Multi-specific binding promolecular proteins and uses thereof

Abstract

The present disclosure provides antigen binding molecules, including proproteins of multispecific binding molecules (MBM). The antigen-binding pre-molecular protein comprises an antigen-binding site, one or more components that inhibit binding of the antigen-binding site to its target, and a protease cleavable linker that is capable of being cleaved by a protease and its cleavage eliminates inhibition of binding of the antigen-binding site to its target. The MBM proprotein and MBM, such as a tandem Fab MBM, produced by cleavage of a protease cleavable linker comprise a T-cell conjugation antigen binding site and a tumor associated antigen binding site. The MBM proprotein further comprises a component that inhibits binding of the T cell-engaging antigen binding site to its target and a protease cleavable linker that can be cleaved by a protease in the tumor environment and its cleavage eliminates the inhibition of binding of the T cell-engaging antigen binding site to its target. The disclosure further provides pharmaceutical compositions comprising the antigen-binding promolecular proteins and methods of using the antigen-binding promolecular proteins in therapy, as well as nucleic acids encoding the antigen-binding promolecular proteins, recombinant cells expressing the antigen-binding promolecular proteins, and methods of using the antigen-binding promolecular proteins in therapy. And methods of producing the antigen-binding promolecular proteins.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to 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 incorporated herein by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML file format, and the sequence listing is hereby incorporated by reference in its entirety. The XML copy created on May 9, 2023 is named RGN-017WO_SL.xml and is 307,242 bytes in size. 3. Background technology

[0005] Selective destruction of tumor cells, while leaving healthy cells and tissues intact, is the goal of cancer immunotherapy. Bispecific antibody therapeutics have been developed to achieve this goal by inducing an immune response against the tumor. In this regard, bispecific antibodies are designed to target components of a tumor-associated antigen ("TAA") and a T cell receptor ("TCR") complex that are preferentially expressed on tumor cells. The simultaneous binding of this antibody to its two targets results in the activation of cytotoxic T cells and subsequent lysis of cells expressing the TAA. Thus, the immune response is redirected to cells expressing the TAA.

[0006] Several bispecific antibody formats have been developed and investigated for their suitability for T cell-mediated immunotherapy. See You et al., 2021, Vaccines 9:724, doi.org / 10.3390 / vaccines9070724.

[0007] The task of generating bispecific molecules suitable for therapy presents several technical challenges related to toxicity, as TAAs are often expressed on normal cells as well as tumor cells. Therefore, there is a need for potent T cell-activating bispecific molecules that selectively release T cell activation in the tumor environment. 4. Summary of the invention

[0008] The present invention generally relates to novel protease-activatable antigen binding molecules ("ABMs"), referred to herein as antigen binding molecule proproteins.

[0009] In general, in its active state, the antigen binding molecule comprises an antigen binding site (ABS) that can be bound to its target. However, in the pre-protein form, the ABS is masked by a masking portion, so that its ability to bind to its target is greatly weakened. The antigen binding molecule pre-protein is configured so that when encountering a protease, such as a protease overexpressed in a tumor environment, the masking portion is cut and a binding molecule with enhanced target binding is produced. This is achieved by including one or more protease cleavable joints ("PCL"), which comprise one or more protease substrate sequences, for example, directly or indirectly between the ABS and the masking portion. The pre-protein can reduce the toxicity and adverse side effects caused by the binding of ABM to normal tissue. In general, the antigen binding molecule pre-protein is described in Section 6.2 and Examples 1 to 43 of Group A numbering, and Examples 1 to 90 of Group B numbering.

[0010] In some embodiments, the antigen binding molecule preprotein of the present disclosure is a multispecific binding molecule (MBM) preprotein. Generally speaking, in its active state, the MBM of the present disclosure comprises a T cell engagement antigen binding site ("TCE ABS") and a tumor-associated antigen binding site ("TAA ABS") that binds to a component of the T cell receptor complex, which can simultaneously bind to their respective targets, thereby stimulating T cells bound by TCE ABS to attack tumor cells expressing TAA bound by TAA ABS. However, when the MBM is in the form of a preprotein, the TCE ABS is masked by the masking portion, thereby greatly reducing its ability to bind to its target. The MBM preprotein is configured so that when a protease is encountered, such as a protease overexpressed in a tumor environment, the masking portion is cleaved and the inhibition of TCE ABS binding is reversed. This is achieved by including a protease cleavable linker ("PCL"), which comprises one or more protease substrate sequences in the MBM preprotein, for example, directly or indirectly located between the masking portion and the TCE ABS. The proprotein can reduce toxicity and adverse side effects caused by the binding of MBM to normal tissues. In general, MBM proproteins are described in Section 6.3 and Group B numbered Examples 1 to 5, 15, 54, and 69 to 72.

[0011] In certain aspects, the MBM preprotein (referred to as a 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, a Type I MBM comprises an anti-TCE ABS masking moiety. In some embodiments, the MBM preprotein is configured such that activation of the MBM preprotein releases an MBM (e.g., a bispecific binding molecule) comprising an intact Fc region. This type of MBM preprotein is referred to as a Type IA MBM preprotein and an exemplary embodiment of a Type IA MBM is depicted in Figures 1A to 1C and described in Section 6.3.1.1 and Group B numbered Examples 6 to 8 and 18 to 24. In other embodiments, the MBM proprotein is configured such that activation of the MBM proprotein releases an MBM lacking an Fc region, such as a tandem Fab or BiTe. This type of MBM proprotein is referred to as a Type IB MBM proprotein and an exemplary embodiment of a Type IB MBM is depicted in FIG. 5A to FIG. 5B and described in Section 6.3.1.2 and Group B numbered Examples 16 and 34 to 40.

[0012] In other aspects, the MBM proprotein (referred to as a Type II MBM), the TCE ABS is sterically hindered from binding to its target due to its proximity to an adjacent domain (e.g., an Fc domain) that sterically hinders its binding to its target. In some embodiments, the MBM proprotein is configured such that activation of the MBM proprotein releases an MBM (e.g., a bispecific binding molecule) comprising an intact Fc region. This type of MBM proprotein is referred to as a Type IIA MBM proprotein and an exemplary embodiment of a Type IIA MBM is depicted in FIG. 3A to FIG. 3F and described in Section 6.3.1.1 and Group B numbered Examples 9 to 14 and 25 to 33. In other embodiments, the MBM proprotein is configured such that activation of the MBM proprotein releases an MBM lacking an Fc region, such as a tandem Fab or BiTe. This type of MBM proprotein is referred to as a Type IIB MBM proprotein and an exemplary embodiment of a Type IIB MBM is depicted in Figure 7 and described in Section 6.3.1.2 and Group B numbered Examples 17 and 41 to 49.

[0013] Section 6.4, Group A, Numbered Examples 29 to 31 and Group B, Numbered Examples 50 to 53 describe exemplary protease-cleavable linkers that can be used in antigen binding molecule proproteins, including MBM proproteins, of the present disclosure.

[0014] The present disclosure further provides a tandem Fab MBM comprising TAA ABS and TCE ABS. The tandem Fab can be produced as a preprotein by activation of a type IB or type IIB MBM, or it can be produced by expression of a tandem Fab protein (with a relevant signal sequence but without a masking moiety). In this case, the TAA ABS and TCE ABS preferably share a common light chain sequence. Section 6.11 and Examples 75 to 83, numbered in Group B, describe exemplary tandem Fab MBMs.

[0015] Section 6.5 and Example 55, numbered in Group B, describe exemplary non-cleavable linkers that can be incorporated into the antigen-binding molecule preproteins (including MBM preproteins) and tandem Fab MBMs of the present disclosure. Section 6.6, Example 23, numbered in Group A, and Examples 56 to 64, numbered in Group B, describe exemplary TAA ABS that can be incorporated into the antigen-binding molecule preproteins (including MBM preproteins) and tandem Fab MBMs of the present disclosure. Section 6.7, Example 22, numbered in Group A, and Examples 64 to 68, numbered in Group B, describe exemplary TCE ABS that can be incorporated into the antigen-binding molecule preproteins (including MBM preproteins) and tandem Fab MBMs of the present disclosure. Section 6.8 describes exemplary anti-TCE ABS that can be incorporated into the antigen-binding molecule preproteins (including MBM preproteins) of the present disclosure. Section 6.9 describes suitable forms of ABS incorporated into the antigen-binding molecule preproteins (including MBM preproteins) of the present disclosure. Section 6.10 and Group B numbered Examples 73 and 74 describe suitable Fc domains that can be incorporated into antigen binding molecule proproteins, including MBM proproteins, of the present disclosure.

[0016] The present disclosure further provides nucleic acids encoding the antigen binding molecule preproteins (including MBM preproteins) and tandem Fab MBMs (in a single nucleic acid or multiple nucleic acids) of the present disclosure, and recombinant host cells and cell lines engineered to express nucleic acids and antigen binding molecule preproteins (including MBM preproteins) of the present disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 6.12, Examples 47 and 48, Group A, and Examples 87 to 92, Group B.

[0017] Also provided are pharmaceutical compositions comprising antigen binding molecule proproteins (including MBM proproteins) and tandem Fab MBMs of the disclosure. Examples of pharmaceutical compositions are described in Section 6.13, Group A numbered Example 44 and Group B numbered Example 84.

[0018] Further provided herein are methods of using the antigen binding molecule proproteins (including MBM proproteins), tandem FabMBMs, and pharmaceutical compositions disclosed herein, e.g., for treating proliferative disorders (e.g., cancer) on which TAAs are expressed. Exemplary methods and indications are described in Section 6.14, Examples 45 and 46, Group A, and Examples 85 and 86, Group B. 5. Description of the drawings

[0019] Figures 1A to 1C Schematic representation of an example of a Type IA MBM preprotein in which the TCE Abs are masked by the anti-TCE Abs. Figure 1A and Figure 1B In the embodiment of , the MBM produced by cleavage of linker B is bivalent, having one TAA ABS and one TCEABS. Figure 1C In some embodiments, the MBM produced by cleavage of linker B is trivalent, having two TAA ABS and one TCEABS. In some embodiments, the trivalent MBM is bispecific. Figures 1A to 1C The ABS in is illustrated as Fab, the ABS may be in other formats, such as scFv or other formats described in Section 6.9. In all embodiments depicted in this figure, the MBM comprising the Fc domain is released after cleavage of linker B.

[0020] Figure 2 The diagram shows Figures 1A to 1C Exemplary activation mechanisms of MBM proproteins.

[0021] Figure 3A Figure 3G is a schematic representation of an example of a Type IIA MBM preprotein in which the Fc region acts as a masking moiety by sterically hindering the binding of TCEABS to its target. Figure 3A and Figure 3B In some embodiments, the MBM produced by cleavage of linker B is trivalent, having two TAA ABS and one TCE ABS. In some embodiments, the trivalent MBM is bispecific. FIG. 3C to FIG. 3F In the embodiment of , the MBM produced by cleavage of linker B is bivalent, having one TAA ABS and one TCEABS. FIG. 3A to FIG. 3F The TAA ABS is shown as Fab, and the ABS may be in other formats, such as scFv or other formats described in Section 6.9. The TCE ABS may be in the form of Fab (e.g., Figure 3A , 3C and 3E) or Fv (e.g., Figure 3B , 3D In all embodiments depicted in this figure, the MBM comprising the Fc domain is released upon cleavage of linker B.

[0022] Figure 4 The diagram shows Figure 3A To Figure 3G for an exemplary activation mechanism of the MBM proprotein.

[0023] FIG. 5A to FIG. 5B An example of a type IB MBM preprotein in which the TCE Abs are masked by anti-TCE Abs is depicted. The MBM resulting from cleavage of linker B is bivalent, has one TAA Abs and one TCE Abs, and lacks an Fc domain. Figure 5A The ABS in the diagram is illustrated as a Fab, the ABS may be in other formats, such as a scFv or other formats described in Section 6.9.

[0024] Figure 6 The diagram shows FIG. 5A to FIG. 5B Exemplary activation mechanisms of MBM proproteins.

[0025] Figure 7 is a diagrammatic representation of an example of a type IIB MBM proprotein in which the Fc region acts as a masking moiety by sterically hindering the binding of the TCE Abs to its target. The MBM produced by cleavage of linker B is bivalent, has one TAA Abs and one TCE Abs, and lacks the Fc domain. Figure 7 The ABS in the diagram is illustrated as a Fab, the ABS may be in other formats, such as a scFv or other formats described in Section 6.9.

[0026] Figure 8 The diagram shows Figure 7 Exemplary activation mechanisms of MBM proproteins.

[0027] Fig. 9 shows the results of a study that showed that in FIG. 3A to FIG. 3F and Figure 7 In constructs with TCE Abs C-terminal to the Fc domain in the configuration shown in , there is a correlation between linker length and the ability of the Fc region to sterically hinder the binding of the anti-CD3 TCE Abs to its target. To demonstrate the effect of linker length, the linker in this study did not contain a substrate and was non-cleavable. Fig. 9 SEQ ID NOs: 1 (1xG4S), 156 (3xG4S) and 153 (5xG4S) are disclosed.

[0028] Fig.10 Gel images showing anti-TAAxCD3 antibodies run on non-reducing and reducing SDS-polyacrylamide gels after protein A purification are shown. The anti-TAAxCD3 bispecific antibodies tested had Figure 7The configuration depicted in , wherein linkers A and B are cleavable linkers as shown or are replaced by the non-cleavable G4S (SEQ ID NO: 1) linker.

[0029] Fig.11 shows the results of a study that demonstrated that Figure 7 Inhibition of CD3 binding on JURKAT cells by Fc steric hindrance in the Type IIB MBM proprotein construct configured as shown in . The curve shows inhibition of CD3 binding by the Type IIB MBM proprotein relative to the anti-TAAxCD3 antibody in IgG format. 6. Specific implementation methods

[0030] 6.1. Definitions

[0031] As used herein, the following terms are intended to have the following meanings:

[0032] ABM proprotein : As used herein, the term "ABM proprotein" (or "antigen binding molecule proprotein") refers to an ABM whose ability to bind to a target recognized by at least one of its ABS is reduced or abolished, for example, due to the presence of a masking moiety. The masking moiety hinders 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 links the masking moiety to the ABM.

