Multispecific binding molecule protein and its use

Abstract

The present disclosure provides a protein of an antigen-binding molecule comprising a multispecific binding molecule (MBM). The antigen-binding molecule protein comprises an antigen-binding site, one or more components that inhibit the binding of the antigen-binding site to its target, and a protease-cleavable linker that can be cleaved by a protease, the cleavage of which removes the inhibition of the binding of the antigen-binding site to its target. The MBM protein and MBM resulting from the cleavage of the protease-cleavable linker, e.g., tandem Fab MBM, comprise a T cell-engaging antigen-binding site and a tumor-associated antigen-binding site. The MBM protein further comprises a component that inhibits the binding of the T cell-engaging antigen-binding site to its target and a protease-cleavable linker that can be cleaved by a protease within the tumor microenvironment, the cleavage of which removes the inhibition of the binding of the T cell-engaging antigen-binding site to its target. The present disclosure further provides a pharmaceutical composition comprising the antigen-binding molecule protein, a method of using the antigen-binding molecule protein in therapy, as well as a nucleic acid encoding the antigen-binding molecule protein, a recombinant cell expressing the antigen-binding molecule protein, and a method of producing the antigen-binding molecule protein.

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 340,891, filed on May 11, 2022, U.S. Provisional Application No. 63 / 481,291, filed on January 24, 2023, and U.S. Provisional Application No. 63 / 489,812, filed on March 13, 2023, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically in XML file format, the entire contents of which are hereby incorporated by reference. The XML copy created on May 9, 2023, is named RGN - 017WO_SL.xml and is 307,242 bytes in size.

Background Art

[0003] 3. Background Selectively destroying tumor cells while leaving healthy cells and tissues intact and undamaged is the goal of cancer immunotherapy. Bispecific antibody therapeutics have been developed to achieve this goal by inducing an immune response against tumors. In this regard, bispecific antibodies are designed to bind to both tumor - associated antigens ( "TAAs") that are preferentially expressed on tumor cells and components of the T - cell receptor ( "TCR") complex. Simultaneous binding of such an antibody to both of its targets results in the activation of cytotoxic T cells and the subsequent lysis of TAA - expressing cells. Thus, the immune response is redirected to TAA - expressing cells.

[0004] Several bispecific antibody formats have been developed and their suitability for T - cell - mediated immunotherapy has been investigated. See Non - Patent Document 1. The task of generating bispecific molecules suitable for therapy poses several technical challenges related to toxicity because TAAs are typically expressed on both normal and tumor cells. Therefore, there is a need for effective T cell-activating bispecific molecules that selectively induce T cell activation in the tumor microenvironment.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] 4. Summary The present invention generally relates to a novel protease-activatable antigen-binding molecule (「ABM」), herein referred to as an antigen-binding molecular proprotein.

[0007] Generally, in their active state, antigen-binding molecules contain an antigen-binding site (ABS) capable of binding to their target. However, in the proprotein form, the ABS is masked by a masking moiety such that its ability to bind to its target is significantly reduced. The antigen-binding molecular proprotein is configured such that upon encounter with a protease, e.g., a protease overexpressed in the tumor microenvironment, the masking moiety is cleaved, generating a binding molecule with enhanced target binding. This is achieved by including one or more protease-cleavable linkers (「PCL」) containing one or more protease substrate sequences, either directly or indirectly, e.g., between the ABS and the masking moiety. The proprotein can thus result in a reduction of toxicity and adverse side effects that may otherwise result from binding of the ABM to normal tissues. Generally, antigen-binding molecular proproteins are described in Section 6.2 and Group A numbered embodiments 1-43, and Group B numbered embodiments 1-90.

[0008] In some embodiments, the antigen-binding molecular proteins of the present disclosure are multispecific binding molecule (MBM) proteins. Generally, in their active states, the MBMs of the present disclosure can simultaneously bind to their respective targets such that T cells bound by the TCE ABS stimulate TAA-expressing tumor cells bound by the TAA ABS to attack. The MBMs include a T cell-engaging antigen-binding site (the "TCE ABS") that binds to a component of the T cell receptor complex and a tumor-associated antigen-binding site (the "TAA ABS"). However, when the MBM is in protein form, the TCE ABS is masked by a masking moiety such that its ability to bind to its target is significantly reduced. The MBM protein is configured such that when it encounters a protease, for example, a protease overexpressed in the tumor microenvironment, the masking moiety is cleaved and the inhibition of TCE ABS binding is reversed. This is achieved, for example, by including a protease-cleavable linker (the "PCL") that contains one or more protease substrate sequences in the MBM protein, either directly or indirectly, for example, between the masking moiety and the TCE ABS. The protein can otherwise result in a reduction of toxicity and adverse side effects that may be caused by the binding of the MBM to normal tissues. Generally, the MBM proteins are described in Section 6.3 and Group B numbered embodiments 1-5, 15, 54, and 69-72.

[0009] In certain embodiments, the MBM protein (referred to herein as type I MBM) comprises an anti-idiotype of a TCE ABS that reversibly masks the binding of the TCE ABS to its target, referred to herein as an “anti-TCE ABS”. Thus, type I MBMs comprise an anti-TCE ABS masking moiety. In some embodiments, the MBM protein is configured such that activation of the MBM protein releases an MBM that comprises a full Fc region (e.g., a bispecific binding molecule). This type of MBM protein is referred to as an IA type MBM protein, and exemplary embodiments of IA type MBMs are depicted in FIGS. 1A-1C and described in Section 6.3.1.1 and Group B numbered embodiments 6-8 and 18-24. In other embodiments, the MBM protein is configured such that activation of the MBM protein releases an MBM that lacks an Fc region, such as a tandem Fab or BiTe. This type of MBM protein is referred to as an IB type MBM protein, and exemplary embodiments of IB type MBMs are depicted in FIGS. 5A-5B and described in Section 6.3.1.2 and Group B numbered embodiments 16 and 34-40.

[0010] In other embodiments, the TCE ABS of the MBM protein (referred to as type II MBM) is sterically hindered from binding to its target by proximity to an adjacent domain, such as an Fc domain, that sterically hinders its binding to the target. In some embodiments, the MBM protein is configured such that activation of the MBM protein releases an MBM that includes a complete Fc region (e.g., a bispecific binding molecule). This type of MBM protein is referred to as a type IIA MBM protein, and exemplary embodiments of type IIA MBM are depicted in FIGS. 3A - 3F and described in Section 6.3.1.1 and Group B numbered embodiments 9 - 14 and 25 - 33. In other embodiments, the MBM protein is configured such that activation of the MBM protein releases an MBM that lacks an Fc region, such as a tandem Fab or BiTe. This type of MBM protein is referred to as a type IIB MBM protein, and exemplary embodiments of type IIB MBM are depicted in FIG. 7 and described in Section 6.3.1.2 and Group B numbered embodiments 17 and 41 - 49.

[0011] Section 6.4, Group A numbered embodiments 29 - 31, and Group B numbered embodiments 50 - 53 describe exemplary protease-cleavable linkers that can be used for antigen-binding molecular proteins that include the MBM proteins of the present disclosure.

[0012] The present disclosure further provides tandem Fab MBMs that include TAA ABS and TCE ABS. The tandem Fab can be produced by activation of a type IB or type IIB MBM as a protein, or it can be generated through expression of a tandem Fab protein (having the relevant signal sequence but no masking portion). In such cases, the TAA ABS and TCE ABS preferably share a common light chain sequence. Section 6.11 and Group B numbered embodiments 75 - 83 describe exemplary tandem Fab MBMs.

[0013] Section 6.5 and Embodiment 55 with Group B numbering describe exemplary non-cleavable linkers that can be incorporated into antigen-binding molecular proteins and tandem Fab MBMs that include the MBM proteins of the present disclosure. Section 6.6, Embodiment 23 with Group A numbering, and Embodiments 56 - 64 with Group B numbering describe exemplary TAA ABSs that can be incorporated into antigen-binding molecular proteins and tandem Fab MBMs that include the MBM proteins of the present disclosure. Section 6.7, Embodiment 22 with Group A numbering, and Embodiments 64 - 68 with Group B numbering describe exemplary TCE ABSs that can be incorporated into antigen-binding molecular proteins and tandem Fab MBMs that include the MBM proteins of the present disclosure. Section 6.8 describes exemplary anti-TCE ABSs that can be incorporated into antigen-binding molecular proteins that include the MBM proteins of the present disclosure. Section 6.9 describes suitable formats for ABSs incorporated into antigen-binding molecular proteins that include the MBM proteins of the present disclosure. Section 6.10 and Embodiments 73 and 74 with Group B numbering describe suitable Fc domains that can be incorporated into antigen-binding molecular proteins that include the MBM proteins of the present disclosure.

[0014] The present disclosure further provides nucleic acids (either in a single nucleic acid or multiple nucleic acids) encoding antigen-binding molecular proteins and tandem Fab MBMs that include the MBM proteins of the present disclosure, and recombinant host cells and cell lines engineered to express antigen-binding molecular proteins that include the nucleic acids and MBM proteins of the present disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 6.12, Embodiments 47 and 48 with Group A numbering, and Embodiments 87 - 92 with Group B numbering.

[0015] Pharmaceutical compositions that include antigen-binding molecular proteins and tandem Fab MBMs that include the MBM proteins of the present disclosure are also provided. Examples of pharmaceutical compositions are described in Section 6.13, Embodiment 44 with Group A numbering, and Embodiment 84 with Group B numbering.

[0016] For example, provided herein are methods of using an antigen-binding molecular protein, tandem Fab MBM, and pharmaceutical composition of the present disclosure's MBM protein for treating a proliferative condition (e.g., cancer) in which TAA is expressed thereon. Exemplary methods and indications are described in Section 6.14, Embodiments 45 and 46 numbered in Group A, and Embodiments 85 and 86 numbered in Group B.

[0017] 5. Brief Description of the Drawings

Brief Description of the Drawings

[0018]

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[0019] 6. DETAILED DESCRIPTION 6.1. Definitions As used herein, the following terms are intended to have the following meanings.

[0020] ABM protein: The term "ABM protein" (or "antigen-binding molecule protein") as used herein refers to an ABM that, due to the presence of a masking moiety, for example, has a reduced or inhibited ability to bind to a target recognized by at least one of its ABSs. The masking moiety impedes the binding of the ABS to its target and is separated from the ABM by proteolytic cleavage, for example, by proteolytic cleavage of a protease-cleavable linker that connects the masking moiety to the ABM.

[0021] ABS chain: Individual ABSs can exist as one polypeptide chain (e.g., in the case of scFv) or in the form of an association of two or more polypeptide chains (e.g., in the case of Fab). As used herein, the term "ABS chain" refers to all or a portion of an ABS present on a single polypeptide chain. The use of the term "ABS chain" is intended for convenience and illustrative purposes only and does not imply a particular configuration or method of production. Further, references to an ABS in the context of describing an ABM, ABM protein, MBM, or MBM protein include an ABS chain unless otherwise indicated by the context. Thus, when describing an ABM, ABM protein, MBM, or MBM protein in which an Fc domain is operably linked to an ABS, the Fc domain can be covalently linked directly or indirectly (e.g., via a linker) through a peptide bond to, for example, (1) the first ABS chain of a Fab (the other components of the Fab are on a second associated ABS chain) or (2) a single ABS chain containing an scFv.

[0022] About, approximately: Terms such as "about" and "approximately" are used before numerical values throughout the specification to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, measurement precision, and / or variations in accuracy, timing, etc.). It should be understood that the disclosure of "about X" or "approximately X", where X is a numerical value, is also a disclosure of "X". Thus, for example, the disclosure of an embodiment where an array has "about X% sequence identity" to another array is also a disclosure of an embodiment where the array has "X% sequence identity" to the other array.

[0023] Activate, activation: Terms such as "activation" and "activate" in the context of the present disclosure with respect to the MBM protein refer to protease-mediated enzymatic cleavage of a protease-cleavable linker, which results in the de-masking of the TCE ABS, and thus, for example, through the release or separation of the TCE ABS from an anti-TCE ABS or Fc domain that sterically hinders the binding of the TCE ABS to its target when the protease-cleavable linker is intact, leads to the production of an ABM or MBM where the ability of the TCE ABS to bind to its target is increased. Sometimes, activation is referred to herein as the "release" of the MBM, masking moiety, or TCE ABS.

[0024] And, or: Unless otherwise indicated, the conjunction "or" is intended to be used in its correct meaning as a Boolean operator that encompasses both the selection of features in an alternative (the selection of A is mutually exclusive from B, A or B) and the selection of combined features (both A and B are selected, A or B). In some places in the text, the term "and / or" is used for the same purpose and should not be interpreted to mean that "or" is used with reference to mutually exclusive alternatives.

[0025] Anti-idiotype antibody, anti-idiotypic antibody: Terms such as "anti-idiotype antibody" and "anti-idiotypic antibody" refer to antibodies that recognize the idiotype of an antigen-binding site, for example, an antigen-binding site specific for a TCR component such as CD3. Anti-idiotype antibodies can specifically bind to the variable region of the antigen-binding site, thereby reducing or preventing the specific binding of the antigen-binding site to its cognate antigen. When associated with a molecule containing an antigen-binding site, anti-idiotype antibodies can function as a masking moiety of the molecule. The antigen-binding component of an anti-idiotype antibody that recognizes the variable region of a T cell-engaging antibody, for example, an antibody that recognizes CD3 or another component of the T cell receptor, is often referred to herein as a "TCE ABS".

[0026] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can bind non-covalently, reversibly, and specifically to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated as CL herein). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that both the variable domain of the light chain portion (VL) and the variable domain of the heavy chain portion (VH) determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2, or CH3) confer important biological properties such as, for example, secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of the constant region domains increases as the constant region domain becomes more distal from the antigen-binding site or the amino terminus of the antibody. The N-terminus is the variable region, the C-terminus is the constant region, and the CH3 and CL domains represent the carboxy termini of the heavy and light chains of the native antibody, respectively. For convenience, and unless otherwise indicated in the context, reference to an antibody also refers to engineered antibodies, including antibody fragments, as well as antigen-binding sites that do not occur naturally and / or have non-natural configurations, for example, molecules having a C-terminal Fab or scFv domain against the CH3 domain.

[0027] Antigen-binding molecule, ABM: As used herein, the terms “antigen-binding molecule” and “ABM” refer to a molecule (e.g., an assembly of multiple polypeptide chains) that includes one or more antigen-binding sites. The ABMs of the present disclosure can be monospecific or multispecific (e.g., bispecific). All of the antigen-binding sites in a monospecific binding molecule bind to the same epitope, whereas a multispecific binding molecule has at least two antigen-binding sites that bind to different epitopes, which can be the same or different target molecules. In some embodiments, the antigen-binding molecule is an antibody.

[0028] Antigen-binding site: The term "antigen-binding site" or "ABS", as used herein, refers to the portion of an antibody, ABM, or MBM protein that has the ability to bind an antigen non-covalently, reversibly, and specifically in the absence of masking. For example, in some embodiments, the ABS can be masked in the context of an ABM or MBM protein, but has the ability to bind an antigen non-covalently, reversibly, and specifically when the ABM or MBM is produced by cleavage of a protease-cleavable linker in the ABM or MBM protein. Examples of antigen-binding sites include single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of an antibody; dAb fragments consisting of VH domains (Ward et al., 1989, Nature 341:544-546); VHH antibodies (also "VHH" or "Nanobody®", Vincke et al., 2012, Methods Mol Biol. 911:15-26), and antibody fragments such as isolated complementarity-determining regions (CDRs), but are not limited thereto. Thus, the term "antibody fragment" encompasses both proteolytic fragments of an antibody (e.g., Fab and F(ab) 2 fragments), as well as engineered proteins containing one or more portions of an antibody (e.g., scFv). Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136).

[0029] Associated: The term "associated" in the context of an ABM or MBM protein refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical bridges, so as to produce a functional MBM protein. Examples of associations that may be present in the MBM proteins of the present disclosure include associations between Fc regions within the Fc domain (homo-dimers or more preferably hetero-dimers as described in Section 6.10.2), associations between the VH and VL regions within a Fab or Fv, and associations between CH1 and CL within a Fab (but are not limited thereto).

[0030] Bivalent: The term "bivalent" in the context of an antigen-binding molecule, as used herein, refers to an antigen-binding molecule having two antigen-binding sites. The domains may be the same or different. Thus, a bivalent antigen-binding molecule can be monospecific or bispecific.

