main methods for activating antibodies relies on specific preferential proteases expressed in the TME. Protease-activated antibodies are based on the introduction of masking domains that block the binding of antibodies to their cognate antigens and are connected by cleavable linkers. Once the antibody reaches the tumor site, the cleavable linker is cleaved by the protease, thereby releasing the masking domain and restoring the antibody binding activity at the tumor site. Since the cleavage is mediated by proteases overexpressed in the TME, in contrast, in healthy tissues with low protease activity, the interaction between the antibody and its target is prevented by the masking domain, thereby limiting on-target off-tumor toxicity.

[0010] Different masking strategies can be used to hinder the paratope-epitope interaction. Affinity-based masks are specific for a given antibody and occupy the antibody paratope so that it cannot interact with the epitope on the antigen. Typically, the interaction must be of weak or medium affinity so that the mask is released from the antibody upon cleavage of the linker. Therefore, for each antibody-mask pair, the affinity of the mask is an important parameter to adjust. Affinity masks can be peptides that are anti-idiotypic antibody fragments. On the other hand, steric-based masks do not interact specifically with the antibody paratope, but inhibit antibody binding through steric hindrance (Bleuez et al., 2022).

[0011] Affinity-based masks were first introduced in 2009. A recombinant epidermal growth factor receptor (EGFR) fragment was fused to a single-chain Fv fragment (scFv) derived from an antibody targeting EGFR via a linker cleavable by a protease expressed in the TME. The masked scFv had poor association with EGFR, while protease treatment of the unmasked scFv restored binding. Since this first example, a variety of affinity-based masked antibodies have emerged. Among them, anti-CD166 (CX-2009), anti-PD-L1 (CX-072), and anti-CD71 (CX-2029) antibodies have entered clinical trials. Anti-CTLA-4 (XT101) demonstrated tumor-selective pharmacodynamic effects and efficacy in preclinical models. For the design of affinity-based masks, the selected peptide must have a suitable affinity for the paratope of the antibody to mask its binding, but also have a sufficiently weak affinity to be released after cleavage. In addition, for each antibody, a specific peptide must be developed.

[0012] Steric masking was recently introduced by Chen et al. in 2017. They demonstrated that fusion of latency-associated peptides (LAPs) to anti-EGFR or anti-TNFα antibodies reduced their binding activity, which could be restored after protease cleavage. Similarly, the addition of polyethylene glycol (PEG) chains to recombinant proteins as well as antibody fragments has been shown to hinder protein-protein interactions and bioactivity by steric hindrance of large PEG chains that nonspecifically mask protein-protein interaction surfaces.

[0013] In healthy tissues, extracellular proteases are usually present at low levels, and their activity is tightly regulated by inhibitors present in the tissue. In tumor tissues, their expression levels and activity can be significantly upregulated. Altered protease expression and activity are hallmarks of cancer, playing an important role in cancer development at multiple stages from tumor formation to metastasis (Vasiajeva et al., 2019). For example, proteases are involved in cancer cell invasion in healthy tissues through degradation of basement membranes and extracellular matrix (ECM).

[0014] Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) belong to proteases that are upregulated in the TME. MMPs are a family of zinc endopeptidases and are involved in cancer development, progression, and angiogenesis. They are upregulated in many cancer types. 23 MMPs have been identified in humans, of which 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 triggers the degradation of ECM components (Mahmood et al., 2011). MMP9 and uPA are commonly used to design activatable antibodies.

[0015] Cytokines are key players in immune responses 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, therapeutic applications by systemic administration often lead to undesirable side effects such as hypotension, flu-like symptoms, nausea, diarrhea, and arrhythmias.

[0016] Among them, interleukin-2 (IL-2) and interleukin-15 (IL-15) are related cytokines that can stimulate immune cells by interacting 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 to be a mediator of T cell expansion, differentiation and survival. IL-15 is known to be a mediator of NK and CD8 memory T cell expansion and differentiation.

[0017] IL-2 is FDA-approved for the treatment of advanced and metastatic melanoma. However, systemic administration of IL-2 is accompanied by severe side effects, which limits its clinical application. In addition, IL-2 is involved in the development of regulatory T cells (Tregs) that inhibit the development of effective anti-tumor immunity.

[0018] Therefore, there is still a need to develop cytokine therapeutics that specifically mediate immune activation at tumor sites without systemic side effects. One way to limit the side effects of IL-2 is to make it specifically active at the tumor site (Puskas et al., 2011). In addition, combining IL-2 with the extracellular domain (ECD) of IL-2Rα can preferentially activate IL-2Rβ and γ expressed by CD8 T cells and NK cells, but limit the activation of Tregs expressing IL-2Rα.

[0019] The combination of cytokines and their receptors as steric shields in activatable antibodies not only keeps the cytokines inactive, but also the attached antibodies inactive outside the tumor. When reaching the TME, the linker is cleaved by proteases overexpressed in the TME to release active cytokines and functional antibodies. This strategy will allow a dual therapeutic effect: activating antibodies and cytokines at the tumor site while limiting undesired activation in healthy tissues and systemic circulation. Summary of the invention

[0020] In various aspects, the present invention provides an antibody fusion protein having the following structure:

[0021] 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). The cytokine is connected to: (i) the N-terminus of L1 and / or L2; (ii) the N-terminus of H1 and / or H2; or (iii) the N-terminus of L1, L2, H1 and / or H2 via a first protease-cleavable linker.

