Novel masked antibody

Abstract

The present invention provides a masked antibody or an antigen-binding fragment thereof containing at least one pair of masked light chain (LC) variable domain and heavy chain (HC) variable domain provided with a mask, wherein the mask contains two peptide masking moieties specifically binding to each other linked to the N-terminals of the light chain and the heavy chain variable domains respectively, at least one masking moiety is bound via a cleavable linker, one of the two masking moieties contains an antigen peptide, and the other contains an sdAb specifically binding to the antigen peptide. The present invention further provides an antibody-drug conjugate (ADC) containing the masked antibody, and a pharmaceutical composition containing the masked antibody or ADC.

Description

Technical Field

[0001] The present invention relates to a novel masked antibody or an antigen-binding fragment thereof containing at least one pair of masked light chain (LC) and heavy chain (HC) variable domains provided with a mask, wherein the mask contains two peptide masking portions specifically binding to each other linked to the N-termini of the light chain and the heavy chain respectively, and at least one masking portion is linked via a cleavable linker. The present invention further provides an antibody-drug conjugate (ADC) containing the masked antibody, and a pharmaceutical composition containing the masked antibody or ADC.

Background Art

[0002] Antibodies are widely used in therapeutic agents for treating diseases such as cancer, autoimmune diseases or inflammatory diseases. Antibodies can specifically recognize and bind to a target antigen and may induce a therapeutic effect. For example, an antibody may specifically bind to a receptor important in a biological process and may exhibit an agonist or antagonist effect on the antigen-receptor. Receptor binding may, for example, interfere with cell proliferation (e.g., tumor growth), or may exhibit an immunomodulatory effect, such as induction of the immune system. Antibodies may exhibit a therapeutic effect alone and / or may be used as a carrier for delivering a drug to a target site (e.g., cancer cells), for example, as part of an ADC.

[0003] In either case, the therapeutic effect depends on the ability of the antibody to bind to the target antigen. A therapeutic antibody must bind to the antigen with sufficient affinity and specificity so that the desired therapeutic effect can be reliably achieved with minimal side effects and / or toxicity. Not only the selectivity of the antibody for the target but also the specificity of the target for the disease (site) to be treated is important. Some target antigens may also be present in non-target cells such as healthy tissues, and binding of the antibody at these sites must be minimized to avoid undesirable side effects of the treatment.

[0004] To avoid the therapeutic antibody binding to sites other than the diseased site, the binding site can be "masked" and the antibody can be modified so that binding to the target antigen is blocked by the mask until the antibody reaches the desired target site. Removal of the "mask" activates the ability of the antibody to bind to the target antigen only at the target site. To ensure that the mask is removed only at the desired target site (e.g., tumor lesion cells), the components of the mask (masking moiety) may be attached to the antibody by a cleavable linker that is cleaved only when the antibody reaches the target. The cleavable linker may include a cleavage site for an enzyme specific to a particular tissue (e.g., a cleavage site recognized by a tumor-specific proteolytic enzyme such as a tumor-specific matrix metalloproteinase present in the extracellular matrix (e.g., MMP2, MMP9 or MMP14), matriptase and urokinase plasminogen activator).

[0005] Cleavage of the cleavable linker at the target site (e.g., tumor site) removes the mask from the antibody, allowing the antigen-binding site of the antibody to be utilized for binding to the target antigen.

[0006] Several antibody masking techniques are known in the art and are reviewed, for example, in Lucchi et al., 2021, ACS Cent. Sci., 7, 724-738 and Lin et al., 2020, Journal of Biomedical Science, 27:76.

[0007] Affinity-based masking strategies known in the art rely on the use of a masking moiety that binds to the antigen-binding site of an antibody and prevent the masked antibody from binding to the target antigen until the mask is removed by cleavage of a cleavable linker used to link the masking moiety to the antibody. Such masking moieties are designed to bind to the antigen-binding site of the masked antibody with a lower affinity than the actual target antigen of the antibody. When the linker holding the masking moiety and the antibody is cleaved, the masking moiety is replaced by the target antigen because the antigen binds to the targeted antibody with a higher affinity than the masking moiety.

[0008] Affinity-based masking methods are described in WO 2004 / 009638 (ISIS Innovation Ltd.). In this method, a therapeutic antibody is provided with a masking moiety that has the potential to be a peptide mimotope that binds reversibly to the antibody's antigen-binding site (referred to as the antibody-binding site (ACS)) early after administration. The masking moiety is linked to the heavy or light chain of the antibody by an enzyme-cleavable linker (a linker having an enzymatic cleavage motif and being readily cleaved by host enzymes in vivo). WO 2009 / 025846 (Cytomx, Univ. California) describes a masked anti-VEGF antibody, where the masking moiety is a peptide having a cysteine residue that is bound to the antibody by a peptide linker cleavable by MMP2 protease. The cysteine of the masking moiety forms a disulfide bridge with a cysteine in the vicinity (or within) the target-binding site of the antibody, thereby masking the antigen-binding site of the antibody. In the cleaved state, the antigen-binding site of the antibody is available for binding to the target protein. US Pat. No. 8,895,702 (Williams et al.) describes a pair of cross-masking antibodies, where a cross-masking epitope that binds to the antigen-binding site of the other antibody is linked to each antibody by a cleavable linker, forming a cross-masking complex until the cleavable linker at the target site is cleaved by MMP9. Affinity-based masking strategies where the masking moiety binds specifically to the antigen-binding site of the antibody have the disadvantage that each antibody requires a different specific mask for its own antigen-binding site, and careful optimization of the affinity of the mask for the antigen-binding site is required.

[0009] Antibody masking technologies have also been developed that provide more generally applicable masks that can be applied to various antibodies regardless of the target antigen. In these methods, the antibody is provided with one or more masking moieties that do not act by binding directly to the antigen-binding site of the antibody. The antibody is provided with one or more masking moieties that do not occupy the antigen-binding site of the antibody but prevent the antibody from binding to the target antigen by sterically interfering with the binding of the antibody. In these methods, peptide masking moieties that do not bind to the antigen-binding site of the antibody but bind to each other to form a mask and are affinity for each other may be linked and extended at the N-terminals of the light chain (LC) and heavy chain (HC) of the antibody in the LC-HC pair. These masking moieties form a mask that protects the antigen-binding site of the antibody by binding to each other when not cleaved, but the mask is removed when cleaved, and the antibody-binding site can bind to its target antigen again. It may be bound to the N-terminals of the LC and HC of the antibody by a cleavable linker in such a way that it becomes possible.

[0010] International Publication No. 2014 / 193973 (DCB USA LLC, Univ. Kaohsiung Medical) describes an antibody masking method that uses the hinge region of a human antibody as a masking moiety. The hinge region to be blocked consists of two peptide arms interconnected by disulfide bonds. Each arm of the hinge region is connected to the heavy and light chains of the antibody binding domain by a cleavable linker (four arms and four cleavable linkers per antibody). International Publication No. 2020 / 229553 (Ultrahuman Six Ltd.) describes a masking method in which two antigen-binding portions, which may be (a part of) an antibody that binds to a disease-specific antigen, are linked to each other by a cleavable peptide linker containing the amino acid sequence of the human hinge region. In the un-cleaved state, the binding of the two antigen-binding portions to their respective target antigens is blocked. International Publication No. 2018 / 107125 (Seattle Genetics Inc.) describes the use of a coiled-coil forming peptide as a masking moiety. The coiled-coil forming peptide is linked to at least one N-terminus of the light and heavy chains of the antibody via a linker containing a protease cleavage site. In the un-cleaved state, the peptides associate to form a coiled-coil structure, thus reducing the binding affinity of the masked antibody light-chain / heavy-chain pair for the target antigen. When exposed to tumor-associated proteases such as MMP2 and MMP9, the coiled-coil peptide is cleaved and the binding of the antibody to the target antigen is restored.

[0011] A problem with existing masking technologies is the potential for the formation of high molecular weight (HMW) aggregates during manufacture by including a mask in the antibody product. For example, HMW aggregates can form when the masking moieties of different antibodies bind to each other rather than to the corresponding masking moieties of the same antibody. As a result, the manufacturing yield of useful masked antibodies can be low and / or additional steps may be required to remove HMW aggregates from the product. Excessive aggregation can also lead to the induction of anti-drug antibodies (ADA).

[0012] Regarding masking methods known in the art, individual steps have been suggested to solve the problem of HMW aggregate formation. International Publication Nos. WO 2020 / 247572 and WO 2020 / 247574 (Seattle Genetics Inc.) describe problems regarding HMW aggregate formation during purification, processing, and storage related to masking methods using (hydrophobic) coiled-coil masks. For example, it has been proposed to store masked antibodies in an aqueous buffer at pH 3.5 to 4.5. In International Publication No. WO 2020 / 028401 (Amgen Inc.), a solution to HMW aggregate formation in the production of masked antibodies also requires changing the pH of the antibody composition.

[0013] Despite the existence of some common masking techniques, there remains a need for a generally applicable antibody masking technique that can be easily manufactured (e.g., without or with limited formation of HMW aggregates). This is particularly true for antibodies used in antibody-drug conjugates (ADCs) where the antibody mask must not interfere with the conjugation of the linker-drug molecule to the masked antibody. Linker-drug molecules often bind via (engineered) cysteines within the antibody protein molecule. In particular, masking techniques that rely on peptide masking moieties that bind to the -S-S- bridge between cysteines or bind via the formation of said bridge may not be well-suited for use in ADCs based on masked antibodies due to the risk of interfering with linker-drug conjugate formation. SUMMARY OF THE INVENTION

[0014] The present invention relates to a novel method for masking an antibody. The present invention provides a masked antibody or an antigen-binding fragment thereof comprising at least one masked light chain and heavy chain pair provided with a mask, wherein the mask contains two peptide masking moieties specifically binding to each other, each linked to the N-terminus of a pair of light and heavy chains, at least one of the masking moieties being linked via a cleavable linker, one of the two masking moieties containing an antigen peptide and the other containing a single domain antibody (sdAb) specifically binding to the antigen peptide.

[0015] The present invention is based on the novel use of an antigen peptide in the masking moiety of a masked antibody and an sdAb specifically binding to the antigen peptide.

[0016] The present invention further provides an antibody-drug conjugate (ADC) based on the masked antibody according to the present invention, the use of the masked antibody or ADC as a medicament, and a pharmaceutical composition containing the masked antibody or ADC.

[0017] More specifically, the present invention is presented as follows.

[0018] An activatable antibody or an antigen-binding fragment thereof containing an antigen-binding site containing a heavy chain variable domain and a light chain variable domain capable of binding to a target protein, wherein the antigen-binding site is provided with a mask suitable for preventing the binding of the antigen-binding site to the target protein, the mask comprising two masking moieties: i. a masking moiety linked to the N-terminus of the heavy chain variable domain, and, ii. a masking moiety linked to the N-terminus of the light chain variable domain, containing, at least one of the masking moieties being linked via a cleavable linker, one of the masking moieties containing an antigen peptide and the other containing a single domain antibody (sdAb) specifically binding to the antigen peptide. An activatable antibody or an antigen-binding fragment thereof.

[0019] The activatable antibody according to the present invention may be used for treatment by administering it to a subject in need of treatment (e.g., a cancer patient undergoing immunotherapy with a therapeutic anti-cancer antibody or an antibody-drug conjugate (ADC)).

[0020] The activatable antibody or an antigen-binding fragment thereof according to the present invention contains an antigen-binding site capable of binding to a target protein in a subject. After the antibody is administered to the subject, the mask is removed in vivo, preferably at the target (disease, e.g., tumor) site, by cleavage of at least one cleavable linker.

[0021] In a preferred embodiment, the activatable antibody or an antigen-binding fragment thereof contains two antigen-binding sites, and masks are provided for these two antigen-binding sites.

[0022] Alternatively, or in combination with the previous preferred embodiment, in a preferred embodiment, the antigen peptide has 4 to 20 amino acid residues, preferably, the antigen peptide has 12 to 20 amino acid residues, and contains the amino acid sequence defined by SEQ ID NO: 51 or consists of the amino acid sequence defined by SEQ ID NO: 51. More preferably, the antigen peptide contains the amino acid sequence defined by SEQ ID NO: 1, SEQ ID NO: 2 (SA variant) or SEQ ID NO: 3 (ST variant) or consists of the amino acid sequence defined by SEQ ID NO: 1, SEQ ID NO: 2 (SA variant) or SEQ ID NO: 3 (ST variant). Even more preferably, the antigen peptide consists of the amino acid sequence defined by SEQ ID NO: 1, SEQ ID NO: 2 (SA variant) or SEQ ID NO: 3 (ST variant).

[0023] Alternatively, or in combination with the previous preferred embodiments, in a preferred embodiment, the nanobody contains the amino acid sequence defined by SEQ ID NO: 4, its humanized version, or a variant of SEQ ID NO: 4 such as the variant shown by SEQ ID NO: 76 (RA variant), SEQ ID NO: 77 (RE variant) or SEQ ID NO: 78 (RS variant), or a humanized version of said variant, or consists of the amino acid sequence defined by SEQ ID NO: 4, its humanized version, or a variant of SEQ ID NO: 4 such as the variant shown by SEQ ID NO: 76 (RA variant), SEQ ID NO: 77 (RE variant) or SEQ ID NO: 78 (RS variant), or a humanized version of said variant. In the RA, RE and RS variants of the B2C nanobody, the original CDR3 (SEQ ID NO: 5) is replaced by the CDR3 shown by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

[0024] The humanized version of the BC2 nanobody may be a nanobody having the amino acid sequence shown by SEQ ID NO: 79 (BC2 Nbヒト化1 ), SEQ ID NO: 80 (BC2 Nbヒト化2 ), or SEQ ID NO: 81 (BC2 Nbヒト化3 ).

[0025] The humanized version of the RA variant nanobody may be a nanobody having the amino acid sequence shown by SEQ ID NO: 82 (RA h1 ), SEQ ID NO: 83 (RA h2 ), or SEQ ID NO: 84 (RA h3 ). The humanized version of the RE variant nanobody may be a nanobody having the amino acid sequence shown by SEQ ID NO: 85 (RE h1 ), SEQ ID NO: 86 (RE h2 ), or SEQ ID NO: 87 (RE h3 ). The humanized version of the RS variant nanobody may be a nanobody having the amino acid sequence shown by SEQ ID NO: 88 (RS h1 ), SEQ ID NO: 89 (RS h2 ), or SEQ ID NO: 90 (RS h3 ).

[0026] In a preferred embodiment, the antigen peptide contains a peptide having the amino acid sequence defined by SEQ ID NO: 2 (SA variant) or its humanized version, or consists of a peptide having the amino acid sequence defined by SEQ ID NO: 2 (SA variant) or its humanized version, and the nanobody contains the amino acid sequence defined by SEQ ID NO: 78 (RS variant).

[0027] Preferably, the humanized nanobody is RS h1 is.

[0028] Alternatively, or in combination with the previous preferred embodiment, in a preferred embodiment, the masking portion containing the sdAb is linked to the N-terminus of the light chain variable domain, and the masking portion containing the antigen peptide is linked to the N-terminus of the heavy chain variable domain.

[0029] In a preferred embodiment, there is a spacer amino acid sequence defined by SEQ ID NO: 9 that connects to the N-terminus of the antigen peptide and is present in front of the antigen peptide.

[0030] Alternatively, or in combination with the previous preferred embodiment, in a preferred embodiment, the cleavable linker contains one or more cleavage sites recognized by one or more tumor-specific proteases. This means that each cleavable linker may contain one or more cleavage sites. Each cleavage site may be recognized by only one or multiple tumor-specific proteases. Preferably, at least one cleavable linker contains a cleavage site recognized by matriptase or a cleavage site recognized by a metalloprotease.

[0031] In a second aspect, the present invention relates to an activatable antibody or an antigen-binding fragment thereof according to the present invention and an antibody-drug conjugate (ADC) containing a linker-drug.

[0032] In a third aspect, the present invention relates to a pharmaceutical composition containing an activatable antibody or an antigen-binding fragment thereof according to the present invention or an ADC according to the present invention and a pharmaceutically acceptable excipient.

[0033] In a fourth aspect, the present invention relates to an activatable antibody or antigen-binding fragment thereof according to the present invention, an ADC according to the present invention, or a pharmaceutical composition according to the present invention for use as a medicament.

[0034] In a fifth aspect, the present invention relates to an activatable antibody or antigen-binding fragment thereof according to the present invention, an ADC according to the present invention, or a pharmaceutical composition according to the present invention for use in the treatment of cancer, autoimmune diseases or infectious diseases, preferably for use in the treatment of cancer.

[0035] In a sixth aspect, the present invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a heavy chain variable domain and a masked portion; and a nucleotide sequence encoding a light chain variable domain and a masked portion, wherein the nucleotide sequences are operably linked to expression control sequences for expression in a host cell, preferably a mammalian host cell. Preferably, the heavy chain variable domain, the light chain variable domain and the masked portion are as defined in connection with the first aspect of the present invention. In a preferred embodiment, the nucleic acid construct is a vector compatible with the host cell.

[0036] In a seventh aspect, the present invention relates to a host cell containing the nucleic acid construct of the present invention.

[0037] In an eighth aspect, the present invention relates to a method for producing an activatable antibody or antigen-binding fragment thereof according to the present invention, the method comprising culturing a host cell according to the present invention under conditions that promote the expression of the activatable antibody or antigen-binding fragment thereof.

[0038] In a ninth aspect, the present invention relates to the use of an antigen peptide as a part of a mask in an activatable antibody or antigen-binding fragment thereof and an sdAb that specifically binds to the antigen peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0039]

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Mode for Carrying Out the Invention

[0040] Detailed Description of the Invention The present invention provides a novel antibody masking method using an antigen peptide and a single domain antibody (sdAb) that specifically binds to the antigen peptide as a part of a masking portion.

[0041] The present invention provides a masked antibody or an antigen-binding fragment thereof containing at least one light chain and heavy chain pair provided with a mask, wherein the mask contains two peptide masking portions that are specifically bound to each other and are respectively linked to the N-terminals of the light chain and heavy chain of the pair, at least one masking portion is bound via a cleavable linker, one of the two masking portions contains an antigen peptide, and the other contains an sdAb that specifically binds to the antigen peptide.

[0042] The present invention thus provides a novel use of an antigen peptide and an sdAb that specifically binds to the antigen peptide as a part of the masking portion in a masked antibody or antigen-binding fragment.

[0043] The use of an sdAb in / as an antibody masking portion has several advantages. The masking effect is based on the specific affinity between the sdAb and its antigen peptide, the binding partner. By binding to each other, the two masking portions protect and block the binding site of the antibody or at least significantly reduce its ability to bind to its target antigen.

[0044] The binding between the masking portions in the mask provided by the present invention does not depend on the formation of any -S-S- bridges formed between the cysteine residues of the two masking portions. In particular, this is an advantage since linker-drugs often bind to the (engineered) cysteine residues of the antibody protein when the masked antibody is part of an ADC. Thus, the masked antibody or masked antigen-binding fragment thereof according to the present invention is particularly useful in an ADC. The masked antibody according to the present invention further has the advantage that relatively high titers thereof can be produced without substantial loss of a substantial portion of the produced masked antibody due to the formation of high molecular weight (HMW) complexes.