[0033] ABS chain : A single ABS may exist as one (e.g., in the case of an scFv) polypeptide chain, or may be formed by the association of more than one polypeptide chain (e.g., in the case of a Fab). As used herein, the term "ABS chain" refers to all or part of the ABS present on a single polypeptide chain. The use of the term "ABS chain" is intended only for convenience and descriptive purposes and does not imply a particular configuration or method of production. In addition, when describing an ABM, ABM preprotein, MBM, or MBM preprotein, reference to the ABS encompasses the ABS chain unless the context indicates otherwise. Thus, when describing an ABM, ABM preprotein, MBM, or MBM preprotein in which an Fc domain is operably linked to an ABS, the Fc domain may be covalently linked (e.g., via a linker) directly or indirectly via a peptide bond to, for example, (1) a first ABS chain of a Fab (the other components of the Fab being on a second associated ABS chain) or (2) a single ABS chain comprising an scFv.

[0034] About: Throughout the specification, the terms "about", "approximately", etc. are used before numbers to indicate that the number is not necessarily exact (e.g., to take into account fractions, variations in measurement accuracy and / or precision, timing, etc.). It should be understood that disclosure of "about X" or "approximately X", where X is a number, is also disclosure of "X". Thus, for example, disclosure of embodiments in which one sequence has "about X% sequence identity" to another sequence is also disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0035] Activate (activate / activation) : The term "activation" and the like used in conjunction with the MBM proprotein of the present disclosure refers to the protease-mediated enzymatic cleavage of the protease-cleavable linker, which results in the unmasking of the TCE ABS, thereby generating an ABM or MBM, and increasing the ability of the TCE ABS to bind to its target, such as when the protease-cleavable linker is intact, by releasing or separating the TCE ABS from the anti-TCE ABS or Fc domain, which sterically hinders the binding of the TCE ABS to its target. Sometimes, activation is referred to herein as "release" of the MBM, masking moiety or TCE ABS.

[0036] and, or : Unless otherwise indicated, the conjunction "or" shall be used in its proper sense as a Boolean logical operator, covering both the selection of features among alternatives (A or B, where the selection of A is mutually exclusive with B) and the selection of features of a union (A or B, where both A and B are selected). In some places in the text, the term "and / or" is used for the same purpose, which should not be interpreted as implying that "or" is used to refer to mutually exclusive alternatives.

[0037] Anti-idiotype antibody (anti-idiotypeantibody / anti-idiotypicantibody) : The term "anti-idiotype antibody (anti-idiotype antibody / anti-idiotypic antibody)" and the like refers to an antibody that recognizes the idiotype of an antigen binding site (e.g., an antigen binding site that is specific to a TCR component such as CD3). Anti-idiotype antibodies are able to 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 comprising an antigen binding site, the anti-idiotype antibody can serve as a masking portion of the molecule. The antigen binding component of an anti-idiotype antibody (which recognizes the variable region of a T cell engaging antibody, such as an antibody that recognizes CD3 or another component of a T cell receptor) is generally referred to herein as "TCE ABS".

[0038] Antibody:As used herein, the term "antibody" refers to a polypeptide (or polypeptide group) of the immunoglobulin family that can non-covalently, reversibly and specifically bind 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 comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a domain (abbreviated herein as CL). The VH region and the VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative 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 carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. 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. Antibodies may have any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Light and heavy chains are divided into structural regions and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distant from the antigen binding site or amino terminus of the antibody. The N-terminus is the variable region, and at the C-terminus is the constant region; the CH3 and CL domains represent the carboxyl termini of the heavy and light chains of natural antibodies, respectively. For convenience, and unless the context otherwise dictates, reference to antibodies also refers to antibody fragments and engineered antibodies, which include non-naturally occurring antigen binding sites and / or antigen binding sites with non-natural configurations, for example, molecules with Fab or scFv domains at the C-terminus of the CH3 domain.

[0039] 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 contains one or more antigen binding sites. The ABMs of the present disclosure may be monospecific or multispecific (e.g., bispecific). The antigen binding sites in a monospecific binding molecule all bind to the same epitope, while a multispecific binding molecule has at least two antigen binding sites that bind to different epitopes, which may be the same or different target molecules. In some embodiments, the antigen binding molecule is an antibody.

[0040] Antigen binding site : As used herein, the term "antigen binding site" or "ABS" refers to the portion of an antibody, ABM, or MBM preprotein that has the ability to non-covalently, reversibly, and specifically bind to an antigen in the absence of masking. For example, in some embodiments, the ABS may be masked in the context of an ABM or MBM preprotein, but when the ABM or MBM is generated by cleavage of a protease-cleavable linker in the ABM or MBM preprotein, the ABS is able to non-covalently, reversibly, and specifically bind to an antigen. Examples of antigen binding sites include antibody fragments, such as, but not limited to, single-chain Fv (scFv), Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments, which consist of a VH domain and a CH1 domain; Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., 1989, Nature 341:544-546), which consist of a VH domain; VHH antibodies (also referred to as "VHH" or Vincke et al., 2012, Methods Mol Biol. 911: 15-26); and isolated complementarity determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins comprising one or more portions of an antibody (e.g., scFv). Antibody fragments may also be incorporated into single domain antibodies, giant antibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

[0041] Association: In the context of ABM or MBM pre-protein, the term "association" refers to a functional relationship between two or more polypeptide chains. Specifically, the term "association" means that two or more polypeptides associate with each other (e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical cross-links), thereby generating a functional MBM pre-protein. Examples of associations that may be present in the MBM pre-protein of the present disclosure include, but are not limited to, associations between Fc regions in an Fc domain (homodimers or, more preferably, heterodimers as described in Section 6.10.2), associations between VH and VL regions in Fab or Fv, and associations between CH1 and CL in Fab.

[0042] Two-price : As used herein, the term "bivalent" used in the context of an antigen binding molecule refers to an antigen binding molecule with two antigen binding sites. These domains can be the same or different. Therefore, a bivalent antigen binding molecule can be monospecific or bispecific.

[0043] cancer : The term "cancer" refers to a disease characterized by uncontrolled (and usually rapid) growth of abnormal cells. Cancer cells can spread locally or spread 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, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penis 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.

[0044] CD3 : The term "CD3" refers to the cluster of differentiation 3 coreceptors (or coreceptor complexes, or polypeptide chains of coreceptor complexes) of the T cell receptor. The amino acid sequence of the polypeptide chain of human CD3 is provided in NCBI Accession Nos. P04234, P07766, and P09693.

[0045] Complementarity determining region:As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within the antibody variable region that confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7: 132-136; Lefranc et al., 2003, Dev. Comp. Immunol. 27: 55-77 ("IMGT" numbering scheme). For example, for the classical form, under Kabat, the CDR amino acid residues in 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 in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, V The CDR amino acids in H are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) (according to "Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0046] Effector function : The term "effector function" refers to the activity of an antibody molecule mediated by binding of a domain other than the antigen binding site in the antibody, usually mediated by the binding of an effector molecule. Effector functions include complement-mediated effector functions, which are mediated by, for example, the binding of the C1 component of complement to an antibody. The activation of complement is important for the opsonization and lysis of cellular pathogens. The activation of complement can also stimulate inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered when the constant domain of an antibody binds to an Fc receptor (FcR). The binding of an antibody to an Fc receptor on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of an antibody can be changed by changing, for example, enhancing or reducing the affinity of the antibody to effector molecules such as Fc receptors or complement components. Binding affinity is usually changed by modifying the effector molecule binding site, and in this case, the site of interest is appropriately positioned and at least a portion of the site is modified in a suitable manner. It is also envisioned that changes in the binding site on the antibody of the effector molecule do not need to significantly change the overall binding affinity, but may change the geometry of the interaction, making the effector mechanism as ineffective as in non-productive binding. It is further envisioned that the effector function can also be changed by modifying sites that are not directly involved in the binding of the effector molecule, but otherwise involved in the performance of the effector function.

[0047] Epitope : An epitope or antigenic determinant is a portion of an antigen that is recognized by an antibody or other antigen binding moiety as described herein. An epitope can be linear or conformational.

[0048] Fab: The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy chain (VH) domain of the antibody, which is operably connected (usually at the N-terminus) to the first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light chain (VL) domain at the N-terminus of the antibody, which is operably connected (usually at the N-terminus) to the second constant domain (referred to herein as C2) that can be paired with the first constant domain. In natural antibodies, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located 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 natural orientation or include domain substitutions or exchanges that promote correct VH and VL pairing. For example, it is possible to replace the CH1 and CL domain pairs in Fab with CH3 domain pairs to promote correctly modified Fab chain pairing in heterodimeric molecules. It is also possible to reverse CH1 and CL so that CH1 is attached to VL and CL is attached to VH, and this configuration is generally referred to as Crossmab. The term "Fab" encompasses single chain Fab.

[0049] Fc domain and Fc region : The term "Fc domain" refers to a portion of a heavy chain that pairs with the corresponding portion 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 an Fc region may be identical or different from each other. In native antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, for example, by knob-and-hole interactions.

[0050] F : The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin containing a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of a heavy chain and a light chain variable domain that are tightly and non-covalently associated. It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer antibody target binding specificity. However, in some cases, even a single variable domain (or half of an Fv containing only three CDRs specific for a target) may have the ability to recognize and bind to a target. The VH-VL dimer mentioned herein is not intended to convey any particular configuration. By way of example and not limitation, VH and VL may be aggregated together in any configuration described herein to form a half antibody, or may each be present on a separate half antibody and aggregated together to form an antigen binding domain when a separate half antibody is associated, for example to form an ABM or MBM preprotein of the present disclosure. When present on a single polypeptide chain (eg, scFv), VH is located at the N-terminus or C-terminus of VL.

[0051] Half Antibody : The term "half antibody" refers to a molecule comprising at least one ABS or ABS chain and which can associate with another molecule comprising an ABS or ABS chain through, for example, a disulfide bridge or a molecular interaction (e.g., a knob-and-hole interaction between Fc heterodimers). A half antibody can be composed of one polypeptide chain or more than one polypeptide chain (e.g., two polypeptide chains of Fab). In a preferred embodiment, the half antibody comprises an Fc region. An example of a half antibody is a molecule comprising a heavy chain and a light chain of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule comprising a first polypeptide comprising a VL domain and a CL domain and a second polypeptide comprising 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 comprising a scFv domain, a CH2 domain, and a CH3 domain.

[0052] A half antibody may comprise more than one ABS, for example a half antibody comprising (in order from N-terminal to C-terminal) a VH1 domain, a CH1 domain, a CH2 domain, a CH3 domain and another VH1 domain or a scFv domain. A half antibody may also comprise an ABS chain that, when associated with another ABS chain in another half antibody, forms a complete ABS.

[0053] Thus, an ABM or MBM preprotein may comprise one, more typically two or even more than two half-antibodies, and a half-antibody may comprise one or more ABS or ABS chains.

[0054] In some ABM or MBM pre-proteins, the first half-antibody will associate, e.g., heterodimerize, with the second half-antibody. In other ABM or MBM pre-proteins, the first half-antibody will be covalently linked to the second half-antibody, e.g., by a disulfide bridge or chemical cross-linking. In still other ABM or MBM pre-proteins, the first half-antibody will associate with the second half-antibody by covalent attachment and non-covalent interactions (e.g., disulfide bridges and knob-in-hole interactions).

[0055] The term "half antibody" is used for descriptive purposes only and does not imply a particular configuration or method of production. Describing half antibodies as the "first" half antibody, the "second" half antibody, the "left" half antibody, the "right" half antibody, etc. is merely for convenience and descriptive purposes.

[0056] Host cells or recombinant host cells: The term "host cell" or "recombinant host cell" refers to, for example, a cell that is genetically engineered by the introduction of a heterologous nucleic acid. It should be understood that such terms refer not only to a specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may actually be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. The host cell may carry the heterologous nucleic acid transiently (e.g., on an extrachromosomal heterologous expression vector) or stably (e.g., by integrating the heterologous nucleic acid into the host cell genome). For the purpose of expressing the ABM or MBM preprotein 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 cells (BHK, e.g., BHK21), Chinese hamster ovary cells (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madara's bovine kidney (MDBK) cells, 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 site-specific integration site derivatives.

[0057] Connectors : As used herein, the term "linker" refers to a protease cleavable linker or a non-cleavable linker.

[0058] Masking part: As used herein, the term "masking moiety" in relation to an MBM preprotein refers to an amino acid sequence in the MBM preprotein that inhibits the ability of a TCE Abs to specifically bind to its target, either by specific interaction with the TCE Abs (e.g., where the masking moiety is an anti-idiotype of the TCE Abs or "anti-TCE Abs"), or by positioning the TCE Abs relative to another component of the MBM preprotein that sterically hinders the binding of the TCE Abs to its target (e.g., by linking the TCE Abs to the Fc region via a short linker). The masking moiety and the TCE Abs are arranged in the MBM preprotein such that cleavage of the protease-cleavable linker reduces the inhibition of the interaction of the TCE Abs with its target, either by generating an MBM lacking the masking moiety or by generating an MBM that alleviates steric restrictions on the ability of the TCE Abs to interact with its target. When used in relation to an antigen binding molecule, the term "masking moiety" more generally refers to an amino acid sequence in the preprotein of an antigen binding molecule that inhibits the ability of an antigen binding site (Abs) in the antigen binding molecule to specifically bind to its target, either by a specific interaction with the Abs (e.g., where the masking moiety is an anti-idiotype of the Abs), or by positioning the Abs relative to another component of the antigen binding molecule that sterically hinders the binding of the Abs to its target (e.g., by linking the Abs to the Fc region via a short linker). In general, the masking moiety and the Abs are arranged in the antigen binding molecule such that cleavage of the protease-cleavable linker reduces the inhibition of the interaction of the Abs with its target, either by generating an antigen binding molecule lacking the masking moiety or by generating a sterically restricted antigen binding molecule that mitigates the ability of the Abs to interact with its target.

[0059] MBM proprotein : As used herein, the term "MBM pre-protein" refers to an MBM whose ability to bind to a target recognized by at least one of its ABS is reduced or abolished, for example, due to the presence of a masking moiety. The masking moiety hinders 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 links the masking moiety to the MBM. Typically, in the MBM pre-proteins of the present disclosure, the TCE ABS are masked, while the TAA ABS are not masked. The TAA ABS can act as a targeting moiety to direct the MBM pre-protein to the tumor site, where recognition of a substrate in the protease-cleavable linker results in linker cleavage, restoring the binding of the TCE ABS to its target and the ability of the MBM to activate T cells against tumor cells expressing the TAA.