[0022] The above antibody fusion protein also has at least a portion of a cognate receptor for a cytokine connected to the N-terminus of H1 and / or H2 or to the N-terminus of L1 and / or L2 via a second protease linker. Different cytokines can be incorporated into the construct of the present invention, such as IL-2 or IL-15. The portion of the cytokine receptor can be the extracellular portion of their respective cognate receptors (such as IL-2Rα, IL-2Rβ or IL-2Rγ, IL-2 or a combination thereof).

[0023] The above antibody fusion proteins also have cytokines and their cognate receptors sequentially linked to the N-terminus of the same light chain or heavy chain of the antibody using the same or different linkers in different orders.

[0024] The components of the invention can be combined in different ways to achieve different levels of masking of i) the antigen binding domain and ii) the cytokine, depending on the intended mode of action of the fusion protein and the potential toxicity of the unmasked antigen binding domain or cytokine.

[0025] In some aspects of the invention, the cytokine sequence can be modified to alter its interaction with various receptors and thus modulate its biological activity when masked or unmasked. Similarly, the receptor sequence can also be modified to alter its interaction with cognate cytokines. In addition, the affinity of the antigen binding domain can be modified to modulate its binding capacity when masked or unmasked.

[0026] If the antigen binding domain contains an Fc domain (e.g., in an antibody), the Fc can be selected based on its ability to engage 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 DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a graphic representation of the mutual masking and activation method by fusing the cytokine receptor complex to the N-terminus of the antibody. The mask can be removed by proteolytic cleavage. The antibody has no binding specificity for the cytokine or cytokine receptor complex.

[0028] Figure 2 Different possible configurations of antibody-cytokine / receptor fusions are shown.

[0029] Figure 3 The different constructs generated are illustrated. The "n°" designation indicates different Novimmune construct configurations.

[0030] Figure 4 Strategies and combinations of elements to achieve the desired mode of action are depicted.

[0031] Figure 5 is a graphic representation of the mutual 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 restored CD47-SIRPα blockade, resulting in increased phagocytic activity of tumor cells and IL-2 signaling that mediates T cell activation and proliferation.

[0032] Figure 6 are examples of vector maps generated for expression of different construct configurations, which are represented schematically.

[0033] 7A to 7F Shown is SDS-PAGE analysis of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9.

[0034] Figure 8 Examples showing the binding profiles of antibody-cytokine / receptor fusions before and after cleavage using the Bio-layer Interferometry (BLI) technique.

[0035] FIG. 9A to FIG. 9AB Shown are the CD47 binding profiles at the surface of peak cells of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9.

[0036] FIG. 10A to FIG. 10Z Display using HEK-Blue TM IL-2 reporter system IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9.

[0037] FIG. 11A to FIG. 11M Display using HEK-Blue TM CD122 / CD132 reporter system for IL-2 signaling activity of antibody-cytokine / receptor fusions before and after proteolytic cleavage by MMP-9. DETAILED DESCRIPTION

[0038] The present disclosure provides a protease-activatable antibody and cytokine or cytokine / receptor fusion in a single construct. Cytokines and / or cytokine / receptors mask antibody binding sites, and conversely, antibodies mask cytokines or cytokine / receptors. Mutual masking simultaneously reduces the biological activity of antibodies and cytokines or cytokine / receptors. Since antibodies have no affinity for cytokines / receptors, mutual masking activity is mediated by steric hindrance. Antibodies are connected to cytokines and / or cytokine / receptors via one or more protease-cleavable joints. After being cleaved by proteases that are upregulated in the TME, both antibodies and cytokines / receptors are released into the TME in a form that fully or partially restores biological activity.

[0039] Thus, the present disclosure allows: (i) mutual dual masking of antibody-cytokine / receptor fusions in circulation and within healthy tissues; (ii) release upon proteolytic cleavage of the active molecule (i.e., antibody, cytokine, cytokine receptor); (iii) unmasked antibody that can engage its target (i.e., specifically bind to its cognate antigen); (iv) biologically active cytokine / receptor that can signal through its cognate receptor; (v) increased therapeutic activity of antibody-cytokine / receptor-cytokine fusions because two different modalities are released compared to previous masking strategies where masking is ineffective after release. (See Figure 1 ).

[0040] Various molecular designs and structures can be used to generate the antibody-cytokine / receptor fusions of the present invention.

[0041] In the first configuration, the cytokine is fused to the N-terminus of the antibody light chain, and the extracellular portion of the cytokine receptor is fused to the N-terminus of the antibody heavy chain. This configuration allows the cytokine to interact with the extracellular portion of the cytokine receptor. Since the N-termini of the heavy and light chains are close to the antibody binding site, this configuration promotes steric hindrance mediated by the cytokine / receptor fusion and mutual inhibition of cytokine activity.