[0045] Thus, in a first aspect, the present invention is an activatable antibody or an antigen-binding fragment thereof containing an antigen-binding site containing a heavy-chain variable domain and a light-chain variable domain capable of binding to a target protein, wherein the antigen-binding site is provided with a mask suitable for preventing the binding between the antigen-binding site and the target protein, and the mask has two masking portions: i. a masking portion linked to the N-terminus of the heavy-chain variable domain, and ii. a masking portion linked to the N-terminus of the light-chain variable domain, and contains at least one of the masking portions is linked via a cleavable linker, one of the masking portions contains an antigen peptide, and the other masking portion contains a single-domain antibody (sdAb) that specifically binds to the antigen peptide, relating to an activatable antibody or an antigen-binding fragment thereof.

[0046] The activatable antibody according to the present invention may be used for treatment by administering it to a subject in need of treatment (e.g., a cancer patient receiving immunotherapy with a therapeutic anti-cancer antibody or an antibody-drug conjugate (ADC)).

[0047] The activatable antibody or an antigen-binding fragment thereof according to the present invention contains an antigen-binding site capable of binding to a target protein in a subject. In the activatable antibody, the antigen-binding site is provided with a mask that prevents binding to the target protein. After the antibody is administered to the subject, the mask is removed in vivo, preferably at the target (disease, e.g., tumor) site where at least one cleavable linker is cleaved, and then the binding site of the antibody can bind to the target protein in the subject.

[0048] Antibody The term "antibody" as used herein preferably refers to an antibody containing two heavy chains and two light chains. The antibodies used in the present invention may be antibodies of isotypes such as IgA, IgE, IgG, or IgM antibodies. Preferably, the antibody is an IgG antibody, more preferably IgG1 or IgG 2 is an antibody. The antibody may be chimeric, humanized or human. Preferably, the antibody is humanized or human. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG 1 monoclonal antibody. The antibody may have a κ (kappa) or λ (lambda) light chain, preferably a κ (kappa) light chain, i.e., a humanized or human IgG 1 -κ antibody.

[0049] The term "antigen-binding fragment" as used herein refers to Fab, Fab’, F(ab’) 2 , Fv, scFv or reduced IgG (rIgG) fragments or fragments of an antibody containing at least the variable binding regions of a light chain (LC) and heavy chain (HC) pair.

[0050] "Humanized" forms of non-human (e.g., rodent) antibodies are antibodies that contain minimal sequences derived from non-human antibodies (e.g., non-human-human chimeric antibodies). Various methods for humanizing non-human antibodies are known in the art. For example, the antigen-binding complementarity-determining regions (CDRs) in the variable domains (also named variable regions or VRs) of the heavy chain (HC) and light chain (LC) are derived from antibodies from non-human species, typically mice, rats or rabbits. These non-human CDRs may be combined with the human framework regions (FRs, i.e., FR1, FR2, FR3 and FR4) of the variable regions of the HC and LC such that the functional properties of the antibody, such as binding affinity and specificity, are at least partially retained. Selected amino acids in the human FRs may be exchanged with the corresponding original non-human species amino acids to further refine the performance of the antibody, such as for improving binding affinity while retaining low immunogenicity. Thus, the humanized variable regions are usually combined with human constant regions. A representative method for humanizing non-human antibodies is the method of Winter and co-workers (Jones et al, 1986, Nature, 321, 522-525; Riechmann et al, 1988, Nature, 332, 323-327; Verhoeyen et al, 1988, Science, 239, 1534-1536). Alternatively, non-human antibodies can be humanized by modifying their amino acid sequences to increase their similarity to antibody variants that are naturally produced in humans. For example, selected amino acids of the original non-human species FRs are exchanged with their corresponding human amino acids to reduce immunogenicity while retaining the binding affinity of the antibody. For further details, see Jones et al, 1986, Nature, 321, 522-525; Riechmann et al, 1988, Nature, 332, 323-327 and Presta, 1992, Curr. Op. Struct. Biol., 2, 593-596.See also the following reviews and references cited in this specification: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma and Immunol., 1, 105-115; Harris, 1995, Biochem. Soc. Transactions, 23, 1035-1038 and Hurle and Gross, 1994, Curr. Op. Biotech., 5, 428-433.

[0051] The CDRs may be determined using the approach of Kabat (in Kabat, E.A. et al, 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, NIH publication no. 91-3242, pp. 662, 680, 689), Chothia (Chothia et al, 1989, Nature 342, 877-883) or IMGT (Lefranc, 1999, The Immunologist, 7, 132-136).

[0052] Typically, an antibody is a monospecific (i.e., specific for one antigen; such an antigen may be common between species or may have a similar amino acid sequence between species) or bispecific (i.e., specific for two different antigens of a species) antibody containing at least one HC and LC variable region that binds to an antigen target, preferably a membrane-bound antigen target that may or may not be internalized. Preferably, in the case of an antibody-drug conjugate, the antibody is internalized by the target cell after binding to the (antigen) target, and then an active effector molecule is released intracellularly.

[0053] Examples of therapeutic antibodies known in the art include blinatumomab (CD19), rituximab (CD20), epratuzumab (CD22), infliximab and brentuximab (CD30), badalizumab (CD33), teturamab (CD37), isatuximab (CD38), vivotuximab (CD44), lorbuvizumab (CD56), borsetuzumab (CD70), mirvetuximab (CD74), polatuzumab (CD79), lobaplizumab (DLL3), fucosylmuramidase (EGFR), oportuzumab (EPCAM), ING-1 (EpCAM), adecatumumab (EpCAM), farletuzumab (FOLR1), glenvacumab (GPNMB), trastuzumab, pertuzumab and margetuximab (HER2), etaracizumab (integrin), anetumab (mesothelin), pancitumumab (MUC1), enfortumab (nectin-4), H8, A1, A3 (5T4), MORAb-66, tisotumab (TF), sacituzumab (TROP2) and urelumab (TNFRSF9, CD137), and the like.

[0054] Masked antibody or antigen-binding fragment The masked antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment as defined above provided with a (removable) mask. The antibody or antigen-binding fragment mask consists of one or more masking moieties that block or at least significantly reduce the ability of the antibody or antigen-binding fragment to bind to the target antigen until the blocking effect of the mask is removed (“demasking”), and the ability of the antibody or antigen-binding fragment to bind to the target antigen is sufficiently restored for the desired therapeutic effect to be achieved. Masked antibodies or antigen-binding fragments are also referred to as activatable antibodies or antigen-binding fragments or antibody or antigen-binding fragment-prodrugs, indicating that they need to be activated by removal of the masking effect of the antibody mask in order to exhibit their therapeutic effect. The terms “masked antibody” and “activatable antibody” are used interchangeably herein. The masking moieties used to create at least one “mask” on the masked antibody or antigen-binding fragment according to the invention are present in pairs; one masking moiety contains an antigenic peptide and the other masking moiety contains an sdAb that specifically binds to the antigenic peptide. One of the paired masking moieties is linked to the HCVR, while the other is linked to the LCVR of at least one HC / LC binding pair of the masked antibody or antigen-binding fragment thereof according to the invention. By binding to each other, the masking moieties form a mask that protects the (at least one) binding site of the antibody or antigen-binding fragment thereof. Thus, an antibody having two antigen-binding sites or an antigen-binding fragment thereof can carry two masks, one on each HCVR / LCVR pair.

[0055] Most target antigens are not completely specific to the target (disease) site, so an antibody or its antigen-binding fragment can be masked to prevent or reduce off-target effects. The mask protects the antigen-binding site in the masked antibody or antigen-binding fragment by shielding it until it reaches its target (disease) site where the masking effect is removed, thereby reducing the ability of the antibody or its antigen-binding fragment to bind to the target antigen. Reduction of binding means that the binding ability of the masked antibody or antigen-binding fragment to its target is significantly reduced by the mask compared to the naked (unmasked) or de-masked (mask removed from the antigen-binding site) antibody or antigen-binding fragment.

[0056] Binding of an antibody or antigen-binding fragment Therapeutic antibodies or antigen-binding fragments are selected based on their selectivity and affinity for the target antigen. Affinity is the strength of the binding interaction between a binding molecule and its ligand (e.g., between an antibody or antigen-binding fragment and its target antigen). The target antigen may be an "antigenic determinant" or "epitope", an antigenic protein (the antigenic part of a protein), that may be presented on the surface of the target cell to which the antibody or antigen-binding fragment binds. In the case of therapeutic antibodies or antigen-binding fragments used for the treatment of cancer, the target cells may be tumor cells.

[0057] The selectivity of an antibody or antigen-binding fragment is a measure of how well the antibody or antigen-binding fragment binds to its target antigen in a mixture of different proteins. In other words, if the epitope to which the antibody or antigen-binding fragment binds is unique to one specific target antigen, the antibody or antigen-binding fragment binds exclusively to the target antigen and does not cross-react with any of the off-target proteins present in the mixture, and thus the selectivity is high.

[0058] The specificity of an antibody or antigen-binding fragment represents the ability of the antibody or antigen-binding fragment to recognize a single epitope. Thus, a monoclonal antibody or antigen-binding fragment that binds to a single epitope is specific. However, if the single epitope to which an antibody or antigen-binding fragment binds appears on multiple proteins, the antibody or antigen-binding fragment may be specific (with respect to the epitope to which it binds with high affinity), but not selective (since the epitope is also present on non-target proteins). Therapeutic antibodies or antigen-binding fragments preferably bind to the target antigen with high affinity and selectivity.

[0059] The binding of an antibody or antigen-binding fragment to a target antigen can be measured and compared by various techniques, and the measured signal represented by a specific parameter is a reflection of the degree to which the antibody or antigen-binding fragment binds to the target antigen. To examine the reduction in binding to the target antigen due to masking of the antigen-binding site of the antibody or antigen-binding fragment, the binding signal of the "naked" antibody or antigen-binding fragment (unmasked antibody or antigen-binding fragment) can be compared with the binding signal of the same antibody or antigen-binding fragment with a mask (masked antibody or antigen-binding fragment) in the same situation (e.g., the same concentration). The difference in the signals obtained between the naked antibody and the masked antibody or antigen-binding fragment, the reduction in the signal of the masked antibody or antigen-binding fragment, is an indicator that the masked antibody or antigen-binding fragment shows a reduction in binding to the target antigen compared to the naked antibody or antigen-binding fragment. Similarly, the recovery of binding can be determined by comparing the signal measured for the masked antibody or antigen-binding fragment with the binding signal obtained for an antibody or antigen-binding fragment from which the masking effect has been removed ("demasked" antibody or antigen-binding fragment). To examine the recovery of binding after demasking, the signal of the demasked antibody or antigen-binding fragment can also be compared with the signal obtained for the naked antibody or antigen-binding fragment.

[0060] One parameter that represents the binding or affinity of an antibody or antigen-binding fragment is the dissociation constant (K D ). For an antibody or antigen-binding fragment, K Dis the dissociation constant (K off ) to the binding rate of the antibody or antigen-binding fragment (K on ) or the bound concentration multiplied by the unbound concentration at equilibrium where the binding and dissociation rates of the binding partners are the same; [A] x [B] / [AB]. The higher the concentration of the binding partners, the more likely they will bind to each other. K D Binding partners with low K D Binding partners with high K require high concentrations to be able to bind to each other. D The smaller the value, the greater the binding affinity between the two binding partners. K D is expressed in molar units. Most antibodies or antigen-binding fragments are available in low micromolar concentrations (10 -6 ) to nanomolar (10 -7 ~10 -9 )K D High affinity antibodies or antigen-binding fragments generally have low nanomolar (10 -9 ), whereas very high affinity antibodies or antigen-binding fragments are considered to be at picomolar concentrations (10 -12 ) is thought to be in low nanomolar concentrations. D is affected by various factors, such as buffer composition, pH and temperature. D ) can be measured, for example, by surface plasmon resonance (SPR).

[0061] A technique suitable for screening for the binding of an antibody or antigen-binding fragment to a (fixed) target antigen is Biolayer Interferometry (BLI). BLI is an optical technique that measures the interference pattern of white light reflected from a sensor chip coated with a biolayer and an internal reference layer; a sensor chip having a fixed and isolated target antigen may be contacted with a solution having a predetermined concentration of an antibody or antigen-binding fragment. When the antibody or antigen-binding fragment in the solution binds to the fixed target antigen on the sensor chip, the optical thickness (number of binding molecules) of the biolayer of the sensor chip changes, causing a wavelength shift in the interference pattern of the reflected light, which is measured in real time. The measured wavelength shift is a direct measurement of the optical thickness (expressed in nm) of the biolayer of the sensor chip and reflects the degree of binding of the antibody or antigen-binding fragment to the (fixed) antigen. BLI can be used, for example, to confirm that an antibody is properly masked; the binding of a masked antibody or antigen-binding fragment to its fixed target antigen can be compared to the binding of an unmasked or de-masked antibody or antigen-binding fragment to obtain an initial indication of the effectiveness of the mask. In such screening, comparing the signals of a naked (unmasked) antibody and a masked antibody revealed a significant signal shift for all masked antibodies according to the present invention tested against a variety of target antigens.

[0062] The binding of an antibody or antigen-binding fragment to a target cell that (over)expresses the target antigen recognized by the antibody or antigen-binding fragment can be measured in vitro in a flow cytometer. Target cells expressing the target antigen may be contacted with different concentrations of an (masked) antibody, antigen-binding fragment or ADC according to the present invention and may be stained with a secondary fluorescently labeled antibody that binds to the first antibody or antigen-binding fragment. The readout results in the median fluorescence intensity (MFI), which is also a measure of the binding of the antibody or antigen-binding fragment to its target antigen expressed on the target cell. The MFI is plotted against concentrations up to and beyond the concentration at which maximum binding (saturation) of different antibodies or antigen-binding fragments occurs to obtain a dose-response curve (DRC). From the dose-response curve, the EC50 The value (the concentration of the antibody or antigen-binding fragment that gives rise to a half-maximal response) can be estimated. EC 50 shift (which may be expressed as the ratio between the EC 50 of the masked and naked (unmasked) antibody or antigen-binding fragment) is a measure of the reduction in target binding due to masking, as long as a complete dose-response curve can be obtained for the masked antibody or antigen-binding fragment. By comparing the EC 50 of the unmasked antibody or antigen-binding fragment (the antibody or antigen-binding fragment from which the mask has been removed) to the EC 50 of the naked (unmasked) antibody or antigen-binding fragment, the degree of recovery of binding after unmasking can be determined. Usually, the EC 50 value is determined by curve fitting with software such as GraphPad Prism (www.graphpad.com). The ratio between the two EC 50 values can also be determined using the same software tool (EC 50 shift or potency shift).

[0063] Masked antibodies or antigen-binding fragments bind very little to the target (which already demonstrates that they are effectively masked). As a result, even at relatively high concentrations, saturation binding may not necessarily be reached in binding experiments, which will complicate the EC 50 determination.

[0064] An alternative to EC 50 determination is to compare the concentration at which minimal binding begins to occur. In the case of the masked antibodies according to the invention, the concentration at which minimal binding can be observed was significantly higher than that of the unmasked or unmasked antibodies.

[0065] For example, with respect to the masked antibodies according to the present invention (e.g., based on trastuzumab as shown in the examples), when tested by flow cytometry for binding in antigen - positive cells, minimal binding was observed only at very high concentrations exceeding 3 μg / mL, and often at 10 - 30 μg / mL or higher, whereas the minimal binding of the naked or unmasked antibody was already observed at 0.04 μg / mL.

[0066] Binding is preferably reduced by at least 10 - fold, preferably 50 - 100 - fold, or even 100 - 500 - fold or greater than 500 - fold. As a result of the reduced binding of the masked antibody to its target antigen expressed on the target cells, cytotoxicity is preferably reduced by a similar factor.

[0067] The reduction in cytotoxicity of the masked antibodies, antigen - binding fragments or ADCs according to the present invention against target cells compared to naked or unmasked antibodies, antigen - binding fragments or ADCs can also be determined. When various masked ADCs according to the present invention were tested for their cytotoxicity compared to an ADC based on a naked antibody, a very significant IC 50 shift was observed. The IC 50 shift of the masked ADC exceeded 30, and a 500 - fold shift was also observed compared to the IC 50 of the same ADC based on the naked antibody. These findings represent a very efficient masking of the binding region of the masked antibodies according to the present invention.

[0068] Also importantly, after demasking (i.e., removal of the mask), binding to the target and the resulting cytotoxicity recover to a level sufficient for the demasked antibody or antigen-binding fragment to exhibit the desired therapeutic effect. Preferably, the demasked antibody or antigen-binding fragment binds to the target antigen as efficiently as the unmasked antibody or antigen-binding fragment. Thus, if the unmasked antibody or antigen-binding fragment binds to the target with a high nanomolar affinity, preferably the demasked antibody or antigen-binding fragment binds similarly. Preferably, the demasked antibody binds to the target antigen in exactly the same way as the corresponding naked antibody. A certain degree of reduction in the binding of the demasked antibody compared to the naked antibody is acceptable. Preferably, the order of binding of the demasked antibody is, for example, as an EC50 shift, 1-2, more preferably 1-1.5, and of course, closest to 1 is most preferred (meaning that the dose-response curves of the demasked antibody and the naked antibody substantially overlap). In the antibodies according to the present invention, it has been found that binding almost completely recovers after demasking when compared to the binding of the naked antibody. The EC 50 shift between the unmasked antibody and the demasked antibody is often 1-2, indicating that binding recovered after demasking. The same is true for the cytotoxicity of the demasked antibody; almost complete recovery of cytotoxicity induction was observed after demasking compared to the naked antibody.

[0069] In the masked antibodies or antigen-binding fragments according to the present invention, the mask contains a pair of peptide masking moieties having specific binding affinity for each other, one masking moiety of the pair contains an antigen peptide, and the other masking moiety contains a single domain antibody (sdAb) that specifically binds to the antigen peptide. The masking moiety is linked to the N-terminus of at least one HC and LC pair of the masked antibody or antigen-binding fragment. The masked antibodies or antigen-binding fragments according to the present invention are effectively masked and, when they reach their target site, they are effectively demasked.

[0070] Masking moiety The masking moiety is the actual (peptide) moiety that (reversibly) binds to an antibody or antigen-binding fragment thereof and can form a mask when complexed with the antibody or antigen-binding fragment. The masked antibody or antigen-binding fragment thereof according to the present invention is provided with a mask containing a set of two peptide masking moieties linked to the N-termini of the heavy-chain variable region (HCVR) and the light-chain variable region (LCVR) that together form the antigen-binding site. One of the two masking moieties of the pair of masking moieties contains an antigen peptide, while the other contains a single-domain antibody (sdAb) that specifically binds to the antigen peptide. At least one of the masking moieties is linked to the N-terminus of the HCVR and / or LCVR via a cleavable linker. By binding to each other, the masking moieties together form a mask that protects the antigen-binding site of the masked antibody or antigen-binding fragment. When the antigen-binding site of the antibody or antigen-binding fragment is protected by the mask, the ability of the masked antibody or antigen-binding fragment to bind to the target antigen is significantly reduced. The antibody or antigen-binding fragment can be demasked by cleavage of at least one cleavable linker. The masking of the antibody or antigen-binding fragment is thus reversible; by cleavage of at least one cleavable linker, the masking effect can be removed. Thus, demasking does not require cleavage of the bond between the sdAb and the antigen peptide that together form the mask (the masking moieties do not have to remain bound to each other when at least one linker is cleaved).