[0060] unit price: As used herein, the term "monovalent" used in the context of an antigen binding molecule refers to an antigen binding molecule with a single antigen binding site.

[0061] Multispecific binding molecules, MBM : The terms "multispecific binding molecule" and "MBM" refer to molecules that contain two or more antigen binding sites. Typically, MBMs contain TAA ABS and TCE ABS.

[0062] Non-cleavable linker A non-cleavable linker refers to a peptide whose amino acid sequence lacks a substrate sequence for a protease (eg, a protease as described in Section 6.4.1 that recognizes and cleaves a specific sequence motif, eg, a substrate as described in Section 6.4.2).

[0063] Operatively connected : The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of fusion proteins or other polypeptides, the term "operably linked" means connecting two or more amino acid segments to produce a functional polypeptide. For example, in the context of the ABM or MBM preprotein of the present disclosure, a separate ABS (or a chain of ABS) can be operably linked via 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 joining two nucleic acids so that the amino acid sequences encoded by the two nucleic acids remain in frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, the promoter or enhancer sequence is operably linked to a coding sequence.

[0064] Polypeptides, peptides and proteins : The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0065] Preprotein A "proprotein" is an inactive protein precursor and can be activated by the proteolytic action of a protease. Thus, a proprotein is "protease-activatable".

[0066] Protease As used herein, the term "protease" refers to any enzyme that catalyzes the hydrolysis of peptide bonds. In general, proteases useful in the present disclosure, e.g., proteases described in Section 6.4.1, recognize and cleave specific sequence motifs, e.g., substrates as described in Section 6.4.2. Preferably, the protease is expressed at a higher level in cancer tissues than in normal tissues.

[0067] 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 (eg, two or three) substrate sequences for one or more proteases.

[0068] Identification : As used herein, the term "recognize" refers to an ABS that finds its epitope and interacts with (eg, binds to) it.

[0069] Single-chain Fab or scFab : As used herein, the term "single-chain Fab" or "scFab" refers to an ABS comprising a VH domain, a CH1 domain, a VL domain, a CL domain and a linker. In some embodiments, the aforementioned domains and linkers are arranged in one of the following orders in the 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 suitably a non-cleavable linker of at least 30 amino acids, preferably 32 to 50 amino acids. Single-chain Fab fragments are generally stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the generation of interchain disulfide bonds by inserting cysteine ​​residues (e.g., at position 44 of the VH domain and position 100 of the VL domain, according to Kabat numbering).

[0070] Single chain Fv or scFv :As used herein, the term "single-chain Fv" or "scFv" 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 includes a polypeptide linker between the VH and VL domains, which enables the scFv to form a desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. (1994), Springer-Verlag, New York, pp. 269-315. VH and VL are arranged in the order VH-VL or VL-VH from N-terminus to C-terminus, usually separated by a linker, such as the linker listed in Table E.

[0071] Spacer : As used herein, the term "spacer" refers to a peptide incorporated into a linker containing a substrate, the amino acid sequence of which is not a substrate for a protease. Spacers can be used to separate substrates from other domains (e.g., ABS) in a molecule. In some aspects, the residues in the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent the cleavage of the N-terminal amino acid.

[0072] Specific (or selective) binding : The term "specific (or selective) binding" to an antigen or epitope refers to a binding reaction that determines the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can, but need not, be mediated by an antibody or antibody fragment. The term "specific binding" does not exclude cross-species 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 may also "specifically bind" to the antigen in one or more other species. Therefore, this cross-species reactivity itself does not change the classification of the antigen binding site as a "specific" binder. In certain embodiments, an antigen binding site of the present disclosure that specifically binds to a human antigen (e.g., "TAA ABS") has cross-species reactivity with one or more non-human mammalian species (e.g., primate species (including but not limited to one or more of cynomolgus monkeys, macaques, and pig-tailed monkeys) or rodent species, such as house mice).

[0073] Subjects : The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, for example, 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.

[0074] Substrate : The term "substrate" refers to the peptide sequence on which the protease will act and in which the protease will cleave peptide bonds.

[0075] TAA ABS : The term "TAA ABS" refers to an ABS that recognizes or specifically binds to an extracellular matrix ("ECM") protein, 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 TAA. The skilled person will recognize that the aforementioned categories of target molecules are not mutually exclusive, and therefore a given target molecule may fall into more than one of the aforementioned categories of target molecules. For example, some molecules may be considered as TAAs and ECM proteins, while other molecules may be considered as TCAs and checkpoint inhibitors.

[0076] T cell antigen, TCA : The term "T cell antigen" or "TCA" refers to a molecule (usually a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of a T lymphocyte and can be used to preferentially target an agent to a specific site. In some embodiments, the site is a cancerous 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.

[0077] 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 signaling through the TCR complex. Exemplary T cell antigens recognized or specifically bound by TCE ABS are CD3 and other components of the TCR complex.

[0078] T cell receptor, TCR : "T cell receptor" or "TCR" is a component of T cells that is responsible for interacting with and sensing the targets of T cell adaptive immunity. In general, TCRs contain heterodimeric protein complexes present on the surface of cells. T cells can be roughly classified as αβ or γδ, depending on the form of somatic rearranged TCR expressed by the T cell on its surface. There are two forms of TCR chain pairs; TCRα and TCRβ are paired with TCRγ and TCRδ. Mature αβ and γδ TCR chain pairs are presented on the cell surface together with many additional CD3 subunits (denoted as ε, γ, δ and ζ). These subunits associate with αβ or γδ TCR into three dimers (εγ, εδ, ζζ). This TCR complex forms a unit that initiates a cell signaling response after TCRαβ or TCRγδ engages with the cognate antigen. The terms "T cell receptor complex" and "TCR complex" refer to a complex of TCRαβ or TCRγδ with CD3.

[0079] Tri-Price : As used herein, the term "trivalent" used in the context of an antigen binding molecule refers to an antigen binding molecule having three antigen binding sites. The antigen binding sites may be all identical, all different, or include two identical antigen binding sites and a third different antigen binding site.

[0080] Tumor : The term "tumor" is used interchangeably with the term "cancer" herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0081] Tumor-associated antigens: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of a cancer cell in its entirety or as a fragment (e.g., MHC / peptide), and it can be used to preferentially target an agent to a cancer cell. In some embodiments, TAAs are markers expressed by normal cells and cancer cells, such as lineage markers. In some embodiments, TAAs are cell surface molecules overexpressed in cancer cells compared to normal cells, such as 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are inappropriately synthesized in cancer cells, such as molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs are exclusively expressed on the cell surface of cancer cells in their entirety or as a fragment (e.g., MHC / peptide), and are not synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses antigens that are specific to cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSAs").

[0082] TAA ABS : As used herein, the term "TAA ABS" refers to an antigen binding site that recognizes or specifically binds to TAA.

[0083] Treatment (treat / treatment / treating) : As used herein, the terms "treat / treatment / treating" refer to reducing or ameliorating the progression, severity and / or duration of a proliferative disorder, or an improvement in one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder caused by the administration of one or more ABM or MBM pre-proteins of the present disclosure. In specific embodiments, the terms "treat / treatment / treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder (such as the growth of a tumor, which may not be discernible to the patient). In other embodiments, the terms "treat / treatment / treating" refer to inhibiting the progression of a proliferative disorder, either physically, such as by stabilizing discernible symptoms, or physiologically, such as by stabilizing physical parameters, or both. In other embodiments, the terms "treat / treatment / treating" refer to a reduction or stabilization of tumor size or cancer cell count.

[0084] Universal light chain, UCL: As used herein, the term "universal light chain" or "ULC" refers to a light chain variable region (VL) that can be paired with more than one heavy chain variable region (VL). In the context of ABS, the term "universal light chain" or "ULC" refers to a light chain polypeptide that can be paired with a heavy chain region of ABS and can also be paired with other heavy chain regions. The ULC may also include a constant domain, for example, the CL domain of an antibody. Universal light chains are also referred to as "common light chains."

[0085] VH : The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of Fv, scFv, dsFv or Fab.

[0086] V L : The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

[0087] 6.2. Antigen Binding Molecule Preprotein

[0088] The present disclosure relates to an antigen binding molecule preprotein comprising an ABS, a masking moiety and a protease cleavable linker (PCL), which is arranged so that the masking moiety reduces or blocks the binding of the ABS to its target, and is configured so that when a protease, such as a protease overexpressed in a tumor environment, is encountered, the masking moiety is cleaved and a binding molecule with enhanced target binding is generated. Typically, the masking moiety of the antigen binding molecule preprotein masks the ABS by steric hindrance (e.g., an Fc domain masking moiety) and / or by binding of a targeting moiety to the ABS (e.g., an anti-ABS binding moiety).

[0089] Thus, the antigen binding molecule preprotein of the present disclosure generally comprises: (a) a masking moiety, and (b) an ABS or a component thereof linked to the masking moiety by a PCL, wherein the ABS is hindered (e.g., sterically hindered) from binding to its target. Exemplary masking moieties include an Fc domain and an anti-ABS binding moiety. The use of an Fc domain as a masking moiety provides a steric hindrance of the ABS, which is released after protease cleavage of the PCL (e.g., by a tumor-specific protease). Specific examples of antigen binding molecule preproteins masked by the Fc domain include type IIA and type IIB MBM preproteins (e.g., as described in Section 6.3.2). The use of an anti-ABS binding moiety as a masking moiety blocks the binding of the ABS to its target, and this blockage is released after protease cleavage of the PCL (e.g., by a tumor-specific protease). Specific examples of antigen binding molecule preproteins masked by anti-ABS binding moieties include type IA and type IB MBM preproteins (e.g., as described in Section 6.3.1.1). The antigen binding molecule proprotein may comprise multiple masking moieties, such as an Fc domain and an anti-ABS binding moiety, wherein one or more of the masking moieties are released upon protease cleavage of PCL.

[0090] PCL comprises one or more substrates that are recognized and cleaved by one or more proteases (e.g., 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 comprise a spacer sequence, e.g., as described in Section 6.4.3. Examples of complete protease-cleavable linkers comprising substrate and spacer sequences are provided in Section 6.4.4.

[0091] Without being bound by theory, it is believed that a PCL linking the ABS and a masking moiety (particularly a masking moiety that sterically hinders the binding of the ABS to its target, such as an Fc domain) has a smaller length (e.g., 30 or less) to provide maximum hindrance to ABS binding prior to protease cleavage of the PCL, thereby minimizing off-target effects of the ABS. Thus, in certain aspects, the length of 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.

[0092] The antigen binding molecule preprotein generally comprises at least two polypeptide chains, wherein the first polypeptide chain comprises a masking moiety, a PCL, and a first component of the ABS (e.g., VH), and the second polypeptide chain comprises a second component of the ABS that is paired with the first component to form the ABS (e.g., VL). In the case where the masking moiety is an Fc domain, the second polypeptide may further comprise an additional Fc domain that is paired with the first Fc domain to form an Fc region (e.g., an Fc heterodimer). The ABS may be, for example, a TAA ABS (e.g., as described in Section 6.6) or a TCE ABS (e.g., as described in Section 6.7).

[0093] In some aspects, the antigen binding molecule preprotein further comprises an additional ABS that is not masked by the masking portion. In this case, the first ABS may be a TCE ABS, and the additional ABS is a TAA ABS; alternatively, the first ABS may be a TAA ABS, and the additional ABS is a TCE ABS. Antigen binding molecule preproteins comprising two or more ABSs may release multispecific binding molecules (e.g., bispecific or trispecific binding molecules) after protease cleavage of PCL. Certain examples of antigen binding molecule preproteins with multiple ABSs are described in Section 6.3 below.

[0094] 6.3.MBM proprotein

[0095] In some embodiments, the present disclosure relates to a MBM preprotein comprising a TCE ABS, a TAA ABS, a masking moiety, and a protease-cleavable linker, which are arranged such that the masking moiety reduces or blocks the binding of the TCE ABS to its target. The MBM preprotein is configured such that upon encountering a protease, such as a protease overexpressed in a tumor environment, the masking moiety is cleaved and produces an MBM in which the inhibition of TCE ABS binding is reversed. Typically, the MBM preprotein of the present disclosure further comprises two Fc domains that associate to form an Fc region, and the TCE ABS is located at the C-terminus of the Fc region.

[0096] Therefore, the MBM preprotein of the present disclosure generally comprises: (a) a first Fc domain and a second Fc domain capable of associating to form an Fc region; (b) a TCE ABS at the C-terminus of the first Fc domain and / or the second Fc domain; (c) a TAA ABS; and (d) a protease cleavable linker (PCL) at the C-terminus of the first Fc domain and / or the second Fc domain. Generally, the TAA ABS in the MBM preprotein is capable of binding to its target regardless of whether the protease cleavable linker is in an uncleaved state, and after the protease cleavable linker is cleaved by a protease, the binding of the TCE ABS to its target is significantly enhanced. After the protease cleavable linker is cleaved, for example, in a tumor environment, the MBM comprising the TAA ABS and the TCE ABS is released.

[0097] In type I MBM proproteins, the TCE Abs are masked by anti-idiotypic antibodies. In type II MBM proproteins, the Fc region acts as a masking moiety by sterically hindering the binding of the TCE Abs to their targets.

[0098] The protease cleavable linker in the type IA, type IIA, type IB and type II MBM preproteins comprises one or more substrates that are recognized and cleaved by one or more proteases, for example, 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 generally further comprises a spacer sequence, for example, as described in Section 6.4.3. Examples of complete protease cleavable linkers comprising substrate and spacer sequences are provided in Section 6.4.4. Depending on the configuration of the MBM preprotein, the Fc region may be retained in the resulting MBM after cleavage of the protease cleavable linker (e.g., after cleavage of the protease cleavable linker in the type IA and type IIA MBM preproteins), or may be released from the resulting MBM after cleavage of the protease cleavable linker (e.g., after cleavage of the protease cleavable linker in the type IB and type IIB MBM preproteins).