[0042] 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. In this exchange configuration, steric hindrance is also promoted as described in the first configuration.

[0043] In other configurations, only the cytokine is fused to the N-terminus of the light chain or the N-terminus of the heavy chain.

[0044] In other configurations, the cytokine is fused to the N-terminus of the light chain and the N-terminus of the heavy chain.

[0045] In other configurations, the first extracellular portion of the cytokine and cytokine receptor is 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 configurations can increase the masking of the cytokine.

[0046] In other configurations, the first extracellular portion of the cytokine and cytokine receptor is 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 configurations can increase the masking of the cytokine.

[0047] In other configurations, the cytokine is fused to the N-terminus of the light chain and two extracellular portions of the cytokine receptor (ie, a first extracellular portion and a second extracellular portion) are fused to the N-terminus of the antibody heavy chain to further block the cytokine activity.

[0048] In other configurations, the cytokine is fused to the N-terminus of the heavy chain, and two extracellular portions of the cytokine receptor (ie, a first extracellular portion and a second extracellular portion) are fused to the N-terminus of the antibody light chain to further block the cytokine activity.

[0049] In each of the above possible configurations, the joint and protease cleavable sequence can be changed to optimize masking and proteolytic cleavage efficiency at the same time. In some embodiments, the antibody fusion protein consists of a first protease cleavable joint. In some embodiments, the antibody fusion protein consists of a first protease cleavable joint and a second protease cleavable joint. In some embodiments, the antibody fusion protein consists of a first protease cleavable joint, a second protease cleavable joint, and a third protease cleavable joint. In some embodiments, the antibody fusion protein consists of three or more cleavable joints. In some embodiments, the cleavable joint is SEQ ID NO: 7 (CM1), SEQ ID NO: 8 (CM2) or SEQ ID NO: 9 (CM3) One or more.

[0050] 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 tissue. In some embodiments, the cleavable linker is cleaved by MMP-9.

[0051] In each of the possible configurations described above, 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.

[0052] In some embodiments, the antibody fusion protein comprises the first part of the cognate receptor of the cytokine. In some embodiments, the antibody fusion protein comprises the second part of the cognate receptor of the cytokine. In some embodiments, the first part of the cognate receptor is any one of the extracellular parts of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15RαSushi 1. In some embodiments, the second part of the cognate receptor is any one of the extracellular parts of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15RαSushi 1. In some embodiments, the first part of the cognate receptor is IL-2Rα, and the second part of the cognate receptor is IL-2Rβ. In some embodiments, the first part of the cognate receptor is IL-2Rβ, and the second part of the cognate receptor is IL-2Rα. In some embodiments, the first part of the cognate receptor is IL-2Rα, and the second part of the cognate receptor is IL-2Rγ. In some embodiments, the first part of the cognate receptor is IL-2Rγ, and the second part of the cognate receptor is IL-2Rα.

[0053] Other configurations combining cytokines and one or several extracellular parts of their receptors can be linked to the N-terminus of the heavy and / or light chains of the antibody using protease-cleavable or non-cleavable linkers to achieve different degrees of masking of the cytokine and the antibody. Figure 2 and Figure 3 A non-exhaustive representation of possible configurations is shown in .

[0054] In some constructs, the cytokine used for N-terminal fusion can be, but is not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, and domain receptors taken from their respective receptors, including IL-2Rα or IL-2Rβ or IL-2Rγ or any combination thereof (if the cytokine used is IL-2). In some embodiments, the mutated cytokine is a mutated IL-2 or a mutated IL-15. In some embodiments, the mutated IL-2 comprises one or more of a C125S mutation, a F42A mutation, a D20T mutation, or a Q126T mutation.

[0055] Any antibody that can be masked according to the present invention can be, for example, but not limited to, an antibody 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, MUC-16. In some embodiments, the antibody fusion protein comprises an antibody specific for CD47, such as a K91 antibody or a K33 antibody.

[0056] Different sequences can be used as linkers and protease-sensitive linkers to connect antibodies and cytokines or cytokine receptor domains.

[0057] The affinity of the antibody or antigen binding component can be modified to optimize the difference in biological activity between the masked and unmasked forms so that potential peripheral toxicity can be minimized while maintaining anti-tumor activity.

[0058] Similarly, the activity of a cytokine can be modified to optimize the difference in biological activity between the masked and unmasked forms so that potential peripheral toxicity can be minimized while maintaining anti-tumor activity.

[0059] The antibody Fc domain can be selected according to its ability to engage Fc receptors and drive effector functions (such as ADCC, ADCP or CDC). The Fc part can also be silenced or enhanced by introducing mutations to further regulate activity. The selection of Fc in combination with different configurations can produce antibody-cytokine receptors with different safety and activity spectra, which can be utilized in the context of the present invention.

[0060] The above-mentioned different components (i.e., the location of cytokine fusion, the presence and number of extracellular parts of cytokine receptors, the affinity of antibodies, the modification of cytokine activity, and the selection of Fc parts) can be combined to find the best configuration according to the antibodies and cytokines used to obtain the desired mode of action and the best safety and efficacy. Mutations that enhance or reduce Fcγ receptor or complement interactions may be performed herein. Mutations that modulate FcRn interactions to change the half-life of antibodies may be performed herein. A list of possible mutations is described in Antibodies 2020, 9(4), 64; https: / / doi.org / 10.3390 / antib9040064, which is incorporated herein in its entirety.