[0071] The antigen peptide used in the antibody masking moiety of the present invention may be a suitable antigen peptide as part of the sdAb / peptide pair. The peptide is selected to contain an epitope sequence (preferably a single epitope sequence) that is specifically recognized by the sdAb of the other masking moiety of the pair of masking moieties that together form the mask.

[0072] Preferably, the selected antigen peptide sequence used in the masking portion is relatively short (such as the peptide tags used in tag - ging techniques based on sdAbs known in the art) so as not to interfere with antibody production and to enable efficient masking and de - masking. Usually, the length of such an antigen peptide sequence is in the order of 4 - 20, preferably 8 - 15 amino acids. More preferably, the antigen peptide sequence has 12 - 15 amino acid residues. Preferably, such a peptide is derived from human proteins to minimize the chance of immunogenicity of the antigen peptide.

[0073] Single - domain antibodies (sdAbs) are (recombinant) antigen - binding fragments with a unique structure composed of a single heavy chain having only one variable domain. The first sdAbs developed were V H H fragments (variable domain) fragments derived from the heavy - chain antibodies of camelids (e.g., llama, camel, dromedary, Bactrian camel, alpaca, vicuña, and guanaco). (S. Muyldermans et al., 2009, Veterinary Immunology and Immunopathology 128, 178 - 183). The circulating heavy - chain antibodies of camelids contain only heavy chains and are naturally lacking in light chains. SdAbs derived from these camelid heavy - chain - only antibodies are generally referred to as "nanobodies" or "V H"H" (Muyldermans S., 2013, Annual 82:775-97. doi: 10.1146 / annurev-biochem-063011-092449). The term "nanobody" was initially adopted by the Belgian company Ablynx because these sdAbs have a nanomolar size ("nanobody" is a registered trademark of Ablynx N.V.). The term nanobody is often used as an equivalent of sdAb and also includes sdAbs from other species. In addition to camels, sdAbs have also been found in cartilaginous fish (e.g., sharks). Sharks and other cartilaginous fish also produce heavy-chain antibodies. These antibodies are called Ig new antigen receptors or IgNAR. IgNAR is composed of two identical heavy chains. The variable domains in these heavy chains are called V-NAR domains. IgNAR also has a single variable domain. The V-NAR domain contains two CDRs, whereas the V H H domain of camelid sdAbs contains three CDRs (Cheong et al., 2020, International journal of biological macromolecules, 147, 369-375). SdAbs can also be obtained from common (human) IgG antibodies (Chen et al., 2009, Methods Mol Biol., 525: 81-99).

[0074] The sdAbs used in the masking moiety according to the present invention may be humanized by methods known in the art (Vincke et al., 2009, The Journal of Biological Chemistry Vol. 284, No. 5, 3273-3284). The SdAbs used in the masking moiety in the masked antibody or antigen-binding fragment according to the present invention are preferably nanobodies or their humanized versions and / or variants.

[0075] SdAbs can be selected and produced in a variety of ways known in the art. Methods for selecting nanobodies are reviewed in S. Muyldermans, 2021, The FEBS Journal, 288, 2084-2102; Hassanzadeh-Ghassabeh et al., 2013, Nanomedicine (Lond), 8(6):1013-26.

[0076] SdAbs are small (12-15 kD) compared to a typical antibody (150-160 kD) composed of two HC / LC pairs. Also, SdAbs are smaller than the antigen-binding fragment of a typical antibody, such as a Fab fragment, which consists of one (half) HC and LC, and has a molecular weight of approximately 50 kD. SdAbs are the smallest antibody fragments that still selectively bind to a specific target antigen.

[0077] SdAbs are used for a variety of purposes, e.g., in therapy (such as the targeting moiety of a cytotoxic drug), but also in diagnostic and imaging applications where the nanobody is conjugated to a tracer (de Meyer et al., 2014, Trends in Biotechnology, Vol. 32, No. 5, 263-270; Bao et al., 2021, EJNMMI Res 11:6, https: / / doi.org / 10.1186 / s13550-021-00750-5). For example, sdAbs against certain peptide tags have been developed for use in the immunoprecipitation of tagged proteins. Such tags include tags developed by, e.g., ChromoTek, Nanotag Biotechnologies, and other companies. Peptide-tag-specific antibodies are also described in Ren et al., 2020, Journal of Chromatography A, 1624, 461227.

[0078] In a preferred embodiment of the present invention, a single polypeptide chain of a variable domain containing the complete antigen-binding ability of a nanobody preferably has an amino acid sequence and structure that can be considered to be composed of four framework regions or "FRs", which are respectively referred to as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4" in the art and in this specification; the framework regions are interrupted by three complementarity-determining regions or "CDRs", which are respectively referred to as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3" in the art. These framework regions and complementarity-determining regions are preferably operably linked in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxy terminus).

[0079] Examples of peptide tags for which sdAbs have been developed include human β-catenin-derived peptides such as the BC2T peptide PDRKAAVSHWQQ (SEQ ID NO: 1) representing a conserved epitope in human β-catenin and nanobodies thereto described in Traenkle et al., 2015, Molecular & Cellular Proteomics, 14(3), 707-723 and European Patent Application Publication No. 3377632. This peptide sequence forms the basis of Spot-Nanobodies that specifically bind to the Spot-Tag® peptide tag, an inactive 12-amino acid peptide tag having the amino acid sequence PDRVRAVSHWSS (SEQ ID NO: 44), and the Spot-tag protein provided by ChromoTek.

[0080] Single-domain antibodies (SdAbs) that recognize peptide tags of just 4 or 5 amino acids (AAs) used as affinity tags are commercially available (e.g., the 4AA peptide tag, the CaptureSelect® C-tag matrix based on E-P-E-A, and the corresponding CaptureSelect® biotin anti-C-tag complex consisting of a 13 kDa camelid antibody fragment (affinity ligand) with high affinity and selectivity for the 4 amino acid “C-tag” peptide tag E-P-E-A (SEQ ID NO: 45), provided by ThermoFisher). Another tag sequence and corresponding sdAb is the “ALFA-tag” described in Goetzke et al., 2019, Nature Communications, 10:4403, https: / / doi.org / 10.1038 / s41467-019-12301-7. The antigen peptide amino acid sequence of the minimal ALFA-tag is SRLEEELRRRLTE (SEQ ID NO: 46). This sequence can form a stable α-helix. NbALFA, an sdAb specific for the ALFA-tag, has been developed, which binds to the peptide with low picomolar affinity. The ALFA-tag and its variants, as well as the sdAb, are also described in International Publication No. WO 2020 / 053239 (Nanotag Biotechnologies).

[0081] Nanobodies generally have low immunogenicity due to their small size and high sequence identity with human IGHV3 family gene products, which means that they rarely generate anti-drug antibodies (ADAs) (Ackaert C et al., 2021, Front. Immunol. 12:632687, doi: 10.3389 / fimmu.2021.632687; Ren et al. (supra); Jovcevska et al., 2020, BioDrugs, 34,11-26; Rossotti M et al., 2021, FEBS J., doi:10.1111 / febs.15809).

[0082] SdAbs are known to have a relatively high affinity for their target antigen peptides (from low nanomolar to picomolar concentrations), which is favorable in many known applications where binding partners must bind to each other even at very low concentrations (low K D ). In the present invention, nanobodies with an affinity of K D below nM (picomolar concentrations) can be used. The K D of such antibodies is typically in the low picomolar range (10 - 30 pM). The K D value for the binding of NbALFA to its ALFA tag is, for example, approximately 26 pM. The affinity of BC2 Nb for its BC2T peptide tag is 1.4 nM (1400 pM).

[0083] The use of high - affinity sdAb / antigen peptide pairs results in efficient masking and demasking of the masked antibodies or antigen - binding fragments according to the present invention. In the masked antibodies or antigen - binding fragments according to the present invention, the masking moieties are in proximity to each other as they are each linked to the N - termini of the light - chain and heavy - chain pairs within the (masked) antibody. Due to this proximity, the masking moieties of the mask may bind to each other even if they have a relatively low affinity for each other. The high affinity between the nanobody and the peptide tag is favorable for other applications, but may not be essential when using them for the masking moieties of the masked antibodies, antigen - binding fragments or ADCs according to the present invention.

[0084] In the context of the present invention, it may be advantageous to use sdAbs that have a relatively low affinity for the specifically recognized antigen peptide sequence. Using sdAbs with a relatively low affinity for the antigen peptide has the advantage that the ability of the masking moieties to form intermolecular bonds (i.e., bonds between the masking moieties on the HC / LC of different antibodies or antigen - binding fragments that may result in HMW complex formation) may be reduced. The percentage of HMW - forming antibodies relative to the total amount of antibody produced (HMW (%)) is preferably less than 10%.

[0085] At the same time, the ability to form intramolecular bonds (i.e., the bond between masking moieties in a heavy and light chain pair within an antibody or antigen-binding fragment for forming a mask) is retained with lower affinity due to the proximity of the two masking moieties when complexed with the HC and LC pair.

[0086] Relatively low affinity means an affinity significantly lower (e.g., 2 - 100 and at least 2 - 10 times) than the high (single) nanomolar concentrations typical for sdAb epitope binding, down to picomolar concentrations. The K of the bond between the Nb / peptide pairs used for the masking moieties of the present invention D can thus be from 10 pM (high affinity) to relatively low affinity, and K D can be as high as 500 nM (0.01 nM - 500 nM).

[0087] Preferably, select sdAbs and antigen peptides with a K value of the affinity between the sdAb and the antigen peptide in the range of 0.1 - 500 nM, more preferably 1 - 100 nM. D

[0088] In a panel of sdAbs raised against a particular epitope sequence, the sdAbs can be selected for lower affinity for the antigen peptide. Alternatively, a particular sdAb initially selected for its high affinity for the antigen peptide can be modified, for example, by introducing a specific point mutation in the binding region of the sdAb to create a variant sdAb with a slightly altered amino acid sequence, thereby reducing its affinity for the peptide. Similarly, the antigen peptide sequence recognized with high affinity by the sdAb can be altered to reduce the binding to the same sdAb.

[0089] Good results were obtained as described by Traenkle et al., 2015, Molecular & Cellular Proteomics, 14(3), 707-723 with a masking moiety based on the β-catenin-based peptide (BC2T) and the nanobody (BC2) against it. The BC2T peptide described by Traenkle et al. has the amino acid sequence PDRKAAVSHWQQ (SEQ ID NO: 1) and represents a conserved linear epitope corresponding to amino acid residues 16 - 27 of β-catenin, although further variants that preserve the epitope are described in European Patent Application Publication No. 3377632, which discloses peptides having the following sequences: X 1 X 2 RX 4 X 5 AX 7 SX 9 WX 11 X 12 (wherein X 1 may be P or A, X 2 may be D or a conservative substitution of D, X 4 may be K or a conservative substitution of K or S, X 5 may be A or R or a conservative substitution of A or R, X 7 may be V or a conservative substitution of V, X 9 may be H or a conservative substitution of H, X 11 and X 12 may independently of each other be Q or a conservative substitution of Q).

[0090] As used herein, the term "conservative substitution" means the substitution of one amino acid for another amino acid such that the substitution results in a silent change. This means that an amino acid residue can be substituted with another amino acid of similar polarity that functions equivalently. The substituent of the amino acid may be selected from another member of the class to which the amino acid belongs (i.e., a conservative substitution). For example, one polar amino acid can be substituted for another polar amino acid, or one positively or negatively charged amino acid can be substituted for another positively or negatively charged amino acid, etc. Amino acid classes include, for example, nonpolar (hydrophobic) amino acids including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids including arginine, lysine, and histidine; and negatively charged (acidic) amino acids including aspartic acid and glutamic acid.

[0091] Certain variant peptides that display amino acids fixed at positions 3(R), 6(A), 8(S), and 10(W) and that are thought to be essential for epitope binding, such as PVRSAALSQWSS (SEQ ID NO: 48), PDRVRAVSHWSS (SEQ ID NO: 49), and ADRVRAVSHWSS (SEQ ID NO: 50), are also disclosed in European Patent No. 3377632.

[0092] Traenkle et al. also describe nanobodies that specifically bind to the BC2T peptide defined by its CDR3 sequence. Of its most performant nanobodies (BC1, BC2, BC6, BC9, and BC13), BC1 and BC2 bind to recombinant β-catenin protein at low nanomolar concentrations (about 1.9 nM and about 3.1 nM) of K D as measured by surface plasmon resonance spectroscopy. BC13 showed only a slightly lower affinity of about 44 nM. For BC6 and BC9, the detected affinities were in the low micromolar range. The CDR3 sequence of the BC2 nanobody is ARGCKRGRYEYDFW (SEQ ID NO: 5).

[0093] The BC2T peptide and its variants, as well as BC2-Nb (and its variants) that specifically bind to the BC2T or variant peptide sequence, can be used in the masking portion to produce the masked antibodies, antigen-binding fragments and / or ADCs according to the present invention.

[0094] On the other hand, in the prior art use of peptide tags and sdAb sequences, pairs were selected for their high-affinity binding, but a very high affinity may not be necessary for use in the masking portion of the masked antibodies, antigen-binding fragments or ADCs according to the present invention. By using antigen peptide / sdAb pairs having a relatively low affinity for each other (1 - 100 nM), it has been found that good results were obtained. Variants of the BC2 nanobody are described in Braun et al., 2016, Sci Rep 6, 19211, https: / / doi.org / 10.1038 / srep19211. Braun et al. mutated the CDR3 region of the BC2 nanobody by exchanging Arg106 with serine (BC2-Nb R106S or the "RS" variant, (SEQ ID NO: 8)) or glutamate (BC2-Nb R106E or the "RE" variant, (SEQ ID NO: 7)). In so doing, the nanobody-peptide three-dimensional structure ("headlock") of the BC2 nanobody was disrupted, resulting in a 10-fold lower binding affinity when measured using surface plasmon resonance spectroscopy (SPR)-based affinity measurements compared to the high-affinity BC2-Nb (K D , approximately 1.4 nM) (K D value, approximately 11 nM; the RE variant showed a K D of 12 nM and the RS variant showed a K D of 9.7 nM). The K D of the RA variant is described in Ren et al., Journal of Chromatography A 1676 (2022) 463274. Ren et al. provided the same 1.4 nM K D for the wild-type BC2-Nb as Braun et al., and compared it to the R106Q variant (K D = 282 nM), the R106A variant (K D= 360.5 nM) and the R106H variant (K D = 335.5 nM) are compared.

[0095] The original BC2-Nb and the RS and RE variants, as well as the variant in which arginine was substituted with alanine (BC2-Nb R106A or the "RA" variant (SEQ ID NO: 6)) were tested in the masking portion of the masked antibody according to the present invention. When the variant Nb was used, for example, as the masking portion of the anti-HER2 antibody trastuzumab, the proportion of the HMW complex was significantly reduced (reduced 5 to 10 times), but the ability of the masking portion to protect the binding site of the masked antibody until at least one of the linkers was cleaved was retained.

[0096] Instead of or in addition to modifying the sdAb sequence to adjust the affinity of the high-affinity peptide / sdAb pair, the sequence of the antigen peptide can be modified for the purpose of reducing the affinity of the peptide-sdAb interaction. For example, in the case of the BC2T peptide, serine at position 8, which is fixed by the formula of the peptide sequence in European Patent No. 3377632, may be substituted with, for example, an alanine or threonine residue in the peptide used in the masking portion of the present invention. Substituting S(8) with X 8 results in this changing the general formula of the peptide to X 1 X 2 RX 4 X 5 AX 7 X 8 X 9 WX 11 X 12 (SEQ ID NO: 51) (wherein X 1 may be P or A, and X 2 may be D or a conservative substitution of D, and X 4 may be K or a conservative substitution of K or S, and X 5 may be A or R or a conservative substitution of A or R, and X 7 may be V or a conservative substitution of V, and X 8 may be S, A, V or T, and X 9 may be H or a conservative substitution of H11 and X 12 is changed to (which may also be a conservative substitution of Q or Q). In the variant formula disclosed in European Patent No. 3377632, the fixed "R" at the 3rd position can be further substituted with "K", and the following formula: X 1 X 2 X 3 X 4 X 5 AX 7 X 8 X 9 WX 11 X 12 (SEQ ID NO: 95) (wherein X 1 , X 2 , X 4 , X 5 , X 7 , X 8 , X 9 , X 11 and X 12 has the above meaning, and X 3 may be R or K), resulting in

[0097] In the preferred peptide according to the present invention, X 8 is A or T, most preferably A, for example, resulting in a peptide having the sequence PDRKAAVAHWQQ (BC2T SA variant, SEQ ID NO: 2) or PDRKAAVTHWQQ (BC2T ST variant, SEQ ID NO: 3), preferably PDRKAAVAHWQQ.

[0098] Variant peptides having a substitution of S at the 8th position to A or T at the 8th position, such as BC2T ST variant or BC2T SA variant, can be used in pairs of masking moieties including BC2-Nb or preferably masking moieties including variants of the BC2-Nb moiety having mutations in the CDR3 region, such as BC2 RE, RS or RA variants. Good results were obtained with a pair of masking moieties containing a masking moiety having a BC2T SA variant peptide and a masking moiety having a BC2-Nb RS variant nanobody.

[0099] Similarly, regarding other nanobodies, ALFA-tags and sequence variants with different affinities from the described sdAbs are described in WO 2020 / 053239. Peptides based on different variant core structures had a K D of about 1 - 5 nM for binding to sdAbs or, if the core structure was different, a K D of about 10 - 50 nM for binding to the same sdAb. An example of a modified ALFA-tag Nb is the Nb PE variant disclosed as SEQ ID NO: 134 in WO 2020 / 053239. Goetzke et al., 2019, Nature Communications, 10:4403, https: / / doi.org / 10.1038 / s41467-019-12301-7 describes that this variant was designed for protein elution. When the original ALFA-tag Nb binds to the tag peptide with a high picomolar affinity (K d is 26 pM), the Nb PE variant ("PE" for peptide elution) binds with a low affinity (K d is 11 nM) as shown in Fig. 6 of the supplement by Goetzke et al. Using the Nb PE variant and the ALFA-tag peptide in the masking portion of an antibody conjugated by a protease-cleavable linker would enable efficient masking and de-masking of the antibody.

[0100] Instead of, or in addition to, using a low-affinity sdAb / antigen peptide pair as a mask to prevent HMW complex formation, prior art methods for removing or reducing the formation of HMW complexes in protein production, purification, and storage can be used as well.

[0101] Linker At least one of the masking moieties (either the antigen peptide or the sdAb or both) may be linked to the antibody by a cleavable linker, preferably a peptide linker that is conditionally cleavable and cleaved only at a target site (a site where the antibody must bind to the target antigen and the mask must be removed, such as within or at the tumor site). Preferably, the cleavable peptide linker is a peptide linker cleavable by a protease. Preferably, both masking moieties are linked to the antibody by a cleavable linker.

[0102] The binding ability of the antibody or antigen-binding fragment may be such that the antibody is already sufficiently restored to have its desired therapeutic effect when only one linker is cleaved. Preferably, all linkers that bind the masking moiety to the antibody or antigen-binding fragment are cleaved and the mask is completely released from the antibody after cleavage.