[0031] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital tract cancer, colon cancer, meninges cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any TAA-positive cancer of any of the aforementioned types.

[0032] CD3: The term "CD3" refers to a cluster of differentiation 3 co-receptors (or co-receptor complex, or polypeptide chains of the co-receptor complex) of the T cell receptor. The amino acid sequences of the polypeptide chains of human CD3 are provided in NCBI accession P04234, P07766, and P09693.

[0033] Complementary Determining Region: The term "complementary determining region" or "CDR", as used herein, refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The exact amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 (the "Chothia" numbering scheme), and the ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (the "IMGT" numbering scheme). For example, for the classical format, under Kabat, the CDR amino acid residues within the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3), and the CDR amino acid residues within the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids within VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues within VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).By combining both the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26 - 35 (CDR-H1), 50 - 65 (CDR-H2), and 95 - 102 (CDR-H3) within human VH, and amino acid residues 24 - 34 (CDR-L1), 50 - 56 (CDR-L2), and 89 - 97 (CDR-L3) within human VL. Under IMGT, the CDR amino acid residues within VH are numbered approximately 26 - 35 (CDR-H1), 51 - 57 (CDR-H2), and 93 - 102 (CDR-H3), and the CDR amino acid residues within VL are numbered approximately 27 - 32 (CDR-L1), 50 - 52 (CDR-L2), and 89 - 97 (CDR-L3) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0034] Effector function: The term "effector function" generally refers to the activity of an antibody molecule mediated by binding through domains of the antibody other than the antigen-binding site, which is mediated by the binding of effector molecules. Effector functions include, for example, complement-mediated effector functions mediated by the binding of the C1 component of complement to an antibody. Complement activation is important for the opsonization and lysis of cellular pathogens. Complement activation can also stimulate an inflammatory response and be involved in autoimmune hypersensitivity. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be induced upon binding of the Fc receptor (FcR) of the constant domain of an antibody. Binding of an antibody to an Fc receptor on the cell surface induces a number of important and diverse biological responses, including the uptake and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, transplacental transfer, and regulation of immunoglobulin production. The effector function of an antibody can be altered, for example, enhanced or reduced, by changing the affinity of the antibody for effector molecules such as Fc receptors or complement components. The binding affinity is generally changed by modifying the effector molecule binding site, in which case it is appropriate to identify the site of interest and modify at least a portion of the site in a suitable manner. Also, a change in the binding site on the antibody for an effector molecule need not significantly change the overall binding affinity, but is also envisioned to change the shape of the interaction that abrogates the effector mechanism, as in non-productive binding. It is further envisioned that the effector function can also be altered by modifying sites that do not directly participate in effector molecule binding but otherwise participate in the performance of the effector function.

[0035] Epitope: An epitope, or antigenic determinant, is a portion of an antigen that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0036] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain contains the variable heavy (VH) domain of an antibody operably linked (typically at the N-terminus) to a first constant domain (referred to herein as C1), and the second polypeptide chain contains the variable light (VL) domain at the N-terminus of an antibody operably linked (typically at the N-terminus) to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, VH is at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is at the N-terminus of the constant domain (CL) of the light chain. The Fab of the present disclosure can be arranged according to the native orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, to promote correct modified Fab-chain pairing in a heterodimeric molecule, it is possible to replace the CH1 and CL domain pairs in the Fab with CH3 domain pairs. It is also possible to reverse CH1 and CL such that CH1 is attached to VL and CL is attached to VH, which is generally a configuration known as Crossmab. The term "Fab" encompasses single-chain Fabs.

[0037] Fc domain and Fc region: The term "Fc domain" refers to the part of a heavy chain that pairs with the corresponding part of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy-chain Fc domains. The two Fc domains within the Fc region may be identical or different from each other. In a native antibody, the Fc domains are typically identical, but one or both Fc domains can advantageously be modified, for example, via a knob-in-hole interaction, to enable heterodimerization.

[0038] Fv: The term "Fv" refers to the smallest antibody fragment derivable from an immunoglobulin that contains the complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain (VH-VL dimer) in a close non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Often, the six CDRs confer target-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv containing only the three CDRs specific for the target) can have the ability to recognize and bind the target. References herein to VH-VL dimers are not intended to convey any particular configuration. By way of non-limiting example, VH and VL can come together in any of the configurations described herein to form a half-antibody, or each can be present on a separate half-antibody and come together to form an antigen-binding domain when the separate half-antibodies associate, for example, to form the ABM or MBM proteins of the present disclosure. When present on a single polypeptide chain (e.g., scFv), VH and be at the N-terminus or C-terminus relative to VL.

[0039] Half-antibody: The term "half-antibody" refers to a molecule that contains at least one ABS or ABS chain and can associate with another molecule containing an ABS or ABS chain through, for example, a disulfide bridge or a molecular interaction (e.g., a knob-in-hole interaction between Fc heterodimers). A half-antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., the two polypeptide chains of a Fab). In a preferred embodiment, the half-antibody contains an Fc region. Examples of half-antibodies are molecules that contain the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule that contains a first polypeptide containing a VL domain and a CL domain and a second polypeptide containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an ABS. Yet another example of a half-antibody is a polypeptide that contains an scFv domain, a CH2 domain, and a CH3 domain.

[0040] A half-antibody can include two or more AB segments, for example, a half-antibody including (in order from the N-terminus to the C-terminus) a VH1 domain, a CH1 domain, a CH2 domain, a CH3 domain, and another VH1 domain or scFv domain. A half-antibody can also include an AB segment that forms a complete AB when associated with another AB segment in a different half-antibody.

[0041] Thus, an ABM or MBM protein can include one, more typically two, or even three or more half-antibodies, and a half-antibody can include one or more AB segments or AB chains.

[0042] In some ABM or MBM proteins, a first half-antibody associates with a second half-antibody, for example, heterodimerizes with the second half-antibody. In other ABM or MBM proteins, a first half-antibody is covalently attached to a second half-antibody, for example, through a disulfide bridge or a chemical cross-link. In still other ABM or MBM proteins, a first half-antibody associates with a second half-antibody through both covalent attachment and non-covalent interactions, for example, disulfide bridges and knob-in-hole interactions.

[0043] The term "half-antibody" is intended for illustrative purposes only and does not mean a particular configuration or production method. Descriptions of half-antibodies as the "first" half-antibody, the "second" half-antibody, the "left" half-antibody, the "right" half-antibody, etc. are for convenience and illustrative purposes only.

[0044] Host cell or recombinant host cell: The terms "host cell" or "recombinant host cell" refer to cells that have been genetically engineered, for example, through the introduction of a heterologous nucleic acid. It should be understood that such terms are intended to refer not only to a particular target cell but also to the progeny of such a cell. Because of either mutation or environmental influences, certain modifications may occur in later generations, and although such progeny may not actually be identical to the parental cell, they are still included within the scope of the term "host cell" as used herein. A host cell can carry a heterologous nucleic acid, for example, transiently on an episomal heterologous expression vector or, for example, stably by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the ABM or MBM proteins of the present disclosure, the host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, for example, derivatives that grow at a higher density than the original cell line, and / or derivatives with modified glycan profiles and / or derivatives with site-specific integration sites.

[0045] Linker: As used herein, the term "linker" refers to a protease-cleavable linker or a non-cleavable linker. Masking moiety: With respect to an MBM protein, the term "masking moiety" as used herein refers to an amino acid sequence in an MBM protein that inhibits the ability of a TCE ABS to specifically bind to its target, either through specific interaction with the TCE ABS (e.g., where the masking moiety is an anti-idiotype or "anti-TCE ABS" of the TCE ABS), or through the disposition of the TCE ABS with respect to another component of the MBM protein that sterically hinders the binding of the TCE ABS to its target (e.g., by connecting the TCE ABS to the Fc region through a short linker). The masking moiety and the TCE ABS are disposed within the MBM protein such that cleavage of a protease-cleavable linker reduces inhibition of the interaction of the TCE ABS with its target, either through generation of an MBM lacking the masking moiety, or through generation of an MBM in which the spatial constraints on the ability of the TCE ABS to interact with its target are relaxed. The term "masking moiety" when used with respect to an antigen-binding molecule more generally refers to an amino acid sequence in an antigen-binding molecule protein that inhibits the ability of an antigen-binding site (ABS) in the antigen-binding molecule to specifically bind to its target, either through specific interaction with the ABS (e.g., where the masking moiety is an anti-idiotype of the ABS), or through the disposition of the ABS with respect to another component of the antigen-binding molecule that sterically hinders the binding of the ABS to its target (e.g., by connecting the ABS to the Fc region through a short linker). In general, the masking moiety and the ABS are disposed within the antigen-binding molecule such that cleavage of a protease-cleavable linker reduces inhibition of the interaction of the ABS with its target, either through generation of an antigen-binding molecule lacking the masking moiety, or through generation of an antigen-binding molecule in which the spatial constraints on the ability of the ABS to interact with its target are relaxed.

[0046] MBM protein: As used herein, the term "MBM protein" refers to an MBM whose ability to bind to a target recognized by at least one of its ABSs is reduced or inhibited, for example, due to the presence of a masking moiety. The masking moiety impedes the binding of the ABS to its target and is separated from the MBM by proteolytic cleavage, for example, by proteolytic cleavage of a protease-cleavable linker that connects the masking moiety to the MBM. Typically, in the MBM proteins of the present disclosure, the TCE ABS is masked while the TAA ABS is not masked. The TAA ABS can function as a targeting moiety for directing the MBM protein to the site of a tumor, and in the presence of a protease that recognizes a substrate within the protease-cleavable linker, results in cleavage of the linker, restoring the ability of the MBM to bind to its target by the TCE ABS and to activate T cells against tumor cells that express the TAA.

[0047] Monovalent: As used herein, the term "monovalent" in the context of an antigen-binding molecule refers to an antigen-binding molecule having a single antigen-binding site. Multispecific binding molecule, MBM: The terms "multispecific binding molecule" and "MBM" refer to a molecule that includes two or more antigen-binding sites. Typically, an MBM includes a TAA ABS and a TCE ABS.

[0048] Non-cleavable linker: A non-cleavable linker refers to a peptide whose amino acid sequence lacks a substrate sequence for a protease that recognizes and cleaves a specific sequence motif, for example, a protease as described in Section 6.4.2.

[0049] Operably linked: The term "operably linked" refers to the functional relationship between two or more peptides or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are linked such that a functional polypeptide is produced. For example, in the context of the ABM or MBM proteins of the present disclosure, separate ABSs (or chains of ABSs) can be operably linked through a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein such as a polypeptide chain of an ABM or MBM of the present disclosure, "operably linked" means that two nucleic acids are joined such that the amino acid sequences encoded by those two nucleic acids are kept in frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0050] Polypeptides, peptides, and proteins: The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0051] Proprotein: A "proprotein" is an inactive protein precursor that can be activated by proteolytic cleavage by a protease. Thus, a proprotein is "protease activatable".

[0052] Protease: The term "protease" as used herein refers to any enzyme that catalyzes the hydrolysis of a peptide bond. In general, proteases useful in the present disclosure, such as those described in Section 6.4.1, recognize and cleave substrates with specific sequence motifs, such as those described in Section 6.4.2. Preferably, the protease is expressed at a higher level in cancer tissue compared to normal tissue.

[0053] Protease-cleavable linker: As used herein, the term "protease-cleavable linker" or "PCL" refers to a peptide whose amino acid sequence contains one or more (e.g., two or three) substrate sequences for one or more proteases.

[0054] Recognize: As used herein, the term "recognize" refers to an ABS that discovers the epitope and interacts (e.g., binds) with the epitope. Single-chain Fab or scFab: As used herein, the term "single-chain Fab" or "scFab" refers to an ABS that includes a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linker are arranged in one of the following orders in an N-terminal to C-terminal orientation: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is preferably a non-cleavable linker of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is typically stabilized via a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine residues (e.g., position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0055] Single-chain Fv or scFv: The term "single-chain Fv" or "scFv" as used herein refers to an ABS comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. Typically, the VH and VL are arranged in an N-terminal to C-terminal order of VH-VL or VL-VH, separated by a linker, e.g., a linker described in Table E, and be.

[0056] Spacer: As used herein, the term "spacer" refers to a peptide of an amino acid sequence that is not a substrate for a protease and is incorporated into a linker that includes a substrate. A spacer can be used to separate a substrate from other domains within a molecule, e.g., an ABS. In some embodiments, the residues within the spacer minimize the action of aminopeptidase and / or exopeptidase to prevent cleavage of the N-terminal amino acid.

[0057] Specifically (or selectively) binds: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of a cognate antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can be mediated by an antibody or antibody fragment, but does not have to be. The term "specifically binds" does not exclude cross-reactivity. For example, an antigen-binding site (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species can also "specifically bind" to that antigen in one or more other species. Thus, such cross-reactivity per se does not change the classification of the antigen-binding site as a "specific" binder. In certain embodiments, the antigen-binding sites of the present disclosure that specifically bind to human antigens (e.g., "TAA ABS") have cross-reactivity with one or more non-human mammalian species, such as primate species (including, but not limited to, one or more of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina), or rodent species, such as Mus musculus.

[0058] Subject: The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals (such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles). In a preferred embodiment, the subject is a human.

[0059] Substrate: The term "substrate" refers to a peptide sequence on which a protease acts and within which the protease cleaves peptide bonds. TAA ABS: The term "TAA ABS" refers to an ABS that recognizes or specifically binds to extracellular matrix ("ECM") proteins, tumor-reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T cell antigens ("TCA"), checkpoint inhibitors, or TAAs. One of ordinary skill in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus, a given target molecule may be classified into two or more of the foregoing categories of target molecules. For example, some molecules may be considered both TAAs and ECM proteins, and other molecules may be considered both TCAs and checkpoint inhibitors.

[0060] T cell antigen, TCA: The term "T cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of T lymphocytes and is useful for the preferential targeting of a pharmacological agent to a particular site. In some embodiments, the site is cancer tissue and / or the T cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, or a checkpoint inhibitor expressed on a T lymphocyte.

[0061] TCE ABS: The term "TCE ABS" refers to an ABS that recognizes or specifically binds to a T cell antigen in a manner that can initiate signal transduction through the TCR complex. Exemplary T cell antigens recognized or specifically bound by a TCE ABS are CD3 and other components of the TCR complex.

[0062] T cell receptor, TCR: The "T cell receptor" or "TCR" is a component of T cells involved in sensing by interacting with targets of T cell adaptive immunity. Generally speaking, the TCR is composed of a heterodimeric protein complex presented on the cell surface. T cells can be broadly classified as αβ or γδ according to the somatic rearrangement TCR forms they express on their surface. There are two forms of TCR chain pairs, the TCRα and TCRβ pair, and the TCRγ and TCRδ pair. The mature αβ and γδ TCR chain pairs are presented on the cell surface in a complex with a number of accessory CD3 subunits designated ε, γ, δ, and ζ. These subunits associate the αβ or γδ TCR as three dimers ( εγ, εδ, ζζ). This TCR complex forms a unit for initiating a cellular signaling response upon engagement with a cognate antigen of TCRαβ or TCRγδ. The terms "T cell receptor complex" and "TCR complex" refer to the complex of TCRαβ or TCRγδ and CD3.

[0063] Trivalent: The term "trivalent" in the context of an antigen-binding molecule, as used herein, refers to an antigen-binding molecule having three antigen-binding sites. The antigen-binding sites can all be the same, all different, or can include two of the same antigen-binding sites and a third different antigen-binding site.

[0064] Tumor: The term "tumor" is used interchangeably herein with the term "cancer" and includes, for example, both solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-cancerous and malignant cancers and tumors.

[0065] Tumor-associated antigen: The term "tumor-associated antigen" or "TAA" generally refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of cancer cells, either as a whole or as a fragment (e.g., MHC / peptide), and is useful for the preferential targeting of pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker. In some embodiments, the TAA is overexpressed in cancer cells compared to normal cells, e.g., a cell surface molecule that is overexpressed 1-fold, 2-fold, 3-fold, or more compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA is expressed exclusively on the cell surface of cancer cells, either as a whole or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" sometimes encompasses antigens that are specific to cancer cells, known in the art as tumor-specific antigens ("TSA").