[0099] In general, the MBM preproteins of the present disclosure contain multiple linkers. Preferably, except for the protease-cleavable linker whose cleavage releases the MBM, the remaining linkers are non-cleavable. Examples of non-cleavable linkers are listed in Section 6.5.

[0100] Exemplary TAA ABSs that can be used in the MBM preproteins of the present disclosure (e.g., the Type IA, Type IIA, Type IB, and Type II MBM preproteins of the present disclosure) are disclosed in Section 6.6. Exemplary TCE ABSs that can be used in the MBM preproteins of the present disclosure (e.g., the Type IA, Type IIA, Type IB, and Type II MBM preproteins of the present disclosure) are disclosed in Section 6.7. Exemplary anti-TCE ABSs that can be used in the Type I MBM preproteins of the present disclosure are disclosed in Section 6.8. Section 6.9 describes suitable forms of ABSs that are incorporated into the MBM preproteins of the present disclosure. Section 6.10 describes suitable Fc domains that can be incorporated into the MBM preproteins of the present disclosure (and which can be used as masking moieties in the Type II MBM preproteins of the present disclosure).

[0101] 6.3.1. Type I MBM preprotein (with anti-idiotypic masking portion)

[0102] The present disclosure provides an MBM preprotein comprising a TCE ABS masked by an anti-TCE ABS, referred to herein as a "type I" MBM preprotein. Typically, both the TCE ABS and the anti-TCE ABS are disposed at the C-terminus of the Fc region, each of which is operably linked to a separate Fc domain. The TCE ABS or the anti-TCE ABS can be separated from an Fc domain of the Fc region by a protease cleavable linker (e.g., as described in Section 6.4). In a type IA MBM preprotein, the Fc region is retained in the MBM activated by cleavage of the protease cleavable linker. In a type IB MBM preprotein, the Fc region is separated from the MBM activated by cleavage of the protease cleavable linker.

[0103] 6.3.1.1. Type IA MBM proprotein

[0104] In general, the type IA MBM pre-protein comprises a first polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a non-cleavable linker and a TCE ABS (or a TCE ABS chain) in an N-terminal to C-terminal orientation, and a second polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a protease-cleavable linker and an anti-TCE ABS (or an anti-TCE ABS chain) in an N-terminal to C-terminal orientation. One or both Fc domains preferably further comprise a TAA ABS (or a TAA ABS chain) at their N-termini. When the TCE ABS, TAA ABS and / or anti-TCE ABS are composed of multiple polypeptide chains, the MBM pre-protein will further comprise other chains, such as the light chain of a Fab.

[0105] After cleavage of the protease-cleavable linker, the anti-TCE ABS is released, resulting in an MBM comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising an Fc domain (with an optional hinge domain at its N-terminus), a non-cleavable linker and a TCE ABS in an N-terminal to C-terminal orientation, and the second polypeptide chain comprising an Fc domain (with an optional hinge domain at its N-terminus) and a portion of a protease-cleavable linker in an N-terminal to C-terminal orientation. One or both Fc domains preferably further comprise a TAA ABS at their N-terminus. When the TCE ABS and / or TAA ABS are composed of multiple polypeptide chains, the resulting MBM will further comprise other chains, such as the light chain of a Fab.

[0106] Preferably, the resulting MBM is bispecific for the targets of TAAs and TCE ABS (e.g., CD3).

[0107] Figures 1A to 1C An exemplary embodiment of a Type IA MBM proprotein is shown. Figure 1A and Figure 1B As shown in , the type IA MBM preprotein may contain only a single TAA ABS. Alternatively, as Figure 1C As shown in , the type IA MBM preprotein may contain two TAAABS. Although depicted as a Fab for illustrative purposes, Figures 1A to 1C The anti-TCE ABS can be a scFv, VHH, VH or other fragment or region from an anti-idiotypic antibody. Figures 1A to 1C The depiction of a single substrate and two spacers in a protease cleavable linker is for illustrative purposes only; as described in Section 6.4, a protease cleavable linker may comprise multiple substrates and more than two spacer sequences.

[0108] 6.3.1.2. Type IB MBM proprotein

[0109] In general, the IB type MBM pre-protein comprises a first polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a non-cleavable linker and an anti-TCE ABS (or an anti-TCE ABS chain) in an N-terminal to C-terminal orientation, and a second polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a protease-cleavable linker, a TCE ABS (or a TCE ABS chain) and a TAA ABS (or a TAA ABS chain) in an N-terminal to C-terminal orientation, and generally the TCE ABS and the TAA ABS are separated by a non-cleavable linker. When the TCE ABS, TAA ABS and / or anti-TCE ABS are composed of multiple polypeptide chains, the MBM pre-protein will further comprise other chains, such as the light chain of a Fab.

[0110] After cleavage of the protease-cleavable linker, the MBM comprising the TCE ABS, the optional linker and the TAA ABS is released. When the TCE ABS and / or the TAA ABS are composed of multiple polypeptide chains, the resulting MBM will further comprise other chains, such as the light chain of a Fab.

[0111] Preferably, the resulting MBM is bispecific for the targets of TAA and TCE ABS (eg, CD3).

[0112] FIG. 5A to FIG. 5B An exemplary embodiment of a type IB MBM proprotein is shown. Although depicted as a Fab for purposes of illustration, Figure 5A The anti-TCE ABS can be a scFv, VHH, VH or other fragment or region from an anti-idiotypic antibody. FIG. 5A to FIG. 5B The depiction of a single substrate and two spacers in a protease cleavable linker is for illustrative purposes only; as described in Section 6.4, a protease cleavable linker may comprise multiple substrates and more than two spacer sequences.

[0113] 6.3.2. Type II MBM preprotein (with sterically hindered masking moiety)

[0114] 6.3.2.1. Type IIA MBM proprotein

[0115] In general, the type IIA MBM preprotein comprises a first polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a non-cleavable linker and a first TCE ABS chain in an N-terminal to C-terminal orientation, and a second polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a protease-cleavable linker and a second TCE ABS chain in an N-terminal to C-terminal orientation. The TCE ABS chain may include VH and VL without corresponding CH1 and CL domain domains, so that a TCE ABS in the form of Fv is formed by the association of the TCE ABS chain. The TCE ABS chain may include VH and VL, with corresponding CH1 and CL domain domains, so that a TCE ABS in the form of Fab is formed by the association of the TCE ABS chain. In some embodiments, the VH and / or VL of the TCE ABS chain attached to the Fc domain comprises a deletion of one or more amino acids at or near its N-terminus, for example, an N-terminal truncation. The VH or VL may have such a deletion (i.e., may be a truncation) precisely at the N-terminus, 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 preprotein of the present disclosure comprises a deletion (e.g., a truncation) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near (e.g., within 10 amino acids) the N-terminus of a reference VH (e.g., a wild-type VH or a VH on which the TCE ABS is based, such as a VH of an antibody or antibody sequence in Table G). In some embodiments, the VL of the TCE ABS of the type IIA MBM pre-protein of the present disclosure comprises a deletion (e.g., truncation) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near (e.g., within 10 amino acids) the N-terminus of a reference VL (e.g., a wild-type VL or a VL on which the TCE ABS is based, such as the VL of an antibody or antibody sequence in Table G).

[0116] The length of the linker separating the Fc domain and the TCE ABS chain is selected to optimize the ability of the Fc domain to sterically hinder the TCE ABS from binding to its target. Preferably, the length of the non-cleavable linker is generally in the range of 5 to 30 amino acids, and in various embodiments is 25 amino acids or less (e.g., ranging between 5 to 25 amino acids), 20 amino acids or less (e.g., ranging between 5 to 20 amino acids), 15 amino acids or less (e.g., ranging between 5 to 15 amino acids) or 10 amino acids or less (e.g., ranging between 5 to 10 amino acids). In a specific embodiment, the length of the non-cleavable linker is 5, 6, 7, 8, 9 or 10 amino acids.

[0117] One or both Fc domains preferably further comprise a TAA ABS (or a TAA ABS chain) at its N-terminus. When the TAA ABS are composed of multiple polypeptide chains, the MBM preprotein will further comprise another chain, such as a light chain of Fab.

[0118] In some embodiments, one of the two Fc domains may comprise a deletion of one or more amino acids at or near the C-terminus (e.g., a C-terminal truncation) relative to a wild-type Fc domain. In some embodiments, one or both Fc domains of the type IIA MBM preprotein of the present disclosure comprise a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near the C-terminus (e.g., a truncation) relative to a wild-type Fc domain. The Fc domain may have such a deletion precisely at the C-terminus (i.e., 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.

[0119] In certain embodiments, the type IIA MBM preprotein of the present disclosure comprises (1) an Fc domain having a deletion (e.g., truncation) of 1, 2, 3, 4, 5, or 6 amino acids at or near the C-terminus relative to the wild-type Fc domain; and (2) a TCE ABS chain having a VH and VL domain having a deletion (e.g., truncation) of 1, 2, 3, 4, 5, or 6 amino acids at or near the N-terminus. For example, the type IIA MBM preprotein may 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 a VH and a VL, each of which has a deletion of 1, 2, or 3 amino acids at or near the N-terminus.

[0120] Upon cleavage of the protease-cleavable linker, the steric hindrance limiting the ability of the TCE ABS to bind to its target is released.

[0121] Preferably, the resulting MBM is bispecific for the targets of TAAs and TCE ABS (e.g., CD3).

[0122] FIG. 3A to FIG. 3F An exemplary embodiment of a type IIA MBM proprotein is shown. Figure 3A and Figure 3B As shown in , the type IIA MBM preprotein may contain only a single TAA ABS. FIG. 3C to FIG. 3F As shown in , type IIA MBM preproteins can contain two TAA Abs. Figure 3A The depiction of a single substrate and two spacers in the protease cleavable linker of Figure 3G is for illustrative purposes only; as described in Section 6.4, a protease cleavable linker can comprise multiple substrates and more than two spacer sequences.

[0123] 6.3.2.2. Type IIB MBM proprotein

[0124] In general, the type IIB MBM preprotein comprises a first polypeptide chain, which comprises an Fc domain (with an optional 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 a TAA ABS chain) in an N-terminal to C-terminal orientation, and a second polypeptide chain, which comprises an Fc domain (with an optional hinge domain at its N-terminus), a protease-cleavable linker and a TCE ABS chain in an N-terminal to C-terminal orientation. When the TAA ABS is composed of multiple polypeptide chains, the MBM preprotein will further comprise other chains, such as the light chain of Fab. The TCE ABS chain may include VH and VL, with corresponding CH1 and CL domains, so that a TCE ABS in the form of a Fab is formed by the association of the TCE ABS chain. In some embodiments, the VH and / or VL of the TCE ABS chain attached to the Fc domain comprises a deletion of one or more amino acids at or near its N-terminus, for example, an N-terminal truncation. The VH or VL may have such a deletion (i.e., may be a truncation) precisely at the N-terminus, 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 IIB MBM preprotein of the present disclosure comprises a deletion (e.g., a truncation) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near (e.g., within 10 amino acids) the N-terminus of a reference VH (e.g., a wild-type VH or a VH on which the TCE ABS is based, such as a VH of an antibody or antibody sequence in Table G). In some embodiments, the VL of the TCE ABS of the Type IIB MBM pre-protein of the present disclosure comprises a deletion (e.g., truncation) or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near (e.g., within 10 amino acids) the N-terminus of a reference VL (e.g., a wild-type VL or a VL on which the TCE ABS is based, such as the VL of an antibody or antibody sequence in Table G).

[0125] The length of the linker separating the Fc domain and the TCEABS chain is selected to optimize the ability of the Fc domain to sterically hinder the TCEABS from binding to its target. Preferably, the length of the linker is generally in the range of 5 to 30 amino acids, and in various embodiments is 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).

[0126] In some embodiments, one of the two Fc domains may comprise a deletion of one or more amino acids at or near the C-terminus (e.g., a C-terminal truncation) relative to a wild-type Fc domain. In some embodiments, one or both Fc domains of the type IIB MBM preprotein of the present disclosure comprise a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids at or near the C-terminus (e.g., a truncation) relative to a wild-type Fc domain. The Fc domain may have such a deletion precisely at the C-terminus (i.e., 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.

[0127] In certain embodiments, the type IIB MBM preprotein of the present disclosure comprises (1) an Fc domain having a deletion (e.g., truncation) of 1, 2, 3, 4, 5, or 6 amino acids at or near the C-terminus relative to the wild-type Fc domain; and (2) a TCE ABS chain having a VH and VL domain having a deletion (e.g., truncation) of 1, 2, 3, 4, 5, or 6 amino acids at or near the N-terminus. For example, the type IIB MBM preprotein may 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 a VH and a VL, each of which has a deletion of 1, 2, or 3 amino acids at or near the N-terminus.

[0128] After cleavage of the protease-cleavable linker, the MBM comprising the TCE ABS, the optional linker and the TAA ABS is released. When the TAA ABS is composed of multiple polypeptide chains, the resulting MBM will further comprise another TAA ABS chain, such as the light chain of a Fab.

[0129] Preferably, the resulting MBM is bispecific for the targets of TAA and TCE ABS (eg, CD3).

[0130] Figure 7 An exemplary embodiment of a type IIB MBM proprotein is illustrated. Figure 7 The depiction of a single substrate and two spacers in a protease cleavable linker is for illustrative purposes only; as described in Section 6.4, a protease cleavable linker may comprise multiple substrates and more than two spacer sequences.

[0131] 6.4. Protease-cleavable linkers

[0132] The MBM proproteins of the present disclosure generally comprise components linked to each other at least via a linker and one or more substrates for a protease, and cleavage thereof results in activation of the MBM.

[0133] Protease cleavable linkers can range from 20 amino acids to 80 or more amino acids, and in certain aspects, non-cleavable peptide linkers range 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.

[0134] The protease cleavable joint comprises one or more substrate sequences of one or more proteases (e.g., one or more of the proteases listed in Section 6.4.1). One or more substrate sequences (e.g., one or more of the substrate sequences listed in Section 6.4.2) are typically flanked by one or more spacer sequences (e.g., spacer sequences as described in Section 6.4.3). Each protease cleavable joint may comprise one, two, three or more substrate sequences. The spacer sequences may be adjacent, overlapping or spaced apart by spacer sequences. Preferably, the C-terminus and N-terminus of the protease cleavable joint contain spacer sequences.