[0103] Peptide linkers used to join different parts of the fusion protein are known in the art. Such linkers may be flexible linkers, rigid linkers, cleavable linkers or combinations thereof (Chen et al., 2013, Adv Drug Deliv Rev., 65(10), 1357-1369). The cleavable linker used in the masked antibody or antibody-binding fragment according to the invention is preferably a peptide linker containing one or more cleavage sites recognized by one or more proteases.

[0104] When using a masked antibody in cancer treatment, the cleavable peptide linker must preferably contain at least one cleavage site recognized by at least one tumor-specific protease. Examples of such proteases known in the art are matriptase (MT-SP1, tumor-associated type II transmembrane serine protease), cathepsin B, urokinase-type plasminogen activator (uPA), which are overexpressed in almost all types of cancer, and / or matrix metalloproteinases (MMP) such as MMP2, MMP9 and MMP14 (Li Yanan et al., 2021, Acta Pharmaceutica Sinica B, 11(8), 2220-2242). Linkers containing tumor-specific protease cleavage sites are also known in the art and, for example, in the context of antibody masking techniques in International Publication No. WO 2018 / 107125 (Seattle Genetics), International Publication No. WO 2009 / 025846, International Publication No. WO 2010 / 081173 and International Publication No. WO 2016 / 118629 (Cytomx Therapeutics LLC) and Choi et al., 2012, Theranostics, 2(2), 156-178.

[0105] The cleavage site is the (minimal) amino acid sequence recognized by a protease, and the actual cleavage occurs within this site. Examples of cleavage sites known in the art are the MMP cleavage site PLGLAG (SEQ ID NO: 13) cleavable by MMP2 and MMP9 (Jiang et al., 2004, PNAS, 101(51), 17867-17872; Trang et al., 2019, Nature biotechnology, 37(7), 761-765.). Other known cleavage sites are, for example, the cleavage site LSGRSDNH (SEQ ID NO: 14) recognized by urokinase-type plasminogen activator (uPA) (European Patent No. 2385955, Cytomx Therapeutics LLC) and the cleavage site of MMP14 having the sequence ISSGLL (SEQ ID NO: 15) (International Publication No. 2016 / 118629, Cytomx Therapeutics LLC). Other cleavage sites such as RQARVVNG (SEQ ID NO: 53) and PMAKK (SEQ ID NO: 54) recognized by matriptase are described in International Publication No. 2017 / 162587 (Hoffmann-La Roche AG). Furthermore, other cleavage sites, PLGVR (SEQ ID NO: 70) and IPVSLR (SEQ ID NO: 52) or IPVSLRSG (SEQ ID NO: 75), are MMP2 cleavable sites described, for example, in International Publication No. 2018 / 107125 (Seattle Genetics Inc.) and Lin et al. (supra). Preferred cleavage sites for use in the masked antibodies or antigen-binding fragments according to the present invention are PLGLAG, IPVSLR(SG), LSGRSDNH and / or PMAKK.

[0106] Preferably, the two masking portions are linked by a linker comprising at least one protease cleavage site. A linker cleaved by multiple proteases may be used to increase activation at a target site, such as a tumor, where at least one masking portion must be cleaved to restore binding of the unmasked antibody. Such a linker may contain cleavage sites recognized by multiple proteases and / or different cleavage sites each recognized by a different protease. Examples of cleavable linker sequences containing multiple cleavage sites recognized by different proteases are described, for example, as "2001" in WO 2016 / 118629 (Cytomx Therapeutics LLC). The sequence "2001" has the AA sequence ISSGLLSGRSDNH (SEQ ID NO: 16) and combines the ISSGLL cleavage site of MMP14 with the LSGRSDNH cleavage site recognized by matriptase and uPA. The cleavage sites can be combined in various orders (WO 2016 / 118629).

[0107] The linkers of both members of the pair of masking portions forming the mask may contain the same (identical) or different (non-identical) cleavage sites. Preferred combinations of linkers used to complex a pair of masking portions forming a mask to an HC / LC pair of an antibody or antigen-binding fragment are combinations of linkers where both linkers contain cleavage sites selected from the group consisting of PLGLAG, LSGRSDNH, IPVSLR(SG), and PMAKK.

[0108] Preferred linker combinations include linkers that complex a masking portion having an SdAb to an antibody, preferably an LC VR, and contain a cleavage site recognized by matriptase, with linkers that complex a masking portion having an antigen peptide to an antibody or antigen-binding fragment, preferably an HC VR of the same HC / LC binding pair, and contain a cleavage site recognized by a metalloprotease, such as MMP2. The masking portion complexed to the LCVR of at least one HC / LC chain pair preferably contains a BC2-Nb RS variant nanobody and is complexed to the LCVR by a linker containing a cleavage site recognized by a metalloprotease, preferably PLGLAG, while the masking portion complexed to the HCVR of the HC / LC chain pair contains a BC2T SA variant peptide and is complexed to the HC by a linker containing a cleavage site recognized by matriptase, preferably LSGRSDNH. Alternatively, the cleavage site recognized by the metalloprotease may be ISSGLL or IPVSLR(SG), and the alternative to the matriptase cleavage site may be PMAKK.

[0109] In addition to the actual protease cleavage site, the linker used to link the masking portion to the HC and / or LC of the masked antibody or antigen-binding fragment according to the invention may contain additional spacer sequences, such as serine and / or glycine-rich stretches, mainly to increase the flexibility of the linker, for example to provide sufficient movement for the masking portions to assume an appropriate orientation for binding to each other and to optimize the degree of protection of the actual mask. The linker may be optimized for use with a particular antibody by varying the spacer sequence (e.g., length) and the position of the cleavage site within the linker.

[0110] Commonly used flexible linkers are, for example, -GGS- or -(GGGGS) nA linker comprising a glycine-rich region such as - (Chen et al. (supra)). In the linker used in the masked antibody according to the present invention, such a glycine ( / serine) -rich spacer sequence may be linked to the actual protease cleavage site at one or both ends of the cleavage site sequence in the cleavable linker. Individual cleavage recognition sequences can be directly linked or separated (and / or adjacent) by a G / S-rich sequence, and individual cleavage sites can be arranged in different orders (e.g., ISSGLL SSGGSGGS LSGRSDNH (SEQ ID NO: 17) or LSGRSDNH GGSGGS ISSGL LSS (SEQ ID NO: 18), described in WO 2016 / 118629).

[0111] Thus, a preferred linker comprises one or more proteolytic cleavage sites adjacent to one or more spacer sequences. Most preferred is a linker in which there is no or a very short adjacent sequence between the N-terminus of the HCVR or LCVR of the antibody or antigen-binding fragment sequence and the (first) cleavage site. Depending on where the linker is cleaved, of course, a portion of the linker sequence may remain bound to the N-terminus of the HC and / or LC of the antibody de-masked after cleavage of the linker. When the cleavage site is close to the N-terminus of the HC and / or LC sequence, there are no or very few amino acids remaining bound to the HC and / or LC after cleavage of the linker. When a spacer sequence is present between the N-terminus of the HC and / or LC and the cleavage site, such a sequence is preferably short, preferably having 0 to 10 or 1 to 5 amino acids, such as the amino acid sequence -GGS-, for example 3 amino acids. An additional spacer sequence, such as a -GGS- or -GGGGS- spacer sequence, may be included at the N'-terminus of the cleavage site sequence. The resulting linker has, for example, the sequence -GGGGS with the MMP cleavage site underlined PLGLAGIt may have GGS-(SEQ ID NO: 11). The resulting linker may be a linker of a suitable length depending on the length and number of cleavage recognition sites used, and the length of the flexible linker present before and / or after the cleavage site sequence. Suitable linkers may generally contain 2 to 30, preferably 5 to 25 amino acids.

[0112] Antibodies or antigen-binding fragments have the same overall three-dimensional structure, but each individual antibody or antigen-binding fragment has a different antigen-binding region. The antigen-binding regions may have different sequences and thus may have different charge distributions and 3D structures. The present invention provides an elegant and uniform method for masking antibodies or antigen-binding fragments that can in principle be used for any antibody or antigen-binding fragment with the same masking moiety to obtain good results, and by adjusting the length of the linker, for example, it may result in a higher yield during production. For that effect, a set of linkers with different lengths can be prepared and tested in different combinations as pairs for linking the masking moiety to the LC and HC. When testing different combinations, the linker set with the best performance may be selected. "The best performance" can be defined based on various measured parameters. For example, a linker pair can be selected that has a high expression level compared to other linkers tested, a very low HMW (%) during production, but still retains the masking and de-masking efficiency.

[0113] For example, a linker can be selected from the set shown in Table 18 (as tested in Example 10 for one specific antibody, tisotumab). In the set shown in Table 18, all of the linkers L14 - L25 contain the protease cleavage site LSGRSDNH but differ in length, and all of the linkers L26, L27, L9, L29, L7, and L30 contain the PLGLAG protease cleavage site but also differ in length. This set of linkers encompasses 324 possible combinations, having the same or different cleavage sites with respect to both masking portions, and enables testing of combinations of linkers that may have the same or different linker lengths. In Example 10, where all 324 combinations of linkers were tested as an example, it was illustrated that the majority could be produced at expression levels exceeding 100 μg / mL. HMW(%) was less than 10% in most cases, but for 198 combinations of linkers, HMW(%) was less than 5%, and for 39 combinations, it was even less than 3%.

[0114] A preferred subset of the combinations of linkers for making these 324 antibodies is the subset of combinations of linkers used to make ADC1 - 12 shown in Table 22 of Example 11.

[0115] This operation may be repeated for all or a subset of the possible combinations that could be selected from Table 18 for the antibody or antigen - binding fragment to be masked. The present invention encompasses a masked antibody or its antigen - binding fragment that complexes a masking moiety with at least one HC / LC pair of the antibody or antigen - binding fragment using a suitable combination of linkers selected from the linkers shown in Table 18.

[0116] Spacer sequences such as QGQSGQG(Qg) (SEQ ID NO: 9) or QVQLVES(Qv) (SEQ ID NO: 10) can also be included in the expressed protein sequence, for example, at the N - terminus of the antigen peptide of the masking moiety (the linker binds to the C - terminus of the peptide).

[0117] When the peptide binds to the HC in the order of N-C, the construct may thus have the following three-dimensional structure: Qg - antigen peptide - linker - HC.

[0118] The Qg spacer sequence is known in the art (e.g., Cytomx International Publication No. WO 2016 / 179285).

[0119] The Qv sequence is based on a conserved sequence found in the framework 1 region of the heavy chain variable domain (VH) sequence in many human antibodies. By introducing a spacer sequence such as the Qg or Qv sequence in front (N-terminus) of the amino acid sequence of the antigen peptide, the production titer of the masked antibody according to the present invention can be increased up to 10-fold, regardless of whether the masking portion having the antigen peptide is linked to the N-terminus of the HC or LC. Good results were obtained with a masked antibody in which the Qg or Qv sequence was placed in front of the BC2T peptide sequence (variant).

[0120] In the masked antibody according to the present invention, at least one of the two masking portions may be linked to the antibody or antigen-binding fragment by a cleavable linker. If the binding ability of the masked antibody or antigen-binding fragment can already be sufficiently restored by cleaving the bond between one masking portion and the antibody or antigen-binding fragment, it may be sufficient to link only one masking portion by a cleavable linker. When only one masking portion is linked to the HC or LC of the antibody or antigen-binding fragment by a cleavable linker and this linker is cleaved, the mask is removed from the antigen-binding site of the antibody or antigen-binding fragment (its binding site is "demasked"), despite the fact that the masking portion may still be bound to the antibody or antigen-binding fragment. When only one masking portion is linked by a cleavable linker, this masking portion is preferably a masking portion containing an sdAb.

[0121] Due to the small size of the mask used with the present invention, the actual binding site of the antibody or antigen-binding fragment is available for binding to its target antigen, and the masking portion does not prevent the antibody or antigen-binding fragment from binding to the target even when only one linker is cleaved. When one of the masking portions, i.e., the sdAb or antigen peptide, is linked by a cleavable (peptide) linker, the other masking portion may also be linked by a peptide linker or, alternatively, may complex with the antibody or antigen-binding fragment. For example, a fusion protein containing an antibody or antigen-binding fragment fused to an sdAb that can complex an antigen peptide acting as a second masking portion can be produced. Alternatively, both the sdAb and the antigen peptide are included in a recombinantly produced (fusion) protein sequence and either one or both are linked via a (cleavable) peptide linker that is part of the expressed fusion protein.

[0122] In the masked antibody or antigen-binding fragment according to the present invention, the antigen peptide and the sdAb may be linked to at least one N-terminus of the light and heavy chain pairs of the antibody or antigen-binding fragment. In a bivalent antibody or antibody fragment (e.g., F(ab’) 2 fragment) having two HC / LC pairs, preferably all (both) HC / LC pairs are provided with a mask.

[0123] In the masked antibody or antigen-binding fragment according to the present invention, the masking portion containing the antigen peptide may be connected to the N-terminus of the LC, the sdAb may be connected to the N-terminus of the HC, or vice versa.

[0124] From a manufacturing perspective, it may be advantageous and preferred to use a masked antibody that connects the masking portion having the sdAb to the N-terminus of the LC and the antigen peptide to the N-terminus of the HC. In the experiments conducted, these antibodies were produced with higher titers (and lower HMW (%)).

[0125] The masked antibody or antigen-binding fragment according to the present invention is particularly suitable for use in antibody-drug conjugates since the mask does not interfere with the complexation of the masked antibody or antigen-binding fragment with the linker-drug. The targeting antibody or antigen-binding fragment that can be used in an antibody-drug conjugate for cancer treatment may be a tumor-targeting antibody or antigen-binding fragment that selectively binds to a tumor-specific or tumor-associated antigen. A tumor-specific antigen is present only on tumor cells, whereas a tumor-associated antigen is an antigen that is expressed (e.g., overexpressed) at a higher level in cancer cells compared to normal (healthy) cells.

[0126] The antigen targets to which the masked antibodies or antigen-binding fragments according to the present invention bind (also named "target proteins" herein) include annexin Al, B7H3, B7H4, BCMA, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242 (cancer antigen 242), CAIX, CCR2, CCR5, CD2, CD3, CD19, CD20, CD22, CD24, CD28, CD30 (tumor necrosis factor 8), CD33, CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44, CD47 (integrin-associated protein), CD56 (neural cell adhesion molecule), CD70, CD71, CD73, CD74, CD79, CD115 (colony stimulating factor 1 receptor), CD123 (interleukin-3 receptor), CD138 (syndecan 1), CD203c (ENPP3), CD303, CD333, CDCP1, CEA, CEACAM, claudin 4, claudin 7, CLCA-1 (C-type lectin-like molecule-1), CLL1, c-MET (hepatocyte growth factor receptor), Cripto, CTLA4, DLL3, EGFL, EGFR, EPCAM, EphA2, EphB3, ETBR (endothelin B receptor), FAP, FcRL5 (Fc receptor-like protein 5, CD307), FGFR3, FOLR1 (folate receptor α), Frβ, GCC (guanylyl cyclase C), GD2, GITR, GLOBO H, GPA33, GPC3, GPNMB, HER2, p95HER2, HER3, HMW-MAA (high molecular weight melanoma-associated antigen), integrin α (e.g. αvβ3 and αvβ5), IGF1R, TM4SF1 (L6), Lewis A-like carbohydrate antigen, Lewis X, Lewis Y (CD174), LGR5, LIV1, mesothelin (MSLN), MN (CA9), MUC1, MUC16, NaPi2b, nectin-4, Notch3, PD-1, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, SIRPα, 5T4 (or TPBG, trophoblast glycoprotein), TF (tissue factor, thromboplastin, CD142), TF-Ag, tag 72, TNFα, TNFR, TROP2 (tumor-associated calcium signal transducer 2), uPAR, VEGFR, and VLA, and may be selected from the group consisting of them.

[0127] The masked antibody or antigen-binding fragment thereof may, where applicable, (1) have a constant region that has been engineered, i.e., for example, to increase the half-life, provide a linker-drug binding site, and / or increase or decrease effector function, and one or more mutations may have been introduced; and / or (2) have a variable region that has been engineered, i.e., for example, to provide a linker-drug binding site, and one or more mutations may have been introduced. The antibody or antigen-binding fragment thereof may be produced recombinantly, synthetically, or by other known suitable methods. Mutations that may decrease the Fc-mediated effector function of the antibody or antigen-binding fragment are, for example, mutations such as those described in Leabman et al., Mabs, 2013, 5(6):896-903.

[0128] ADC The present invention further provides an antibody-drug conjugate based on the masked antibody or antigen-binding fragment according to the present invention. The ADC according to the present invention contains a masked antibody or antigen-binding fragment according to the present invention to which one or more linker-drug compounds are conjugated. Methods for producing ADCs are known in the art. The masked antibody or antigen-binding fragment according to the present invention can be used in the production of ADCs according to conventional methods.

[0129] To synthesize the conjugate according to the present invention, one or more linker-drug compounds may be conjugated to the masked antibody or antigen-binding fragment according to the present invention. The "drug" in the ADC is a pharmacologically active moiety; for example, a cytotoxic moiety or an immunomodulatory moiety or a moiety that exerts a desired pharmacological effect directly or indirectly on or through cells to which the antibody or antigen-binding fragment of the ADC can bind.

[0130] Suitable linker-drugs for use in an ADC are known in the art. For example, linker-drugs based on duocarmycin (such as trastuzumab-based SYD985) are disclosed in WO 2011 / 133039 and Elgersma et al., 2015, Molecular Pharmaceutics, 12, 1813-1835. Elgersma et al. disclose the synthesis and use of seco-DUBA-based linker-drugs (linker-drug 2 or LD2 (SYD980)).

[0131] The linker-drug compound may be conjugated to a suitable polypeptide via a reactive natural amino acid residue present in the polypeptide, such as lysine or cysteine, or via the N-terminus or C-terminus. Alternatively, natural or unnatural reactive amino acid residues may be genetically engineered into a suitable polypeptide or reactive groups may be introduced via post-translational modification.

[0132] The ADC according to the invention may be produced by conjugating a linker-drug compound to a masked antibody or an antigen-binding fragment thereof via the lysine ε-amino group of the antibody or antigen-binding fragment, preferably using an intermediate containing an amine-reactive group such as an activated ester. Such methods are known for producing conventional ADCs. Alternatively, the ADC may be produced by conjugating it to a linker via the free thiol of the side chain of cysteine generated via reduction of an interchain disulfide bond using methods and conditions known in the art. See, for example, Doronina et al, 2006, Bioconjugate Chem. 17, 114-124. The production method involves modifying the thiol obtained by a linker containing a maleimide, a linker containing a Michael acceptor such as an α-haloacetamide or ester, after partial reduction of the solvent-exposed interchain disulfide. The cysteine conjugation strategy results in a linker-drug containing up to two linkers per reduced disulfide.

[0133] Preferred antibodies according to the invention are human IgG-type antibodies. Most human IgG molecules have four solvent-exposed disulfide bonds, which are equal to an integer number of linked linker moieties from zero to eight per antibody. The exact number of linker-drug molecules per antibody (or antigen-binding fragment) is determined by the degree of disulfide reduction and the number of molar equivalents of linker-drug containing the linker in the subsequent conjugation reaction. Complete reduction of all four disulfide bonds results in a homogeneous construct with eight linker moieties per antibody, while partial reduction usually results in a heterogeneous mixture with zero, two, four, six, or eight linked moieties per antibody.