[0066] TAA ABS: As used herein, the term "TAA ABS" refers to an antigen-binding site that recognizes or specifically binds to a TAA. To treat, treatment, treating: As used herein, the terms “to treat,” “treatment,” and “treating” refer to a reduction or amelioration in the progression, severity, and / or duration of a proliferative disorder, or an improvement in one or more symptoms (preferably, one or more distinguishable symptoms) of a proliferative disorder resulting from administration of one or more ABM or MBM proteins of the present disclosure. In certain embodiments, the terms “to treat,” “treatment,” and “treating” refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be distinguishable by a patient. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to inhibition of the progression of a proliferative disorder, either physically, e.g., by stabilization of distinguishable symptoms, physiologically, e.g., by stabilization of physical parameters, or both. In other embodiments, the terms “to treat,” “treatment,” and “treating” refer to a reduction or stabilization of tumor size or cancer cell number.

[0067] Universal light chain, UCL: The term “universal light chain” or “ULC,” as used herein, refers to a variable light chain region (VL) that can pair more with a heavy chain variable region (VL) than with itself. In the context of an ABS, the term “universal light chain” or “ULC” refers to a light chain polypeptide that can pair with the heavy chain region of an ABS and can also pair with other heavy chain regions. The ULC can also include a constant domain, e.g., the CL domain of an antibody. The universal light chain is also known as the “common light chain.”

[0068] VH: The term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, or Fab. VL: The term “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

[0069] 6.2. Antigen-binding molecular protein The present disclosure relates to an antigen-binding molecular proprotein comprising an antigen-binding site (ABS), a masking moiety, and a protease-cleavable linker (PCL), wherein the masking moiety is arranged to reduce or block the binding of the ABS to its target, and is configured such that upon encountering a protease, e.g., a protease overexpressed in the tumor microenvironment, the masking moiety is cleaved and a binding molecule with enhanced target binding is produced. Typically, the masking moiety of the antigen-binding molecular proprotein masks the ABS via steric hindrance (e.g., an Fc domain masking moiety) and / or via binding of a targeting moiety to the ABS (e.g., an anti-ABS binding moiety).

[0070] Accordingly, the antigen-binding molecular proprotein of the present disclosure generally comprises (a) a masking moiety and (b) an ABS or a component thereof connected to the masking moiety via a PCL, wherein the ABS or the component thereof is hindered (e.g., sterically hindered) from binding to its target. Exemplary masking moieties include an Fc domain and an anti-ABS binding moiety. Use of an Fc domain as a masking moiety provides steric hindrance to the ABS, which is relieved after protease cleavage of the PCL (e.g., by a tumor-specific protease). Specific examples of Fc domain masking antigen-binding molecular proproteins include type IIA and type IIB MBM proproteins (such as described in Section 6.3.2). Use of an anti-ABS binding moiety as a masking moiety blocks the binding of the ABS to its target, which is relieved after protease cleavage of the PCL (e.g., by a tumor-specific protease). Specific examples of anti-ABS binding moiety masking antigen-binding molecular proproteins include type IA and type IB MBM proproteins (such as described in Section 6.3.1.1). The antigen-binding molecular proprotein can comprise multiple masking moieties, e.g., an Fc domain and also an anti-ABS binding moiety, wherein one or more of the masking moieties are relieved after protease cleavage of the PCL.

[0071] PCL contains one or more substrates that are recognized and cleaved by one or more proteases, such as one or more of the proteases described in Section 6.4.1. Exemplary substrate sequences are described in Section 6.4.2. PCL may further include a spacer sequence, as described, for example, in Section 6.4.3. Examples of protease-cleavable linkers containing substrate and spacer sequences are provided in Section 6.4.4.

[0072] Without being bound by theory, a PCL that connects an ABS and a masking moiety (especially a masking moiety that sterically hinders the binding of the ABS, such as an Fc domain, to its target) of a small length (e.g., 30 or less) provides the greatest hindrance to ABS binding prior to protease cleavage of the PCL, thereby minimizing the off-target effects of the ABS. Thus, in certain embodiments, the PCL is 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, or 8 or less amino acids in length.

[0073] An antigen-binding molecular proprotein generally includes at least two polypeptide chains. The first polypeptide chain includes a masking moiety, a PCL, and a first component of the ABS (e.g., VH), and the second polypeptide chain includes a second component of the ABS (e.g., VL) that pairs with the first component to form the ABS. When the masking moiety is an Fc domain, the second polypeptide may further include an additional Fc domain that pairs with the first Fc domain to form an Fc region (e.g., an Fc heterodimer). The ABS can be, for example, a TAA ABS (as described, for example, in Section 6.6) or a TCE ABS (as described, for example, in Section 6.7).

[0074] In some embodiments, the antigen-binding molecule protein further comprises additional ABSs that are not masked by the masking moiety. In such cases, the first ABS can be a TCE ABS, while the additional ABSs are TAA ABSs, or the first ABS can be a TAA ABS, while the additional ABSs are TCE ABSs. An antigen-binding molecule protein comprising two or more ABSs can release a multispecific binding molecule (e.g., a bispecific or trispecific binding molecule) upon protease cleavage of the PCL. Certain examples of antigen-binding molecule proteins having multiple ABSs are described in Section 6.3 below.

[0075] 6.3. MBM proteins In some embodiments, the disclosure relates to an MBM protein comprising a TCE ABS, a TAA ABS, a masking moiety, and a protease-cleavable linker, wherein the masking moiety is arranged to reduce or block the binding of the TCE ABS to its target. The MBM protein is configured such that upon encountering a protease, e.g., a protease that is overexpressed in the tumor microenvironment, the masking moiety is cleaved, producing an MBM in which the inhibition of TCE ABS binding is reversed. Typically, the MBM proteins of the disclosure further comprise two Fc domains that associate to form an Fc region, and the TCE ABS is C-terminal to the Fc region.

[0076] Accordingly, the MBM proteins of the present disclosure generally include: (a) a first Fc domain and a second Fc domain that can associate to form an Fc region; (b) a C-terminal TCE ABS for the first Fc domain and / or the second Fc domain; (c) a TAA ABS; and (d) a C-terminal protease-cleavable linker (PCL) for the first Fc domain and / or the second Fc domain. Typically, the TAA ABS in the MBM protein can bind to its target regardless of whether the protease-cleavable linker is in an uncleaved state, while the binding of the TCE ABS to its target is significantly enhanced after protease cleavage of the protease-cleavable linker. For example, after cleavage of the protease-cleavable linker in the tumor microenvironment, the MBM containing the TAA ABS and the TCE ABS is released.

[0077] In type I MBM proteins, the TCE ABS is masked by an anti-idiotypic antibody. In type II MBM proteins, the Fc region acts as a masking moiety by sterically hindering the binding of the TCE ABS to its target.

[0078] The protease-cleavable linkers in type IA, IIA, IB, and type II MBM proteins contain one or more substrates that are recognized and cleaved by one or more proteases, such as one or more of the proteases described in Section 6.4.1. Exemplary substrate sequences are described in Section 6.4.2. The protease-cleavable linker typically further comprises a spacer sequence, such as described in Section 6.4.3. Examples of the entire protease-cleavable linker, including the substrate and spacer sequences, are provided in Section 6.4.4. Depending on the configuration of the MBM protein, the Fc region can be retained in the MBM obtained after cleavage of the protease-cleavable linker, for example, after cleavage of the protease-cleavable linker in type IA and IIA MBM proteins, or can be released from the MBM obtained after cleavage of the protease-cleavable linker, for example, after cleavage of the protease-cleavable linker in type IB and IIB MBM proteins.

[0079] Generally, the MBM proteins of the present disclosure contain multiple linkers. Preferably, the remaining linkers other than the protease-cleavable linker whose cleavage releases the MBM are non-cleavable. Examples of non-cleavable linkers are described in Section 6.5.

[0080] Exemplary TAA ABSs that can be used with the MBM proteins of the present disclosure (e.g., type IA, IIA, IB, and type II MBM proteins of the present disclosure) are disclosed in Section 6.6. Exemplary TCE ABSs that can be used with the MBM proteins of the present disclosure (e.g., type IA, IIA, IB, and type II MBM proteins of the present disclosure) are disclosed in Section 6.7. Exemplary anti-TCE ABSs that can be used with the type I MBM proteins of the present disclosure are disclosed in Section 6.8. Section 6.9 describes suitable formats for the ABSs incorporated into the MBM proteins of the present disclosure. Section 6.10 describes suitable Fc domains that can be incorporated into the MBM proteins of the present disclosure (and that can be used as masking moieties in the type II MBM proteins of the present disclosure).

[0081] 6.3.1. Type I MBM proteins (having an anti-idiotypic masking moiety) The present disclosure provides MBM proteins that include TCE ABSs masked by anti-TCE ABSs, referred to herein as "type I" MBM proteins. Typically, both the TCE ABS and the anti-TCE ABS are disposed C-terminally relative to the Fc region, and each is operably linked to a separate Fc domain. The TCE ABS or the anti-TCE ABS can be separated from one of the Fc domains of the Fc region by a protease-cleavable linker, for example, as described in Section 6.4. In type IA MBM proteins, the Fc region is retained by the MBM activated by cleavage of the protease-cleavable linker. In type IB MBM proteins, the Fc region is separated from the MBM activated by cleavage of the protease-cleavable linker.

[0082] 6.3.1.1. Type IA MBM proteins Generally, an IA type MBM protein comprises a first polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), a non-cleavable linker, and a TCE ABS (or a TCE ABS chain), and a second polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), a protease-cleavable linker, and an anti-TCE ABS (or an anti-TCE ABS chain). One or both of the Fc domains preferably further contain a TAA ABS (or a TAA ABS chain) at their N-terminus. When the TCE ABS, TAA ABS, and / or anti-TCE ABS are composed of multiple polypeptide chains, the MBM protein further comprises other chain(s), e.g., the light chain of a Fab.

[0083] After cleavage of the protease-cleavable linker, the anti-TCE ABS is released, and an MBM is produced that comprises a first polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), a non-cleavable linker, and a TCE ABS, and a second polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), and a portion of the protease-cleavable linker. One or both of the Fc domains preferably further contain a TAA ABS at their N-terminus. When the TCE ABS and / or TAA ABS are composed of multiple polypeptide chains, the resulting MBM further comprises other chain(s), e.g., the light chain of a Fab.

[0084] Preferably, the resulting MBM is bispecific for the targets of TAA and TCE ABS, e.g., CD3. Figures 1A - 1C illustrate exemplary embodiments of type IA MBM proteins. As shown in Figures 1A and 1B, a type IA MBM protein can contain only a single TAA ABS. Alternatively, as shown in Figure 1C, a type IA MBM protein can contain two TAA ABSs. Although depicted as Fabs for purposes of illustration, the anti - TCE ABSs in Figures 1A - 1C can be scFvs, VHHs, VHs, or other fragments or regions from anti - idiotypic antibodies. The depiction of a single substrate and two spacers in the protease - cleavable linker in Figures 1A - 1C is for purposes of illustration only, and as described in Section 6.4, a protease - cleavable linker can contain multiple substrates and three or more spacer sequences.

[0085] 6.3.1.2.Type IB MBM proteins Generally, a type IB MBM protein comprises a first polypeptide chain containing, in an N - to - C orientation, an Fc domain (optionally having a hinge domain at its N - terminus), a non - cleavable linker, and an anti - TCE ABS (or anti - TCE ABS chain), and a second polypeptide chain containing, in an N - to - C orientation, an Fc domain (optionally having a hinge domain at its N - terminus), a protease - cleavable linker, a TCE ABS (or TCE ABS chain), and a TAA ABS (or TAA ABS chain), and generally, the TCE ABS and TAA ABS are separated by a non - cleavable linker. If the TCE ABS, TAA ABS, and / or anti - TCE ABS are composed of multiple polypeptide chains, the MBM protein further comprises other chain(s), e.g., the light chain of a Fab.

[0086] After cleavage of the protease - cleavable linker, the MBM containing the TCE ABS, optional linker, and TAA ABS is released. If the TCE ABS and / or TAA ABS are composed of multiple polypeptide chains, the resulting MBM further comprises other chain(s), e.g., the light chain of a Fab.

[0087] Preferably, the resulting MBM is bispecific for targets of TAA and TCE ABS (e.g., CD3). Figures 5A - 5B illustrate exemplary embodiments of type IB MBM proteins. Although depicted as Fabs for purposes of illustration, the anti - TCE ABS of Figure 5A can be an scFv, VHH, VH, or other fragment or region from an anti - idiotypic antibody. The depiction of a single substrate and two spacers in the protease - cleavable linker of Figures 5A - 5B is for purposes of illustration only, and as described in Section 6.4, the protease - cleavable linker can include multiple substrates and three or more spacer sequences.

[0088] 6.3.2. Type II MBM proteins (having a sterically - hindering masking portion) 6.3.2.1. Type IIA MBM proteins Generally, a type IIA MBM protein comprises a first polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), a non-cleavable linker, and a first TCE ABS chain, and a second polypeptide chain containing, in an N-terminal to C-terminal orientation, an Fc domain (optionally having a hinge domain at its N-terminus), a protease-cleavable linker, and a second TCE ABS chain. The TCE ABS chain can include a VH and a VL without the domains of the corresponding CH1 and CL domains such that the TCE ABS in the form of an Fv is formed by the association of the TCE ABS chains. The TCE ABS chain can include a VH and a VL having the domains of the corresponding CH1 and CL domains such that the TCE ABS in the form of a Fab is formed by the association of the TCE ABS chains. In some embodiments, the VH and / or VL of the TCE ABS chain attached to the Fc domain includes a deletion of one or more amino acids, e.g., N-terminal truncation, at or near their N-termini. The VH or VL can have such a deletion precisely at the N-terminus (i.e., it can be a cleavage), or in some cases, within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the N-terminus. In some embodiments, the VH of the TCE ABS of the type IIA MBM protein of the present disclosure has a deletion (e.g., cleavage) or includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at or near (e.g., within 10 amino acids of) the N-terminus of a reference VH, such as the VH of an antibody or antibody sequence in Table G, e.g., a wild-type VH or a VH on which the TCE ABS is based. In some embodiments, the VL of the TCE ABS of the type IIA MBM protein of the present disclosure includes a deletion (e.g., cleavage) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at or near (e.g., within 10 amino acids of) the N-terminus of a reference VL, such as the VL of an antibody or antibody sequence in Table G, e.g., a wild-type VL or a VL on which the TCE ABS is based.

[0089] The length of the linker that separates the Fc domain and the TCE ABS chain is selected to optimize the ability of the Fc domain to sterically hinder the binding of the TCE ABS to its target. Preferably, the length of the non-cleavable linker is typically in the range of 5 to 30 amino acids, and in various embodiments, 25 amino acids or less (e.g., in the range of 5 to 25 amino acids), 20 amino acids or less (e.g., in the range of 5 to 20 amino acids), 15 amino acids or less (e.g., in the range of 5 to 15 amino acids), or 10 amino acids or less (e.g., in the range of 5 to 10 amino acids). In certain embodiments, the non-cleavable linker is 5, 6, 7, 8, 9, or 10 amino acids in length.

[0090] One or both of the Fc domains preferably further comprise a TAA ABS (or TAA ABS chain) at its N-terminus. When the TAA ABS is composed of multiple polypeptide chains, the MBM protein further comprises other chains, such as the light chain of the Fab.

[0091] One of both of the Fc domains may, in some embodiments, include a deletion of one or more amino acids (e.g., C-terminal truncation) at or near the C-terminus as compared to the wild-type Fc domain. In some embodiments, one or both of the Fc domains of the type IIA MBM proteins of the present disclosure include a deletion (e.g., truncation) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at or near the C-terminus as compared to the wild-type Fc domain. The Fc domain may have such a deletion exactly at the C-terminus (i.e., it may be a truncation), or in some cases, within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus.

[0092] In certain embodiments, the type IIA MBM protein of the present disclosure comprises: (1) an Fc domain having a deletion (e.g., cleavage) of 1, 2, 3, 4, 5, or 6 amino acids at or near the C-terminus compared to the wild-type Fc domain; and (2) a TCE ABS chain having VH and VL domains having a deletion (e.g., cleavage) of 1, 2, 3, 4, 5, or 6 amino acids at or near the N-terminus. For example, the type IIA MBM protein can comprise: (1) two Fc domains each having a deletion of 4, 5, or 6 amino acids at or near the C-terminus; and (2) a TCE ABS comprising VH and VL each having a deletion of 1, 2, or 3 amino acids at or near the N-terminus.

[0093] After cleavage of the protease-cleavable linker, the steric hindrance that restricts the ability of the TCE ABS to bind to its target is relieved. Preferably, the resulting MBM is bispecific for the TAA and the target of the TCE ABS (e.g., CD3).