[0135] Exemplary protease cleavable linker sequences are listed in Section 6.4.4

[0136] 6.4.1. Proteases

[0137] Exemplary proteases whose substrate sequences can be incorporated into protease-cleavable linkers are listed in Table A below.

[0138]

[0139]

[0140] In specific embodiments, the protease is matrix metalloproteinase (MMP)-2, MMP-9, legumain asparaginyl endopeptidase, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloproteinase (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 protease, urokinase / urokinase-type plasminogen activator (uPA), disintegrin and metalloproteinase (ADAM) 10, ADAM12, ADAM17, ADAM containing thrombospondin motif (ADAMTS), ADAMTS5, β-secretase (BACE), granzyme A, granzyme B, guanidinobenzoatase, heparin, protease, protease 2, transmembrane peptidase, enkephalinase, 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, γ-secretase component, antiplasmin cleaving enzyme (APCE), decysin 1. Apoptosis-related cysteine ​​peptidase or N-acetylated α-linked acid dipeptidase-like 1.

[0141] 6.4.2. Substrate

[0142] Exemplary substrate sequences that are cleavable by tumor proteases and that can be incorporated into a protease-cleavable linker are listed in Table B below.

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] 6.4.3. Spacers

[0149] Exemplary spacer sequences that can be incorporated into a protease-cleavable linker are listed below in Table C. In addition to the spacer sequences listed in Table C, any non-cleavable linker sequence described in Section 6.5 (e.g., a non-cleavable linker sequence listed in Table E) or a portion thereof can be used as a spacer sequence.

[0150]

[0151]

[0152] In some embodiments, as 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.

[0153] 6.4.4. Exemplary protease-cleavable linkers

[0154] Exemplary protease-cleavable linkers comprising one or more substrate sequences and a spacer sequence are listed in Table D below.

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] In certain aspects, the protease cleavable linker comprises an amino acid sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitutions compared to a sequence listed in Table D. Thus, in some embodiments, the protease cleavable linker comprises or consists of any amino acid sequence in Table D having 1-5 amino acid substitutions compared to a sequence listed in Table D.

[0161] 6.5. Non-cleavable linkers

[0162] In certain aspects, the present disclosure provides MBM preproteins, wherein two or more components of the MBM preprotein are linked to each other via a peptide linker. As an example and not limitation, the linker can be used to link (a) an ABS and an Fc domain; (b) two ABS; or (c) different domains within an ABS (e.g., VH and VL domains in a scFv).

[0163] Preferably, all linkers in the MBM proprotein are non-cleavable linkers (NCLs), except for the protease-cleavable linker whose cleavage results in MBM activation.

[0164] The non-cleavable linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects, the non-cleavable peptide linker has a length ranging 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.

[0165] In particular aspects, 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.

[0166] In some embodiments of the foregoing, the length of the non-cleavable linker is in the range of 5 amino acids to 50 amino acids, such as 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 embodiments of the foregoing, the length of the non-cleavable linker is in the range of 6 amino acids to 50 amino acids, such as 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 yet other embodiments of the foregoing, the length of the non-cleavable linker is in the range of 7 amino acids to 50 amino acids, such as 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.

[0167] Charged (eg, charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred.

[0168] Examples of flexible non-cleavable linkers that can be used in the MBM preproteins of the present disclosure include those disclosed by 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 or contain repeating sequences of glycine and serine, such as G n S (SEQ ID NO: 265) or SG n(SEQ ID NO: 266), wherein n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the non-cleavable linker is or comprises a monomer or multimer of a repeating sequence of G4S (SEQ ID NO: 1), such as (GGGGS) n (SEQ ID NO: 150).

[0169] Polyglycine non-cleavable linkers can be suitably used in the MBM preproteins of the present disclosure. In some embodiments, the peptide non-cleavable linker comprises 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).

[0170] Exemplary non-cleavable linker sequences are listed in Table E below.

[0171]

[0172]

[0173]

[0174] In certain aspects, the MBM preprotein of the present disclosure may comprise a polypeptide chain comprising 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 links the ABS to the CH2 domain and can be said to constitute a type of linker. Exemplary hinge domains are listed in Section 6.10.3.

[0175] 6.6.TAA ABS

[0176] The MBM pre-protein of the present disclosure comprises 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 a "TAA ABS". The skilled person will recognize that the aforementioned categories of target molecules are not mutually exclusive, and thus a given target molecule may fall into more than one of the aforementioned categories of target molecules. For example, some molecules may be considered as TAAs and ECM proteins, while other molecules may be considered as TCAs and checkpoint inhibitors. 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. Particular aspects relate to MBM pre-proteins comprising at least one ABS that specifically binds to a TAA. In certain embodiments, TAAs are preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, TAAs are lineage markers.

[0177] It is contemplated that any type of tumor and any type of ECM antigen, tumor reactive lymphocyte antigen, cell surface molecule of tumor or viral lymphocyte, TCA, checkpoint inhibitor or TAA can be targeted by the MBM pre-protein of the present disclosure. Exemplary cancer types that can be targeted include acute lymphocytic leukemia, acute myeloid leukemia, cholangiocarcinoma, B cell leukemia, B cell lymphoma, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, triple negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma and urinary bladder cancer. However, one skilled in the art will recognize that TAAs and other target molecules associated with the tumor microenvironment are known for nearly any type of cancer.

[0178] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, nectin (e.g., connexin-4), tenascin, collagen (e.g., type X collagen), and matrixin.

[0179] Other target molecules are cell surface molecules of tumor or viral lymphocytes, for example T cell co-stimulatory 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.

[0180] 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 is a non-blocking or weakly blocking ligand-receptor binding. Examples of non-blocking or weakly blocking anti-PD1 antibodies include antibodies having VH / VL amino acid sequences of SEQ ID No: 2 / 10 of PCT Publication No. WO2015 / 112800A1; SEQ ID No: 16 / 17 of U.S. Patent No. 11,034,765B2; and SEQ ID No. 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 weakly 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 US Publication No. US2022 / 0056126A1.

[0181] 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 upon which TAA ABSs may be based.

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] In some aspects, the TAA ABS competes with the antibodies listed in Table F for binding to TAAs. In further aspects, the TAA ABS comprises CDRs having CDR sequences of anti-TAA antibodies listed in Table F. In some embodiments, the TAA ABS comprises all 6 CDR sequences of the anti-TAA antibodies listed in Table F. In other embodiments, the TAA ABS comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3 and a light chain CDR sequence of a universal light chain. In further aspects, the TAA ABS comprises a VH comprising the amino acid sequence of a VH of an anti-TAA antibody listed in Table F. In some embodiments, the TAA ABS further comprises a VL comprising the amino acid sequence of a VL of an anti-TAA antibody listed in Table F. In other embodiments, the TAA ABS further comprises a universal light chain VL sequence.

[0193] Additional TAAs that can be targeted by MBM preproteins 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 incorporated herein by reference in its entirety.

[0194] Still further exemplary TAAs include fibroblast activation protein (FAP), A1 domain of tenascin-C (TNCA1), A2 domain of tenascin-C (TNC A2), 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 / C D3-ζ chain, the MAGE family of tumor antigens (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), the GAGE ​​family of tumor antigens (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, gp100 Pmel117, 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 tumor family antigens, Imp-1, P1A, EBV-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-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate enzyme-specific antigen (PSA), PAP, LAGA-1a, p53, prostate-specific protein (prostein), PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin 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).

[0195] 6.7.TCE ABS

[0196] The MBM pre-protein of the present disclosure comprises at least a T cell engaging ABS specifically binding to a T cell receptor complex component, referred to herein as a "TCE ABS". Exemplary targets of TCE ABS are CD3 and T cell receptors (e.g., TCRαβ or TCRγδ). Preferably, the TCE ABS target is a human T cell receptor complex component. The epitope of the TCE ABS may be a single polypeptide (e.g., CD3ε) or a multimeric component of a T cell receptor complex (e.g., TCRαβ dimer or TCRγδ dimer).

[0197] Exemplary CD3 and TCR antibodies or antibody sequences upon which TAA ABSs may be based are listed in Table G below.

[0198]

[0199]

[0200]

[0201] In some aspects, TCE ABS competes with the T cell engagement (TCE) antibodies listed in Table G for binding to the target (e.g., CD3 or T cell receptor) of the TCE antibody. In other aspects, TCE ABS comprises CDRs having CDR sequences of TCE antibodies listed in Table G. In some embodiments, TCE ABS comprises all 6 CDR sequences of TCE antibodies listed in Table G. In other embodiments, TCE ABS comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) and a light chain CDR sequence of a universal light chain. In other aspects, TCE ABS comprises VH, which comprises the amino acid sequence of the VH of the TCE antibody listed in Table G. In some embodiments, TCE ABS further comprises VL, which comprises the amino acid sequence of the VL of the TCE antibody listed in Table G. In other embodiments, TCE ABS further comprises a universal light chain VL sequence.

[0202] 6.8. Anti-TCE ABS

[0203] In certain embodiments, the MBM preprotein of the present disclosure comprises an anti-TCE ABS. The anti-TCE ABS is a fragment of an anti-idiotypic antibody that can specifically bind to TCE-ABS. Various types of antibody fragments can be used in the context of the anti-TCE ABS of the present disclosure, including, for example, scFv, Fab, VHH and VH. In some embodiments, the anti-TCE ABS is an scFv of an anti-idiotypic antibody. In some embodiments, the anti-TCE ABS is a VHH derived from an anti-idiotypic antibody. In some embodiments, the anti-TCE ABS is a VH derived from an anti-idiotypic antibody.

[0204] 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; WO 94 / 04678).

[0205] Anti-idiotypic antibodies are antibodies directed against the antigen combining or variable region (called the idiotype or Id) of another antibody molecule. In principle, immunization with an antibody molecule expressing the paratope (antigen combining site) of a given antigen should produce a group of anti-antibodies, some of which share the complementary structure of the paratope with the antigen.

[0206] Exemplary methods for producing anti-idiotypic antibodies are found in U.S. Patent No. 10,150,817 B2 and Example 1 of WO 2017 / 162587 A1.

[0207] Exemplary anti-CD3 anti-idiotypic ABS that can be used as anti-TCE ABS of the present disclosure are scFvs designated 4.15.64 and 4.32.63, which are anti-idiotypic of the anti-CD3 ABS designated CH2527. The sequences of 4.15.64, 4.32.63 and CH2527 are disclosed in WO 2017 / 162587 A1.

[0208] 6.9.ABS Form

[0209] In certain aspects, the ABS in the MBM preprotein of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to the antigenic determinant. In one embodiment, the ABS is an immunoglobulin molecule or a fragment thereof, particularly an IgG class immunoglobulin molecule, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include but are not limited to VH (or V H ) fragment, VL (or V L) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies and tetrabodies.

[0210] 6.9.1.Fab

[0211] Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain.The Fab domain may comprise constant domain and variable region sequences from any suitable species, and may therefore be murine, chimeric, human or humanized.

[0212] The Fab domain typically comprises a CH1 domain attached to a VH domain, which is paired with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain is paired with the VL domain to form the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding site. The disulfide bond between the two constant domains can further stabilize the Fab domain.

[0213] For the MBM preprotein of the present disclosure, particularly when the light chain of the ABS is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same ABS and minimize abnormal pairing of Fab domains belonging to different ABS. For example, the Fab heterodimerization strategy shown in Table H below can be used:

[0214]

[0215] Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is promoted by exchanging the VL and VH domains of the Fab with each other or exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0216] Correct Fab pairing can also be promoted 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 one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0217] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of the variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13: 1619-1633 provides definitions of framework residues based on the Kabat, Chothia and IMGT numbering schemes.

[0218] 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 a combination of various interactions. Complementarity between protein surfaces is widely described in the literature as lock and key fits, knobs into sockets, protrusions and cavities, donors and acceptors, etc., all of which imply the nature of the structural and chemical matching between the two interacting surfaces.

[0219] In one embodiment, the one or more modifications introduced introduce new hydrogen bonds at the interface of the Fab component. In one embodiment, the one or more modifications introduced introduce new salt bridges at the interface of the Fab component. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of both of which are hereby incorporated by reference.

[0220] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 domain and the CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0221] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0222] In some embodiments, the Fab domain may contain modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In one embodiment, 39K, 62E modifications are introduced in the VH domain, H172A, F174G modifications are introduced in the CH1 domain, 1R, 38D, (36F) modifications are introduced in the VL domain, and L135Y, S176W modifications are introduced in the CL domain. In another embodiment, 39Y modifications are introduced in the VH domain and 38R modifications are introduced in the VL domain.

[0223] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond to improve the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0224] The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with the constant domain of the T cell receptor, replacing the CL domain with the b domain of the T cell receptor, and pairing these domain replacements with additional charge-charge interactions between the VL domain and the VH domain by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0225] Instead of or in addition to using a Fab heterodimerization strategy 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 pre-protein of the present disclosure. In various embodiments, the use of a common light chain as described herein reduces the number of inappropriate species in the MBM pre-protein compared to the use of an original homologous VL. In various embodiments, the VL domain of the ABS is identified from a monospecific antibody comprising a common light chain. In various embodiments, the VH region of the ABS in the MBM pre-protein comprises human heavy chain variable gene segments that are rearranged in vivo in mouse B cells that have been previously engineered to express a limited human light chain library, or a single human light chain homologous to a human heavy chain, and in response to exposure to an antigen of interest, an antibody library containing multiple human VHs is produced that is homologous to one or one of two possible human VLs, wherein the antibody library is specific for the antigen of interest. Common light chains are those derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, and include somatically mutated (eg, affinity matured) versions. See, eg, US Pat. No. 10,412,940.

[0226] 6.9.2.scFv

[0227] Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as a single-chain polypeptide, and retain the specificity of the intact antibody from which it is derived. Typically, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for joining the VH and VL chains of the scFv are the non-cleavable linkers identified in Section 6.5.

[0228] Unless otherwise indicated, a scFv used herein may have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0229] The scFv may comprise VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[0230] To generate a nucleic acid encoding an scFv, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.5 (typically repeats 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 contiguous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, e.g., 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).