[0134] In a preferred embodiment, the invention relates to an ADC in which the linker-drug compound complexes with an antibody or an antigen-binding fragment thereof masked via a cysteine residue of the antibody or antigen-binding fragment.

[0135] Since antibodies contain many lysine residues and cysteine disulfide bonds, conventional conjugation usually produces heterogeneous mixtures that pose challenges with respect to analytical characterization and manufacturing. Furthermore, the individual components of these mixtures exhibit different physicochemical properties and pharmacologies with respect to their pharmacokinetics, efficacy, and safety profiles, hindering a rational approach to optimizing this modality.

[0136] To improve the uniformity of the conjugate, the masked antibody or antigen-binding fragment used to produce the masked ADC according to the present invention may be modified to enable site-specific conjugation of the linker-drug molecule. Methods for site-specific drug conjugation with an antibody (or antigen-binding fragment) are comprehensively reviewed by C.R. Behrens and B. Liu, mAbs, 2014, 6 (1), 1-8, International Publication No. WO 2005 / 084390 and International Publication No. WO 2006 / 034488. Site-specific ADCs are preferably produced by conjugating a linker-drug compound to an antibody or its antigen-binding fragment via the side chain of an engineered cysteine residue at a suitable position of a mutated antibody or antigen-binding fragment. The engineered cysteine is usually capped with another thiol such as cysteine or glutathione to form a disulfide. To effect linker-drug binding, the caps of these capped residues need to be removed. The binding of the linker-drug to the engineered residue is achieved either by (1) reducing both native interchain and variant disulfides and then performing standard conjugation of the uncapped engineered cysteine with the linker-drug using a mild oxidizing agent such as CuSO 4 or dehydroascorbic acid to re-oxidize the native interchain cysteine and then performing standard conjugation of the uncapped engineered cysteine with the linker-drug or (2) using a mild reducing agent that reduces variant disulfides at a higher rate than interchain disulfide bonds and then performing standard conjugation of the uncapped engineered cysteine with the linker-drug. Suitable methods for site-specific conjugation of linker-drugs can be found, for example, in International Publication No. WO 2015 / 177360, which describes methods of reduction and re-oxidation, International Publication No. WO 2017 / 137628, which describes methods using mild reducing agents, and International Publication No. WO 2018 / 215427 and Coumans et al., 2020, Bioconjugate Chem 31(9), 2136-2146, https: / / doi.org / 10.1021 / acs.bioconjchem.0c00337, which describe methods of conjugating both reduced interchain cysteine and uncapped engineered cysteine.

[0137] Pharmaceutical composition

[0138]

[0139] Medical use In a further aspect, the invention provides a masked antibody or antigen-binding fragment thereof or a masked ADC according to the invention or a composition according to the invention for use as a medicament, preferably for the treatment of cancer, an autoimmune disease or an infectious disease.

[0140] The masked antibody, masked antigen-binding fragment, masked ADC or composition according to the invention are hereinafter collectively referred to as the product for use according to the invention. In one aspect, the product for use according to the invention is for use in the treatment of solid tumours or haematological malignancies. In a second aspect, the product for use according to the invention is for use in the treatment of autoimmune diseases. In a third aspect, the product for use according to the invention is for use in the treatment of infectious diseases such as bacteria, viruses, parasites or other infectious agents.

[0141] Cancer, in the context of the present invention, is preferably a tumor that expresses an antigen targeted by a product for use according to the present invention. Such tumors may be solid tumors or hematological malignancies. Examples of tumors or hematological malignancies that may be treated by the product for use according to the present invention as defined above include, but are not limited to, breast cancer; brain cancer (e.g., glioblastoma); head and neck cancer; thyroid cancer; parotid gland cancer; adrenal gland cancer (e.g., neuroblastoma, paraganglioma or pheochromocytoma); bone cancer (e.g., osteosarcoma); soft tissue sarcoma (STS); eye cancer (e.g., uveal melanoma); esophageal cancer; gastric cancer; small intestine cancer; colorectal cancer; transitional cell carcinoma (e.g., bladder, penile, ureteral or kidney cancer); ovarian cancer; uterine cancer; vaginal, vulvar and cervical cancer; lung cancer (particularly non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)); melanoma; mesothelioma (particularly malignant pleural and peritoneal mesothelioma); liver cancer (e.g., hepatocellular carcinoma); pancreatic cancer; skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma or dermatofibrosarcoma protuberans); testicular cancer; prostate cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); myelodysplastic syndrome (MDS); blastic plasmacytoid dendritic cell neoplasm (BPDCN); Hodgkin lymphoma; non-Hodgkin lymphoma (NHL) (including follicular lymphoma (FL), CNS lymphoma and diffuse large B-cell lymphoma (DLBCL)); light chain amyloidosis; plasmacytic leukemia; and multiple myeloma (MM).

[0142] An autoimmune disease in the context of the present invention is preferably an autoimmune disease associated with an antigen targeted by a product for use according to the present invention. An autoimmune disease represents a condition resulting from an abnormal immune response against normal somatic cells and tissues. There are widely diverse at least 80 types of autoimmune diseases. Some diseases are organ-specific and are limited to certain specific tissues affected, while other diseases are similar to systemic inflammatory diseases that affect many tissues throughout the body. The appearance and severity of these signs and symptoms occur over time and may vary depending on the location and type of the inflammatory response. Examples of autoimmune diseases that may be treated by a product for use according to the present invention as defined above include, but are not limited to, rheumatoid arthritis; juvenile dermatomyositis; psoriasis; psoriatic arthritis; lupus; sarcoidosis; Crohn's disease; eczema; nephritis; uveitis; polymyositis; neuritis including Guillain-Barré syndrome; encephalitis; meningitis; systemic sclerosis; autoimmune skeletal muscle and connective tissue diseases; neurodegenerative diseases including Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and Kawasaki disease and Henoch-Schönlein vasculitis of large blood vessels, medium-sized vessels, and small vessels; cold and warm agglutinin diseases; autoimmune hemolytic anemia; type 1 diabetes; Hashimoto's thyroiditis; Graves' disease; Graves' ophthalmopathy; adrenalitis; hypophysitis; pemphigus vulgaris; Addison's disease; ankylosing spondylitis; Behçet's syndrome; celiac disease; Goodpasture's syndrome; myasthenia gravis; sarcoidosis; scleroderma; primary sclerosing cholangitis, acquired epidermolysis bullosa, and bullous pemphigoid may also be included.

[0143] In the context of the present invention, the infectious disease is preferably an infectious disease associated with the antigen targeted by the product for use according to the present invention. Such infectious diseases may be bacterial, viral, parasitic or other infectious diseases. Examples of infectious diseases that may be treated by the product for use according to the present invention as defined above include, but are not limited to, malaria; toxoplasmosis; pneumocystis jirovecii; melioidosis; bacillary dysentery; listeriosis; cyclospora; mycobacterium leprae; tuberculosis; and prevention of infectious diseases in immunocompromised individuals such as HIV-positive individuals, individuals undergoing immunosuppressive therapy or individuals with congenital deficiencies such as cystic fibrosis or benign proliferative diseases (e.g., hydatidiform mole or endometriosis).

[0144] The product for use according to the present invention described herein may be for use in the manufacture of a pharmaceutical as described herein. The product for use according to the present invention described herein is preferably for a method of treatment, and the product for use is administered in a therapeutically effective amount to a subject, preferably a subject in need thereof. Thus, alternatively or in combination with other aspects, in one aspect, the present invention relates to the use of the product for use according to the present invention for the manufacture of a pharmaceutical for the treatment of cancer, an autoimmune disease or an infectious disease, particularly for the treatment of cancer. For representative but non-limiting cancers or other diseases treated by the present invention, see above.

[0145] Alternatively or in combination with other aspects, in one aspect, the present invention is a method of treating cancer, an autoimmune disease or an infectious disease, particularly cancer, comprising administering to a subject in need of said treatment a therapeutically effective amount of the product for use according to the present invention. For representative but non-limiting cancers or other diseases treated by the present invention, see above.

[0146] The product for use according to the present invention is for administration to a subject. The product for use according to the present invention can be used in the above-mentioned treatment method by administering an effective amount of the composition to a subject in need thereof. The term "subject" as used herein refers to all animals classified as mammals, including but not limited to primates and humans. The subject is preferably a human. The expression "therapeutically effective amount" means an amount sufficient to produce a desired response or to improve a condition or symptom. The therapeutically effective amount for a particular subject may vary depending on factors such as the condition being treated, the overall health of the subject, the method of administration, the route of administration and the dosage, as well as the severity of side effects.

[0147] Combined use In a further aspect, the present invention provides a product for use according to the present invention, wherein the use is combined with one or more other therapeutic agents. The product for use according to the present invention may be used simultaneously or sequentially with one or more other therapeutic agents. Suitable chemotherapeutic agents include alkylating agents such as nitrogen mustard, hydroxyurea, nitrosourea, tetrazine (e.g., temozolomide) and aziridine (e.g., mitomycin); drugs that interfere with DNA damage response such as PARP inhibitors, ATR and ATM inhibitors, CHK1 and CHK2 inhibitors, DNA-PK inhibitors and WEE1 inhibitors; antimetabolites such as folic acid antagonists (e.g., pemetrexed), fluoropyrimidines (e.g., gemcitabine), deoxynucleoside analogs and thiopurine; microtubule inhibitors such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics such as anthracyclines and bleomycin; demethylating agents such as decitabine and azacitidine; histone deacetylase inhibitors; all-trans retinoic acid; and arsenic trioxide. Suitable radiotherapeutic agents include 131 I-metaiodobenzylguanidine (MIBG), as sodium phosphate 32 P, 223 Ra chloride, 89 Sr chloride and 153It contains radioisotopes such as Sm diamine tetramethylene phosphonate (EDTMP). Agents suitable for use as hormonal therapeutics include inhibitors of hormone synthesis such as aromatase inhibitors and GnRH analogs; hormone receptor antagonists such as selective estrogen receptor modulators (e.g., tamoxifen and fulvestrant) and anti-androgens such as bicalutamide, enzalutamide and flutamide; CYP17A1 inhibitors such as abiraterone; and somatostatin analogs.

[0148] Targeted therapeutics are therapeutics that interfere with specific proteins involved in tumor formation and growth, and may be small molecule drugs; proteins such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids such as ADCs. Examples of targeted small molecule drugs include TLR ligands such as everolimus, temsirolimus and rapamycin, mTor inhibitors; kinase inhibitors such as imatinib, dasatinib and nilotinib; VEGF inhibitors such as sorafenib and regorafenib; EGFR / HER2 inhibitors such as gefitinib, lapatinib and erlotinib; and CDK4 / 6 inhibitors such as palbociclib, ribociclib and abemaciclib. Examples of peptide or peptide derivative targeted therapeutics include proteasome inhibitors such as bortezomib and carfilzomib.

[0149] Suitable anti-inflammatory drugs include D-penicillamine, azathioprine and 6-mercaptopurine, cyclosporine, anti-TNF biological agents (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab or certolizumab pegol), leflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acitretin and JAK inhibitors (e.g., tofacitinib, baricitinib or upadacitinib).

[0150] Immunotherapeutic agents include agents that induce, enhance, or suppress an immune response, such as cytokines (IL-2 and IFN-α); immunomodulatory imide drugs, such as thalidomide, lenalidomide, pomalidomide, or imiquimod; therapeutic cancer vaccines, such as talimogene laherparepvec; cell-based immunotherapeutic agents, such as dendritic cell vaccines, adoptive T cells, or chimeric antigen receptor-modified T cells; and therapeutic antibodies that can induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) via their Fc regions when bound to membrane-bound ligands on cells.

[0151] In the context of the present invention, treatment preferably is preventing, reversing, curing, ameliorating, and / or delaying cancer, an autoimmune disease, or an infectious disease. This may mean that the severity of at least one symptom of cancer, an autoimmune disease, or an infectious disease is reduced and / or at least one parameter associated with cancer, autoimmunity, or an infectious disease is improved.

[0152] In the context of the present invention, a subject may be considered to be alive and / or disease-free. Alternatively, a disease or condition may be arrested or delayed. In the context of the present invention, an improvement in quality of life and an observed reduction in pain may mean that a subject requires less pain medication than at the start of treatment. "Less" in this context may mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less. A subject may no longer require pain medication. An improvement in quality of life and an observed reduction in pain may be recognized, detected, or evaluated at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more after treatment in a subject, and may be compared to the quality of life and observed reduction in pain at the start of treatment of said subject.

[0153] Nucleic acid construct In a further aspect, the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a heavy chain variable domain, a protease-cleavable peptide linker and a masking moiety; and / or a nucleotide sequence encoding a light chain variable domain, a cleavable peptide linker and a masking moiety, wherein the nucleotide sequence is operably linked to an expression control sequence for expression in a host cell, preferably a mammalian host cell. The heavy and light chains may be expressed from the same expression vector or different expression vectors (different expression cassettes).

[0154] Preferably, the expression control sequence includes a promoter and, optionally, other regulatory elements such as a terminator, an enhancer, a polyadenylation signal, a signal sequence for secretion, etc. Such nucleic acid constructs are particularly useful for the production of an activatable antibody or an antigen-binding fragment thereof of the invention using recombinant techniques for expressing a nucleotide sequence encoding an activatable antibody in a suitable host cell as described in Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. By operably linked is meant that the DNA sequences being linked are usually contiguous and, where two protein coding regions are to be joined, are in the same reading frame contiguously.

[0155] In a preferred embodiment, the nucleotide sequence defined above is codon-optimized with respect to the host cell in which the activatable antibody or antigen-binding fragment thereof according to the invention is produced.

[0156] In a preferred embodiment, the nucleic acid construct is a vector that is compatible with the host cell.

[0157] Host cell In a further aspect, the invention relates to a host cell comprising the nucleic acid construct defined above. Preferably, the host cell is a host cell for the production of the activatable antibody or antigen-binding fragment thereof according to the invention. The host cell may be a host cell capable of producing the antigen-binding protein of the invention, for example a prokaryotic cell host such as Escherichia coli or a (cultured) mammalian, plant, insect, fungal or yeast host cell including, for example, CHO cells, BHK cells, human cell lines (including HeLa, COS, HEK293 and PER.C6), Sf9 cells and Sf+ cells. However, a preferred host cell for the production of the activatable antibody or antigen-binding fragment thereof according to the invention is a mammalian cell, more preferably HEK293 or CHO cells.

[0158] Production The masked antibodies or antigen-binding fragments according to the present invention can be produced by recombinant DNA techniques known in the art for the production of recombinant antibodies or antigen-binding fragments. Essentially, such methods involve creating a DNA construct (e.g., an expression vector) containing the DNA coding sequence of the antibody or antigen-binding fragment, the coding sequence of the masking moiety, and, optionally, the coding sequence of a peptide linker containing any spacer sequences, all in the appropriate order and orientation and together with the usual expression control elements known in the art. Such constructs can be introduced into a host (e.g., bacteria, yeast, or mammals) suitable for the production of the masked antibody or antigen-binding fragment in cell culture in a suitable medium. Most monoclonal antibodies or antigen-binding fragments produced for use as biotherapeutics are produced in mammalian cell cultures such as Chinese hamster ovary (CHO) cells that stably express the antibody or antigen-binding fragment. The antibody or antigen-binding fragment can be harvested and purified from the cell culture. Thus, a further aspect of the present invention is a method for producing an activatable antibody or antigen-binding fragment thereof according to the present invention, the method comprising culturing the host cell as defined above under conditions that promote the expression of the activatable antibody or antigen-binding fragment thereof. The method preferably comprises (a) culturing the host cell as defined above under conditions that promote the expression of the activatable antibody or antigen-binding fragment thereof; and, optionally, (b) purifying the activatable antibody or antigen-binding fragment thereof from at least one of the host cell and the medium. Suitable conditions may include the use of a suitable medium, the presence of suitable food and / or suitable nutrients, a suitable temperature, and, optionally, the presence of a suitable inducer or compound (e.g., if the nucleotide sequence of the present invention is under the control of an inducible promoter); all of which may be selected by one of ordinary skill in the art. Under such conditions, the amino acid sequence of the present invention may be expressed constitutively or transiently or only when appropriately induced.Next, the activatable antibody or antigen-binding fragment thereof of the present invention may be isolated from a host cell / host organism and / or the medium in which the host cell or host organism is cultured, using protein isolation and / or purification techniques known per se, such as (fractional) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using a specific cleavable amino acid sequence fused to the amino acid sequence of the present invention) and / or fractional immunological techniques (i.e., using an antibody against the antigen-binding protein to be isolated).

[0159] Use In a further aspect, the present invention relates to the use of an antigen peptide and an sdAb that specifically binds to the antigen peptide as a part of the mask in the activatable antibody or antigen-binding fragment thereof according to the present invention.

[0160] In this specification and its claims, the verb "comprising" and its conjugations are used in a non-limiting sense, meaning that it includes the items following the term, but does not exclude items not specifically mentioned. In addition, the mention of an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one element. For this reason, the indefinite article "a" or "an" usually means "at least one".

[0161] The term "about" or "approximately", when used in relation to a numerical value (e.g., about 10), preferably means that the value may be a predetermined value that is greater than or less than 1% of the value.

[0162] When considering the parameters of a substance in the context of the present invention, unless otherwise specified, the parameters are determined, measured or become apparent under physiological conditions. Physiological conditions are known to those skilled in the art and contain an aqueous solvent system, atmospheric pressure, a pH value between 6 and 8, room temperature (RT) to about 37 °C (about 20 °C to about 40 °C) and a suitable concentration of buffer salts or other components.

[0163] All patents and references cited in this specification are hereby incorporated by reference in their entirety into this specification.

[0164] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example

[0165] Example 1 Transient Expression, Purification, and HMW(%) Determination of Masked Antibodies (Materials and Methods) Design of Masked Antibody Chains The masked antibody chain binds the HAVT20 leader sequence (Boel et al., 2000, J. of Immunol. Meth., 239, 153-166, SEQ ID NO: 43) to the N-terminus of a masking moiety containing BC2-nanobody (or a variant thereof) or BC2T peptide (or a variant thereof), followed by binding a linker containing the substrate sequence of L9 (SEQ ID NO: 11), which is a matrix metalloproteinase-2 (MMP2) linker sequence, or L13 (SEQ ID NO: 12), a scrambled form of L9 that cannot be cleaved by MMP2. Optionally, a spacer sequence (Qg: SEQ ID NO: 9, or Qv: SEQ ID NO: 10) is sandwiched in between, and finally, the heavy chain (HC) or light chain (LC) of trastuzumab (HC: SEQ ID NO: 21, HC-P41C: SEQ ID NO: 22, LC: SEQ ID NO: 23), an anti-HER2 antibody; ING-1 (HC: SEQ ID NO: 24, HC-P41C: SEQ ID NO: 25, LC: SEQ ID NO: 26), an anti-EpCAM antibody; or adecatumumab (HC: SEQ ID NO: 27, HC-P41C: SEQ ID NO: 28, LC: SEQ ID NO: 29); sacituzumab (HC: SEQ ID NO: 30, HC-P41C: SEQ ID NO: 31, LC: SEQ ID NO: 32), an anti-TROP2 antibody; MORAb-066 (HC: SEQ ID NO: 33, HC-P41C: SEQ ID NO: 34, LC: SEQ ID NO: 35), an anti-TF antibody; or tisotumab (HC: SEQ ID NO: 36, HC-P41C: SEQ ID NO: 37, LC: SEQ ID NO: 38); urelumab (HC: SEQ ID NO: 39, LC: SEQ ID NO: 40), an anti-CD137 antibody; or tremelimumab (HC: SEQ ID NO: 41, LC: SEQ ID NO: 42), an anti-CTLA4 antibody, is bound.