[0094] Figures 3A - 3F illustrate exemplary embodiments of the type IIA MBM protein. As shown in Figures 3A and 3B, the type IIA MBM protein can comprise only a single TAA ABS. Alternatively, as shown in Figures 3C - 3F, the type IIA MBM protein can comprise two TAA ABSs. The depiction of a single substrate and two spacers in the protease-cleavable linker in Figures 3A - 3G is for illustrative purposes only, and as described in Section 6.4, the protease-cleavable linker can comprise multiple substrates and three or more spacer sequences.

[0095] 6.3.2.2. Type IIB MBM Protein Generally, a type IIB MBM protein comprises a first polypeptide chain that, in an N-terminal to C-terminal orientation, includes an Fc domain (optionally having a hinge domain at its N-terminus), a first protease-cleavable linker, an anti-TCE ABS chain, an optional non-cleavable linker, and a TAA ABS (or TAA ABS chain), and a second polypeptide chain that, in an N-terminal to C-terminal orientation, includes an Fc domain (optionally having a hinge domain at its N-terminus), a protease-cleavable linker, and a TCE ABS chain. When the TAA ABS is composed of multiple polypeptide chains, the MBM protein further includes other chain(s), e.g., the light chain of a Fab. The TCE ABS chain can include a VH and a VL having domains of the corresponding CH1 and CL domains such that the TCE ABS in the form of a Fab is formed by the association of the TCE ABS chains. In some embodiments, the VH and / or VL of the TCE ABS chain attached to the Fc domain includes a deletion of one or more amino acids, e.g., N-terminal truncation, near or at their N-termini. The VH or VL can have such a deletion precisely at the N-terminus (i.e., it can be a truncation), or optionally within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the N-terminus. In some embodiments, the VH of the TCE ABS of the type IIB MBM protein of the present disclosure has a deletion (e.g., truncation) or includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids near or at the N-terminus (e.g., within 10 amino acids) of a reference VH, such as the VH of an antibody or antibody sequence in Table G, e.g., a wild-type VH or a VH based on which the TCE ABS is derived. In some embodiments, the VL of the TCE ABS of the type IIB MBM protein of the present disclosure includes a deletion (e.g., truncation) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids near or at the N-terminus (e.g., within 10 amino acids) of a reference VL, such as the VL of an antibody or antibody sequence in Table G, e.g., a wild-type VL or a VL based on which the TCE ABS is derived.

[0096] The length of the linker that separates the Fc domain and the TCE ABS chain is selected to optimize the ability of the Fc domain to sterically hinder the binding of the TCE ABS to its target. Preferably, the length of the linker is typically in the range of 5 to 30 amino acids, and in various embodiments, 25 amino acids or less (e.g., in the range of 5 to 25 amino acids), 20 amino acids or less (e.g., in the range of 5 to 20 amino acids), 15 amino acids or less (e.g., in the range of 5 to 15 amino acids), or 10 amino acids or less (e.g., in the range of 5 to 10 amino acids).

[0097] In some embodiments, one of both of the Fc domains can include a deletion of one or more amino acids (e.g., C-terminal truncation) at or near the C-terminus as compared to the wild-type Fc domain. In some embodiments, one or both of the Fc domains of the type IIB MBM protein of the present disclosure include a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (e.g., truncation) at or near the C-terminus as compared to the wild-type Fc domain. The Fc domain can have such a deletion exactly at the C-terminus (i.e., can be a truncation), or optionally, within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus.

[0098] In certain embodiments, the type IIB MBM protein of the present disclosure includes (1) an Fc domain having a deletion of 1, 2, 3, 4, 5, or 6 amino acids (e.g., truncation) at or near the C-terminus as compared to the wild-type Fc domain, and (2) a TCE ABS chain having VH and VL domains having a deletion of 1, 2, 3, 4, 5, or 6 amino acids (e.g., truncation) at or near the N-terminus. For example, the type IIB MBM protein can include (1) two Fc domains each having a deletion of 4, 5, or 6 amino acids at or near the C-terminus, and (2) a TCE ABS including VH and VL each having a deletion of 1, 2, or 3 amino acids at or near the N-terminus.

[0099] After cleavage of the protease-cleavable linker, the MBM comprising the TCE ABS, optional linker, and TAA ABS is released. When the TAA ABS is composed of multiple polypeptide chains, the resulting MBM further comprises other TAA ABS chains, such as the light chain of a Fab.

[0100] Preferably, the resulting MBM is bispecific for the targets of the TAA and TCE ABS (e.g., CD3). Figure 7 illustrates an exemplary embodiment of a type IIB MBM protein. The depiction of a single substrate and two spacers in the protease-cleavable linker of Figure 7 is for illustrative purposes only, and as described in Section 6.4, the protease-cleavable linker can include multiple substrates and three or more spacer arrays.

[0101] 6.4. Protease-Cleavable Linker The MBM proteins of the present disclosure typically include at least components that are connected to each other by a linker having one or more substrates for a protease, the cleavage of which results in activation of the MBM.

[0102] The protease-cleavable linker can range from 20 amino acids to 80 amino acids or more. In certain embodiments, the non-cleavable peptide linker ranges from 20 amino acids to 60 amino acids, 20 amino acids to 40 amino acids, 30 amino acids to 50 amino acids, 20 amino acids to 80 amino acids, or 30 amino acids to 70 amino acids in length.

[0103] The protease-cleavable linker contains one or more substrate sequences for one or more proteases, such as one or more of the proteases described in Section 6.4.1. One or more substrate sequences, such as one or more of the substrate sequences described in Section 6.4.2, are typically adjacent to one or more spacer sequences, such as spacer sequences as described in Section 6.4.3. Each protease-cleavable linker can contain 1, 2, 3 or more substrate sequences. The spacer sequences can be adjacent, overlapping, or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease-cleavable linker contain spacer sequences.

[0104] Exemplary protease-cleavable linker sequences are described in Section 6.4.4. 6.4.1. Protease Exemplary proteases whose substrate sequences can be incorporated into the protease-cleavable linker are described in Table A below.

[0105]

Table 1-1

[0106]

Table 1-2

[0107] In certain embodiments, the protease is matrix metalloprotease (MMP)-2, MMP-9, legumain, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane-type 1 matrix metalloprotease (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3 / 4), cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urokinase / urokinase-type plasminogen activator (uPA), disintegrin and metalloprotease (ADAM)10, ADAM12, ADAM17, ADAM with thrombospondin motif (ADAMTS), ADAMTS5, beta-secretase (BACE), granzyme A, granzyme B, guanidino benzoatase, hepsin, matriptase, matriptase 2, meprin, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor converting enzyme (TACE), kallikrein-related peptidase (KLK)3, KLK5, KLK7, KLK11, hepatitis C virus NS3 / 4 protease (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, gamma-secretase component, antiplasmin cleavage enzyme (APCE), decysin 1, apoptosis-related cysteine peptidase, or N-acetylated alpha-linked acidic dipeptidase-like 1.

[0108] 6.4.2. Substrate Exemplary substrate sequences that can be cleaved by tumor proteases and incorporated into protease-cleavable linkers are set forth in Table B below.

[0109]

Table 2-1

[0110]

Table 2-2

[0111]

Table 2-3

[0112]

Table 2-4

[0113]

Table 2-5

[0114] 6.4.3. Spacer Exemplary spacer sequences that can be incorporated into a protease-cleavable linker are set forth in Table C below. In addition to the spacer sequences set forth in Table C, any of the non-cleavable linker sequences described in Section 6.5, e.g., the non-cleavable linker sequences described in Table E, or a portion thereof, can be used as the spacer sequence.

[0115]

Table 3-1

[0116]

Table 3-2

[0117] In some embodiments, when used in Table C above, n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 6.4.4. Exemplary Protease-Cleavable Linkers Exemplary protease-cleavable linkers that include one or more substrate sequences and spacer sequences are set forth in Table D below.

[0118] [Table 4-1]

[0119] [Table 4-2]

[0120] [Table 4-3]

[0121] [Table 4-4]

[0122] [Table 4-5]

[0123] In certain embodiments, the protease-cleavable linker includes an amino acid sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitutions as compared to the sequences set forth in Table D. Thus, in some embodiments, the protease-cleavable linker includes, or consists of, any amino acid sequence in Table D having 1-5 amino acid substitutions as compared to the sequences set forth in Table D.

[0124] 6.5. Non-Cleavable Linkers In certain embodiments, the present disclosure provides an MBM protein in which two or more components of the MBM protein are connected to each other by a peptide linker. By way of example and not limitation, linkers can be used to connect (a) an ABS and an Fc domain, (b) two ABSs, or (c) different domains within an ABS (e.g., the VH and VL domains within an scFv).

[0125] Preferably, all linkers in the MBM protein other than the protease-cleavable linker whose cleavage results in activation of the MBM are non-cleavable linkers (NCLs).

[0126] The non-cleavable linker can range from 2 amino acids to 60 amino acids or more. In certain embodiments, the non-cleavable peptide linker ranges from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids in length.

[0127] In certain embodiments, the non-cleavable linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length and, optionally, is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0128] In some of the foregoing embodiments, the non-cleavable linker ranges from 5 amino acids to 50 amino acids in length, e.g., 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids in length. In other of the foregoing embodiments, the non-cleavable linker ranges from 6 amino acids to 50 amino acids in length, e.g., 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids in length. In still other of the foregoing embodiments, the non-cleavable linker ranges from 7 amino acids to 50 amino acids in length, e.g., 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids in length.

[0129] Charges (e.g., charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred. Examples of flexible non-cleavable linkers that can be used in the MBM proteins of the present disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible non-cleavable linkers are repeats of glycine and serine, e.g., G n S (SEQ ID NO: 265) or SG n (SEQ ID NO: 266) monomers or multimers, or those containing them, where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-cleavable linker is G 4 S (SEQ ID NO: 1) monomer or multimer of its repeat, e.g., (GGGGS) n (SEQ ID NO: 150) or contains it.

[0130] Polyglycine non-cleavable linkers can be suitably used in the MBM proteins of the present disclosure. In some embodiments, the peptide non-cleavable linker contains two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 267), five consecutive glycines (5Gly) (SEQ ID NO: 268), six consecutive glycines (6Gly) (SEQ ID NO: 269), seven consecutive glycines (7Gly) (SEQ ID NO: 270), eight consecutive glycines (8Gly) (SEQ ID NO: 271), or nine consecutive glycines (9Gly) (SEQ ID NO: 272).

[0131] Exemplary non-cleavable linker sequences are described in Table E below.

[0132]

Table 5-1

[0133]

Table 5-2

[0134]

Table 5-3

[0135] In certain embodiments, the MBM proteins of the present disclosure can include a polypeptide chain containing an ABS (or ABS chain), a hinge domain, and a CH2 domain, and a CH3 domain, in an N-terminal to C-terminal orientation. Thus, the hinge domain can be said to connect the ABS to the CH2 domain and constitute a type of linker. Exemplary hinge domains are described in Section 6.10.3.

[0136] 6.6.TAA ABS The MBM proteins of the present disclosure include at least one ABS that specifically binds to an extracellular matrix (“ECM”) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (“TCA”), a checkpoint inhibitor, or a tumor-associated antigen (TAA), referred to herein as “TAA ABS”. One of ordinary skill in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus, a given target molecule may be classified into two or more of the foregoing categories of target molecules. For example, some molecules may be considered both a TAA and an ECM protein, and other molecules may be considered both a TCA and a checkpoint inhibitor. Preferably, the ECM antigen, tumor-reactive lymphocyte antigen, cell surface molecule of a tumor or viral lymphocyte, TCA, checkpoint inhibitor, or TAA is a human antigen. The antigen may or may not be present on normal cells. Certain embodiments are directed to MBM proteins that include at least one ABS that specifically binds to a TAA. In certain embodiments, the TAA is preferentially expressed or upregulated on tumor cells as compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0137] Any type of tumor and any type of ECM antigen, tumor-reactive lymphocyte antigen, cell surface molecule of tumor or viral lymphocytes, TCA, checkpoint inhibitor, or TAA is expected to be targeted by the MBM protein of the present disclosure. Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, biliary tract cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, lung squamous cell carcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and other bladder cancers. However, those skilled in the art will recognize that TAA and other target molecules related to the tumor microenvironment are known for substantially any type of cancer.

[0138] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin EGF-like, lectin, fibronectin, notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matricin.

[0139] Other target molecules are cell surface molecules of tumor or viral lymphocytes, such as T cell costimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0140] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In some embodiments where the target molecule is a checkpoint inhibitor, the TAA ABS does not block or poorly blocks ligand-receptor binding. Examples of non-blocking or poorly blocking anti-PD1 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NO: 2 / 10 of PCT Publication No. WO2015 / 112800A1, 16 / 17 of U.S. Patent No. 11,034,765B2, SEQ ID NOs: 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 of U.S. Patent No. 10,294,299B2. Examples of non-blocking or poorly blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 23 / 24, 3 / 4, and 11 / 12 of U.S. Publication No. 2022 / 0056126A1.

[0141] In certain embodiments, the target molecule is a TAA. Exemplary TAAs are listed in Table F below, along with references to exemplary antibodies or antibody sequences to which the TAA ABS can be based.

[0142]

Table 6-1

[0143]

Table 6-2

[0144]

Table 6-3

[0145]

Table 6-4

[0146]

Table 6-5

[0147]

Table 6-6

[0148]

Table 6-7

[0149]

Table 6-8

[0150]

Table 6-9

[0151]

Table 6-10

[0152] In some embodiments, the TAA ABS competes with the antibodies described in Table F for binding to TAA. In a further embodiment, the TAA ABS comprises a CDR comprising the CDR sequences of the anti-TAA antibodies described in Table F. In some embodiments, the TAA ABS comprises all six CDR sequences of the anti-TAA antibodies described in Table F. In other embodiments, the TAA ABS comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, and the light chain CDR sequences of the universal light chain. In a further embodiment, the TAA ABS comprises a VH comprising the amino acid sequence of the VH of the anti-TAA antibodies described in Table F. In some embodiments, the TAA ABS further comprises a VL comprising the amino acid sequence of the VL of the anti-TAA antibodies described in Table F. In other embodiments, the TAA ABS further comprises a universal light chain VL sequence.

[0153] Additional TAAs that can be targeted by the MBM protein are disclosed in Table K below and, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly Table 1. Table 1 of Hafeez et al. is hereby incorporated by reference in its entirety.

[0154] Additional exemplary TAAs include fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), the extra domain B of fibronectin (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2, and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family of tumor antigens, Imp-1, P1A, Epstein-Barr virus-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (heteromultimer associated with TCR), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, Ephrin B2, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and A1 domain of tenascin-C (TnC A1) are included.

[0155] 6.7.TCE ABS The MBM proteins of the present disclosure include at least T cell engaging ABSs that specifically bind to T cell receptor complex components, referred to herein as "TCE ABSs". Exemplary targets for TCE ABSs are CD3 and the T cell receptor (e.g., TCRαβ or TCRγδ). Preferably, the TCE ABS target is a human T cell receptor complex component. The epitopes of TCE ABSs can be individual polypeptides (e.g., CD3 epsilon) or multimeric components of the T cell receptor complex (e.g., TCRαβ dimer or TCRγδ dimer).

[0156] Exemplary CD3 and TCR antibodies or antibody sequences that can be the basis for TAA ABSs are described in Table G below.

[0157]

Table 7-1

[0158]

Table 7-2

[0159]

Table 7-3

[0160] In some embodiments, the TCE ABS competes with a T cell engaging (TCE) antibody described in Table G for binding to a target of the TCE antibody (e.g., CD3 or the T cell receptor). In further embodiments, the TCE ABS comprises a CDR comprising the CDR sequences of the TCE antibody described in Table G. In some embodiments, the TCE ABS comprises all six CDR sequences of the TCE antibody described in Table G. In other embodiments, the TCE ABS comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3), and the light chain CDR sequences of a universal light chain. In further embodiments, the TCE ABS comprises a VH comprising the amino acid sequence of the VH of the TCE antibody described in Table G. In some embodiments, the TCE ABS further comprises a VL comprising the amino acid sequence of the VL of the TCE antibody described in Table G. In other embodiments, the TCE ABS further comprises a universal light chain VL sequence.