[0061] 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.

[0062] In an ideal scenario, the construct effectively blocks both cytokines and antibodies, and thus high affinity antibodies and cytokines that retain full activity can be incorporated. High affinity antibodies and fully active cytokines can also be used if the antibodies have limited toxicity in the periphery and cytokine blockade is effective.

[0063] If this cannot be achieved or if the desired mode of action is more antibody driven, then the focus can be placed on effective antibody masking, enabling the use of potent antibodies that have high toxicity or other tendencies in the periphery. In this case, if optimal cytokine masking is difficult to achieve with optimal antibody blockade, then a lower potency cytokine can be intentionally incorporated into the fusion construct.

[0064] In contrast, if cytokine function is the primary driver of the desired mode of action, the focus may be on effective cytokine blockade and incorporation of lower affinity antibodies, thereby limiting their undesired effects in the periphery.

[0065] Additionally, the choice of an active or less active or inactive Fc portion brings another layer of optimizing tumor activity and limiting peripheral toxicity. For example, if the focus of the construct is on a cytokine component and the antibody is not completely blocked, a high affinity antibody can still be used if the Fc is silenced to limit off-tumor side effects.

[0066] The combination of elements and strategies to achieve the desired mode of action achieved by the present invention is described in Figure 4 middle.

[0067] In one embodiment, the antibody used is a high affinity anti-human CD47 antibody, and the cytokine receptor complex is IL-2 and IL-2Rα and / or IL-2Rβ and / or IL-2Rγ. CD47 is overexpressed in a variety of cancers, but is also commonly expressed in healthy tissues including red blood cells (RBCs). The interaction of CD47 with the transmembrane signal regulatory protein-α (SIRPα) expressed on the surface of macrophages inhibits phagocytosis. Therefore, by blocking the interaction of CD47 with SIRPα, phagocytic cells are activated and can mediate phagocytosis. However, anti-CD47 antibodies will bind to and block CD47 on each cell, resulting in undesirable toxicity and poor pharmacokinetic properties, which are observed when anti-CD47 monoclonal antibodies are administered to patients. Therefore, according to the present invention, CD47 antibodies masked with cytokine / receptor complexes will not effectively bind to CD47 in peripherally restricted toxicity and improved pharmacokinetic properties, and after activation by proteolytic cleavage in TME, CD47-SIRPα interactions can be effectively blocked. This blockade enhances the activity of phagocytes 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 toxicity in the periphery. The mutual masking and activation method using CD47 antibodies and IL-2 / IL-2Rα as an example is shown in Figure 5 middle.

[0068] The compositions of the present invention are not limited to monoclonal antibodies of any isotype or 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 VHH).

[0069] 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.

[0070] In some embodiments, the antibody fusion protein is a bispecific antibody, wherein a first antigen binding domain binds to a first antigen and a second antigen binding domain binds to a second antigen, wherein the first antigen and the second antigen are not the same antigen.

[0071] While the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0072] Example

[0073] Example 1. Design and molecular cloning of antibody-cytokine / receptor fusions

[0074] Anti-CD47 antibodies K91 and K33 (see, USSN 17 / 701,573 (NOVI-048 / 001 US, the contents of which are hereby incorporated by reference in their entirety) were used to design several antibody-cytokine / receptor fusions (see, Table 1). The sushi domains of IL-2 (SEQ ID NO: 1) or IL-15 (SEQ ID NO: 5) or IL-2Rα (SEQ ID NO: 2) or IL-2Rβ (SEQ ID NO: 3) and / or IL-2Rγ (SEQ ID NO: 4) or IL-15Rα (SEQ ID NO: 6) were fused at the N-terminus of the antibody light chain or heavy chain using different sets of connecting linkers (Linkers 1, 2, 3, 4, 5 and 6) and linkers cleavable by the selected protease. Cleavable moiety 1 (CM1) refers to a cleavable moiety that can be cleaved by the tumor protease MMP-9 (VHMPLGFLGP; SEQ ID NO: 6). NO:7); cleavable portion 2 (CM2) refers to a linker cleavable by uPA (TSTSGRSANPRG; SEQ ID NO:8), and cleavable portion 3 (CM3) refers to a linker cleavable by uPA, matriptase, legumin, MMP-2 / 7 / 9 / 14 (EAGRSANHTPAGLTGP; SEQ ID NO:9). The cleavable linker is preceded and followed by a flexible glycine serine (GS) linker, which is kept short to facilitate steric hindrance. In some constructs containing IL-2 as a cytokine component, mutations were introduced into the IL-2 sequence to stabilize IL-2 or alter its interaction with different components of IL-2R. Some control constructs (n860 and n900) without a cleavable linker were also designed.

[0075] The different constructs generated are described in Table 1 and shown in Figure 3 middle.

[0076] Table 1. Structure and composition of masked antibody constructs. Detailed structure of a masked antibody with a masking domain, constructs are numbered for ease of understanding .