[0166] The sequence of BC2-Nb is shown in SEQ ID NO: 4, the sequence of its CDR3 is shown in SEQ ID NO: 5, and the sequences of CDR3 variants are shown in SEQ ID NOs: 6-8. The sequence of the BC2T peptide is shown in SEQ ID NO: 1, and the sequences of BC2T peptide variants are shown in SEQ ID NOs: 2-3. Optionally, a 7-amino acid residue spacer sequence QGQSGQG (Qg, SEQ ID NO: 9) or QVQLVES (Qv, SEQ ID NO: 10) was included in the masking portion containing the BC2T peptide in front of (the N-terminus of) the BC2T sequence to enable efficient signal peptide cleavage and enhanced expression of the antibody chain. Cleavage of the leader sequence corresponded to the cleavage site predicted using the SignalP program https: / / services.healthtech.dtu.dk / service.php?SignalP, Bendtsen, Jannick Dyrlov, et al. "Improved prediction of signal peptides: SignalP 3.0." Journal of molecular biology 340.4 (2004): 783-795.

[0167] To enable site-specific conjugation for the development of antibody-drug conjugates, a single cysteine residue was introduced into the heavy chain variable domain by substituting the residue at position 41 according to Kabat numbering with a cysteine residue. The obtained amino acid sequences of the masked heavy and light chains were reverse translated into cDNA sequences and codon optimized for expression in human (Homo sapiens) cells. The cDNA constructs of the heavy and light chains were chemically synthesized by the vendor (Geneart, Thermo Fisher Scientific) and obtained from the vendor.

[0168] Expression vector construction For the expression of antibody chains, the mammalian expression vector pcDNA3.4-TOPO (Invitrogen, Thermo Fisher Scientific) containing the CMV:BGHpA expression cassette was used. The genes for the heavy chain (HC) or light chain (LC) were synthesized by synthetic oligo assembly and / or PCR products and cloned into pcDNA3.4-TOPO, respectively. After transformation into Escherichia coli K12 DH10B T1R and growth, large-scale production of the antibody chain expression vector for transfection was carried out using the EndoFree Plasmid Maxi kit according to the manufacturer's (Qiagen) instructions.

[0169] Transient expression in mammalian cells Commercially available Expi293F cells (Gibco, Thermo Fisher Scientific) were co-transfected with the antibody chain expression vector prepared as described above using the FectoPRO transfection agent (Polyplus-transfection) according to the manufacturer's instructions as follows: One day before transfection, the cells were seeded at 1×10 6 viable cells (VC) / mL in 270 mL of Expi293 expression medium; the day after seeding, 240 μg of the antibody chain expression vector was mixed with 240 μL of the FectoPRO transfection agent (1:1 ratio) and added to the cells. The cells were cultured according to the manufacturer's (Gibco, Thermo Fisher Scientific) instructions. Small-scale batches were obtained at linear scalability using the equal volume ratio as described above. Six days after transfection, the cell culture supernatant was harvested by centrifugation at 4,000 g for 15 minutes, and the clarified harvest was filtered through an MF75 filter (Nalgene). Filtration was omitted for small-scale batches (3 mL). The antibody concentration was determined using a Protein A biosensor and trastuzumab as calibration substances according to the manufacturer's (Sartorius) instructions by ForteBio Octet QK384.

[0170] (Masked) Antibody purification and determination of titer and HMW The (masked) antibody was purified using the following procedure. The harvest from the expression containing the (masked) antibody produced by Expi293 cells was captured on Protein A resin (column or plate), and after elution, it was adjusted to pH 5.5 - 6.0 or rebuffered into a buffer of pH 6.0. The purification process was carried out as follows: The Protein A step (MabSelect® SuRe, Cytiva) started with disinfecting the resin (column or plate) with 0.1 M NaOH for 15 minutes and rinsing with purified water (PW). Then the resin was equilibrated with PBS at pH 7.4. The (masked) antibody containing the harvest was loaded onto MabSelect® SuRe resin at a ratio of 25 mg or less of the (masked) antibody per 1 ml. The resin was washed with the equilibration buffer (PBS pH 7.4) and then with 25 mM NaAc (pH 5.0). Then the (masked) antibody was eluted with 25 mM acetate pH 3.0. The concentration of the purified (masked) antibody was measured by the Lambert-Beer law using the theoretical extinction coefficient based on the amino acid sequence of the (masked) antibody. After elution, the (masked) antibody was adjusted to pH 5.5 - 6.0 or rebuffered into a buffer of pH 6.0.

[0171] For the purity and soluble protein aggregation analysis of the (masked) antibody, size exclusion chromatography (SEC) with UV detection was used. The (masked) antibody was diluted to a concentration of 1 mg / mL and injected onto a TSKgel UP-SW3000 column equipped in-line with a TSKgel UPSW Direct Connect guard column, using 400 mM NaCl containing 15% 2-propanol, 150 mM PO 4 buffer pH 6.2 as the mobile phase. The ratio of the main peak (monomer) to the high molecular weight (HMW) species (e.g., soluble aggregates) was determined based on the peak area relative to the total peak area of all protein-related peaks.

[0172] For the antibody purified using the column, the integrity of the (masked) antibody was confirmed by SDS-PAGE.

[0173] Example 2 Production of various (half-)masked antibodies 2A: Masked trastuzumab antibody variants (SET1: Masked antibodies S101 - S112) To examine whether various variants of the masked trastuzumab antibody can be produced at acceptable titers, variants with different methods of linking the masking moiety to the antibody were produced. The masked antibodies were based on the anti-HER2 antibody trastuzumab (Ab) or trastuzumab with a modified HC sequence in which proline at position 41 was replaced with cysteine (41C). All antibodies had a masking moiety containing the BCT2 peptide (SEQ ID NO: 1) or BC2-Nb (SEQ ID NO: 4).

[0174] The cleavable sequence contains the MMP2-cleavable sequence PLGLAG (SEQ ID NO: 13), with GS-rich regions adjacent to both ends, resulting in the following sequence GGGGGSPLGLAGGGS (SEQ ID NO: 11, hereinafter referred to as "L9" in this specification).

[0175] The antibodies differed depending on whether the masking moiety with BC2-Nb was bound to the HC and the masking moiety with the BCT2 peptide was bound to the LC, or vice versa.

[0176] Table 1 shows the production titers of various masked antibodies. The "HC" column in Table 1 shows the various masking moieties (with spacer sequences (Qg or Qv) present before the nanobody BC2-Nb or peptide BCT2) at the N-terminus of the peptide and the linker (L9) in the heavy chain of the produced masked antibodies, and the "LC" column shows the various masking moieties and the linker in the light chain that combined with the heavy chain to form the masked antibody. Antibodies in which both HC and LC contained the same BC2 nanobody masking moiety could also be produced at high titers.

[0177] Generally, it was found that the production of antibodies (antibodies S103, S104, S107, S108, and S110) having a masking portion containing a nanobody in the LC increased the titer and decreased the formation rate of the HMW complex. Table 1 shows the ratio of the HMW complex in the product for masked antibodies having various combinations of the masking portions. For comparison, "half-masked" antibodies containing only the HC or LC with the masking portion were also produced. Antibodies containing the same masking portion in both the HC and LC are also included. Apparently, the half-masked antibodies and the antibodies with a double Nb mask had a reduced formation of the HMW complex.

[0178]

Table 1

[0179] A schematic diagram of an antibody having an Nb on the HC and a peptide on the LC, and the reverse antibody, is shown in Fig. 1A, and a schematic diagram of the formation of the HMW complex between the masked antibodies is shown in Fig. 1B.

[0180] 2B: Comparison of the production yield (titer (μg / mL)) and HMW (%) of the masked trastuzumab of the variant and wild-type BC2T / BC2-Nb A hypothesis was established that the HMW formation might correlate with the binding strength (affinity) between the masking portions on the LC and HC. To test this hypothesis, a set of antibodies (SET-2) was produced using masking portions based on the BC2-Nb and BC2T peptides.

[0181] The antibody used was trastuzumab, and the masking portion was attached to the N-terminus of the HC and LC using the L9 linker sequence described in Example 1 for the ligation of the (mutated) BC2T peptide and the (mutated) BC2-Nb.

[0182] Based on the idea that introducing mutations into the amino acid sequences of BC2-Nb and / or BC2 peptides may reduce the binding affinity between the nanobody and the peptide, thereby decreasing the aggregation that forms high molecular weight (HMW) complexes, BC2 / BC2T variants were created. The mutations tested are shown in Table 2. The mutations in Nb are in the CDR3 of Nb. The CDR3 of BC2-Nb is shown in SEQ ID NO: 5. The sequences of the mutated CDR3s of the BC2-Nb variants are shown in SEQ ID NOs: 6-8 in Table 2.

[0183]

Table 2

[0184] Each masking portion having a BC2T peptide or its variant was combined with each masking portion having a BC2-Nb or its variant, resulting in 3×4 = 12 combinations. All combinations were tested for the masking portions of Nb or peptide on HC or LC, and a total of 12×2 = 24 different masked antibodies were obtained.

[0185] Table 3 shows the results (post-production titer (μg / mL) and HMW (%)) of all 24 masked antibodies combined with the tested masking portions.

[0186]

Table 3

[0187] It is clear from Table 3 that the production titers of the antibodies (S201 - S211) with the masking portion having Nb on LC are higher than those of the antibodies (S212 - S222) with Nb on HC.

[0188] It is even clearer that mutations in the nanobody array and / or peptide array reduce the rate of HMW complex formation (presumably because the binding affinity between the masking moieties is reduced by the mutation). See, for example, the results of antibodies S209, S205, S201, S203, S211 and S207. This effect can be observed not only in antibodies where the Nb is on the LC, but also in antibodies where the Nb is on the HC (especially in the case of BC2-Nb R106A and R106S mutants such as S214 and S220).

[0189] 2C: Production of Masked Antibodies Against Diverse Targets (SET3) To demonstrate that the masked antibodies according to the present invention can be produced based on antibodies against diverse targets, various masked antibodies (SET-3) were produced. SET3 included mAbs targeting EpCAM, TROP2, tissue factor (TF), CD137 and CTLA4. The set included unmasked (naked) antibodies, as well as masked versions of the masked antibodies and antibodies having the 41C mutation in their HC (prepared for later use in site-directed ligation of the linker-drug molecule in the production of masked antibody-drug conjugates).

[0190] All of the masked antibodies in SET3 had the following masking moieties: BC2-Nb was linked to linker sequence L9 at the N-terminus of the LC of each antibody, and the masking moiety at the N-terminus of the HC of each antibody had a Qg sequence in front of the BC2T peptide and was linked to the HC by the L9 sequence.

[0191] Results of Antibody Production 30 mL batches were produced for all of the masked antibodies.

[0192] HMW (%) was relatively low for all wild-type mAbs (0.6 - 2.7) and masked wild-type and 41C chisotuzumab (4.1 and 3.7, respectively).

[0193] The masked ING-1 mAb targeting EpCAM had the highest HMW (%) (65.5). The masked wild-type and 41C-Adekizumab (which also targets EpCAM) also showed relatively high HMW (%) (33.7 and 30.9, respectively), as shown in Table 4.

[0194] 2D: Production Yield (mg) and HMW (%) of Half-Masked Antibodies Against Various Targets (EpCAM, TROP2, TF, CD137, and CTLA4) Half-masked antibodies could also be produced, as evidenced by the data shown in Table 5.

[0195] Antibodies against various targets were half-masked, which means that the masking portion containing BC2-Nb was linked to the LC of the antibody using the L9 linker sequence, or the masking portion containing the BC2T antigen peptide was linked to the HC of the antibody. When linking to the HC, the Qg spacer sequence was included before the peptide sequence.

[0196] For various antibodies, two variants of the masked HC were produced: one had the wild-type sequence of the antibody HC, and one had the 41C HC mutation (a 41C mutation that introduced a cysteine that can be used for site-specific conjugation of the linker-drug molecule when the antibody is used in an ADC).

[0197] [Table 4]

[0198] [Table 5]

[0199] Synthesis of Antibody-Drug Conjugates Based on the Antibodies of Example 3 (Masked or Half-Masked) An ADC based on a masked antibody, in which the duocarmycin-based linker-drug LD2 (Elgersma et al., 2015 (supra)) was site-specifically complexed with the HC41 cysteine introduced into the HC sequence of the antibody, was synthesized according to the following protocol as disclosed in Coumans et al., 2020 (supra).

[0200] A solution of cysteine-engineered antibody (10 - 15 mg / mL, pH 5, 100 mM histidine) was treated with 2-(diphenylphosphino)benzenesulfonic acid (diPPBS, 16 - 32 equivalents, 10 mM in water), and the resulting mixture was incubated at RT for 16 - 24 h. The excess diPPBS was removed by centrifugal concentrator (Vivaspin filter, 30 kDa cutoff, PES) or carbon filtration using 4.2 mM histidine, 50 mM trehalose, pH 6. The pH of the resulting antibody solution was raised to approximately 7.4 with Tris (1 M in water, pH 8) or maintained at pH 6, then N,N-dimethylacetamide (DMA) was added, followed by addition of a solution of linker-drug (10 mM in DMA). The final concentration of DNA was 5 - 10%. The resulting mixture was incubated in the dark at RT for 2 - 3 h or overnight if at pH 6. To remove the excess linker-drug, activated carbon was added and the mixture was incubated at RT for 0.5 h or more. The activated carbon was removed with a 0.2 μm PES filter, and the resulting ADC was formulated using a Vivaspin centrifugal concentrator (30 kDa cutoff, PES) in 4.2 mM histidine, 50 mM trehalose, pH 6.

[0201] Using the above site-directed complexation method, the ADCs shown in Table 6 were produced, all of which were produced by site-directed complexation, had linker-drug 2 (LD2), and had a drug-to-antibody ratio (DAR) of 2.

[0202]

Table 6

[0203] Example 4. Biolayer Interferometry (BLI) Experiment to Compare the Target Binding of Masked Antibodies and Naked (Unmasked) Antibodies Materials and Methods Enzymatic Demasking of Masked Antibodies / ADCs by Protease Treatment A stock solution of 24 mM APMA (p-aminophenylmercury acetate, sigma-aldrich, A-9563) in DMSO was prepared. rhMMP2 (R&D systems, 902-MP, 0.1 mg / mL) was thawed to RT. 10 μL of the enzyme was activated at 37 °C for 1 hour with 0.44 μL of fresh APMA solution. After incubation, the activated enzyme solution was diluted to 3.0 ng / μL with 329 μL of TCNB (50 mM TRIS, 10 mM CaCl 2 , 150 mM NaCl, 0.05% (w / v) BRIJ®-35, pH 7.5) buffer.

[0204] The masked mAb or ADC was diluted with TCNB buffer and then the activated enzyme was added. Final conditions: substrate 0.4 mg / mL with a substrate-to-enzyme molar ratio of approximately 300. The mixture was incubated at 37 °C with gentle shaking. After 2 hours, the mixture was cooled to RT, harvested for SDS-page analysis, and then stored at -80 °C until further use.

[0205] Biolayer Interferometry (BLI) The biotinylated target protein was immobilized on the SAX biosensor chip by biotin-streptavidin interaction or on Ni-NTA for the immobilization of His-tagged target protein. The masked antibody was tested at a 50-fold dilution by immersing the SAX / Ni-NTA sensor chip immobilized with the analyte. Protein A binding was estimated with the same sample to correct for the difference in concentration between the analyte's stock solutions. The binding levels of the masked antibody (analyte) to the immobilized target on the SAX or Ni-NTA chip and the Protein A sensor chip were measured 10 seconds after binding and used for analysis because they are in the linear part of the binding curve and are proportional to the concentration of the analyte.

[0206] Materials · 384-well tilted bottom microplate, Forte bio, Cat# 18-5076 · High-precision streptavidin (SAX) dip and read sensor chip, Forte Bio, 18-5118 · Protein A sensor chip, Forte Bio, 18-5012 · HBS-EP+ buffer, GE Healthcare, 10x: BR-1006-69, # 30964 · Biotin in HBS-EP+ at 1 mg / mL (Example 4B) · Ni-NTA biosensor, Forte Bio, 18-5102 (Example 4B) · HBS-P+, GE Healthcare (Example 4B)

[0207] Example 4A (SET2 antibody) · Biotinylated human HER2 (Acro Biosystems, Cat. HE2-H822R) immobilized on a SAX biosensor chip as the target protein · Masked antibodies at the concentrations shown in Table 7

[0208] [Table 7]

[0209] Example 4B (SET3 antibody) As the target protein, the following reagents were immobilized on the sensor chip: · Biotinylated antigens EpCAM, TROP2 and CD137 (4-1BB) immobilized on a SAX biosensor chip Biotinylated human TROP2 / TACSTD2 protein, His Avitag, Cat. No. TR2-H82E5, 200 μg / mL Biotinylated human EpCAM / TROP1 protein, Avitag His tag, Cat. No. EPM-H82E8, 200 μg / mL Biotinylated human CTLA4 protein, His Avitag, Cat No. CT4-H82E1, 200 μg / mL Biotinylated human 4-1BB / TNFRSF9 protein, His Avitag, Cat No. 41B-H82E6, 200 μg / mL · Human coagulation factor III / tissue factor protein, His-tag, Cat. No. TF3-H52H5, 600 μg / mL (His-tagged tissue factor was immobilized on Ni-NTA biosensor chip) · The concentrations of the SET3 nanobody-peptide masked trastuzumab constructs tested are shown in Table 10.

[0210]

Table 10

[0211] Example 4A Reduction of binding of masked SET2 antibody to HER2 compared to trastuzumab To test the effect on the masking efficiency of the mutations introduced into the masking portion of the masked antibody of SET2, the binding ability of antibodies S201 - S222 to HER2 (the target antigen of the masked trastuzumab antibody) was tested. Two masked antibodies from SET1 (S101 and S103) were compared with wild-type trastuzumab (TRA) (concentration 21 mg / mL) as a positive control and anti-PSMA SYD1030 (HC S41C) (aPSMA) (concentration 1 mg / mL) as a negative control. The experiment was carried out as described in the "Materials and Methods" above.

[0212] Results: Tables 8 and 9 show the binding levels as the likelihood of antibody binding to the HER2 and Protein A sensor chips immobilized on the SAX sensor chip after 130 seconds compared to the baseline. The results are shown as the optical thickness of the biosensor layer of the sensor chip as a wavelength shift in nm units. The point of 130 seconds corresponds to 10 seconds of binding since the baseline is created in the first 120 seconds. The last column shows the quotient of HER2 / concentration, by which the binding between various nanobody-peptide trastuzumab constructs can be compared.

[0213] All masked trastuzumab constructs were found to have significantly reduced binding to HER2 compared to unmasked trastuzumab, indicating that the modification of the nanobody and / or peptide used in the masking portion of the masked antibody in SET2 does not negatively affect the ability of the masked trastuzumab to mask the binding to the target HER2. Together with the data in Table 3 for production titer and HMW (%), these data show that efficiently manufacturable masked antibodies (indicated by high titer and relatively low HMW (%)) still have favorable masking characteristics (meaning that the binding of the masked antibody to the target is significantly reduced compared to the positive control trastuzumab).