[0161] 6.8. Anti-TCE ABS In certain embodiments, the MBM protein of the present disclosure comprises an anti-TCE ABS. The anti-TCE ABS is a fragment of an anti-idiotype antibody that can specifically bind to a TCE-ABS. For example, various types of antibody fragments including scFv, Fab, VHH, and VH can be used in the context of the anti-TCE ABS of the present disclosure. In some embodiments, the anti-TCE ABS is an scFv of an anti-idiotype antibody. In some embodiments, the anti-TCE ABS is a VHH derived from an anti-idiotype antibody. In some embodiments, the anti-TCE ABS is a VH derived from an anti-idiotype antibody.

[0162] The anti-TCE ABS can be derived from a single domain antibody composed of a single VH or VL domain that exhibits sufficient affinity for the target. In a specific embodiment, the single domain antibody is a camelid VHH domain (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38, WO94 / 04678).

[0163] An anti-idiotype antibody is an antibody directed against the antigen-combining region or variable region (called the idiotype or Id) of another antibody molecule. In principle, immunization with an antibody molecule expressing a paratope (antigen-combining site) against a given antigen should produce a group of anti-antibodies, some of which share with the antigen a complementary structure to the paratope.

[0164] In U.S. Patent No. 10,150,817B2 and Example 1 of WO2017 / 162587A1, exemplary methods for producing anti-idiotype antibodies. Exemplary anti-CD3 anti-idiotype ABSs that can be used as the anti-TCE ABSs of the present disclosure are scFvs designated as 4.15.64 and 4.32.63, which are anti-idiotypes of an anti-CD3 ABS designated as CH2527. The sequences of 4.15.64, 4.32.63, and CH2527 are disclosed in WO2017 / 162587A1.

[0165] 6.9. ABS format In certain embodiments, the ABSs in the MBM proteins of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the ABS is an immunoglobulin molecule or fragment thereof, particularly an immunoglobulin molecule of the IgG class, more specifically, IgG 1 or IgG 4 immunoglobulin molecules. Antibody fragments include, but are not limited to, VH (or V H ) fragments, VL (or V L ) fragments, Fab fragments, F(ab’) 2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetra-bodies.

[0166] 6.9.1. Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can include constant domain sequences and variable region sequences from any suitable species and can thus be murine, chimeric, human, or humanized.

[0167] Fab domains typically include a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding site. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0168] For the MBM proprotein of the present disclosure, when the light chain of the ABS is not a common or universal light chain in particular, it is advantageous to use a Fab heterodimerization strategy to enable the correct association of Fab domains belonging to the same ABS and minimize the abnormal pairing of Fab domains belonging to different ABSs. For example, the Fab heterodimerization strategy shown in Table H below can be used.

[0169]

Table 8

[0170] Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is facilitated, for example, by exchanging the VL and VH domains of the Fab or by exchanging the CH1 and CL domains of the Fab, as described in WO2009 / 080251.

[0171] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications in the CH1 domain of the Fab and one or more amino acid modifications in the CL domain, and / or by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The amino acids to be modified are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with other Fab components.

[0172] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by residue Kabat numbering. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0173] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or combinations of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which imply the nature of the structural and chemical correspondence between two interacting surfaces.

[0174] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab components. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0175] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, thereby introducing a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0176] In some embodiments, the Fab domain comprises 143Q and 188V substitutions within the CH1 domain and 113T and 176V substitutions within the CL domain, which helps to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0177] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In an embodiment, 39K and 62E modifications are introduced into the VH domain, H172A and F174G modifications are introduced into the CH1 domain, 1R, 38D, (36F) modifications are introduced into the VL domain, and L135Y and S176W modifications are introduced into the CL domain. In another embodiment, a 39Y modification is introduced into the VH domain and a 38R modification is introduced into the VL domain.

[0178] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of Fab component pairing. For example, the engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0179] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, in Wu et al., 2015, MAbs 7:364-76, the CH1 domain was replaced with the constant domain of the T cell receptor and the CL domain was replaced with the b domain of the T cell receptor, and these domain replacements were described in terms of pairing with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification into the VL domain and a 39K modification into the VH domain.

[0180] Instead of, or in addition to, using Fab heterodimerization strategies to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABS in the MBM proteins of the present disclosure. In various embodiments, using the common light chain described herein reduces the number of inappropriate species in the MBM protein as compared to using the original cognate VL. In various embodiments, the VL domain of the ABS is identified from a single-specificity antibody that includes a common light chain. In various embodiments, the VH region of the ABS in the MBM protein includes human heavy chain variable gene segments that are rearranged in vivo within mouse B cells that have been pre-engineered to express a limited human light chain repertoire or a single human light chain, are homologous to the human heavy chain, and in response to exposure to the antigen of interest, generate an antibody repertoire that includes one or more human VHs that are homologous to one of two possible human VLs, and this antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence and includes somatic mutants (e.g., affinity matured forms). See, for example, U.S. Patent No. 10,412,940.

[0181] 6.9.2.scFv A single-chain Fv or "scFv" antibody fragment contains the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as a single-chain polypeptide, and retains the specificity of the intact antibody from which they are derived. Generally, an scFv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFv are the non-cleavable linkers identified in Section 6.5.

[0182] As used herein, unless specified otherwise, an scFv may have the VL variable region and the VH variable region in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may include VL-linker-VH, or may include VH-linker-VL.

[0183] An scFv can include VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences. To create a nucleic acid encoding an scFv, DNA fragments encoding VH and VL are operably linked to a fragment encoding another linker, such as a linker described in Section 6.5 (typically, a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 156)), such that the VH and VL sequences can be expressed as a continuous single-chain protein having a VL region and a VH region joined by a flexible linker (see, for example, Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, McCafferty et al., 1990, Nature 348:552-554).

[0184] 6.10. Fc Region The MBM proteins of the present disclosure typically include a pair of Fc domains that associate to form an Fc region. In native antibodies, the Fc region includes a hinge region at their N-terminus to form the constant domain. Throughout the present disclosure, unless otherwise specified, references to Fc domains include Fc domains having a hinge domain at their N-terminus.

[0185] The Fc domain can be derived from any suitable species operably linked to the ABS or a component thereof. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the ABS or a component thereof is fused to an IgG Fc molecule. The ABS or a component thereof can be fused to the N-terminus and / or C-terminus of the IgG Fc domain.

[0186] The Fc domain can be derived from any suitable class of antibodies, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4. Exemplary sequences of Fc domains from IgG1, IgG2, IgG3, and IgG4 are provided in Table Y below.

[0187] [Table 9]

[0188] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 336. When the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 336 (e.g., 90% - 99% sequence identity to SEQ ID NO: 336), the Fc domain may also comprise one or more of the amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.10.1), and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.10.2).

[0189] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 337. When the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 337 (e.g., 90% - 99% sequence identity to SEQ ID NO: 337), the Fc domain may also comprise one or more of the amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.10.1), and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.10.2).

[0190] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 338. If the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 338 (e.g., 90% - 99% sequence identity to SEQ ID NO: 338), the Fc domain may also comprise one or more of the amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.10.1), and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.10.2).

[0191] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 339. If the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 339 (e.g., 90% - 99% sequence identity to SEQ ID NO: 339), the Fc domain may also comprise one or more of the amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.10.1), and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.10.2).

[0192] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules, such as the MBM proteins of the present disclosure and the MBMs produced by their activation, the Fc domains may advantageously be different to allow for heterodimerization, as described in Section 6.10.2 below.

[0193] In natural antibodies, the heavy chain Fc domains of IgA, IgD, and IgG are composed of two heavy chain constant domains (CH2 and CH3), and the domains of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4). These dimerize to create the Fc region.

[0194] In the MBM proteins of the present disclosure, the Fc region and / or the Fc domains therein can include heavy chain constant domains from one or more different classes of antibodies, e.g., from one, two, or three different classes.

[0195] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG1. One or both of the Fc domains of the Fc region may have a deletion of one or more amino acids compared to the naturally occurring IgG1 CH3 sequence. In some embodiments, the Fc domains of the present disclosure have a deletion (e.g., truncation) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at or near the C-terminus of the CH3 domain compared to naturally occurring IgG1.

[0196] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG2. One or both of the Fc domains of the Fc region may have a deletion of one or more amino acids compared to the naturally occurring IgG2 CH3 sequence. In some embodiments, the Fc domains of the present disclosure have a deletion (e.g., truncation) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at or near the C-terminus of the CH3 domain compared to naturally occurring IgG2.

[0197] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3. One or both of the Fc domains of the Fc region may have a deletion of one or more amino acids as compared to the naturally occurring IgG3 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has a deletion (e.g., cleavage) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at or near the C-terminus of the CH3 domain as compared to naturally occurring IgG3.

[0198] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4. One or both of the Fc domains of the Fc region may have a deletion of one or more amino acids as compared to the naturally occurring IgG4 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has a deletion (e.g., cleavage) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at or near the C-terminus of the CH3 domain as compared to naturally occurring IgG4.

[0199] In one embodiment, the Fc region comprises a CH4 domain derived from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG, and a CH4 domain derived from IgM.

[0200] It will be understood that the heavy chain constant domains for use in the production of the Fc region of the MBM proteins of the present disclosure can include variants of the naturally occurring constant domains described above. Such variants can include one or more amino acid mutations as compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0201] Certain variant constant domains contemplated herein include those having one or more amino acid deletions (e.g., truncations) at or near the C-terminus (e.g., within 10 amino acids thereof) as compared to the wild-type constant domain. In some embodiments, the constant domain of the present disclosure includes a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at or near the C-terminus as compared to the wild-type constant domain.

[0202] IgM and IgA are naturally present in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when the J chain is incorporated and as a hexamer when the J chain is absent. IgA exists as monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to a C-terminal constant domain known as the tailpiece. The tailpiece contains cysteine residues that form disulfide bonds between the heavy chains within the polymer and is thought to play an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the MBM proteins of the present disclosure do not include the tailpiece.

[0203] The Fc domain incorporated into the MBM protein of the present disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond architecture, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.10.1.

[0204] The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric MBM proteins, for example, by enabling heterodimerization, which is the preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization enables the production of MBM proteins in which different polypeptide components are connected to each other by Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.10.2.

[0205] It will be understood that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or can be combined with other modifications to alter the properties of the MBM protein.

[0206] 6.10.1. Fc Domain with Altered Effector Function In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

[0207] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically, human FcγRIIIa, FcγRI or FcγRIIa, and most specifically, human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0208] In one embodiment, the Fc domain or Fc region (e.g., one or both Fc domains of MBM protomers that can associate to form the Fc region) comprises amino acid substitutions at positions selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions selected from the group of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In such an embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In certain embodiments, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0209] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU indexing), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0210] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising the D265A, N297A mutations (EU numbering) for reducing effector function.

[0211] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table 1 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequences shown below:

[0212]

Table 10-1

[0213]

Table 10-2

[0214]

Table 10-3

[0215]

Table 10-4

[0216]

Table 10-5

[0217] In certain embodiments, IgG4 having reduced effector function includes the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 and may be referred to herein as IgG4s or hIgG4s.

[0218] For the heterodimeric Fc region, it is possible to incorporate combinations of the variant IgG4 Fc sequences described above, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or the bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or the bolded portion thereof).

[0219] 6.10.2. Fc Heterodimerization Variant Certain MBM proteins are involved in dimerization between two Fc domains operably linked to non-identical N-terminal regions, unlike natural immunoglobulins. For example, one Fc domain is connected to a Fab and the other Fc domain is connected to an ABS or a component thereof. Insufficient heterodimerization of the two Fc domains to form the Fc region can be an obstacle to increasing the yield of the desired heterodimer molecule and can pose purification challenges. Various approaches available in the art can be used to enhance the dimerization of the Fc domains that may be present in the MBM proteins of the present disclosure, as disclosed in, for example, EP1870459A1, U.S. Patent No. 5,582,996, U.S. Patent No. 5,731,168, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441, U.S. Patent No. 7,183,076, U.S. Patent Application Publication No. 2006 / 204493A1, and PCT Publication No. WO2009 / 089004A1.

[0220] The present disclosure provides an MBM protein comprising an Fc heterodimer, i.e., an Fc region comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the foregoing section.

[0221] Heterodimerization of two different heavy chains in the CH3 domain results in the desired MBM protein, while homodimerization of the same heavy chain will reduce the yield of the desired MBM protein. Thus, in a preferred embodiment, the polypeptides that associate to form the MBM proteins of the present disclosure will comprise CH3 domains having modifications that favor heterodimeric association compared to unmodified Fc domains.

[0222] In a specific embodiment, the modification that promotes the formation of the Fc heterodimer is a so-called "knob-into-hole" or "knob-in-hole" modification that includes a "knob" modification on one of the Fc domains and a "hole" modification on the other Fc domain. The knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, US7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide to promote heterodimer formation and hinder homodimer formation, so that the protrusion can be placed within the cavity. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0223] Thus, in some embodiments, the amino acid residues within the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned within the cavity within the CH3 domain of the second subunit, and the amino acid residues within the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit in which the protrusion within the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and the cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0224] In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first Fc domain, additionally, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue). In the second Fc domain, additionally, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). In a particular embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0225] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote the association of the first and second Fc domains of the Fc region.

[0226] As an alternative to, or in addition to, the use of an Fc domain engineered to promote heterodimerization, the Fc domain can be engineered to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide comprises a modified Fc domain that inhibits its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, e.g., U.S. Patent No. 8,586,713. As such, an MBM protan protein comprises a first CH3 domain and a second Ig CH3 domain, and the first and second Ig CH3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the MBM protan protein to Protein A as compared to a corresponding MBM protan protein lacking the amino acid difference. In one embodiment, the first CH3 domain binds to Protein A and the second CH3 domain comprises a mutation / modification that reduces or abolishes Protein A binding, e.g., an H95R modification (H435R according to EU numbering by IMGT exon numbering). The second CH3 may further comprise a Y96F modification (Y436F according to EU by IMGT). Modifications of this class are referred to herein as "star" mutations.

[0227] In some embodiments, the Fc can comprise one or more mutations (e.g., knob and hole mutations) to promote heterodimerization, as well as star mutations to facilitate purification. 6.10.3. Hinge domain The MBM protein of the present disclosure can include an Fc domain that includes a hinge domain at its N-terminus. The hinge region can be a native hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge domain" refers to a monomeric hinge domain in the context of a single or monomeric polypeptide chain, and in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer MBM protein formed by the association of two Fc domains), can include two associated hinge sequences on separate polypeptide chains, which can be naturally occurring or non-naturally occurring hinge sequences. Sometimes, the two associated hinge sequences are referred to as the "hinge reion".

[0228] The native hinge region is typically the hinge region found between the Fab domain and the Fc domain in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such a hinge can include hinge regions from other species such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions can include the complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region can include a portion or repeating unit of the native hinge, with each repeating unit being derived from the native hinge region. In a further alternative, the native hinge region can be altered by converting one or more cysteines or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine residues. By such means, the number of cysteine residues in the hinge region can be increased or decreased. Other modified hinge regions can be completely synthetic and can be designed to have desired properties such as length, cysteine composition, and flexibility.

[0229] Several modified hinge regions are already described, for example, in U.S. Patent No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are hereby incorporated by reference.

[0230] In one embodiment, the MBM protein of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge domain at their N-terminus. In various embodiments, positions 233-236 within the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered according to EU numbering.

[0231] In some embodiments, the MBM protein of the present disclosure comprises a modified hinge region that reduces the binding affinity for Fcγ receptors as compared to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0232] In one embodiment, the MBM protein of the present disclosure includes an Fc region in which each Fc domain has an intact hinge domain at its N-terminus, each Fc domain and hinge domain are derived from IgG4, and each hinge domain includes a modified sequence CPPC (SEQ ID NO: 329). The core hinge region of human IgG4 includes the sequence CPSC (SEQ ID NO: 330) as compared to IgG1 that includes the sequence CPPC (SEQ ID NO: 334). The serine residue present in the IgG4 sequence results in an increase in flexibility in this region, and thus, a proportion of the molecules form disulfide bonds (intra-chain disulfides) within the same protein chain rather than cross-linking to other heavy chains within the IgG molecule to form inter-chain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). By changing the serine residue to a proline to obtain the same core sequence as IgG1, complete formation of inter-chain disulfides within the IgG4 hinge region becomes possible, and thus, the heterogeneity in the purified product is reduced. This altered isotype is called IgG4P.