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] In order to produce antibody-cytokine / receptor fusions, expression plasmids encoding these different constructs are generated. The expression vector contains an origin of replication, a kanamycin resistance gene, and two expression cassettes under the transcriptional control of a human cytomegalovirus promoter (hCMV) for expression of HC and LC in mammalian cells with or without a cleavable masking domain. There is also an SV40 promoter and glutamine synthetase for expression in CHO cells. Examples of expression vectors and diagrams of corresponding configurations are shown in Figure 6In. The synthetic sequence consisting of a masking domain, a flexible linker, a cleavable linker and another flexible linker was purchased from Eurofins and flanked with suitable restriction enzyme sites for molecular cloning into expression vectors. 10 μg of expression vector pNOVI K91 (allowing expression of high-affinity anti-CD47 antibody K91) or pNOVI K33 (allowing expression of low-affinity anti-CD47 antibody K33) and 10 μg of synthetic DNA inserts encoding the different constructs listed in Table 1 were digested with 10 units of appropriate restriction enzymes for 1 hour at 37°C. The digested vector was dephosphorylated by incubation with 40 units of alkaline phosphatase at 37°C for 15 minutes. The vector and insert were then loaded onto E-Gel Agarose with SYBR Safe DNA Gel Stain, 1.2% (Invitrogen) and purified twice using the MiniElute gel extraction kit (Qiagen) according to the Qiagen protocol. After purification, 15ng to 30ng of insertion DNA was connected to 40ng to 50ng of carrier using a rapid DNA ligation kit (Roche). Control was connected with a single DNA carrier and a single DNA insert. 30 μL of E. coli XL1 competent cells were slowly thawed on ice and added to the ligation reaction and kept on ice for 30 minutes. Then, heat shock was carried out by incubating the cells at 42°C for 1 minute, and then incubating on ice for 2 minutes. Cells were recovered for 1 hour at 37°C under 1250rpm stirring in the SOC culture medium (Invitrogen) of 500 μL. The cells were then spread on LB agar plates containing kanamycin and incubated at 37°C, ON. Some colonies were picked randomly, plated on Masterplate to separate each clone, and cultivated in 5mL of LB culture medium containing kanamycin at 37°C, ON. DNA was extracted using a small amount of QIAprep Spin preparation kit (Qiagen). DNA digestion was analyzed to check the presence of insert, and clones with expected insert size were subsequently sequenced. A mixture containing approximately 200 ng DNA and 1 μM primers in a final volume of 5 μL was used for Sanger sequencing. The sequences were aligned to the 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. The purified DNA was sequenced as described above.

[0089] Example 2. Expression and purification of antibody-cytokine / receptor fusions

[0090] The expression vector of Example 1 was transiently transfected into Expi293 cells, and the corresponding antibody-cytokine / receptor fusion was purified and characterized. Expi293 was cultured in Expi293 expression medium (ThermoFisher) containing 25 mg / L gentamicin (Gibco) at 37°C, >80% relative humidity, 8% CO2, and 120 rpm agitation. On the day of transfection, cells were plated at 3×10 6 Cell / mL dilution. Polyethyleneimine (PEI) (Polysciences) transfection reagent was used to transfect 50mL cells. DNA mixture was prepared with 1.3mL NaCl and 62.5μg DNA. DNA mixture was added dropwise to PEI mixture prepared with 1.3mL NaCl and 250μL PEI and incubated for 10 minutes at RT. Then, mixture DNA / PEI was transferred dropwise to Expi293 cells. At 37°C,>80% relative humidity, 8% CO2 and 120rpm stirring, the cells of transfection were incubated. After 6 days of cultivation, the supernatant was recovered and filtered on 0.22μm membrane using Sartoclear Dynamics Lab V kit (Sartorius). Antibodies were purified by affinity chromatography using FcXL affinity matrix (ThermoFisher). The appropriate amount of FcXL pre-washed 3 times in phosphate buffered saline (PBS) was added to the supernatant and incubated at 4°C, 15rpm, ON. The sample was then centrifuged at 2000rpm and 4°C for 10 minutes to reclaim the resin and discard the flow-through. The resin was washed twice with PBS, transferred to an Amicon Pro device (Merck), washed with PBS again and centrifuged at 200g for 5 minutes. Then, eluted with the 50mM glycine pH3.5 elution buffer neutralized with 1 / 10 (v:v) 1M Tris-HCl pH7.5. Three elution fractions of 3mL were used, recovered, and transferred to an Amicon membrane 50kDa (Merck) pre-equilibrated with 25mM histidine, 125mM NaCl pH6.0 formulation buffer. 3 step dilutions / concentrations were carried out with formulation buffer, centrifuged at 3500rpm between each step. Desalination and concentrated samples were then recovered and transferred to a LoBind tube (Eppendorf). Antibody concentration was measured by Nanodrop.