[0214] [Table 8]

[0215] [Table 9]

[0216] Example 4B Reduction of Binding of Masked SET3 Antibody to Target Compared to Naked (Unmasked) Antibody To examine the masking efficiency of masked antibodies against diverse targets, SET3 antibodies S300 - S317 were tested for their binding ability to targets using a Biolayer Interferometer (BLI). As described in Example 2C, SET3 included mAbs targeting EpCAM (ING - 1 and adecatumumab), TROP2 (sacituzumab), TF (MORAb - 066 and tisotumab), and CD137 (urelumab). SET3 included naked (unmasked) antibodies, masked wild - type antibodies, and masked antibodies having a P41C mutation in the HC (prepared for later use in site - directed ligation of linker - drug molecules in the manufacture of antibody - drug conjugates based on the masked antibodies).

[0217] The experiment was conducted as described in the “Materials and Methods” above.

[0218] Results Tables 11A - 11F show the binding levels as wavelength shifts in nm indicating the possibility of antibody binding to the immobilized target on the SAX sensor chip or Ni - NTA sensor chip and Protein A sensor chip after 70 seconds. The 70 - second time point corresponds to 10 - second binding as a baseline is established in the first 60 seconds. The last column shows the quotient of target binding / concentration, by which binding between various nanobody - peptide antibody constructs can be compared.

[0219] The results of the binding of the naked, masked, masked (P41C), and MMP2 - treated versions of each antibody tested to the target were corrected for differences in concentration (target binding / Protein A binding × 1000) and are similarly shown in FIGS. 8A - F.

[0220] It was observed that compared to the wild - type antibodies, all of the masked antibodies had a significantly reduced binding to the target.

[0221] The masked antibodies were also treated with MMP2, and it was analyzed whether demasking occurred in the presence of MMP2. For all antibodies, the binding levels of the wild-type antibodies were similar to those of the MMP2-treated masked antibodies, thereby demonstrating the concept of the MMP2-cleavable nanobody-masked construct.

[0222]

Table 11A

[0223]

Table 11B

[0224]

Table 11C

[0225]

Table 11D

[0226]

Table 11E

[0227]

Table 11F

[0228] Example 5 Cell Binding and Cytotoxicity of Masked Antibodies, Half-Masked Antibodies, and Demasked Antibodies Materials and Methods Determination of Cell Binding Masked antibodies, semi-masked antibodies, naked (unmasked) antibodies, and de-masked (treated with MMP2 according to the protocol described in Example 4) antibodies, as well as the cell binding of the ADCs, were tested on the human HER2-positive tumor cell line SK-BR-3. Cells were used at approximately 90% confluence at the time of the assay, detached with trypsin-Versene (EDTA) (Lonza) for 5 - 10 minutes, washed, and resuspended in ice-cold FACS buffer (1× PBS, 0.1 w / v% BSA, 0.02 v / v% sodium azide (NaN 3 )) to a concentration of 1 × 10 6 cells / mL.

[0229] Staining was performed at 4°C in 96-well round-bottom microtiter plates using ice-cold reagents / solutions to prevent modulation and internalization of surface antigens. 100,000 cells / well were added to the 96-well plates (100 μL / well) and centrifuged at 300 × g for 3 minutes. The supernatant was discarded, and the cells were stained with 50 μL of each antibody or ADC for 30 minutes. Serial dilutions were made in ice-cold FACS buffer. The cells were washed three times by centrifugation at 300 × g for 3 minutes and resuspended in 50 μL of a 6000-fold diluted secondary F(ab’) 2 goat anti-human IgG (Fc fragment specific) antibody APC conjugate (Jackson ImmunoResearch).

[0230] After a 30-minute incubation on ice, the cells were washed again and resuspended in 150 μL of ice-cold FACS buffer for FACS analysis. For data analysis, the fluorescence intensity was determined by flow cytometry (BD Biosciences) and presented as the median fluorescence intensity (MFI-median).

[0231] The dose-response binding curves were fitted to a non-linear regression with variable slope (4-parameter) in GraphPad Prism version 9. EC 50 values were calculated in GraphPad Prism.

[0232] Determination of in vitro cytotoxicity The in vitro cytotoxicity of masked, unmasked, demasked, and / or semi-masked anti-HER2 ADCs was determined in the human HER2-positive tumor cell line SK-BR-3 and the human HER2-negative tumor cell line Jurkat NucLight Red. Cells in complete growth medium were placed in 96-well plates (90 μL / well) and incubated at 37 °C, 5% CO 2 at the following cell densities: 6500 SK-BR-3 and 3000 Jurkat NucLight Red per 90 μL per well.

[0233] After overnight incubation, 10 μL of ADC was added. Serial dilutions of the ADC were made in the medium. Cell viability was evaluated 6 days later using the CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's (Promega Corporation, USA) instructions.

[0234] Survival was calculated by dividing the luminescence measured at each ADC concentration by the mean of the untreated cells (growth medium only) and multiplying by 100.

[0235] IC 50 values were calculated for each dose-response curve of log(inhibitor) vs response by variable slope (4-parameter) fit available in GraphPad Prism version 9.

[0236] Example 5A Cell Binding of Masked and Demasked SET2 (S201–S211) Antibodies to SK-BR-3 Cells Cell binding experiments were performed with antibodies to SET2. SET2 antibodies are shown in Table 3 and include antibodies with mutations in the BC2-Nb and / or BC2T peptide sequences. Demasking by MMP2 and cell binding were performed as described (M&M supra).

[0237] The purpose of this experiment was to examine whether target binding would recover after the de - masking of masked antibodies, as compared to the target binding of naked (un - masked) trastuzumab. At the same time, the effect of differences in mutations in the BC2 - Nb and / or BCT2 peptide sequences on the masking efficiency of the SET2 antibody (S201 - S211) was examined and compared with S103 (which is masked with a masking moiety similar to the SET2 antibody but has no mutations in the BC2 - Nb and BC2T peptide sequences).

[0238] EC of cell HER2 target binding in SK - BR - 3 cells after de - masking of masked antibodies by MMP2, compared with the binding of naked (un - masked) trastuzumab 50 The EC shift is shown in Table 12. Reliable EC values for the target binding of masked Ab could not be determined because the dose - response curve did not show a maximum (did not reach saturation binding). Table 12 means the binding EC, which is the antibody concentration at 50% binding of all binding in HER2 - positive SK - BR - 3 cells 50 and the EC shift compared with naked (un - masked) trastuzumab. It shows the binding characteristics of all de - masked SET2 antibodies and de - masked S103, expressed as an EC shift 50 and the EC shift compared with naked (un - masked) trastuzumab. It shows the binding characteristics of all de - masked SET2 antibodies and de - masked S103, expressed as an EC shift 50 It shows the binding characteristics of all de - masked SET2 antibodies and de - masked S103, expressed as an EC shift

[0239] Figures 2A - 2L show the dose - dependent binding of masked and MMP2 - treated (de - masked) S103 and S201 - S211 in HER2 - positive SK - BR - 3 cells measured by flow cytometry, compared with naked (un - masked) trastuzumab binding. The data show the MFI (median fluorescence intensity) of one experiment

[0240] The dose - response curves shown in Figures 2A - 2L and the results shown in Table 12 indicate that the antibody was effectively de - masked after MMP2 treatment and that HER2 target binding was almost completely restored in most de - masked antibodies (compared with naked (un - masked) trastuzumab).

[0241]

Table 12

[0242] Masked antibodies bind little to the target even at very high concentrations, so masked antibodies do not reach saturation binding (incomplete dose-response curves as shown in Figures 2A - 2L), and a reliable EC 50 value could not be determined.

[0243] Comparing the concentration at which the minimum binding can be observed for masked, naked (unmasked), and demasked (MMP2-treated) antibodies, the difference in binding is clear. The minimum binding of unmasked or demasked antibodies is observed at a low concentration of 0.04 μg / mL, while masked antibodies showed minimum binding at high concentrations ranging from 3.7 μg / mL (S103 and S204) to 33 μg / mL (S210). For most of the masked antibodies tested, the minimum binding could only be observed at a high concentration of 11 μg / mL. When the estimated EC 50 was calculated manually, values exceeding 300 μg / mL were obtained for the binding of masked antibodies.

[0244] Example 5B Cytotoxicity Induced by Masked / Demasked ADC-S105 in HER2-Positive SK-BR-3 Cells The ADC-S105 shown in Table 6 is a masked trastuzumab (P41C)-based ADC based on the masked antibody S105 in Table 1, with a masking moiety containing BC2 nanobody (BC2-Nb-L9-HC(P41C)) on the heavy chain and a masking moiety containing BC2T peptide after the Qg sequence (Qg-BC2T-L9-LC) on the LC. The two masking moieties are linked to the antibody by an MMP2-cleavable linker L9.

[0245] The cytotoxicity of masked and demasked (MMP2-treated) ADC-S105 was tested in HER2-positive SK-BR-3 cells to examine whether the ADC was efficiently masked and whether the decrease in cell viability after demasking of ADC-S105 by MMP2 protease was equivalent to that of naked (unmasked trastuzumab 41C-based) ADC. For comparison, unmasked ADCs based on trastuzumab and rituximab (as isotype control) were also tested (ADC-TRA41C and ADC-RIT41C shown in Table 6).

[0246] Cell viability was measured with the CellTiter-Glo® (CTG) luminescence assay kit after 6 days of treatment.

[0247] Figure 3 shows the dose-response curves for cytotoxicity induced by masked and MMP2-treated (unmasked) ADC-S105 compared to ADC-TRA41C and isotype control ADC-RIT41C. The data represent the viability of two experiments. Table 13 shows the IC 50 determined by CTG readout after 6 days of treatment of HER2-positive SK-BR-3 cells with masked ADC-S105 and its demasked (MMP2-treated) ADC. The IC 50 efficacy shift induced by the concept of masking is associated with ADC-TRA41C. Masked ADC-S105 shows a 73-fold IC 50 shift compared to ADC-TRA41C, indicating effective masking of trastuzumab-mediated cell killing. When ADC-S105 is demasked after MMP2 protease treatment, almost complete recovery of cytotoxicity induction is observed (demasked ADC-S105 shows a 2-fold IC 50 shift compared to ADC-TRA41C).

[0248]

Table 13

[0249] Cell binding of masked / MMP2-treated ADC-S423 and ADC-S426 (non-cleavable linker L13) to HER2-positive SK-BR-3 cells compared to ADC-TRA41C The cell binding of masked and MMP2-treated ADCs ADC-S423 and ADC-S426 to HER2-positive SK-BR-3 cells was compared to the binding of ADC-TRA41C (with / without MMP2 treatment). Dose-dependent binding in HER2-positive SK-BR-3 cells was measured by flow cytometry.

[0250] To examine whether cleavage of the L9 linker that connects the masking moiety to the S423 antibody in ADC-S423 was MMP2-dependent, ADC-S426 was similarly tested. For the masked S426 antibody, the same masking moiety used for S423 was linked to the antibody by a scrambled version of the L9 linker (L13: GGGGS LALGPGGGS, SEQ ID NO: 12). L13 does not contain the MMP2-cleavable site present in L9. (Trang et al. Nature biotechnology 37.7 (2019): 761-765, Jiang et al. Proceedings of the National Academy of Sciences 101.51 (2004): 17867-17872).

[0251] Both ADCs are shown in Table 6. For comparison, a trastuzumab-based naked (unmasked) ADC (ADC-TRA41C) was also treated with MMP2.

[0252] The results are shown in Figure 5 and show dose-dependent binding of masked and MMP2-treated (demasked) ADC-S423 and ADC-S426 in HER2-positive SK-BR-3 cells compared to ADC-TRA41C binding (with / without MMP2 treatment). Data represent the MFI (median fluorescence intensity) of one experiment.

[0253] The masked ADC-S423 showed a 540-fold EC shift compared to ADC-TRA41C, with an EC less than 2, indicating almost complete recovery of HER2 target binding when the mask was removed after MMP2 protease treatment. ADC-S426 showed a 137-fold EC shift compared to ADC-TRA41C and did not show recovery of HER2 target binding after MMP2 protease treatment. 50 The trastuzumab-mediated binding on HER2-positive SK-BR-3 cells was effectively masked by the two masked ADCs (ADC-S423 and ADC-S426). For ADC-S423 after demasking by MMP2 treatment, the binding was restored. The data also showed that linker L13 was insensitive to MMP2 as the binding did not recover after MMP2 treatment. 50 Example 7 Cytotoxicity of Masked / MMP2-Treated ADC-S423 and ADC-S426 in HER2-Positive SK-BR-3 Cells 50 To examine the effect of MMP2 protease-mediated demasking on the cell viability of masked ADC-S423, the cytotoxicity of masked and demasked (MMP2-treated) ADC-S423 and ADC-S426 was tested in HER2-positive SK-BR-3 cells and compared to naked (unmasked) trastuzumab-based ADC-TRA41C and isotype control ADC-RIT41C. Cell viability was measured using the CellTiter-Glo® (CTG) luminescence assay kit after 6 days of treatment.

[0254] The results are shown in Figure 4, where cytotoxicity (shown as % cell survival) was induced in HER2-positive SK-BR-3 cells with various concentrations of ADC ADC-S423, ADC-S426, ADC-TRA41C, and isotype control ADC-RIT41C.

[0255] HER2-positive SK-BR-3 cells

[0256]

[0257] ​​IC obtained by CTG readout after 6 days of treatment of HER2-positive SK-BR-3 cells with masked ADC-S423 and ADC-S426 and their respective MMP2-treated ADCs 50 is shown in Table 14.

[0258] Masked ADC-S423 shows a 36-fold IC 50 shift compared to ADC-TRA41C, but almost complete recovery of cytotoxicity induction is observed upon de-masking after MMP2 protease treatment (1.3-fold IC 50 shift of de-masked ADC-S423 compared to ADC-TRA41C).

[0259] The scrambled linker in (ADC-)L026S426 cannot be cleaved by MMP2, as can be seen by 30-fold and 27-fold shifts equivalent to those of masked and MMP2-treated ADC-S426 compared to ADC-TRA41C.

[0260] [Table 14]

[0261] Example 8 Cell binding of half-masked Ab (A) and half-demasked ADC (B) to HER2-positive SK-BR-3 cells, and cytotoxicity of half-(de)masked ADC (C) to HER2-positive SK-BR-3 cells To investigate whether half-masked Abs or ADCs can still bind to the target and induce cell death as effectively as the original (unmasked) Abs or ADCs, the cell binding and cytotoxicity of half-masked antibodies and ADCs were tested in HER2-positive SK-BR-3 cells. "Half-masked" in this experiment means that the masking moiety containing the Nb or antigen peptide complexes with the HC or LC of the "half-masked" antibody.

[0262] 8A: Cell binding of half-masked Ab to HER2-positive SK-BR-3 cells The cell binding of the half-masked antibodies S109 and S110 was compared with that of trastuzumab. The dose-dependent binding of the antibodies to HER2-positive SK-BR-3 cells was measured by flow cytometry. The data shown in Figure 7A represent the median fluorescence intensity (MFI) of two experiments. Table 15 shows the EC 50 and the EC compared with trastuzumab 50 shift, showing the binding characteristics of the half-masked antibodies S109 and S110. The binding of the half-masked Ab to the HER2 target was significantly reduced by more than 4-fold in potency compared with trastuzumab, but binding was still possible.

[0263]

Table 15

[0264] 8B: Cell binding of (half-demasked) ADC-S424 and ADC-S425 compared with ADC-TRA41C in HER2-positive SK-BR-3 cells.

[0265] For ADC, ADC-S424, and ADC-S425, complete masking is provided, and one masking moiety is attached to a non-cleavable linker (detailed in Table 16). In ADC-S424, the BC2T antigen peptide is attached to the HC by a non-cleavable linker, and in ADC-S425, the Nb BC2 is attached to the LC by a non-cleavable linker. ADC, ADC-S424, and ADC-S425 are completely masked before MMP2 treatment and are "half-demasked" after MMP2 treatment. Cell binding experiments in HER2-positive SK-BR-3 cells were performed as described above for the half-masked antibodies S109 and S110. MMP2 treatment of ADC-S424 and ADC-S425 was performed as described above.

[0266] For comparison, a trastuzumab-based naked (unmasked) ADC (ADC-TRA41C) and an isotype control (ADC-RIT41C) were obtained.

[0267] The results of dose-dependent binding of masked and MMP2-treated (half-demasked) ADC-S424 and ADC-S425 in HER2-positive SK-BR-3 cells compared to ADC-TRA41C and ADC-RIT41C binding are shown in Figure 7B. Data represent the MFI (median fluorescence intensity) of one experiment.

[0268] Masked ADC-S424 and ADC-S425 bound very little to cells, as shown by a very flat dose-response curve. After MMP-2 treatment, which removed only one masking moiety, cell binding was greatly restored for half-demasked ADC-S424 and ADC-S425, but clearly did not fully recover to the level of naked ADC-TRA41C. The results are shown in Table 16.

[0269]

Table 16

[0270] 8C:Cytotoxicity of half-(de)masked ADCs in HER2-positive SK-BR-3 cells compared to ADC-TRA41C and isotype control ADC-RIT41C The cytotoxicity induced by half-masked ADCs ADC-S109, ADC-S110, ADC-S111 (detailed in Table 6) and ADC-S424 and ADC-S425 in HER2-positive SK-BR-3 cells was compared to ADC-TRA41C and isotype control ADC-RIT41C.

[0271] ADCs ADC-S109, ADC-S110 and ADC-S111 are based on antibodies with only one masking moiety (containing Nb BC2). Therefore, these ADCs are "half-masked". ADCs ADC-S109, ADC-S110 and ADC-S111 were not treated with MMP2.

[0272] The cytotoxicity of ADC-S424 and ADC-S425 was determined before and after MMP2 treatment. After MMP2 treatment, the masking moiety conjugated via a non-cleavable linker remained bound to the antibody in ADC-S424 and ADC-S425 (the ADCs are "half-masked").

[0273] The results are shown in Figure 7B (for ADC-S109, ADC-S110 and ADC-S111) and Figure 7C (for ADC-S424 and ADC-S425). Cell viability was measured with the CellTiter-Glo® (CTG) luminescence assay kit after 6 days of treatment. The data represent the viability of two experiments.

[0274] Table 17 shows the IC 50 determined by the CTG readout after 6 days of treatment of HER2-positive SK-BR-3 cells with half-masked ADCs ADC-S109, ADC-S110, ADC-S111, ADC-S424, ADC-S425 and ADC-TRA41C (the ADCs are shown in Table 6). The shift in IC 50 potency induced by the concept of half-masking is associated with ADC-TRA41C.

[0275]

Table 17

[0276] After target binding, the half-masked ADCs ADC-S109, ADC-S110, ADC-S111 induce efficient cell death but have a two-fold reduced potency compared to ADC-TRA41C and reach a cell killing potency almost similar to that of the unmasked ADC, indicating that cleavage of one linker is sufficient. No differences in binding or cytotoxic potency were observed in relation to the differences in masking of the half-masked Abs and ADCs ADC-S109, ADC-S110, ADC-S111.