[0233] 6.10.3.1. Chimeric hinge sequence The hinge domain can be a chimeric hinge domain. For example, the chimeric hinge can include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0234] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 331), previously disclosed as SEQ ID NO: 8 in WO2014 / 121087, which is hereby incorporated by reference in its entirety, or ESKYGPPCPPCPAPPVA (SEQ ID NO: 332), previously disclosed as SEQ ID NO: 9 in WO2014 / 121087. Such chimeric hinge sequences can be suitably linked to the IgG4 CH2 region (e.g., incorporated into an IgG4 Fc domain, e.g., a human or mouse Fc domain, and further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.10.1).

[0235] 6.10.3.2. Hinge Sequences with Reduced Effector Function In a further embodiment, the hinge region can be modified to reduce effector function, as described, for example, in WO2016 / 161010A2, which is hereby incorporated by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, where the positions are numbered according to EU numbering (shown in Figure 1 of WO2016 / 161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an empty position.

[0236] Position 236 is empty in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0237] The hinge modifications within positions 233 - 236 can be combined with position 228 occupied by P. Position 228 is originally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing the exchange of heavy - light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226 - 229 are occupied by C, P, P, and C respectively ( "CPPC" is disclosed as SEQ ID NO: 329).

[0238] Exemplary hinge regions have residues 226 - 236, sometimes referred to as the middle (or core) and lower hinge, and are occupied by modified hinge sequences designated GGG-(233 - 236), GG--(233 - 236), G---(233 - 236) and no G (233 - 236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG - GPSVF (SEQ ID NO: 333) (previously disclosed as SEQ ID NO: 1 in WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 26) (previously disclosed as SEQ ID NO: 2 in WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 106) (previously disclosed as SEQ ID NO: 3 in WO2016161010A2) or CPPCPAP----GPSVF (SEQ ID NO: 152) (previously disclosed as SEQ ID NO: 4 in WO2016161010A2).

[0239] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinge) adjacent to the designated region. Such additional constant region segments that are present are typically of the same isotype, preferably a human isotype, but may also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3 or hybrids thereof with different domains. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016161010A2.

[0240] In certain embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., incorporated into an IgG4 Fc domain, e.g., a human or mouse Fc domain, and further modified in the CH2 and / or CH3 domains to reduce effector function as described, for example, in Section 6.10.1).

[0241] 6.11. Tandem Fab Certain aspects of the present disclosure relate to tandem Fab MBM. Tandem Fab MBM includes both a TCE ABS, e.g., a TCE ABS as described in Section 6.7, and a TAA ABS, e.g., a TAA ABS as described in Section 6.6, in the form of Fab domains, e.g., Fab domains as described in Section 6.9.1. The TCE ABS and the TAA ABS can be separated by a non-cleavable linker as described, for example, in Section 6.5.

[0242] In some embodiments, the present disclosure provides a tandem Fab MBM comprising: (1) a first polypeptide chain comprising, in an N-terminal to C-terminal orientation, a VH of a TCE ABS operably linked to a CH1 domain, an optional linker, e.g., a non-cleavable linker, and a VH of a TAA ABS operably linked to the CH1 domain; (2) a second polypeptide chain comprising a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain comprising a VL of a TCE ABS operably linked to a CL domain. In some embodiments, the tandem Fab MBM comprises: (1) a first polypeptide chain comprising, in an N-terminal to C-terminal orientation, (a) a VH of a TCE ABS, (b) a CH1 domain, (c) a non-cleavable linker, (d) a VH of a TAA ABS, and (c) a CH1 domain; (2) a second polypeptide chain comprising (a) a VL of a TAA ABS and (b) a CL domain; and (3) a third polypeptide chain comprising (a) a VL of a TCE ABS and (b) a CL domain.

[0243] In other embodiments, the present disclosure provides a tandem Fab MBM comprising: (1) a first polypeptide chain comprising, in an N-terminal to C-terminal orientation, a VH of a TAA ABS operably linked to a CH1 domain, an optional linker, e.g., a non-cleavable linker, and a VH of a TCE ABS operably linked to the CH1 domain; (2) a second polypeptide chain comprising a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain comprising a VL of a TCE ABS operably linked to a CL domain. In some embodiments, the tandem Fab MBM comprises: (1) a first polypeptide chain comprising, in an N-terminal to C-terminal orientation, (a) a VH of a TAA ABS, (b) a CH1 domain, (c) a non-cleavable linker, (d) a VH of a TCE ABS, and (c) a CH1 domain; (2) a second polypeptide chain comprising (a) a VL of a TAA ABS and (b) a CL domain; and (3) a third polypeptide chain comprising (a) a VL of a TCE ABS and (b) a CL domain.

[0244] In some embodiments, CH1 of TCE ABS and CH1 of TAA ABS are the same. In further embodiments, CL of TCE ABS and CL of TAA ABS are the same. In still further embodiments, VL of TAA ABS and TCE ABS are identical and are a universal light chain VL. Exemplary universal light chains that can be incorporated into TAA ABS and TCE ABS are described in Table J below.

[0245]

Table 11

[0246] The optional linker is preferably selected from the linkers disclosed in section

[0112] . In some embodiments, the linker has an amino acid length of less than 50 amino acids (e.g., 5 - 45 amino acids in length, 10 - 35 amino acids in length, or 5 - 25 amino acids in length) and contains a glycine - serine sequence, such as G4S (SEQ ID NO: 1) or a multimer thereof.

[0247] 6.12. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the MBM proteins and tandem Fab MBMs of the present disclosure. In some embodiments, the MBM proteins and tandem Fab MBMs are encoded by a single nucleic acid. In other embodiments, the MBM proteins and tandem Fab MBMs can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0248] A single nucleic acid can encode an MBM protoprotein or tandem Fab MBM that contains a single polypeptide chain, an MBM protoprotein or tandem Fab MBM that contains two or more polypeptide chains, or a portion of an MBM protoprotein or tandem Fab MBM that contains three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an MBM protoprotein or tandem Fab MBM that contains three, four, or more polypeptide chains, or three polypeptide chains of an MBM protoprotein that contains four or more polypeptide chains). To control expression separately, open reading frames encoding two or more polypeptide chains can be placed under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element, separated by an internal ribosome entry site (IRES) sequence, and allow translation into separate polypeptides.

[0249] In some embodiments, an MBM protoprotein or tandem Fab MBM that contains two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the MBM protoprotein or tandem Fab MBM can be less than or equal to the number of polypeptide chains in the MBM protoprotein or tandem Fab MBM (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0250] The nucleic acids of the present disclosure can be DNA or RNA (e.g., mRNA). In another aspect, the present disclosure provides host cells and vectors containing the nucleic acids of the present disclosure. The nucleic acids can be present in a single vector, as described in more detail below herein, or in separate vectors present in the same host cell or separate host cells.

[0251] 6.12.1. Vector The present disclosure provides a vector comprising a nucleotide sequence encoding an MBM protein, tandem Fab MBM, or a component thereof, such as one or two of the polypeptide chains of the half-antibody of the MBM protein. The vector includes, but is not limited to, a virus, plasmid, cosmid, lambda phage, or yeast artificial chromosome (YAC).

[0252] A number of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous sarcoma virus, MMTV or MoMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0253] Additionally, cells with stably integrated DNA into the chromosome can be selected by introducing one or more markers that enable the selection of transfected host cells. The marker can provide, for example, prototropy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0254] When a DNA sequence containing an expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can vary or be optimized based on the specific expression vector and mammalian host cell used, as described herein.

[0255] 6.12.2. Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0256] In one embodiment, the host cell is genetically engineered by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such a sequence. Such cassettes can include a promoter, an open reading frame with or without an intron, and a termination signal. Additional factors necessary or useful for bringing about expression, such as an inducible promoter, may also be used.

[0257] The present disclosure also provides a host cell comprising a vector described herein. The cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0258] 6.13. Pharmaceutical Compositions The MBM proteins and tandem Fab MBMs of the present disclosure can be in the form of a composition comprising an MBM protein or tandem Fab MBM, and one or more carriers, excipients, and / or diluents. The composition can be formulated for a particular use such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.), as well as the excipients, diluents, and / or carriers used, will depend on the intended use of the MBM protein and tandem Fab MBM, and in the case of therapeutic use, on the mode of administration.

[0259] In the case of therapeutic use, the composition can be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition can be administered to the patient by various routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. The most suitable route of administration in any given case will depend on the particular MBM protein or tandem Fab MBM, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0260] The pharmaceutical composition can conveniently be presented in unit dosage form containing a predetermined amount of the MBM protein or tandem Fab MBM of the present disclosure per dose. The amount of MBM protein or tandem Fab MBM contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder or in liquid form containing an amount of MBM protein or tandem Fab MBM suitable for a single administration. The dry powder unit dosage form can be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. The unit dosage in liquid form can conveniently be supplied in the form of a syringe pre-filled with an amount of MBM protein suitable for a single administration.

[0261] The pharmaceutical composition may also be bulk-supplied since it contains an amount of MBM protein or tandem Fab MBM suitable for multiple administrations. The pharmaceutical composition may be prepared for storage as a lyophilized formulation or an aqueous solution by mixing an MBM protein or tandem Fab MBM having a desired purity with any optional pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as “carriers”), i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic surfactants, antioxidants, and various other additives, typically used in the art. See Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the dosages and concentrations employed.

[0262] Buffers serve to maintain the pH within a range close to physiological conditions. They can be present at a wide variety of concentrations, but will typically be present at concentrations in the range of about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., sodium citrate - disodium citrate mixtures, citric acid - trisodium citrate mixtures, citric acid - sodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid - sodium succinate mixtures, succinic acid - sodium hydroxide mixtures, succinic acid - disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid - sodium tartrate mixtures, tartaric acid - potassium tartrate mixtures, tartaric acid - sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid - sodium fumarate mixtures, disodium fumarate mixtures, sodium fumarate - disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconic acid - sodium glyconate mixtures, gluconic acid - sodium hydroxide mixtures, gluconic acid - potassium glyconate mixtures, etc.), oxalate buffers (e.g., oxalic acid - sodium oxalate mixtures, oxalic acid - sodium hydroxide mixtures, oxalic acid - potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid - sodium lactate mixtures, lactic acid - sodium hydroxide mixtures, lactic acid - potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid - sodium acetate mixtures, acetic acid - sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0263] A preservative may be added to delay the growth of microorganisms and may be added in an amount in the range of about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyl dimethyl benzyl ammonium chloride, benzalconium halide (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, e.g., sugar alcohols of three or more valences (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol, etc.). A stabilizer, in its function, refers to a broad category of excipients that can range from a bulking agent to an additive and serves to solubilize a therapeutic agent or prevent denaturation or adhesion to the container wall.Typical stabilizers can be polyhydric sugar alcohols (enumerated above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate, etc.), low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins, etc.), hydrophilic polymers (e.g., polyvinylpyrrolidone, etc.), monosaccharides (e.g., xylose, mannose, fructose, glucose, etc.), disaccharides (e.g., lactose, maltose, sucrose, and trehalose, etc.), trisaccharides (e.g., raffinose, etc.), and polysaccharides (e.g., dextran, etc.). The stabilizer may be present in an amount in the range of 0.5 to 10% by weight per weight of the MBM protein or tandem Fab MBM.

[0264] Adding a nonionic surfactant or detergent (also known as a "wetting agent") can help solubilize the glycoprotein and protect the glycoprotein from agitation-induced aggregation, thereby allowing the formulation to be exposed to a shear plane under load without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pluronic® polyols. The nonionic surfactant can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0265] As various additional excipients, diluents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents may be mentioned.

[0266] The MBM proteins and tandem Fab MBMs of the present disclosure can be formulated, for example, as pharmaceutical compositions comprising an MBM protein or tandem Fab MBM containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical composition or a sterile composition comprising an MBM protein and tandem Fab MBM of the present disclosure, an MBM protein or tandem Fab MBM preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0267] For example, formulations of MBM protein and tandem Fab MBM can be prepared by mixing the MBM protein or tandem Fab MBM with a physiologically acceptable carrier, excipient, or stabilizer in the form of, for example, a lyophilized powder, slurry, aqueous solution, lotion, or suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wlkins, New York, N.Y., Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY, Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0268] The effective amount for a particular subject can vary depending on factors such as the condition being treated, the overall health of the subject, the route and dosage of administration, and the severity of side effects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0269] The compositions of the present disclosure can also be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Exemplary routes of administration for the MBM protein and tandem Fab MBM include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other common routes by injection or infusion. General administration can typically represent a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered via non-conventional routes, such as local, epithelial, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the MBM protein and tandem Fab MBM are administered by infusion. In another embodiment, the MBM protein or tandem Fab MBM of the present disclosure is administered subcutaneously.

[0270] 6.14. Therapeutic Indications and Methods of Treatment The MBM proteins and tandem Fab MBMs of the present disclosure can be used for the treatment of any proliferative disorder (e.g., cancer) that expresses TAA. In certain embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, appendiceal cancer, astrocytoma, basal cell cancer, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal cancer, fetal tumor, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary, midline tract carcinoma associated with the NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, throat cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0271] The following Table K shows exemplary indications for which MBM proteins and tandem Fab MBMs targeting specific TAAs can be used.

[0272]

Table 12-1

[0273]

Table 12-2

[0274]

Table 12-3

[0275] Additional TAAs and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is hereby incorporated by reference in its entirety.

[0276] 7. Numbered Embodiments Although various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is illustrated by the numbered embodiments described below.

[0277] In the following numbered embodiments, the TAA ABS preferably binds to a mammalian TAA, the TCE ABS preferably binds to a mammalian TCE, the Fc domain preferably is derived from a mammalian antibody, and the subject is preferably a mammal. More preferably, the mammal is a human.

[0278] The present disclosure is illustrated by the numbered embodiments of Group A and Group B described below. Unless otherwise specified, any feature of a particular group of numbered embodiments is applicable to the other group of numbered embodiments with changes to the points to be changed.

[0279] Group A numbered embodiments 1. A binding molecule comprising: (a) a first Fc domain; (b) a second Fc domain; (c) a first component of a first antigen-binding site (ABS) connected to the first Fc domain via a protease-cleavable linker (PCL). (i) The PCL is 25 amino acids or less in length and / or (ii) the first ABS is sterically hindered from binding to its target, the binding molecule.

[0280] 2. The binding molecule according to embodiment 1, wherein the binding of the first ABS to its target is enhanced after protease cleavage of the PCL. 3. The binding molecule according to embodiment 1, wherein the first ABS is sterically hindered from binding to its target before protease cleavage of the PCL, and the steric hindrance is released after protease cleavage of the PCL.

[0281] 4. The binding molecule according to any one of embodiments 1 to 3, wherein the PCL is 20 amino acids or less in length. 5. The binding molecule according to any one of embodiments 1 to 3, wherein the PCL is 15 amino acids or less in length.

[0282] 6. The binding molecule according to any one of embodiments 1 to 3, wherein the PCL is 10 amino acids or less in length. 7. The binding molecule according to any one of embodiments 1 to 6, wherein after protease cleavage of the PCL, the first ABS enhances its binding to its target by at least 10-fold.

[0283] 8. The binding molecule according to any one of embodiments 1 to 6, wherein after protease cleavage of the PCL, the first ABS enhances its binding to its target by at least 100-fold. 9. The binding molecule according to any one of embodiments 1 to 8, wherein the first ABS is a Fab.

[0284] 10. The binding molecule according to any one of Embodiments 1 to 8, wherein the first ABS is Fv. 11. The binding molecule according to any one of Embodiments 1 to 10, wherein the first component is VH.

[0285] 12. The binding molecule according to any one of Embodiments 1 to 10, wherein the first component is VL. 13. The binding molecule according to any one of Embodiments 1 to 12, wherein the binding molecule comprises at least two polypeptide chains, the first polypeptide chain comprises a first Fc domain and a first component of a first ABS, and the second polypeptide chain comprises a second Fc domain.

[0286] 14. The binding molecule according to Embodiment 13, wherein the second polypeptide chain comprises a second component of the first ABS. 15. The binding molecule according to Embodiment 14, wherein the first component and the second component associate to form the first ABS.

[0287] 16. The binding molecule according to Embodiment 14 or 15, wherein the first component is VH and the second component is VL. 17. The binding molecule according to Embodiment 16, wherein (1) the first polypeptide chain further comprises a C-terminal CH1 for VH, and (2) the second polypeptide chain further comprises a C-terminal CL for VL.

[0288] 18. The binding molecule according to any one of Embodiments 13 to 15, further comprising a third polypeptide chain comprising a third component of the first ABS. 19. The binding molecule according to any one of Embodiments 1 to 18, which is a multispecific binding molecule protein comprising a second ABS.