[0091] Example 3. Characterization of Antibody-Cytokine / Receptor Fusions

[0092] Purity, molecular size and integrity of antibody-cytokine / receptor fusions were evaluated by SDS-PAGE. Under denaturing and reducing conditions, purified antibody-cytokine / receptor fusions were loaded onto NuPAGE gels (Invitrogen). 5 μg of protein diluted in PBS was incubated together with NuPAGE LDS 4X buffer (Invitrogen) containing 4% beta-mercaptoethanol at 95°C for 5 minutes. Migration was carried out at 150V for 45 minutes in 1X MES NuPAGE running buffer (Invitrogen). Gel was stained with Coomassie blue to determine antibody integrity, and 0.2M sodium phosphate pH 6.8 mobile phase was used to assess aggregation state by SEC-UPLC using Acquity UPLC BEH SEC post (Waters).

[0093] The characterization of different antibody-cytokine / receptor fusions is summarized in Table 2.

[0094] Table 2. Yield and aggregation levels of antibody-cytokine / receptor fusions .

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] The overall construct with a single IL-15 on LC or on LC and HC showed lower expression levels. Better expression levels were observed using a combined masking of IL-15 on LC and IL-15Rα on HC, indicating better stability due to possible interactions between IL-15 and IL-15Rα. Masked constructs with IL-2 or IL-2Rα showed good expression levels. Constructs fused with IL-2 to LC and HC showed high aggregation levels (42%, 53%). These constructs also showed unexpected patterns in SDS-PAGE analysis. Constructs with IL-2 on LC and IL-2Rα on HC showed good expression and low aggregation levels (<1%), indicating good stability due to possible interactions 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 many constructs, since their molecular weights were significantly different from IgG, the aggregation level could not be determined as they displayed unusual characteristics on SEC-UPLC (indicated as ND in Table 2).

[0101] Based on this initial characterization, antibody-cytokine / receptor fusions were selected for further biological characterization.

[0102] Example 4. Proteolytic cleavage of the masking domain

[0103] Evaluation of proteolytic cleavage of antibody-cytokine / receptor fusions. 3 μg of antibody was treated with 10 units of hMMP-9 (Abcam) in a final volume of 20 μL of a buffer containing 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 20 μM ZnCl2 (pH 7.5). The reaction was carried out at 37°C for 4 to 5 hours. Cleavage of the masking domain was evaluated by SDS-PAGE analysis under reducing and denaturing conditions as described above.

[0104] The antibody-cytokine / receptor fusion was incubated with recombinant MMP-9 and the cleavage efficacy was observed by SDS-PAGE under denaturing and reducing conditions ( FIG. 7 ). mAbs K91 (n46) and K33 (n22) were used as controls. As shown in the figure, before cleavage, mAbs K91 and K33 showed two bands corresponding to HC (about 48 kDa) and LC (about 23 kDa), respectively. Fig. 7A and FIG. 7C to FIG. 7E and Fig. 7E). n361 was not efficiently cleaved after MMP-9 treatment (about 60 kDa), and bands corresponding to masked HC and LC with strong intensity still existed. In contrast, n41, n281, and n291 were more efficiently cleaved, and bands corresponding to unmasked HC and LC appeared, as well as a band that may correspond to IL-2 (about 15 kDa). IL-2Rα, which was not visible on the gel, could co-migrate with LC, and their molecular weights were similar (about 24 kDa for IL-2Rα and about 23 kDa for LC) ( Fig. 7A In conclusion, all constructs were cleaved by proteases and showed expected bands on SDS-PAGE gels ( FIG. 7B to FIG. 7D and Figure 7F As expected, the non-cleavable constructs n860 and n900 remained intact after protease cleavage ( Fig. 7E ).

[0105] Example 5. Antibody Binding Spectra Before and After Proteolytic Cleavage of Antibody-Cytokine / Receptor Fusions

[0106] The ability of the masking domain to inhibit antibody binding activity was evaluated by biolayer interferometry (BLI). Wild-type mAbs K91 (n46) and K33 (n22) were used as positive controls. Binding experiments were performed to evaluate masking efficiency and binding recovery after cleavage of the masking domain. BLI was performed on an Octet RED96 system (Sartorius). His-tagged human CD47 was diluted in kinetic buffer (KB) (Sartorius) at 2.5 μg / mL and loaded on a HIS1K biosensor (anti-His tag antibody biosensor, Sartorius) for 300 seconds. The loaded biosensor was immersed in an antibody diluted at 15 μg / mL in KB for 300 seconds to monitor association. Then, the biosensor was transferred to KB to dissociate for 60 seconds. The binding spectrum was then analyzed with ForteBio data analysis software.

[0107] Examples of binding profiles obtained with cleaved and uncleaved antibody-cytokine / receptor fusions are shown in Figure 8 The binding capacity of all constructs is shown in Table 3. Some constructs were not characterized after cleavage (indicated as ND).

[0108] All constructs showed lower binding 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 component.