[0277] Table 18 shows the IC determined by CTG readout after 6-day treatment of HER2-positive SK-BR-3 cells with ADC ADC-S424, ADC-S425, and ADC-TRA41C (ADCs are shown in Table 6). 50 The IC induced by the concept of half-masking 50 The shift in potency is associated with the corresponding masked ADC (ADC-TRA41C does not have a maximum in the DRC and the IC 50 cannot be determined).

[0278]

Table 18

[0279] After MMP2 treatment, the half-demasked ADCs ADC-S424 and ADC-S425 also induce efficient cell death compared to their fully masked (before MMP2 treatment) counterparts, but the potency is 11-fold lower compared to ADC-TRA41C.

[0280] Example 9 HMW Measurement in the Production of Masked ADCs with Various DARs To investigate the effect of mutations in the BC2-Nb sequence and BC2T peptide sequence on HMW (%) in the production of ADCs with various drug-to-antibody ratios (DARs). ADCs with various DARs were synthesized based on the masked antibodies S103 and S211. S103 has a mask based on BC2-Nb and BCT2 peptide, and S211 has a mask with mutations introduced into BC2-Nb and BCT2 peptide. S103 is detailed in Table 1 and S211 is detailed in Table 3. For convenience, both antibodies are shown again in Table 19.

[0281]

Table 19

[0282] ADCs based on the masked antibodies S103 and S211 with the linker-drug LD2 (Elgersma et al. (supra)) having various DARs were synthesized by the following procedure.

[0283] The solution of the masked antibody was mixed with buffer (4.2 mM histidine, 50 mM trehalose, pH 6), 1 M TRIS*HCl (pH 8), 25 mM EDTA solution, and different aliquots of 2 mM TCEP solution. Different aliquots of TCEP were used to obtain a certain range of DAR after complexation (different amounts of TCEP produce different amounts of thiols available for complexation, and the amount of thiols determines the amount of conjugated linker-drug).

[0284] The final conditions were 2 mg / mL antibody, 10 mM TRIS, 1 mM EDTA, and TCEP (1 - 1.6 molar equivalents of the antibody). After adding N,N-dimethylacetamide (DMA), a solution of linker-drug (10 mM in DMA, 3 molar equivalents of linker-drug with respect to TCEP) was added. The final concentration of DMA was 5%.

[0285] The resulting mixture was incubated at RT for 3 hours in the dark, and then the samples were taken for analysis by size exclusion chromatography (SEC) for macromolecules by the following method.

[0286] For the soluble protein aggregation analysis of the ADC, an SEC method with UV detection was used. The (masked) antibody was diluted to a concentration of 1 mg / mL and injected into a Waters Acquity UPLC BEH200 SEC with 100 mM PO 4 buffer pH 7.5 containing 10% 2-propanol as the mobile phase. The ratio of the main peak (monomer) to the high molecular weight (HMW) species (e.g., soluble aggregates) was determined based on the peak area relative to the total peak area of all protein-related peaks.

[0287] The average DAR of the ADC was determined by HIC chromatography as described in Coumans et al, 2020 (supra).

[0288] Figure 9 shows the polymer-to-average DAR of the ADCs tested. The tested DAR is shown on the X-axis, and the percentage of HMW of the resulting product is shown on the Y-axis.

[0289] It is clear that the HMW(%) of ADC-S211 is lower at the tested DAR compared to ADC-S103.

[0290] Example 10 Selection of Linker (Masked Anti-TF Antibody) All possible combinations of the linkers exemplified in Table 18 were used to produce a set of 324 masked antibodies in a method similar to the method described in Example 1. The masking portion used in each case was a pair of masking portions containing a masking portion having a BC2T SA mutant peptide linked to each LC of the anti-TF antibody tisotumab and a masking portion having a BC2-Nb RS mutant nanobody linked to each HC. In each antibody, a Qg sequence was present before the sequence of the antigen peptide.

[0291] In the set of linkers in Table 20, linkers 14 - 25 all contain the protease cleavage site LSGRSDNH but have different lengths, and linkers L26, L27, L9, L29, L7, and L30 all contain the PLGLAG protease cleavage site but also have different lengths. This set of linkers allows for testing of combinations of linkers having the same cleavage site for the two masking portions but different linker lengths. The set also allows for combinations of linkers with different cleavage sites and / or lengths, and of course includes combinations using exactly the same linker for the two masking portions. By testing all possible combinations (18×18 = 324 combinations), a linker pair can be selected that optimizes, for example, the expression level and / or HMW(%).

[0292] For all the antibodies produced, the amount of antibody obtained (μg / mL) and HMW(%)(the percentage of all antibodies lost due to the formation of high molecular weight complexes) were determined.

[0293]

Table 20

[0294] All antibodies were tested for their binding to immobilized target tissue factor antigen in BLI, essentially as described in Example 4.

[0295] The amount of antigen obtained for each antibody is shown in Figure 10A.

[0296] The HMW (%) of each antibody is shown in Figure 10B.

[0297] The results of the BLI binding experiment are shown in Figure 10C as binding to tissue factor / binding to Protein A × 1000.

[0298] The results for the control antibody S312 (naked chimeric antibody) are similarly shown in the tables of Figures 10A - 10C.

[0299] By setting a predefined cut - off for each measured parameter and combining the results of the three graphs, the selection of linker combinations for this particular antibody can be defined.

[0300] It is evident that the majority of the antibodies (264 out of 324) could be produced at expression levels exceeding 100 μg / mL. For the majority of the antibodies, HMW (%) was less than 10%, but for 86 linker combinations, HMW (%) was less than 5%, and for 13 combinations, it was even less than 3%.

[0301] When comparing the binding in the BLI experiment to that of the parental antibody, a significant decrease in binding was observed for all masked antibodies tested; the binding parameter for the parental antibody chimeric antibody was approximately 750, and for all masked antibodies, the binding parameter was less than 150, and for nearly 50% of the antibodies, it was even less than 40.

[0302] Combining three parameters (expression level > 100 μg / mL, HMW(%) < 4% and BLI binding parameter < 40), 52 antibodies passed the test.

[0303] Even when the criterion for HMW(%) was set to less than 3%, 14 antibodies met all the criteria. Ten out of these 14 could be produced at an expression level exceeding 200 μg / mL.

[0304] Of course, further comparisons can be made for a subset of the antibodies selected as described above, for example, in a cell binding assay and / or using measurement of cytotoxicity.

[0305] The experiments show that good results can be obtained by varying the length of the linker and by combining linkers with the same or different protease cleavage sites.

[0306] ADC of the antibody selections of Examples 11 and 12 (from Example 10) Twelve of the masked antibodies tested in Example 10 were selected for conjugation with DUBA to produce masked antibody-drug conjugates (masked ADCs).

[0307] The selected antibodies and their linkers are shown in Table 21.

[0308] The ADCs were prepared based on these antibodies by subjecting the antibodies to typical selective reduction and DUBA conjugation conditions as described in Example 3.

[0309] For all the masked antibodies, a very slight increase in HMW(%) was observed, and the final formulated form contained 3 - 8% HMW species with a DAR of 1.9. The unmasked ADC had 1.4% HMW and a DAR of 1.9 as well.

[0310] [Table 21]

[0311] Example 12 In Vitro Characterization of Selected Masked Anti-TF ADCs of 12 The 12 ADCs shown in Table 21 were tested in vitro.

[0312] The cytotoxicity of the 12 ADCs was tested essentially as described in the materials and methods of Example 5. Since the antibodies used for ADC1 to ADC13 were the anti-tissue factor (TF) antibody tisotumab, the cytotoxicity was determined in TF-expressing cells (instead of the HER2-positive tumor cell line used in Example 5). The cytotoxicity was tested using three cell lines: HCT-116, BxPC3, and FaDu, and their antigen (TF) densities were approximately 30,000, approximately 200,000, and approximately 400,000, respectively, and were classified as low, "moderate," and high. The TF antigen expression on the surface of human tumor cell lines was determined by APC-conjugated secondary F(ab’)2 goat anti-human IgG (Fc fragment specific) antibody according to the manufacturer's (Biocytex, Marseille, France) protocol using antibody S312 (naked tisotumab) and the human IgG Calibrator kit.

[0313] There appeared to be a correlation between the antigen expression level and the activity of the masked ADCs.

[0314] In the low-expression HCT116 cell line, the masked ADCs had curves that overlapped with the isotype (unbound) control ADCs, suggesting that at low levels of target density (e.g., healthy tissue), target-mediated activity is hardly predicted.

[0315] In the moderate- and high-expression BxPC3 and FaDu, the masked ADCs showed higher activity than the isotype control (smaller IC between the masked and unmasked / half-unmasked ADCs) 50(As shown by the window), at higher antigen concentrations (e.g., in tumors), the masked ADCs can start to exert an anti-tumor effect (or at least start to show tumor accumulation) even without being demasked.

[0316] The half-demasked ADCs have an IC 50 value equivalent to that of the fully demasked ADCs.

[0317] All ADCs (masked vs. demasked) lack cytotoxicity against TF-negative BT474 and IncuCyte® Jurkat NucLight® Red (data not shown).

[0318] Differences in activity and masking efficiency were observed among individual masked ADCs (with different linkers used for the connection between the masking moiety and the antibody), but all ADCs were effectively masked.

[0319] Demasking and partial demasking restored the cell-killing ability of the ADCs.

[0320] The results of the cytotoxicity tests are presented as IC 50 shifts in Table 22.

[0321] For each ADC (ADC1 - 12), the IC 50 shift is provided by comparing with the dose - response curve of unmasked ADC13 (the first column). This value provides an indicator of the efficiency of the mask.

[0322] In the next column, the IC 50 shift of the demasked ADCs (protease - treated) compared to unmasked (naked) ADC13 is shown. Here the values are very small, indicating that the demasked ADCs bind to the target antigen in the same way as the naked ADCs (which never had a mask in the first place).

[0323] The following two columns provide the results of the treatment of ADC1-12 by only one protease (MMP2 or matriptase). For ADCs (ADC1, ADC2, ADC5, ADC6, ADC11 and ADC12) constructed by a pair of linkers, where one linker contains an MMP cleavage site and the other linker contains a matriptase cleavage site, cleavage by one or the other protease results in a semi-(de)masked ADC in that only one linker is cleaved and only one masking moiety is thus cleaved from the antibody. For only those semi-(de)masked ADCs, the IC 50 shift is shown in Table 22 compared to the dose-response curve of unmasked ADC13. As can be understood from Table 22, the semi-(de)masking is equivalent to the dose-response of the fully de-masked antibody and similarly has an equivalent dose-response to unmasked ADC13 (IC 50 shift shown by the low value). This clearly shows that after cleavage of only one linker of the masked ADC, the binding to the target antigen has already been restored.

[0324] From this table, it is clear that the (semi-)demasking of the masked ADCs shifts the dose-response curve, indicating that all masked ADCs are effectively masked and efficiently demasked.

[0325]

Table 22

[0326] Example 13 In Vivo Testing of Masked ADCs Xenograft studies were performed at an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited CRO using well-established protocols developed and maintained at these CROs.

[0327] The masked anti-TF ADCs 1-13 were tested in vivo in a patient-derived xenograft (PDX) model; the non-small cell lung cancer model LXFA629. TF expression in this model was evaluated using immunohistochemistry (IHC). Tumor tissues were fixed with formalin, embedded in paraffin, and used for TF IHC using a rabbit monoclonal anti-TF antibody of Cell Signaling Technologies number #97438. IHC staining showed TF 2+ expression levels.

[0328] LXFA629 tumors were cut into fragments (fragments 3-4 mm in length) and unilaterally SC transplanted into the flanks of immunodeficient CES1c KO SCID mice. When the volume of the tumor graft reached 80-250 mm 3 of the target, the mice were assigned to treatment groups (n = 6 / group) such that the median and mean of the tumor volumes of the groups were equivalent. The mice were then, the next day, given a single IV injection via the tail vein of a dose of 2 or 2.4 mg / kg ADC in a buffer containing 4.2 mM histidine, 50 mM trehalose, pH 6.0 (buffer SOL092).

[0329] The antitumor activity of the ADC was evaluated by measuring tumor size twice a week until day 42. The length and width of the tumor were measured with calipers, and the tumor volume was calculated using the formula

[0330]

Number

[0331] (Simpson-Herren et al., 1970).

[0332] When the tumor of an individual mouse reached 2000 mm 3 the mouse was sacrificed, and complete group data were not shown from that point onwards.

[0333] All masked ADCs showed efficacy in the same range as the efficacy of the unmasked anti-TF ADC.

[0334] The results are shown in Figure 11; each data point represents the mean of the absolute tumor volumes (mm 3 ) for all mice in each group.

[0335] Figure 12 shows the absolute tumor volumes of individual mice in each group on the last day of the experiment (day 42). The line indicates the mean.

[0336] Example 14 Use of alternative Nb / peptide tags in the masking moiety The masking moiety was designed based on the "ALFA tag" as described in WO 2020 / 053239.

[0337] For the design of the masking moiety, variants with lower affinity for the ALFA tag peptide were selected. This variant, Nb PE is disclosed in SEQ ID NO: 134 of WO 2020 / 053239 (SEQ ID NO: 91). The second masking moiety was based on a peptide sequence containing the ALFA tag (SEQ ID NO: 92).

[0338] Based on the use of these sequences in the masking moiety, the anti-HER2 antibody trastuzumab was masked and thereby the peptide tag was linked to the N-terminus of the HC using a linker containing the "Qg" spacer sequence and the PLGLAG cleavage site, and Nb PE was linked to the LC using a linker containing the same cleavage site. A set of linkers (L12, L9, L7, L34 and L5) was used in all possible combinations and a set of 25 masked antibodies was obtained. The linkers used are shown in Table 23.

[0339]

Table 23

[0340] Masked antibodies were produced and purified. After purification, the expression levels and the percentage of HMW complexes were determined as described in Example 1. The expression levels of all the produced masked antibodies exceeded 100 μg / mL, and most of the antibodies exceeded 200 μg / mL. The HMW (%) was very low (about 1%) for all combinations of linkers tested. The expression levels are shown in Table 24, and the HMW (%) is shown in Table 25.

[0341]

Table 24

[0342]

Table 25-1

[0343] Using the BLI method essentially as described in Example 4, the binding of the masked antibodies to the immobilized HER2 protein was measured. The BLI measurements showed that effective protection could be achieved by masking the trastuzumab antibody with the masking moiety based on the ALFA tag (Nb PE ) and the ALFA tag peptide (tag ALFA).

[0344] The results of the BLI binding of all 25 antibodies are shown in Table 25. All the antibodies were efficiently masked (binding to HER-2 was significantly reduced), but the masking seemed to be most efficient with the shorter linkers (L5, L34, and L7). For comparison, the BLI value obtained for unmasked (naked) trastuzumab was 331.2.

[0345]

Table 25-2

[0346]

Table 26-1

[0347]

Table 26-2

[0348]

Table 26-3

[0349]

Table 26-4

[0350]

Table 26-5

[0351]

Table 26-6

[0352]

Table 26-7

[0353]

Table 26-8

[0354]

Table 26-9

Claims

**Claim 1** An activatable antibody or an antigen-binding fragment thereof containing an antigen-binding site that contains a heavy-chain variable domain and a light-chain variable domain capable of binding to a target protein, wherein the antigen-binding site is provided with a mask suitable for blocking the binding between the antigen-binding site and the target protein, and the mask has two masking portions: i. a masking portion linked to the N-terminus of the heavy-chain variable domain, and ii. a masking portion linked to the N-terminus of the light-chain variable domain, and at least one of the masking portions is bound via a cleavable linker, wherein one masking portion contains an antigen peptide and the other masking portion contains a single-domain antibody (sdAb) that specifically binds to the antigen peptide, an activatable antibody or an antigen-binding fragment thereof. **Claim 2** The activatable antibody or an antigen-binding fragment thereof according to claim 1, wherein all the masking portions are bound via cleavable linkers. **Claim 3** The activatable antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the cleavable linker is a peptide linker cleavable by a protease. **Claim 4** The activatable antibody or an antigen-binding fragment thereof according to any one of the preceding claims, wherein the sdAb is a nanobody. **Claim 5** The activatable antibody or an antigen-binding fragment thereof according to any one of the preceding claims, which contains two antigen-binding sites, and masks are provided for these two antigen-binding sites. **Claim 6** The activatable antibody or an antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen peptide has 4 to 20 amino acid residues. **Claim 7** The activatable antibody or an antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen peptide has 12 to 20 amino acid residues and contains the amino acid sequence defined by SEQ ID NO: 51 or consists of the amino acid sequence defined by SEQ ID NO:

51. **Claim 8** The activatable antibody or an antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen peptide contains the amino acid sequence defined by SEQ ID NO: 1, SEQ ID NO: 2 (SA variant) or SEQ ID NO: 3 (ST variant). **Claim 9** The nanobody contains the amino acid sequence defined by SEQ ID NO: 4, its humanized version, the amino acid sequence defined by SEQ ID NO: 76 (RA variant), SEQ ID NO: 77 (RE variant) or SEQ ID NO: 78 (RS variant), a variant of SEQ ID NO: 4 having the amino acid sequence, or a humanized version of the variant, and is the activatable antibody or antigen-binding fragment thereof according to any one of claims 4 to 8.

10. The antigen peptide contains a peptide having the amino acid sequence defined by SEQ ID NO: 2 (SA variant) or consists of a peptide having the amino acid sequence defined by SEQ ID NO: 2 (SA variant), and the nanobody contains the amino acid sequence defined by SEQ ID NO: 78 (RS variant) or its humanized version, and is the activatable antibody or antigen-binding fragment thereof according to claim 9.

11. The masking portion containing the sdAb is linked to the N-terminus of the light chain variable domain, and the masking portion containing the antigen peptide is linked to the N-terminus of the heavy chain variable domain, and is the activatable antibody or antigen-binding fragment thereof according to any one of the preceding claims.

12. The cleavable linker contains one or more cleavage sites recognized by one or more tumor-specific proteases, and is the activatable antibody or antigen-binding fragment thereof according to any one of the preceding claims.

13. At least one cleavable linker contains a cleavage site recognized by matriptase or a cleavage site recognized by a metalloprotease, and is the activatable antibody or antigen-binding fragment thereof according to any one of the preceding claims.

14. An antibody-drug conjugate (ADC) containing the activatable antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a linker-drug.

15. A pharmaceutical composition containing the activatable antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the ADC according to claim 14 and a pharmaceutically acceptable additive.

16. The activatable antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the antibody-drug conjugate according to claim 14 or the pharmaceutical composition according to claim 15 for use as a medicament.

17. An activatable antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, an antibody-drug conjugate according to claim 14, or a pharmaceutical composition according to claim 15, for use in the treatment of cancer, an autoimmune disease or an infectious disease, preferably for use in the treatment of cancer.

18. A nucleotide sequence encoding a heavy chain variable domain, a protease-cleavable peptide linker, and a masking moiety; and / or A nucleotide sequence encoding a light chain variable domain, a cleavable peptide linker, and a masking moiety; A nucleic acid construct containing The nucleotide sequence is operably linked to an expression control sequence for expression in a host cell, preferably a mammalian host cell.

19. A host cell containing the nucleic acid construct according to claim 18.

20. A method for producing an activatable antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the method comprising culturing the host cell according to claim 19 under conditions that promote the expression of the activatable antibody or antigen-binding fragment thereof.

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