[0289] 20. The binding molecule according to Embodiment 19, wherein (a) the first ABS is a TCE ABS and the second ABS is a TAA ABS, or (b) the first ABS is a TAA ABS and the second ABS is a TCE ABS.

[0290] 21. The binding molecule according to embodiment 20, wherein the first ABS is TCE ABS. 22. The binding molecule according to embodiment 21, wherein the TCE ABS can bind to CD3, TCRαβ, or TCRγδ.

[0291] 23. The TAA ABS is (a) any TAA identified in Section 6.6, or (b) AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplakin-3, and is the binding molecule according to any one of embodiments 20 to 22.

[0292] 24. The binding molecule according to any one of embodiments 1 to 23, wherein the first Fc domain and the second Fc domain associate to form an Fc heterodimer. 25. The binding molecule according to embodiment 24, wherein the Fc heterodimer comprises a knob-in-hole mutation, for example, (i) the first Fc domain comprises one or more knob mutations and the second Fc domain comprises one or more hole mutations, or (ii) the first Fc domain comprises one or more hole mutations and the second Fc domain comprises one or more knob mutations.

[0293] 26. The binding molecule according to any one of embodiments 1 to 25, wherein the first Fc domain and / or the second Fc domain comprises a star mutation. 27. The binding molecule according to any one of embodiments 1 to 26, wherein the tandem Fab is produced after protease cleavage of PCL.

[0294] 28. The binding molecule according to any one of embodiments 1 to 26, wherein the multispecific binding molecule comprising the first Fc domain and the second Fc domain is produced after protease cleavage of PCL.

[0295] 29. The binding molecule according to any one of embodiments 1 to 28, wherein PCL comprises a substrate sequence cleavable by any protease described in Table A. 30. The binding molecule according to any one of embodiments 1 to 29, wherein PCL comprises one or more substrate sequences selected from the substrate sequences described in Table B.

[0296] 31. The binding molecule according to any one of embodiments 1 to 30, wherein PCL comprises an amino acid sequence of any of the PCL sequences selected from the sequences described in Table D. 32. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3A.

[0297] 33. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3B. 34. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3C.

[0298] 35. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3D. 36. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3E.

[0299] 37. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3F. 38. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 7.

[0300] 39. The binding molecule according to any one of embodiments 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 90% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

[0301] 40. The binding molecule according to any one of embodiments 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

[0302] 41. The binding molecule according to any one of embodiments 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

[0303] 42. The binding molecule according to any one of embodiments 1 to 41, wherein the first Fc domain and / or the second Fc domain comprises one or more amino acid substitutions that reduce effector function (e.g., as described in Section 6.10.1).

[0304] 43. The binding molecule according to any one of embodiments 1 to 41, wherein the first Fc domain and / or the second Fc domain comprises one or more amino acid substitutions that promote heterodimerization (such as described in Section 6.10.2).

[0305] 44. A pharmaceutical composition comprising the binding molecule according to any one of embodiments 1 to 43 and an excipient. 45. A method of treating cancer, comprising administering to a subject suffering from cancer an effective amount of the binding molecule according to any one of embodiments 1 to 43 or the pharmaceutical composition according to embodiment 44.

[0306] 46. The method according to embodiment 45, wherein the cancer is associated with the expression of an epitope bound by a TAA ABS, such as described in Table K. 47. A nucleic acid or plurality of nucleic acids encoding the binding molecule according to any one of embodiments 1 to 43.

[0307] 48. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the binding molecule according to any one of embodiments 1 to 43 under the control of one or more promoters.

[0308] 49. A method of producing a binding molecule, comprising: (a) culturing the cell according to embodiment 48 under conditions in which the binding molecule is expressed; and (b) recovering the binding molecule from the cell culture.

[0309] 50. The method according to embodiment 49, further comprising concentrating the binding molecule and / or purifying the binding molecule. Group B numbered embodiments 1. A multispecific binding molecule (MBM) protein, comprising: (a) a first Fc domain and a second Fc domain that can associate to form an Fc region; and (b) a C-terminal T cell engaging antigen binding site ("TCE ABS") for the first Fc domain and / or the second Fc domain, (c) at least one antigen binding site ("TAA ABS") capable of binding to a tumor associated antigen, (d) a C-terminal protease cleavable linker (PCL) for the first Fc domain or the second Fc domain, and comprising, The TAA ABS in the MBM protein is capable of binding to its target when the PCL is in an uncleaved state, and the binding of the TCE ABS to its target is enhanced after protease cleavage of the PCL, MBM protein.

[0310] 2. The MBM protein according to embodiment 1, wherein cleavage of the PCL releases the MBM comprising the TAA ABS and the TCE ABS. 3. The MBM protein according to embodiment 1 or embodiment 2, wherein the TCE ABS in the MBM protein is masked by an anti-idiotype antibody.

[0311] 4. The MBM protein according to embodiment 1 or embodiment 2, wherein the binding of the TCE ABS in the MBM protein to its target is sterically hindered by the Fc domain.

[0312] 5. The MBM protein according to any one of embodiments 1 to 4, wherein the MBM comprises the first and second Fc domains. 6. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 1A.

[0313] 7. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 1B. 8. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 1C. 9. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3A.

[0314] 10. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3B. 11. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3C. 12. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3D.

[0315] 13. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3E. 14. The MBM protein according to embodiment 5, having the configuration depicted in FIG. 3F. 15. The MBM protein according to any one of embodiments 1 to 4, wherein the MBM lacks the first and second Fc domains.

[0316] 16. The MBM protein according to embodiment 15, having the configuration depicted in FIG. 5. 17. The MBM protein according to embodiment 15, having the configuration depicted in FIG. 7. 18. A multispecific binding molecule (MBM) protein, optionally the MBM according to any one of embodiments 1 to 8, comprising: (a) A first polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional first linker, (ii) A first Fc domain, (iii) A second linker, and (iv) A first polypeptide chain comprising a T cell engaging antigen binding site ("TCE ABS") or a component thereof, (b) A second polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional third linker, (ii) A second Fc domain capable of heterodimerizing with the first Fc domain to form an Fc heterodimer, (iii) A fourth linker that is a protease-cleavable linker ("PCL"), and (iv) A second polypeptide chain comprising an antigen-binding site that is an anti-idiotype of TCE ABS (「anti-TCE ABS」) or a component thereof, An MBM protein, wherein the first polypeptide chain and / or the second polypeptide chain comprises an antigen-binding site (「TAA ABS」) that can bind to a tumor-associated antigen or a component thereof.

[0317] 19. The MBM protein according to embodiment 18, wherein the TAA ABS(s), TCE ABS, and anti-TCE ABS are in the form of Fab, and the TAA ABS chain(s), TCE ABS chain, and anti-TCE ABS chain each comprise a VH domain that associates with their respective VL domains on separate polypeptide chains.

[0318] 20. The MBM protein according to embodiment 18, wherein the VL domain is a universal light chain VL domain. 21. The MBM protein according to any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of a scFv.

[0319] 22. The MBM protein according to any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of a VHH. 23. The MBM protein according to any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of a VH.

[0320] 24. The MBM protein according to any one of embodiments 18 to 20, wherein the linker other than PCL is a non-cleavable linker (「NCL」). 25. An MBM protein that is optionally an MBM according to any one of embodiments 1 to 5 and 9 to 14, (a) A first polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional first linker, (ii) A first Fc domain, (iii) A second linker, and (iv) A first polypeptide chain comprising a T cell-engaging antigen-binding site (“TCE ABS”) component comprising VH, and (b) A second polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional third linker, (ii) A second Fc domain capable of heterodimerizing with a first Fc domain to form an Fc heterodimer, (iii) A fourth linker, and (iv) A second polypeptide chain comprising a T cell-engaging antigen-binding site (“TCE ABS”) component comprising VL, and (A) The second linker or the fourth linker is a protease-cleavable linker (“PCL”), (B) The first polypeptide chain and / or the second polypeptide chain comprises an antigen-binding site (“TAA ABS”) capable of binding to a tumor-associated antigen, (C) The TCE ABS is a MBM protein whose binding to its target is sterically hindered.

[0321] 26. The MBM protein according to embodiment 25, wherein the TCE ABS is a Fab. 27. The MBM protein according to embodiment 26, wherein the first polypeptide chain comprises a C-terminal CH1 domain with respect to the VH of the TCE ABS, and the second polypeptide chain comprises a C-terminal CL domain with respect to the VL of the TCE ABS.

[0322] 28. The MBM protein according to embodiment 25, wherein the TCE ABS is an Fv. 29. The MBM protein according to embodiment 28, wherein the first polypeptide chain lacks the C-terminal CH1 domain with respect to the VH of the TCE ABS, and the second polypeptide chain lacks the C-terminal CL domain with respect to the VL of the TCE ABS.

[0323] 30. The MBM protein according to any one of embodiments 25 to 29, wherein the TAA ABS(s) is / are in Fab form, and the TAA ABS chain(s) each comprise a VH domain that associates with its respective VL domain on a separate polypeptide chain.

[0324] 31. The MBM protein according to embodiment 30, wherein the VL domains on separate polypeptide chains are universal light chain VL domains. 32. The MBM protein according to any one of embodiments 25 to 31, wherein the linker other than PCL is a non-cleavable linker ("NCL").

[0325] 33. The MBM protein according to any one of embodiments 25 to 32, wherein the second linker or the fourth linker is 25 or fewer amino acids, 20 or fewer amino acids, 15 or fewer amino acids, or 10 or fewer amino acids.

[0326] 34. An MBM protein, optionally an MBM according to any one of embodiments 1 to 8, 15, and 16, comprising: (a) a first polypeptide chain, in an N-terminal to C-terminal orientation, (i) an optional first linker, (ii) a first Fc domain, (iii) a second linker, and (iv) a first polypeptide chain comprising an antigen-binding site that is an anti-TCE ABS of the second polypeptide chain ("anti-TCE ABS") or an anti-idiotype of a component thereof; and (b) a second polypeptide chain, in an N-terminal to C-terminal orientation, (i) an optional third linker, (ii) a second Fc domain capable of heterodimerizing with the first Fc domain to form an Fc heterodimer, (iii) a fourth linker that is a protease-cleavable linker ("PCL"), (iv) a T cell-engaging antigen-binding site ("TCE ABS") or a component thereof, (v) an optional fifth linker, and (vi) a second polypeptide chain comprising an antigen-binding site ("TAA ABS") or a component thereof capable of binding to a tumor-associated antigen.

[0327] 35. The TAA ABS, TCE ABS, and anti-TCE ABS are in the form of Fab, and the TAA ABS chain, TCE ABS chain, and anti-TCE ABS chain each contain a VH domain that associates with its respective VL domain on a separate polypeptide chain, the MBM protein according to embodiment 34.

[0328] 36. The VL domain is a universal light chain VL domain, the MBM protein according to embodiment 35. 37. The anti-TCE ABS is in the form of a scFv, the MBM protein according to embodiment 34.

[0329] 38. The anti-TCE ABS is in the form of a VHH, the MBM protein according to embodiment 34. 39. The anti-TCE ABS is in the form of a VH, the MBM protein according to embodiment 34.

[0330] 40. A linker other than PCL is a non-cleavable linker ("NCL"), the MBM protein according to any one of embodiments 34 to 39. 41. Optionally, a multispecific binding molecule (MBM) protein that is the MBM according to any one of embodiments 1 to 4, 15, and 17, (a) A first polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional first linker, (ii) A first Fc domain, (iii) A second linker that is a protease-cleavable linker ("PCL"), and (iv) A first polypeptide chain comprising a T cell-engaging antigen-binding site ("TCE ABS") component comprising a VH, (b) A second polypeptide chain, in an N-terminal to C-terminal orientation, (i) An optional third linker, (ii) A second Fc domain that can heterodimerize with the first Fc domain to form an Fc heterodimer, (iii) A fourth linker that is a protease-cleavable linker (“PCL”), and (iv) a second polypeptide chain comprising a T cell-engaging antigen-binding site (“TCE ABS”) component comprising a VL, An MBM protein, wherein the first polypeptide chain or the second polypeptide chain comprises an antigen-binding site (“TAA ABS”) or a component thereof that can bind to a tumor-associated antigen and is C-terminal to the TCE ABS chain.

[0331] 42. The MBM protein according to embodiment 41, wherein the TCE ABS is a Fab. 43. The MBM protein according to embodiment 42, wherein the first polypeptide chain comprises a C-terminal CH1 domain with respect to the VH of the TCE ABS, and the second polypeptide chain comprises a C-terminal CL domain with respect to the VL of the TCE ABS.

[0332] 44. The MBM protein according to embodiment 41, wherein the TCE ABS is an Fv. 45. The MBM protein according to embodiment 44, wherein the first polypeptide chain lacks a C-terminal CH1 domain with respect to the VH of the TCE ABS, and the second polypeptide chain lacks a C-terminal CL domain with respect to the VL of the TCE ABS.

[0333] 46. The MBM protein according to any one of embodiments 41 to 45, wherein the TAA ABS is in the form of a Fab, and the TAA ABS chain comprises a VH domain that associates with a VL domain on a separate polypeptide chain.

[0334] 47. The MBM protein according to embodiment 46, wherein the VL domain on the separate polypeptide chain is a universal light chain VL domain. 48. The MBM protein according to any one of embodiments 41 to 47, wherein the linker other than the PCL is a non-cleavable linker (“NCL”).

[0335] 49. The MBM protein according to any one of embodiments 41 to 48, wherein the second linker or the fourth linker is 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, or 10 amino acids or less.

[0336] 50. The MBM protein according to any one of embodiments 1 to 49, wherein each PCL contains a substrate sequence cleavable by any protease described in Table A. 51. The MBM protein according to any one of embodiments 1 to 50, wherein each PCL contains one or more substrate sequences selected from the substrate sequences described in Table B.

[0337] 52. The MBM protein according to any one of embodiments 1 to 51, wherein each PCL contains one or more spacer sequences selected from the substrate sequences described in Table C, and optionally, the PCL contains, or consists of, any one of the following amino acid sequences.

[0338] (a) GGGGSGGGGSGGGGSISSGLLSGRSDNHGGSGGS (SEQ ID NO: 205); (b) GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS (SEQ ID NO: 206); (c) ISSGLLSGRSDNH (SEQ ID NO: 230), and (d) A variant of any one of the foregoing having 1 to 5 amino acid substitutions.

[0339] 53. The MBM protein according to any one of embodiments 1 to 52, wherein each PCL contains the amino acid sequence of any one of the PCL sequences selected from the substrate sequences described in Table D.

[0340] 54. The MBM protein according to any one of embodiments 1 to 53, wherein all linkers within the molecule other than the PCL(s) are non-cleavable linkers ("NCL"). 55. The MBM protein according to embodiment 54, wherein the NCL is selected from Table E.

[0341] 56. The TAA ABS is (a) any TAA specified in Section 4.5, or (b) AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplakin-3, and is the MBM proprotein according to any one of Embodiments 1 to 55 that can bind thereto.

[0342] 57. The TAA ABS is the MBM proprotein according to any one of Embodiments 1 to 56, wherein (a) (i) it contains a CDR, or (ii) the VH and VL sequences of the antibody described in Table F, or (b) it competes with the antibody described in Table F for binding to the TAA.

[0343] 58. The MBM protein according to any one of embodiments 1 to 57, wherein the TAA is CEACAM5. 59. The MBM protein according to any one of embodiments 1 to 57, wherein the TAA is EpCAM.

[0344] 60. The MBM protein according to any one of embodiments 1 to 57, wherein the TAA is HER2. 61. The MBM protein according to any one of embodiments 1 to 5571, wherein the TAA is PMSA.

[0345] 62. The MBM protein according to any one of embodiments 1 to 57, wherein the TAA is STEAP2. 63. The MBM protein according to any one of embodiments 1 to 57, wherein the TAA is EGFR.

[0346] 64. The MBM protein according to any one of embodiments 1 to 57, wherein the TCE ABS can bind to a component of the T cell receptor (TCR) complex. 65. The MBM protein according to embodiment 64, wherein the component of the TCR complex is CD3.

[0347] 66. The MBM protein according to embodiment 64, wherein the component of the TCR complex is TCRαβ. 67. The MBM protein according to embodiment 64, wherein the component of the TCR complex is TCRγδ.

[0348] 68. The MBM protein according to any one of embodiments 1 to 67, wherein the TCE ABS comprises (a) (i) a CDR, or (ii) VH and VL sequences of an antibody described in Table G, or (b) competes with an antibody described in Table G for binding to its target.