[0109] The binding activity of the antibody-cytokine / receptor fusions before and after cleavage was also evaluated by flow cytometry. Peak cells derived from human cells HEK 293 expressing CD47 were used to evaluate the masking efficacy and the recovery of binding to CD47 by flow cytometry. Peak cells were diluted to 1.2×10 in cold FACS buffer (PBS, 2% BSA). 6 cells / mL. 5 Cells / well were added to a 96-well V-bottom plate. The plate was then centrifuged at 1300rpm for 5 minutes at 4°C. The supernatant was removed and the cells were washed twice with FACS buffer. A series of dilutions of antibody-cytokine / receptor fusions that were pre-digested with MMP-9 or not digested as described above were prepared in FACS buffer. Including irrelevant antibodies. Then, 150 μL of each diluted antibody was added to the corresponding well and incubated at 4°C for 30 minutes. The cells were washed twice with FACS buffer, and 100 μL of mouse anti-human Fc-PE conjugated secondary antibodies were added and the plate was incubated at 4°C for 20 minutes. The cells were then washed twice with FACS buffer. Finally, 150 μL of SYTOX blue dead cell stain (ThermoFisher) diluted to 1 / 5000 in FACS buffer was added, and then detected by flow cytometry with Cytoflex (Beckman Coulter). Each well was collected 10,000 times. The data was analyzed with FlowJo software.

[0110] Binding of antibody-cytokine / receptor fusions corresponding to the different constructs to cell surface CD47 before and after proteolytic cleavage is shown in FIG. 9 .

[0111] As expected, wild-type antibody K91 n46 showed strong binding to spike cells both before and after proteolytic cleavage ( FIG. 9A to FIG. 9R and Figures 9T to 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 a strong reduction in binding to peak cells prior to cleavage, with reduced EC50s for binding compared to mAb K91 n46 ( FIG. 9B to FIG. 9AB ). Masked n2 showed weaker binding reduction ( Fig. 9A After cleavage, the unmasked antibodies showed different levels of binding recovery. In addition, constructs without a cleavable linker (n860 and n900) showed reduced binding profiles that were not altered by proteolytic cleavage ( Figure 9U and Figure 9X ).

[0112] Binding on the peak cell surface confirmed the masking efficacy provided by the domain fused to the N-terminal mAb through steric masking. The GS linker was required to achieve efficient cleavage and effective recovery of binding.

[0113] Table 3. Binding profiles of masked cytokine-antibody constructs. Antibody binding masking was evaluated by flow cytometry. 50 Evaluate combined recovery separately .

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Example 6. IL-2 signaling by antibody-cytokine / receptor fusions before and after protease treatment active

[0122] HEK-Blue expressing high affinity trimeric IL-2R (IL-2Rα, IL-2Rβ and IL-2Rγ) TM The IL-2 reporter system was used to evaluate the IL-2 signaling capacity of antibody-cytokine / receptor fusions before and after proteolytic cleavage. This cell line expresses human JAK3 / STAT5 and a STAT5-inducible SEAP reporter gene. Binding of IL-2 to IL-2R results in the secretion of SEAP, which can be expressed using QUANTI-Blue TM Solution monitoring. Add 20 μL of serially diluted antibody to each well of a flat-bottom 96-well plate, to which 180 μL of HEK-Blue TM IL-2 cells (about 100'000 cells) and incubate at 37°C in a CO2 incubator for 24 hours. Transfer 20 μL of supernatant to a plate containing 180 μL of QUANTI-Blue TM A flat-bottom 96-well plate containing uninduced HEK-Blue TM Control wells for IL-2 cells. 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.

[0123] The activity measured using a dose response of the antibody-cytokine / receptor fusions before and after protease treatment is shown in Figure 10. The results summarized in Table 4 show that the IL-2 signaling activity in uncleaved antibody-cytokine / receptor fusions is reduced when compared to cleaved antibody-cytokine / receptor fusions for most of the constructs listed. IL-2 mutant candidates with lower affinity for the IL-2R subunit showed reduced signaling before and after cleavage ( Figures 10N to 10Q and Figure 10Z ). Constructs without a cleavable linker (n860 and n900) showed only minor differences before and after protease cleavage ( Figure 10T and Figure 10W ).

[0124] Table 4. IL-2 activity profile of cytokine-antibody constructs before and after proteolytic cleavage. HEKBlueIL-2 cells (Invivogen) were used to evaluate the masking and restoration of IL-2 activity. IL-2EC 50 Separately evaluate the restoration of IL-2 activity .

[0125]

[0126]

[0127]

[0128]

[0129] Example 7. Antibody-cytokine / receptor fusion pairs express only IL-2R before and after protease treatment Β and IL-2R Γ IL-2 signaling activity of cells .

[0130] Using HEK-Blue TM The CD122 / CD132 reporter system was used to evaluate the IL-2 signaling capacity of antibody-cytokine / receptor fusions before and after proteolytic cleavage in the presence of low- to medium-affinity dimeric IL-2Rs (IL-2Rβ and IL-2Rγ). 20 μL of serially diluted antibody was added to each well of a flat-bottom 96-well plate, to which 180 μL of HEK-Blue TM CD122 / CD132 cells (about 100'000 cells) were plated and incubated at 37°C in a CO2 incubator for 24 hours. 20 μL of supernatant was transferred to a 180 μL QUANTI-Blue TM A flat-bottom 96-well plate containing uninduced HEK-Blue TM Control wells for CD122 / CD132 cells. SEAP levels were determined after incubation at 37°C for 1 to 3 hours by reading the absorbance at 620 to 655 nm using a spectrophotometer.