[0231] 6.10. Fc Region

[0232] The MBM preproteins of the present disclosure generally include a pair of Fc domains that associate to form an Fc region. In native antibodies, the Fc region contains a hinge region at their N-terminus to form a constant domain. Throughout the present disclosure, unless otherwise indicated, reference to an Fc domain encompasses an Fc domain having a hinge domain at its N-terminus.

[0233] The Fc domain may be derived from any suitable species that is operably linked to the ABS or its components. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the ABS or its components are fused to an IgG Fc molecule. The ABS or its components may be fused to the N-terminus or C-terminus or both of the IgG Fc domain.

[0234] The Fc domain can be derived from an antibody of any suitable class, 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.

[0235]

[0236]

[0237] In some aspects, 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. In cases where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 336 (e.g., having between 90% and 99% sequence identity to SEQ ID NO: 336), the Fc domain may further comprise one or more 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).

[0238] In some aspects, 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. In cases where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 337 (e.g., having between 90% and 99% sequence identity to SEQ ID NO: 337), the Fc domain may further comprise one or more 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).

[0239] In some aspects, 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. In cases where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 338 (e.g., having between 90% and 99% sequence identity to SEQ ID NO: 338), the Fc domain may further comprise one or more 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).

[0240] In some aspects, 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. In cases where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 339 (e.g., having between 90% and 99% sequence identity to SEQ ID NO: 339), the Fc domain may further comprise one or more 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).

[0241] The two Fc domains within the Fc region may be identical or different from each other. In native antibodies, the Fc domains are usually identical, but for the purpose of generating multispecific binding molecules, such as the MBM preproteins of the present disclosure and the MBMs generated by their activation, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.10.2 below.

[0242] In natural antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerize to produce the Fc region.

[0243] In the MBM preproteins of the present disclosure, the Fc region and / or the Fc domain therein may comprise heavy chain constant domains from antibodies of one or more different classes (eg, one, two, or three different classes).

[0244] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1. One or two Fc domains of the Fc region may have one or more amino acid deletions relative to the naturally occurring IgG1 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions (e.g., truncations) at or near the C-terminus of the CH3 domain relative to naturally occurring IgG1.

[0245] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2. One or two Fc domains of the Fc region may have one or more amino acid deletions relative to the naturally occurring IgG2 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions (e.g., truncations) at or near the C-terminus of the CH3 domain relative to naturally occurring IgG2.

[0246] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3. One or two Fc domains of the Fc region may have one or more amino acid deletions relative to the naturally occurring IgG3 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions (e.g., truncations) at or near the C-terminus of the CH3 domain relative to naturally occurring IgG3.

[0247] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4. One or two Fc domains of the Fc region may have one or more amino acid deletions relative to the naturally occurring IgG4 CH3 sequence. In some embodiments, the Fc domain of the present disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletions (e.g., truncations) at or near the C-terminus of the CH3 domain relative to naturally occurring IgG4.

[0248] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0249] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0250] It should be understood that the heavy chain constant domain of the Fc region used to generate the MBM preprotein of the present disclosure may include variants of the above-mentioned naturally occurring constant domains. Such variants may include one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain whose sequence is different from the wild-type constant domain. It should be understood that the variant constant domain may 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.

[0251] Certain variant constant domains contemplated herein include those with a deletion (e.g., truncation) of one or more amino acids at or near the C-terminus (e.g., within 10 amino acids) relative to a wild-type constant domain. In some embodiments, the constant domains of the present disclosure comprise a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at or near the C-terminus relative to a wild-type constant domain.

[0252] IgM and IgA naturally exist in the human body as covalent polymers of the common H2L2 antibody unit. When IgM is incorporated into the J chain, it appears as a pentamer; or when IgM lacks the J chain, it appears as a hexamer. IgA appears in monomer and dimer forms. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, called the tailpiece. The tailpiece contains cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and are believed to play an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the MBM preprotein of the present disclosure does not include a tailpiece.

[0253] The Fc domain incorporated into the MBM preproteins of the present disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, modified disulfide bond structure, or altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.10.1.

[0254] The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric MBM pre-proteins, for example by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains relative to identical Fc domains. Heterodimerization allows the generation of MBM pre-proteins in which the different polypeptide components are linked to each other through Fc regions containing Fc domains of different sequences. Examples of heterodimerization strategies are illustrated in Section 6.10.2.

[0255] It will be appreciated that any of the above modifications may be combined in any suitable manner to achieve desired functional properties and / or combined with other modifications to alter the properties of the MBM preprotein.

[0256] 6.10.1. Fc domains with altered effector functions

[0257] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0258] In a specific embodiment, 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, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the following groups: complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0259] In one embodiment, the Fc domain or Fc region (e.g., one or two Fc domains of a MBM preprotein that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group consisting 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 an amino acid substitution at a position selected from the group consisting 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 one such embodiment, the Fc domain or region is an Igd Fc domain or region, in particular 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, amino acid substitution is P329A or P329G, particularly P329G (according to the numbering of Kabat EU index). In one embodiment, Fc domain or Fc region comprises amino acid substitution at position P329 and another amino acid substitution at the position selected from E233, L234, L235, N297 and P331 (according to the numbering of KabatEU index). In a more specific embodiment, another amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, Fc domain or Fc region comprises amino acid substitution at position P329, L234 and L235 (according to the numbering of KabatEU index). In a more specific embodiment, Fc domain comprises amino acid mutation L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").

[0260] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a specific embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in the first and second Fc domains in the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0261] In one embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0262] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to an Fc receptor. An exemplary IgG4 Fc domain with reduced binding to an Fc receptor may comprise an amino acid sequence selected from Table 1 below: In some embodiments, the Fc domain includes only the bold portion of the sequence shown below:

[0263]

[0264]

[0265]

[0266]

[0267] In a specific embodiment, the IgG4 with reduced effector function comprises the bold portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4.

[0268] For heterodimeric Fc regions, it is possible to incorporate a combination of the above-mentioned variant IgG4 Fc sequences, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or its bold portion) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or its bold portion), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or its bold portion) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or its bold portion).

[0269] 6.10.2. Fc Heterodimerization Variants

[0270] Some MBM pre-proteins require dimerization between two Fc domains, which, unlike native immunoglobulins, are operably linked to non-identical N-terminal regions, for example, one Fc domain is linked to Fab and the other Fc domain is linked to Abs or a component thereof. Insufficient heterodimerization of the two Fc domains to form the Fc region may become an obstacle to increasing the yield of the desired heterodimeric molecule and poses a challenge to purification. A variety of methods available in the art can be used to enhance dimerization of Fc domains that may be present in the MBM proproteins of the present disclosure, such as disclosed in EP 1870459A1; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0271] The present disclosure provides MBM preproteins comprising Fc heterodimers (i.e., Fc regions comprising heterologous, non-identical Fc domains). Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class or subclass, and preferably the constant region of an antibody of the IgG (IgG1, IgG2, IgG3 and IgG4) class, as described in the previous section.

[0272] Heterodimerization of two different heavy chains at the CH3 domain produces the desired MBM pre-protein, while homodimerization of the same heavy chain will reduce the yield of the desired MBM pre-protein. Therefore, in a preferred embodiment, the polypeptides that associate to form the MBM pre-protein of the present disclosure will contain a CH3 domain with a modification that favors heterodimer association relative to an unmodified Fc domain.

[0273] In a specific embodiment, the modification that promotes the formation of Fc heterodimers is a so-called "knob-into-hole" or "knob-into-hole" modification, comprising a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described in, for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621 and Carter, 2001, Immunol Meth 248:7-15. In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing small amino acid side chains at the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size to the protuberances are created on the interface of the second polypeptide by replacing larger amino acid side chains with smaller side chains (eg, alanine or threonine).

[0274] Therefore, in some embodiments, the amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue with a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, which is a cavity that can be positioned in the CH3 domain of the second subunit, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue with a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity. Preferably, the amino acid residue with 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 with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). Protrusions and cavities can be prepared by changing the nucleic acid encoding a polypeptide, for example, by site-specific mutagenesis, or by peptide synthesis. Exemplary substitutions are Y470T.

[0275] In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbered according to the Kabat EU index). In another embodiment, in the first Fc domain, additionally, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0276] In some embodiments, electrostatic steering (eg, 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.

[0277] Alternatively or additionally, using an Fc domain modified to promote heterodimerization, the Fc domain may be modified to allow a purification strategy that enables selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling a purification method for producing heterodimeric proteins. See, e.g., U.S. Patent No. 8,586,713. Thus, the MBM preprotein comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the MBM preprotein to protein A compared to a corresponding MBM preprotein lacking the amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). This class of modifications is referred to herein as "star" mutations.

[0278] In some embodiments, the Fc may contain one or more mutations (eg, knob and hole mutations) to promote heterodimerization and a star mutation to promote purification.

[0279] 6.10.3. Hinge domain

[0280] The MBM preprotein of the present disclosure may include an Fc domain, which includes a hinge domain at its N-terminus. The hinge region may be a natural or modified hinge region. The hinge region is usually found at the N-terminus of the Fc region. Unless otherwise specified in the context, the term "hinge domain" refers to a hinge sequence that occurs naturally or non-naturally, which is a monomer hinge domain in the context of a single or monomeric polypeptide chain and may include two associated hinge sequences located on separate polypeptide chains in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer MBM preprotein formed by the association of two Fc domains). Sometimes, two associated hinge sequences are referred to as "hinge regions".

[0281] The native hinge region is the hinge region usually found between the Fab and Fc domains in naturally occurring antibodies. The modified hinge region is any hinge whose length and / or composition are different from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may include a complete hinge region derived from antibodies of different classes or subclasses from the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region may include a part of a native hinge or a repeating unit, wherein each unit in the repeating unit is derived from a native hinge region. In another alternative, the native hinge region may be changed by converting one or more cysteine ​​or other residues into neutral residues, such as serine or alanine, or by converting appropriately placed residues into cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region may increase or decrease. Other modified hinge regions may be fully synthetic and may be designed to have desired properties, such as length, cysteine ​​composition and flexibility.

[0282] Many modified hinge regions have been described in, for example, US Pat. Nos. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971, and WO 2005 / 003171, and are incorporated herein by reference.

[0283] In one embodiment, the MBM proprotein of the present disclosure comprises an Fc region, wherein one or both Fc domains have a complete hinge domain at their N-termini.

[0284] In various embodiments, positions 233-236 within the hinge region can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where the positions are numbered by EU numbering.

[0285] In some embodiments, the MBM pre-proteins of the present disclosure comprise a modified hinge region that reduces binding affinity to an Fcγ receptor relative to a wild-type hinge region of the same isotype (eg, human IgG1 or human IgG4).

[0286] In one embodiment, the MBM preprotein of the present disclosure comprises an Fc region, wherein each Fc domain has a complete hinge domain at its N-terminus, wherein each Fc domain and hinge domain are derived from IgG4 and each hinge domain comprises a modified sequence CPPC (SEQ ID NO: 329). Compared with IgG1 containing the sequence CPPC (SEQ ID NO: 334), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 330). The serine residues present in the IgG4 sequence lead to increased flexibility in this region, so that a portion of the molecules form disulfide bonds (intrachain disulfide bonds) within the same protein chain, rather than bridging to other heavy chains in the IgG molecule to form interchain disulfide bonds. (Angel et al., 1993, Mol Immunol 30 (1): 105-108). The serine residues are changed to proline to obtain the same core sequence as IgG1, so that the interchain disulfide bonds are completely formed in the IgG4 hinge region, thereby reducing the heterogeneity of the purified product. This altered isotype is called IgG4P.

[0287] 6.10.3.1. Chimeric Hinge Sequences

[0288] The hinge domain may be a chimeric hinge domain.

[0289] For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0290] In specific embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 331) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 332) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to an IgG4 CH2 region (e.g., by incorporation of an IgG4 Fc domain, e.g., a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.10.1).

[0291] 6.10.3.2. Hinge sequences with reduced effector function

[0292] In another embodiment, the hinge region may be modified to reduce effector function, for example as described in WO2016161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, wherein the position is numbered by EU numbering (as shown in FIG. 1 of WO2016161010A2). These sections may be represented as GGG-, GG--, G---, or ----, where "-" represents an unoccupied position.

[0293] Position 236 is not occupied in typical human IgG2, but is occupied in other typical human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0294] Hinge modifications within positions 233-236 can be combined with position 228 occupied by P. Position 228 is naturally 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 beneficial for stabilizing IgG4 antibodies and reducing the exchange of heavy chain light chain pairs between exogenous antibodies and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P and C, respectively (such as "CPPC" disclosed in SEQ ID NO: 329).

[0295] Exemplary hinge regions have residues 226-236, sometimes referred to as the mid-base (or core), and the lower hinge, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and non-G(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAP GG-GPSVF (SEQ ID NO: 333) (previously disclosed as SEQ ID NO: 1 of WO2016161010A2), CPPCPAP G--GPSVF (SEQ ID NO: 26) (previously disclosed as SEQ ID NO: 2 of WO2016161010A2), CPPCPAP G--GPSVF (SEQ ID NO: 106) (previously disclosed as SEQ ID NO: 3 of WO2016161010A2) or CPPCPAP----GPSVF (SEQ ID NO: 152) (previously disclosed as SEQ ID NO: 4 of WO2016161010A2).

[0296] The above-mentioned modified hinge region can be incorporated into the heavy chain constant region, which usually includes CH2 and CH3 domains, and which may have additional hinge segments (e.g., upper hinges) flanking the specified region. Such additional constant region segments that are present usually have the same isotype, preferably a human isotype, although they can be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but it can also be human IgG1, IgG2 or IgG3 or a hybrid with different isotypes in its domains. Exemplary sequences of human IgG1, IgG2 and IgG4 are shown in WO2016161010A2. Figures 2 to 4 middle.

[0297] In specific embodiments, the modified hinge sequence may be linked to an IgG4 CH2 region (e.g., by incorporation of an IgG4 Fc domain, such as a human or murine Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.10.1).

[0298] 6.11. Tandem Fab

[0299] Certain aspects of the present disclosure relate to tandem Fab MBMs. The tandem Fab MBMs comprise TCE ABS (e.g., TCE ABS as described in Section 6.7) and TAA ABS (e.g., TAA ABS as described in Section 6.6), both in the form of Fab, e.g., Fab domains as described in Section 6.9.1. The TCE ABS and TAA ABS may be separated by a non-cleavable linker, e.g., as described in Section 6.5.