[0349] 69. The MBM protein according to any one of embodiments 1 to 68, which produces a bispecific binding molecule ("BBM") after protease cleavage of the PCL. 70. The MBM protein according to embodiment 69, wherein the BBM is divalent.

[0350] 71. The MBM protein according to embodiment 69, wherein the BBM is trivalent. 72. The MBM protein according to any one of embodiments 1 to 71, wherein the Fc region is a heterodimer.

[0351] 73. The MBM protein according to embodiment 72, wherein the Fc heterodimer contains a knob-in-hole mutation. 74. The MBM protein according to any one of embodiments 1 to 72, wherein at least one Fc domain contains a star mutation when compared to the wild-type Fc domain.

[0352] 75. The MBM protein according to any one of embodiments 1 to 74, wherein the TCE ABS contains a VL having one or more amino acid deletions at the N-terminus. 76. The MBM protein according to any one of embodiments 1 to 74, wherein the TCE ABS contains a VH having one or more amino acid deletions at the N-terminus.

[0353] 77. The MBM protein according to any one of embodiments 1 to 76, wherein the first Fc domain contains one or more amino acid deletions at the C-terminus relative to the wild-type Fc domain. 78. The MBM protein according to embodiment 77, wherein the first Fc domain contains one amino acid deletion at the C-terminus relative to the wild-type Fc domain.

[0354] 79. The MBM protein according to embodiment 77, wherein the first Fc domain contains two amino acid deletions at the C-terminus relative to the wild-type Fc domain. 80. The MBM protein according to embodiment 77, wherein the first Fc domain contains three amino acid deletions at the C-terminus relative to the wild-type Fc domain.

[0355] 81. The MBM protein according to embodiment 77, wherein the first Fc domain contains a deletion of four amino acids at the C-terminus relative to the wild-type Fc domain. 82. The MBM protein according to embodiment 77, wherein the first Fc domain contains a deletion of five amino acids at the C-terminus relative to the wild-type Fc domain.

[0356] 83. The MBM protein according to embodiment 77, wherein the first Fc domain contains a deletion of six amino acids at the C-terminus relative to the wild-type Fc domain. 84. The MBM protein according to any one of embodiments 1 to 83, wherein the second Fc domain contains a deletion of one or more amino acids at the C-terminus relative to the wild-type Fc domain.

[0357] 85. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of one amino acid at the C-terminus relative to the wild-type Fc domain. 86. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of two amino acids at the C-terminus relative to the wild-type Fc domain.

[0358] 87. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of three amino acids at the C-terminus relative to the wild-type Fc domain. 88. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of four amino acids at the C-terminus relative to the wild-type Fc domain.

[0359] 89. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of five amino acids at the C-terminus relative to the wild-type Fc domain. 90. The MBM protein according to embodiment 84, wherein the second Fc domain contains a deletion of six amino acids at the C-terminus relative to the wild-type Fc domain.

[0360] 91. A tandem Fab MBM comprising: (1) a first polypeptide chain comprising a VH of a TCE ABS operably linked to a CH1 domain, an optional linker, such as a non-cleavable linker, and a VH of a TAA ABS operably linked to the CH1 domain, in an N-terminal to C-terminal orientation; (2) a second polypeptide chain comprising a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain comprising a VL of a TCE ABS operably linked to a CL domain.

[0361] 92. A tandem Fab MBM comprising: (1) a first polypeptide chain comprising, in an N-terminal to C-terminal orientation, (a) a VH of a TCE ABS, (b) a CH1 domain, (c) a non-cleavable linker, (d) a VH of a TAA ABS, and (c) a CH1 domain; (2) a second polypeptide chain comprising (a) a VL of a TAA ABS and (b) a CL domain; and (3) a third polypeptide chain comprising (a) a VL of a TCE ABS and (b) a CL domain.

[0362] 93. A tandem Fab MBM comprising: (1) a first polypeptide chain comprising a VH of a TAA ABS operably linked to a CH1 domain, an optional linker, such as a non-cleavable linker, and a VH of a TCE ABS operably linked to the CH1 domain, in an N-terminal to C-terminal orientation; (2) a second polypeptide chain comprising a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain comprising a VL of a TCE ABS operably linked to a CL domain.

[0363] 94. A tandem Fab MBM comprising: (1) a first polypeptide chain having an N-terminal to C-terminal orientation and containing (a) the VH of TAA ABS, (b) the CH1 domain, (c) a non-cleavable linker, (d) the VH of TCE ABS, and (c) the CH1 domain; (2) a second polypeptide chain containing (a) the VL of TAA ABS and (b) the CL domain; and (3) a third polypeptide chain containing (a) the VL of TCE ABS and (b) the CL domain.

[0364] 95. The tandem Fab MBM according to any one of embodiments 91 to 94, wherein the CH1 of TCE ABS and the CH1 of TAA ABS are the same. 96. The tandem Fab MBM according to any one of embodiments 91 to 95, wherein the CL of TCE ABS and the CL of TAA ABS are the same.

[0365] 97. The tandem Fab MBM according to any one of embodiments 91 to 9674, wherein the VLs of TAA ABS and TCE ABS are identical and are a universal light chain VL.

[0366] 98. The tandem Fab MBM according to any one of embodiments 91 to 97, wherein TAA ABS is as defined in any one of embodiments 50 to 57. 99. The tandem Fab MBM according to any one of embodiments 91 to 98, wherein TCE ABS is as defined in any one of embodiments 64 to 68.

[0367] 100. A pharmaceutical composition comprising the MBM protein according to any one of embodiments 1 to 90 or the tandem Fab MBM according to any one of embodiments 91 to 99, and an excipient.

[0368] 101. A method for treating cancer, comprising administering to a subject suffering from cancer an effective amount of the MBM protein according to any one of Embodiments 1 to 90, the tandem Fab MBM according to any one of Embodiments 91 to 99, or the pharmaceutical composition according to Embodiment 100.

[0369] 102. The method according to Embodiment 101, wherein the cancer is associated with the expression of an epitope bound by a TAA ABS, as described, for example, in Table K. 103. A nucleic acid or plurality of nucleic acids encoding the MBM protein according to any one of Embodiments 1 to 90 or the tandem Fab MBM according to any one of Embodiments 91 to 99.

[0370] 104. A cell engineered to express the MBM protein according to any one of Embodiments 1 to 90 or the tandem Fab MBM according to any one of Embodiments 91 to 99.

[0371] 105. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the MBM protein according to any one of Embodiments 1 to 90 or the tandem Fab MBM according to any one of Embodiments 91 to 99 under the control of one or more promoters.

[0372] 106. A method for producing an MBM protein or tandem Fab MBM, comprising: (a) culturing the cell according to Embodiment 104 or Embodiment 105 under conditions in which the MBM is expressed; and (b) recovering the MBM protein or tandem Fab MBM from the cell culture, if applicable.

[0373] 107. The method according to Embodiment 106, further comprising concentrating the MBM protein or tandem Fab MBM, if applicable. The method according to embodiment 106 or embodiment 107, further comprising purifying the MBM protein or tandem Fab MBM when applicable.

Examples

[0374] 8. Examples 8.1. Example 1: Influence of linker length on Fc masking of the C-terminal antigen-binding site Anti-TAA×CD3 bispecific antibodies having the configuration depicted in FIG. 3B with different linker lengths (5, 15, and 25 amino acids) at the C-terminus to the Fc domain were generated, except that linker B was a non-cleavable linker (see FIG. 3B). The goal of this study was to determine the influence of the linker that separates the Fc domain from the C-terminal ABS or ABS chain on the ability of the ABS to bind its target, in this case CD3, on JURKAT cells. A conventional anti-TAA×CD3 bispecific antibody was used as a control for CD3 binding.

[0375] Flow cytometry analysis was utilized to determine the binding of the TAA×CD3 bispecific antibody to JURKAT cells, followed by detection with an allophycocyanin (APC)-labeled anti-human IgG antibody. Briefly, 1×10 5 cells / well were incubated with serial dilutions of the anti-TAA×CD3 bispecific antibody for 30 minutes at 4°C. After incubation, the cells were washed twice with cold PBS containing 1% filtered FBS, and an APC-labeled anti-human secondary antibody was added to the cells and incubated for an additional 30 minutes. Wells containing secondary only were used as controls.

[0376] After incubation, the cells were washed and resuspended in 200 μL of cold PBS containing 1% filtered FBS and analyzed by flow cytometry on a BD FACS Canto II.

[0377] The results are shown in Figure 9. As is apparent in Figure 9, the ability of the CD3 ABS to bind to its target correlated with the linker length, with shorter linker lengths maximizing steric hindrance by the Fc and resulting in the greatest inhibition of CD3 binding, and longer linker lengths being more permissive for CD3 binding (although still reduced compared to conventional bispecific antibodies).

[0378] 8.2. Example 2: Fc steric hindrance reduces anti-TAA×CD3 bispecific antibody target binding Anti-TAA×CD3 bispecific antibodies having the configuration depicted in Figure 7 were generated by transient transfection in Expi293 cells, and linkers A and B were either cleavable linkers as exemplified or non-cleavable G4S linkers (SEQ ID NO: 1) that had been replaced. Figure 10 shows the expression of anti-TAA×CD3 bispecific antibodies (Fc-PCL-CD3-TAA) having cleavable linkers A and B, and anti-TAA×CD3 bispecific antibodies (Fc-G4S-CD3-TAA) that do not contain cleavable linkers A and B after single-step Pro A purification.

[0379] Flow cytometry analysis was utilized as described in Section 8.1, whereby the binding of the TAA×CD3 bispecific antibody to JURKAT cells was evaluated. Compared to anti-TAA×CD3 bispecific antibodies in the IgG configuration, all anti-TAA×CD3 bispecific antibodies with Fc steric hindrance showed inhibition of CD3 binding on JURKAT cells (Figure 11A). Similar constructs with cleavage at the C-terminus of the Fc by 4 or 6 amino acids and / or at the N-terminus of the VH / VL by 1 or 2 amino acids also inhibited CD3 binding to JURKAT cells (data not shown).

[0380] 9. Citation of references All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes. In case of conflict between one or more of the teachings incorporated by reference herein and the present disclosure, the teachings of this specification shall govern.

Claims

**Claim 1** A binding molecule comprising: (a) a first Fc domain; (b) a second Fc domain; and (c) a first component of a first antigen-binding site (ABS) connected to the first Fc domain via a protease-cleavable linker (PCL), wherein (i) the PCL is 25 amino acids or shorter in length and / or (ii) the binding of the first ABS to its target is sterically hindered, the binding molecule. **Claim 2** The binding molecule according to claim 1, wherein the binding of the first ABS to its target is enhanced after protease cleavage of the PCL. **Claim 3** The binding molecule according to claim 1, wherein the first ABS is sterically hindered from binding to its target prior to protease cleavage of the PCL, and the steric hindrance is released after protease cleavage of the PCL. **Claim 4** The binding molecule according to any one of claims 1 to 3, wherein the PCL is 20 amino acids or shorter in length. **Claim 5** The binding molecule according to any one of claims 1 to 3, wherein the PCL is 15 amino acids or shorter in length. **Claim 6** The binding molecule according to any one of claims 1 to 3, wherein the PCL is 10 amino acids or shorter in length. **Claim 7** The binding molecule according to any one of claims 1 to 6, wherein after protease cleavage of the PCL, the first ABS enhances its binding to its target by at least 10-fold. **Claim 8** The binding molecule according to any one of claims 1 to 6, wherein after protease cleavage of the PCL, the first ABS enhances its binding to its target by at least 100-fold. **Claim 9** The binding molecule according to any one of claims 1 to 8, wherein the first ABS is a Fab. **Claim 10** The binding molecule according to any one of claims 1 to 8, wherein the first ABS is an Fv. **Claim 11** The binding molecule according to any one of claims 1 to 10, wherein the first component is a VH. **Claim 12** The binding molecule according to any one of claims 1 to 10, wherein the first component is a VL. **Claim 13** The binding molecule according to any one of claims 1 to 12, wherein the binding molecule comprises at least two polypeptide chains, a first polypeptide chain comprising the first Fc domain and the first component of the first ABS, and a second polypeptide chain comprising the second Fc domain. **Claim 14** The binding molecule according to claim 13, wherein the second polypeptide chain comprises a second component of the first ABS.

15. The binding molecule according to claim 14, wherein the first component and the second component associate to form the first ABS.

16. The binding molecule according to claim 14 or 15, wherein the first component is VH and the second component is VL.

17. The binding molecule according to claim 16, wherein (1) the first polypeptide chain further comprises a C-terminal CH1 for the VH, and (2) the second polypeptide chain further comprises a C-terminal CL for the VL.

18. The binding molecule according to any one of claims 13 to 15, further comprising a third polypeptide chain comprising a third component of the first ABS.

19. The binding molecule according to any one of claims 1 to 18, which is a multispecific binding molecule comprising a second ABS.

20. The binding molecule according to claim 19, wherein (a) the first ABS is a TCE ABS and the second ABS is a TAA ABS, or (b) the first ABS is a TAA ABS and the second ABS is a TCE ABS.

21. The binding molecule according to claim 20, wherein the first ABS is the TCE ABS.

22. The binding molecule according to claim 21, wherein the TCE ABS can bind to CD3, TCRαβ, or TCRγδ.

23. The TAA ABS is (a) any TAA specified in Section 6.6, or (b) A binding molecule according to any one of claims 20 to 22, which is capable of binding to AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEA CAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplakin-3. [

24. ] The binding molecule according to any one of claims 1 to 23, wherein the first Fc domain and the second Fc domain associate to form an Fc heterodimer. [

25. ] The binding molecule according to claim 24, wherein the Fc heterodimer comprises a knob-in-hole mutation, e.g., (i) the first Fc domain comprises one or more knob mutations and the second Fc domain comprises one or more hole mutations, or (ii) the first Fc domain comprises one or more hole mutations and the second Fc domain comprises one or more knob mutations. [

26. ] The binding molecule according to any one of claims 1 to 25, wherein the first Fc domain and / or the second Fc domain comprises a star mutation.

27. The binding molecule according to any one of claims 1 to 26, wherein the tandem Fab is produced after protease cleavage of the PCL.

28. The binding molecule according to any one of claims 1 to 26, wherein the multispecific binding molecule comprising the first Fc domain and the second Fc domain is produced after protease cleavage of the PCL.

29. The binding molecule according to any one of claims 1 to 28, wherein the PCL comprises a substrate sequence cleavable by any protease described in Table A.

30. The binding molecule according to any one of claims 1 to 29, wherein the PCL comprises one or more substrate sequences selected from the substrate sequences described in Table B.

31. The binding molecule according to any one of claims 1 to 30, wherein the PCL comprises an amino acid sequence of any of the PCL sequences selected from the sequences described in Table D.

32. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3A.

33. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3B.

34. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3C.

35. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3D.

36. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3E.

37. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG. 3F.

38. The binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in FIG.

7.

39. The binding molecule according to any one of claims 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 90% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

40. The binding molecule according to any one of claims 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

41. The binding molecule according to any one of claims 1 to 38, wherein the first Fc domain and / or the second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337, and 338.

42. The binding molecule according to any one of claims 1 to 41, wherein the first Fc domain and / or the second Fc domain comprises one or more amino acid substitutions that reduce effector function (as described, for example, in Section 6.10.1).

43. The binding molecule according to any one of claims 1 to 41, wherein the first Fc domain and / or the second Fc domain comprises one or more amino acid substitutions that promote heterodimerization (as described, for example, in Section 6.10.2).

44. A pharmaceutical composition comprising the binding molecule according to any one of claims 1 to 43 and an excipient.

45. A method of treating cancer comprising administering to a subject afflicted with cancer an effective amount of the binding molecule according to any one of claims 1 to 43 or the pharmaceutical composition according to claim 44.

46. The method according to claim 45, wherein the cancer is associated with the expression of an epitope bound by the TAA ABS, as described, for example, in Table K.

47. A nucleic acid or nucleic acids encoding the binding molecule according to any one of claims 1 to 43.

48. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the binding molecule according to any one of claims 1 to 43 under the control of one or more promoters.

49. A method of producing a binding molecule, comprising: (a) culturing the cell according to claim 48 under conditions in which the binding molecule is expressed; and (b) recovering the binding molecule from the cell culture.

50. The method according to claim 49, further comprising concentrating and / or purifying the binding molecule.

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