[0131] The activity measured using a dose response of the antibody-cytokine / receptor fusions before and after protease treatment is shown in Figure 11. The results summarized in Table 5 indicate that mutations of IL-2 reduced interaction with IL-2Rγ and reduced IL-2 signaling activity when compared to the trimeric IL-2R complex.

[0132] Table 5. IL-2 activity profile of cytokine-antibody constructs before and after proteolytic cleavage. HEKBlueCD122 / CD132 cells (Invivogen) were used to evaluate the masking and restoration of IL-2 activity. IL-2EC 50 Separately evaluate the restoration of IL-2 activity .

[0133]

[0134]

[0135] These examples show that in antibody-cytokine / receptor fusions, antibody binding and cytokine activity are mutually impaired. Proteolytic cleavage and release of both antibody and cytokine can restore full binding and signaling activity.

[0136] Example 8. Sequences used to design masking domains

[0137]

[0138]

[0139] Sequences in italics indicate leader sequences

[0140] References

[0141] Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: amechanistic review of the pipeline. Nat Rev Drug Discov. 2019Aug; 18(8):585-608.

[0142] Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJT. The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov. 2010Apr;9(4):325–38.

[0143] Lucchi R,Bentanachs J,Oller-Salvia B.The Masking Game:Design ofActivatable Antibodies and Mimetics for Selective Therapeutics and CellControl.ACS.

[0144] Bleuez C,Koch WF,Urbach C,Hollfelder F,Jermutus L.Exploiting proteaseactivation for therapy.Drug Discov Today.2022Jun;27(6):1743–54.

[0145] Chen IJ,Chuang CH,Hsieh YC,Lu YC,Lin WW,Huang CC,et al.Selectiveantibody activation through protease-activated pro-antibodies that maskbinding sites with inhibitory domains.Sci Rep.2017Sep 14;7:11587.

[0146] Vasiljeva O,Hostetter DR,Moore SJ,Winter MB.The multifaceted roles oftumor-associated proteases and harnessing their activity for prodrugactivation.Biol Chem.2019

[0147] 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.2018Feb 12;8:24.

[0148] Berraondo P,Sanmamed MF,Ochoa MC,Etxeberria I,Aznar MA,Perez-GraciaJL,et al.Cytokines in clinical cancer immunotherapy.Br J Cancer.2019Jan120(1):6–15.

[0149] Puskas J,Skrombolas D,Sedlacek A,Lord E,Sullivan M,FrelingerJ.Development of an attenuated interleukin-2fusion protein that can be activated by tumor-expressed proteases.Immunology.2011Jun:133(2):206-20.

Claims

1. An antibody fusion protein having 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); wherein the cytokine is linked to: (i) the N-terminus of said L1 and / or said L2; (ii) the N-terminus of said H1 and / or said H2; or (iii) the N-terminus of said L1, said L2, said H1 and / or said H2; and Wherein the first antigen binding domain and the second antigen binding domain are not specific for the cytokine.

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

3. The antibody fusion protein of claim 2, further comprising at least a first portion of a cognate receptor for a cytokine linked via the second protease linker. The antibody fusion protein according to claim 3 , wherein the second protease linker is connected to the N-terminus of the H1 and / or the H2. The antibody fusion protein according to claim 3 , wherein the second protease linker is connected to the N-terminus of the L1 and / or the L2. 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 the cognate receptor for the cytokine.

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

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

10. The antibody fusion of claim 9, wherein the third protease linker is attached to the N-terminus of H1, H2, L1, L2, the first portion of the cognate receptor of the cytokine, or any of the cytokine. The antibody fusion protein according to 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, a mutated IL-2 or a mutated IL-15.

13. The antibody fusion protein according to any one of the preceding claims, wherein the at least first portion of the cognate receptor is any one of the extracellular portion of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15RαSushi 1.

14. The antibody fusion protein according to any one of the preceding claims, wherein the at least second portion of the cognate receptor is any one of the extracellular portion of IL-2Rα, IL-2Rβ, IL-2Rγ, IL-15RαSushi 1.

15. The antibody fusion protein of any one of the preceding claims, wherein the antibody fusion protein is specific for CD47. The antibody fusion protein according to claim 15 , wherein the antibody protein fusion protein comprises K91 or K33 antibody.

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

18. The antibody fusion protein of any one of the preceding claims, wherein the antibody fusion protein further comprises a modified Fc domain.

19. The antibody fusion protein according to claim 18, wherein 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.

20. The antibody fusion protein of any 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, wherein the first antigen and the second antigen are not the same antigen.

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

22. The masking method according to 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. The masking method of 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 a matrix metalloproteinase is determined by surface plasmon resonance or by bio-layer interferometry.

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

26. The masking method of 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. The masking method according to any one of claims 21 to 23, wherein the cytokine activity before and after cleavage by a matrix metalloproteinase is determined by measuring the cytokine activity using a cytokine signaling cell reporter system.

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

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

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

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

33. The method of treating a human disease according to claim 32, wherein the cancer is one of bladder cancer, breast cancer, colorectal 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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