[0300] In some aspects, the present disclosure provides a tandem Fab MBM comprising (1) a first polypeptide chain, which comprises, in an N-terminal to C-terminal orientation, 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 a CH1 domain; (2) a second polypeptide chain, which comprises a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain, which comprises 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.

[0301] In other aspects, the present disclosure provides a tandem Fab MBM comprising (1) a first polypeptide chain, which comprises, in an N-terminal to C-terminal orientation, 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 a CH1 domain; (2) a second polypeptide chain, which comprises a VL of a TAA ABS operably linked to a CL domain; and (3) a third polypeptide chain, which comprises 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.

[0302] In some embodiments, the CH1 of the TCE ABS and the CH1 of the TAA ABS are the same. In other embodiments, the CL of the TCE ABS and the CL of the TAA ABS are the same. In yet other embodiments, the VL of the TAA ABS and the TCE ABS are the same and are universal light chain VL. Exemplary universal light chains that can be incorporated into TAA ABS and TCE ABS are listed in Table J below.

[0303]

[0304] The optional linker is preferably selected from the linkers disclosed in Section

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

[0305] 6.12. Nucleic Acids and Host Cells

[0306] In another aspect, the present disclosure provides nucleic acids encoding the MBM preprotein and tandem Fab MBM of the present disclosure. In some embodiments, the MBM preprotein and tandem Fab MBM are encoded by a single nucleic acid. In other embodiments, the MBM preprotein and tandem Fab MBM may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0307] A single nucleic acid may encode a MBM preprotein or tandem Fab MBM comprising a single polypeptide chain, a MBM preprotein or tandem Fab MBM comprising two or more polypeptide chains, or a portion of a MBM preprotein or tandem Fab MBM comprising more than two polypeptide chains (e.g., a single nucleic acid may encode two polypeptide chains of a MBM preprotein or tandem Fab MBM comprising three, four or more polypeptide chains, or three polypeptide chains of a MBM preprotein comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding the two or more polypeptide chains may be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding the two or more polypeptides may also be controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into separate polypeptides.

[0308] In some embodiments, a MBM preprotein or tandem Fab MBM comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the MBM preprotein or tandem Fab MBM may be equal to or less than the number of polypeptide chains in the MBM preprotein or tandem Fab MBM (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0309] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA).

[0310] In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids can be present in a single vector or in separate vectors in the same host cell or in separate host cells, as described in more detail below.

[0311] 6.12.1. Carrier

[0312] The present disclosure provides vectors comprising nucleotide sequences encoding the MBM preprotein, tandem Fab MBM or components thereof described herein, such as one or two polypeptide chains of a half antibody of the MBM preprotein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages or yeast artificial chromosomes (YACs).

[0313] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (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.

[0314] In addition, the cells that have been stably integrated into their chromosomes by the introduction of one or more markers that allow the transfected host cells to be selected can be selected. The marker can provide, for example, prototropy, biocide resistance (for example, antibiotic) or resistance to heavy metals such as copper to auxotrophic hosts. The selectable marker gene can be directly connected to the DNA sequence to be expressed, or is introduced into the same cell by co-transformation. Other elements can also be required for the optimal synthesis of mRNA. These elements can include splicing signals and transcriptional promoters, enhancers and termination signals.

[0315] Once an expression vector or DNA sequence containing a construct for expression has been prepared, the expression vector can be transfected or introduced into an appropriate host cell. Various techniques can be used to achieve this goal, 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 for recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the description of the present invention, depending on the specific expression vector and mammalian host cell used.

[0316] 6.12.2. Cells

[0317] The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

[0318] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0319] In one embodiment, host cells are genetically engineered by using expression cassettes. The phrase "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such sequence. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or helpful to affect expression may also be used, such as, for example, inducible promoters.

[0320] The present disclosure also provides host cells comprising the vectors described herein.

[0321] 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.

[0322] 6.13. Pharmaceutical compositions

[0323] The MBM preprotein and tandem Fab MBM of the present disclosure may be in the form of a composition comprising the MBM preprotein or tandem Fab MBM and one or more carriers, excipients and / or diluents. The composition may be formulated for a specific use, such as for veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend on the intended use of the MBM preprotein and tandem Fab MBM and the mode of administration for therapeutic use.

[0324] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administering it to the patient). The pharmaceutical composition may be administered to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical or local. The most appropriate route of administration in any given case will depend on the particular MBM proprotein or tandem Fab MBM, the subject, and the nature and severity of the disease, as well as the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0325] The pharmaceutical composition may conveniently be presented in a unit dosage form containing a predetermined amount of the MBM proprotein or tandem Fab MBM of the present disclosure per dose. The amount of MBM proprotein or tandem Fab MBM contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses may be in the form of a lyophilized dry powder containing an amount of MBM proprotein or tandem Fab MBM suitable for a single administration, or in liquid form. The dry powder unit dosage form may be packaged in a kit together with a syringe, an appropriate amount of diluent, and / or other components for administration. The unit dose in liquid form may be conveniently provided in the form of a syringe pre-filled with an amount of MBM proprotein suitable for a single administration.

[0326] Pharmaceutical compositions may also be supplied in bulk containing an amount of MBM proprotein or tandem Fab MBM suitable for multiple administrations.

[0327] Pharmaceutical compositions for storage as lyophilized formulations or aqueous solutions can be prepared by mixing MBM preprotein or tandem Fab MBM having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (all of which are referred to herein as "carriers") commonly employed in the art (i.e., buffers, stabilizers, preservatives, isotonicity agents, nonionic detergents, antioxidants and other miscellaneous additives). See Remington's Pharmaceutical Sciences, 16th edition (Osol ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0328] Buffers help maintain the pH value within a range close to physiological conditions. They can be present in a variety of concentrations, but are generally present in a concentration ranging from about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium 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-monosodium fumarate mixtures, etc.), In some embodiments, the present invention provides the present invention relates to a kind of buffer solution (for example, a mixture of gluconic acid-sodium gluconate, a mixture of gluconic acid-sodium hydroxide, a mixture of gluconic acid-potassium gluconate, etc.), a buffer solution (for example, a mixture of gluconic acid-sodium gluconate, a mixture of gluconic acid-sodium hydroxide, a mixture of gluconic acid-potassium gluconate, etc.), a buffer solution (for example, a mixture of oxalic acid-sodium oxalate, a mixture of oxalic acid-sodium hydroxide, a mixture of oxalic acid-potassium oxalate, etc.), a buffer solution (for example, a mixture of lactic acid-sodium lactate, a mixture of lactic acid-sodium hydroxide, a mixture of lactic acid-potassium lactate, etc.), and a buffer solution (for example, a mixture of acetic acid-sodium acetate, a mixture of acetic acid-sodium hydroxide, etc.). In addition, a phosphate buffer, a histidine buffer and a trimethylamine salt such as Tris can be used.

[0329] Preservatives can be added to retard microbial growth, and can be added in an amount within the range of about 0.2%-1% (w / v). Suitable preservatives for the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide and iodide), hexamethylammonium chloride, and alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents, sometimes referred to as "stabilizers", can be added to ensure the isotonicity of the liquid composition of the present disclosure, and include polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a large class of excipients, whose functions range from bulking agents to dissolving therapeutic agents or additives that help prevent denaturation or adhesion to the container wall. Typical stabilizers may be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents such as urea. , glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.5 wt % to 10 wt % per weight of the MBM preprotein or tandem Fab MBM.

[0330] Nonionic surfactants or detergents (also referred to as "wetting agents") can be added to help dissolve glycoproteins and protect glycoproteins from the aggregation caused by stirring, which also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamer (184, 188, etc.) and pluronic polyols. Nonionic surfactants can exist in the range of about 0.05 mg / mL to about 1.0 mg / mL, for example, about 0.07 mg / mL to about 0.2 mg / mL.

[0331] Additional miscellaneous excipients include extenders (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and solubilizing agents.

[0332] The MBM proproteins and tandem Fab MBMs of the present disclosure can be formulated as pharmaceutical compositions comprising the MBM proproteins or tandem Fab MBMs, e.g., containing one or more pharmaceutically acceptable excipients or carriers. To prepare pharmaceutical or sterile compositions comprising the MBM proproteins and tandem Fab MBMs of the present disclosure, the MBM proprotein or tandem Fab MBM formulations can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0333] For example, formulations of MBM proproteins and tandem Fab MBMs can be prepared by mixing the MBM proprotein 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, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wlkins, New York, NY; 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, NY).

[0334] The effective amount for a particular subject may vary depending on a variety of factors, such as the condition being treated, the subject's overall health, 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).

[0335] The compositions of the present disclosure may also be administered by one or more routes of administration using one or more methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending on the desired outcome. The selected routes of administration of the MBM preprotein and the tandem Fab MBM include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other general routes of administration, such as by injection or infusion. General administration may represent modes of administration other than enteral and topical administration (usually administered by injection), and include but are not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure may be administered by non-general routes, such as topical, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical. In one embodiment, the MBM preprotein and the tandem Fab MBM are administered by infusion. In another embodiment, the MBM proprotein or tandem Fab MBM of the present disclosure is administered subcutaneously.

[0336] 6.14. Therapeutic indications and treatment methods

[0337] MBM proproteins and tandem Fab MBMs disclosed herein can be used to treat any proliferative disorder (e.g., cancer) that expresses TAAs. In specific embodiments, the cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, primary unknown cancer, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal carcinoma, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma (Ewing sarcoma), eye cancer, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasm syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, 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 carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumors, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms tumor.

[0338] Table K below shows exemplary indications for which MBM proproteins and tandem Fab MBMs targeting specific TAAs may be used.

[0339]

[0340]

[0341]

[0342]

[0343] 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 incorporated herein by reference in its entirety.

[0344] 7. Numbered Examples

[0345] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the present disclosure.The present disclosure is illustrated by way of the numbered embodiments set forth below.

[0346] In the subsequently 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.

[0347] The present disclosure is exemplified by the embodiments set forth below as Group A and Group B. Unless otherwise stated, the features of any one of a particular group of numbered embodiments are applicable mutatis mutandis to the other group of numbered embodiments.

[0348] Embodiments of Group A

[0349] 1. A binding molecule comprising:

[0350] (a) the first Fc domain;

[0351] (b) a second Fc domain; and

[0352] (c) a first component linked to the first antigen binding site (ABS) of said first Fc domain via a protease cleavable linker (PCL),

[0353] wherein (i) the PCL is 25 amino acids or less in length, and / or (ii) the first ABS is sterically hindered from binding its target.

[0354] 2. The binding molecule of embodiment 1, wherein said binding of said first ABS to its target is enhanced upon protease cleavage of said PCL.

[0355] 3. The binding molecule of embodiment 1, wherein the first ABS is sterically hindered from binding its target prior to protease cleavage of the PCL, and the steric hindrance is released after protease cleavage of the PCL.

[0356] 4. The binding molecule of any one of embodiments 1 to 3, wherein the PCL is 20 amino acids or less in length.

[0357] 5. The binding molecule of any one of embodiments 1 to 3, wherein the PCL is 15 amino acids or less in length.

[0358] 6. The binding molecule of any one of embodiments 1 to 3, wherein the PCL is 10 amino acids or less in length.

[0359] 7. The binding molecule of any one of embodiments 1 to 6, wherein binding of the first ABS to its target is enhanced at least 10-fold following protease cleavage of the PCL.

[0360] 8. The binding molecule of any one of embodiments 1 to 6, wherein binding of the first ABS to its target is enhanced at least 100-fold following protease cleavage of the PCL.

[0361] 9. The binding molecule of any one of embodiments 1 to 8, wherein the first ABS is a Fab.

[0362] 10. The binding molecule of any one of embodiments 1 to 8, wherein the first ABS is an Fv.

[0363] 11. The binding molecule of any one of embodiments 1 to 10, wherein the first component is VH.

[0364] 12. The binding molecule of any one of embodiments 1 to 10, wherein the first component is VL.

[0365] 13. The binding molecule of any one of embodiments 1 to 12, wherein the binding molecule comprises at least two polypeptide chains, wherein a first polypeptide chain comprises the first Fc domain and the first component of the first ABS, and a second polypeptide chain comprises the second Fc domain.

[0366] 14. The binding molecule of embodiment 13, wherein the second polypeptide chain comprises the second component of the first ABS.

[0367] 15. The binding molecule of embodiment 14, wherein said first component and said second component associate to form said first ABS.

[0368] 16. A binding molecule according to embodiment 14 or 15, wherein the first component is VH and the second component is VL.

[0369] 17. The binding molecule of embodiment 16, wherein (1) the first polypeptide chain further comprises the CH1 C-terminus of the VH, and (2) the second polypeptide chain further comprises the CL C-terminus of the VL.

[0370] 18. The binding molecule of any one of embodiments 13 to 15, further comprising a third polypeptide chain comprising the third component of the first ABS.

[0371] 19. The binding molecule of any one of embodiments 1 to 18, which is a multispecific binding molecule comprising a second ABS.

[0372] 20. The binding molecule of embodiment 19, wherein (a) the first ABS is TCE ABS and the second ABS is TAA ABS or (b) the first ABS is TAA ABS and the second ABS is TCE ABS.

[0373] 21. The binding molecule of embodiment 20, wherein the first ABS is a TCE ABS.

[0374] 22. The binding molecule according to embodiment 21, wherein the TCE ABS is capable of binding to CD3, TCRαβ or TCRγδ.

[0375] 23. The binding molecule of any one of embodiments 20 to 22, wherein said TAA ABS is capable of binding to:

[0376] (a) any TAA identified in Section 6.6; or

[0377] (b) AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-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 proteins (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 urothelial-specific membrane protein 3 (uroplakin-3).

[0378] 24. The binding molecule of any one of embodiments 1 to 23, wherein said first Fc domain and said second Fc domain associate to form an Fc heterodimer.

[0379] 25. A binding molecule according to embodiment 24, wherein the Fc heterodimer comprises knob-hole mutations, for example, wherein (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.

[0380] 26. The binding molecule of any one of embodiments 1 to 25, wherein said first Fc domain and / or said second Fc domain comprises a star mutation.

[0381] 27. The binding molecule according to any one of embodiments 1 to 26, wherein the tandem Fab is produced after protease cleavage of said PCL.

[0382] 28. The binding molecule of any one of embodiments 1 to 26, wherein the multispecific binding molecule comprising the first Fc domain and the second Fc domain is generated following protease cleavage of the PCL.

[0383] 29. The binding molecule of any one of embodiments 1 to 28, wherein the PCL comprises a substrate sequence cleavable by any protease listed in Table A.

[0384] 30. The binding molecule of any one of embodiments 1 to 29, wherein said PCL comprises one or more substrate sequences selected from said substrate sequences listed in Table B.

[0385] 31. The binding molecule of any one of embodiments 1 to 30, wherein the PCL comprises an amino acid sequence of any one of the PCL sequences selected from the sequences listed in Table D.

[0386] 32. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3A The configuration depicted in .

[0387] 33. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3B The configuration depicted in .

[0388] 34. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3C The configuration depicted in .

[0389] 35. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3D The configuration depicted in .

[0390] 36. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3E The configuration depicted in .

[0391] 37. The binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 3F The configuration depicted in .

[0392] 38. A binding molecule according to any one of embodiments 1 to 31, wherein the binding molecule has Figure 7 The configuration depicted in .

[0393] 39. The binding molecule of any one of embodiments 1 to 38, wherein said first Fc domain and / or said second Fc domain has at least about 90% sequence identity to any one of SEQ ID NOs: 335, 336, 337 and 338.

[0394] 40. The binding molecule of any one of embodiments 1 to 38, wherein said first Fc domain and / or said second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337 and 338.

[0395] 41. The binding molecule of any one of embodiments 1 to 38, wherein said first Fc domain and / or said second Fc domain has at least about 95% sequence identity to any one of SEQ ID NOs: 335, 336, 337 and 338.

[0396] 42. The binding molecule of any one of embodiments 1 to 41, wherein said first Fc domain and / or said second Fc domain comprises one or more amino acid substitutions that reduce effector function (eg, as described in Section 6.10.1).

[0397] 43. A 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 (e.g., as described in Section 6.10.2).

[0398] 44. A pharmaceutical composition comprising the binding molecule according to any one of embodiments 1 to 43 and an excipient.

[0399] 45. A method of treating cancer, comprising administering an effective amount of the binding molecule according to any one of embodiments 1 to 43 or the pharmaceutical composition according to embodiment 44 to a subject suffering from cancer.

[0400] 46. ​​The method of embodiment 45, wherein the cancer is associated with expression of an epitope bound by a TAA ABS, e.g., as shown in Table K.

[0401] 47. A nucleic acid or multiple nucleic acids encoding the binding molecule according to any one of embodiments 1 to 43.

[0402] 48. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding a binding molecule according to any one of embodiments 1 to 43 under the control of one or more promoters.

[0403] 49. A method for producing a binding molecule, the method comprising:

[0404] (a) culturing the cell according to embodiment 48 under conditions where the binding molecule is expressed; and

[0405] (b) recovering the binding molecule from the cell culture.

[0406] 50. The method according to embodiment 49 further comprises enriching the binding molecules and / or purifying the binding molecules.

[0407] Embodiments of Group B

[0408] 1. A multispecific binding molecule (MBM) proprotein comprising

[0409] (a) a first Fc domain and a second Fc domain, which are capable of associating to form an Fc region;

[0410] (b) a T cell engaging antigen binding site ("TCE ABS") at the C-terminus of the first Fc domain and / or the second Fc domain;

[0411] (c) at least one antigen binding site capable of binding to a tumor associated antigen ("TAA ABS"); and

[0412] (d) a protease cleavable linker (PCL) at the C-terminus of the first Fc domain or the second Fc domain, wherein when the PCL is in an uncleaved state, the TAA ABS in the MBM pre-protein is able to bind to its target, and the binding of the TCE ABS to its target is enhanced after protease cleavage of the PCL.

[0413] 2. The MBM proprotein of embodiment 1, wherein cleavage of the PCL releases the MBM comprising the TAA ABS and the TCE ABS.

[0414] 3. The MBM preprotein according to embodiment 1 or embodiment 2, wherein the TCEABS in the MBM preprotein are masked by anti-idiotypic antibodies.

[0415] 4. The MBM preprotein of embodiment 1 or embodiment 2, wherein the TCEABS in the MBM preprotein are sterically hindered from binding to their target by the Fc domain.

[0416] 5. The MBM proprotein of any one of embodiments 1 to 4, wherein the MBM comprises a first and a second Fc domain.

[0417] 6. The MBM proprotein according to embodiment 5, having Figure 1A The configuration depicted in .

[0418] 7. The MBM proprotein according to embodiment 5, having Figure 1B The configuration depicted in .

[0419] 8. The MBM proprotein according to embodiment 5, having Figure 1C The configuration depicted in .

[0420] 9. The MBM proprotein according to embodiment 5, having Figure 3A The configuration depicted in .

[0421] 10. The MBM proprotein according to embodiment 5, having Figure 3B The configuration depicted in .

[0422] 11. The MBM proprotein according to embodiment 5, having Figure 3C The configuration depicted in .

[0423] 12. The MBM proprotein according to embodiment 5, having Figure 3D The configuration depicted in .

[0424] 13. The MBM proprotein according to embodiment 5, having Figure 3E The configuration depicted in .

[0425] 14. The MBM proprotein according to embodiment 5, having Figure 3F The configuration depicted in .

[0426] 15. The MBM proprotein of any one of embodiments 1 to 4, wherein the MBM lacks the first and second Fc domains.

[0427] 16. The MBM proprotein of embodiment 15, having the configuration depicted in FIG5 .

[0428] 17. The MBM proprotein according to embodiment 15, having Figure 7 The configuration depicted in .

[0429] 18. A multispecific binding molecule (MBM) proprotein, which is optionally the MBM according to any one of embodiments 1 to 8, comprising:

[0430] (a) a first polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0431] (i) an optional first linker;

[0432] (ii) a first Fc domain;

[0433] (iii) a second connector; and

[0434] (iv) a T cell engaging antigen binding site ("TCE ABS") or a component thereof; and

[0435] (b) a second polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0436] (i) an optional third linker;

[0437] (ii) a second Fc domain, which is capable of heterodimerizing with the first Fc domain to form an Fc heterodimer;

[0438] (iii) a fourth linker which is a protease cleavable linker ("PCL"); and

[0439] (iv) an antigen binding site which is an anti-idiotype of said TCE ABS ("anti-TCE ABS") or a component thereof,

[0440] The first polypeptide chain and / or the second polypeptide chain comprises an antigen binding site ("TAA ABS") or a component thereof that is capable of binding to a tumor associated antigen.

[0441] 19. The MBM preprotein of embodiment 18, wherein the TAA ABS, TCE ABS and anti-TCE ABS are in the form of Fab, and wherein the TAA ABS chain, TCE ABS chain and anti-TCE ABS chain comprise a VH domain associated with their respective VL domains on separate polypeptide chains.

[0442] 20. The MBM preprotein of embodiment 18, wherein the VL domain is a universal light chain VL domain.

[0443] 21. The MBM proprotein of any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of a scFv.

[0444] 22. The MBM proprotein of any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of a VHH.

[0445] 23. The MBM proprotein of any one of embodiments 1 to 18, wherein the anti-TCE ABS is in the form of VH.

[0446] 24. The MBM proprotein of any one of embodiments 18 to 20, wherein the linker other than the PCL is a non-cleavable linker ("NCL").

[0447] 25. A multispecific binding molecule (MBM) proprotein, which is optionally the MBM according to any one of embodiments 1 to 5 and 9 to 14, comprising:

[0448] (a) a first polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0449] (i) an optional first linker;

[0450] (ii) a first Fc domain;

[0451] (iii) a second connector; and

[0452] (iv) a T cell engaging antigen binding site ("TCE ABS") component comprising VH;

[0453] (b) a second polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0454] (i) an optional third linker;

[0455] (ii) a second Fc domain, which is capable of heterodimerizing with the first Fc domain to form an Fc heterodimer;

[0456] (iii) a fourth connector; and

[0457] (iv) a T cell engaging antigen binding site ("TCE ABS") component comprising a VL, wherein (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 capable of binding to a tumor associated antigen ("TAA ABS"), and (C) the binding of the TCE ABS to its target is sterically hindered.

[0458] 26. The MBM proprotein of embodiment 25, wherein the TCE ABS is a Fab.

[0459] 27. The MBM proprotein of embodiment 26, wherein the first polypeptide chain comprises a C-terminal CH1 domain of the VH of the TCE ABS, and the second polypeptide chain comprises a C-terminal CL domain of the VL of the TCE ABS.

[0460] 28. The MBM proprotein of embodiment 25, wherein the TCE ABS is Fv.

[0461] 29. The MBM preprotein of embodiment 28, wherein the first polypeptide chain lacks the C-terminal CH1 domain of the VH of the TCE ABS, and the second polypeptide chain lacks the C-terminal CL domain of the VL of the TCE ABS.

[0462] 30. The MBM pre-protein of any one of embodiments 25 to 29, wherein the TAA ABS is in the form of a Fab, and wherein the TAA ABS chains each comprise a VH domain associated with their respective VL domains on a separate polypeptide chain.

[0463] 31. The MBM proprotein of embodiment 30, wherein the VL domains on separate polypeptide chains are universal light chain VL domains.

[0464] 32. The MBM proprotein of any one of embodiments 25 to 31, wherein the linker other than the PCL is a non-cleavable linker ("NCL").

[0465] 33. The MBM preprotein of 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.

[0466] 34. A multispecific binding molecule (MBM) proprotein, which is optionally the MBM according to any one of embodiments 1 to 8, 15 and 16, comprising:

[0467] (a) a first polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0468] (i) an optional first linker;

[0469] (ii) a first Fc domain;

[0470] (iii) a second connector; and

[0471] (iv) an antigen binding site which is an anti-idiotype of said TCE ABS of said second polypeptide chain ("anti-TCEABS") or a component thereof; and

[0472] (b) a second polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0473] (i) an optional third linker;

[0474] (ii) a second Fc domain, which is capable of heterodimerizing with the first Fc domain to form an Fc heterodimer;

[0475] (iii) a fourth linker, which is a protease cleavable linker ("PCL");

[0476] (iv) T cell engaging antigen binding sites ("TCE ABS") or components thereof;

[0477] (v) an optional fifth linker; and

[0478] (vi) an antigen binding site ("TAA ABS") or a component thereof that is capable of binding to a tumor associated antigen.

[0479] 35. The MBM pre-protein of embodiment 34, wherein the TAA ABS, TCE ABS and anti-TCE ABS are in the form of Fab, and wherein the TAA ABS chain, TCE ABS chain and anti-TCE ABS chain comprise a VH domain associated with their respective VL domains on separate polypeptide chains.

[0480] 36. The MBM preprotein of embodiment 35, wherein the VL domain is a universal light chain VL domain.

[0481] 37. The MBM proprotein of embodiment 34, wherein the anti-TCE ABS is in the form of a scFv.

[0482] 38. The MBM proprotein of embodiment 34, wherein the anti-TCE ABS is in the form of a VHH.

[0483] 39. The MBM proprotein of embodiment 34, wherein the anti-TCE ABS is in the form of VH.

[0484] 40. The MBM proprotein of any one of embodiments 34 to 39, wherein the linker other than the PCL is a non-cleavable linker ("NCL").

[0485] 41. A multispecific binding molecule (MBM) proprotein, which is optionally the MBM according to any one of embodiments 1 to 4, 15 and 17, comprising:

[0486] (a) a first polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0487] (i) an optional first linker;

[0488] (ii) a first Fc domain;

[0489] (iii) a second linker which is a protease cleavable linker ("PCL"); and

[0490] (iv) a T cell engaging antigen binding site ("TCE ABS") component comprising VH;

[0491] (b) a second polypeptide chain comprising, in the N-terminal to C-terminal direction:

[0492] (i) an optional third linker;

[0493] (ii) a second Fc domain, which is capable of heterodimerizing with the first Fc domain to form an Fc heterodimer;

[0494] (iii) a fourth linker which is a protease cleavable linker ("PCL"); and

[0495] (iv) a T cell engaging antigen binding site ("TCE ABS") component comprising a VL, wherein the first polypeptide chain or the second polypeptide chain comprises an antigen binding site ("TAA ABS") or a component thereof at the C-terminus of the TCE ABS chain that is capable of binding to a tumor associated antigen.

[0496] 42. The MBM proprotein of embodiment 41, wherein the TCE ABS is a Fab.

[0497] 43. The MBM proprotein of embodiment 42, wherein the first polypeptide chain comprises a C-terminal CH1 domain of the VH of the TCE ABS, and the second polypeptide chain comprises a C-terminal CL domain of the VL of the TCE ABS.

[0498] 44. The MBM proprotein of embodiment 41, wherein the TCE ABS is Fv.

[0499] 45. The MBM preprotein of embodiment 44, wherein the first polypeptide chain lacks the C-terminal CH1 domain of the VH of the TCE ABS, and the second polypeptide chain lacks the C-terminal CL domain of the VL of the TCE ABS.

[0500] 46. ​​The MBM preprotein of any one of embodiments 41 to 45, wherein the TAA ABS is in the form of a Fab, and wherein the TAA ABS chain comprises a VH domain associated with a VL domain on a separate polypeptide chain.

[0501] 47. The MBM proprotein of embodiment 46, wherein the VL domains on separate polypeptide chains are universal light chain VL domains.

[0502] 48. The MBM proprotein of any one of embodiments 41 to 47, wherein the linker other than the PCL is a non-cleavable linker ("NCL").

[0503] 49. The MBM preprotein of any one of embodiments 41 to 48, 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.

[0504] 50. The MBM proprotein of any one of embodiments 1 to 49, wherein each PCL comprises a substrate sequence cleavable by any protease listed in Table A.

[0505] 51. The MBM proprotein of any one of embodiments 1 to 50, wherein each PCL comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

[0506] 52. The MBM proprotein of any one of embodiments 1 to 51, wherein each PCL comprises one or more spacer sequences selected from the substrate sequences listed in Table C, optionally wherein the PCL comprises or consists of any one of the following amino acid sequences:

[0507] (a)GGGGSGGGGSGGGGSISSGLLLSGRSDNHGGSGGS (SEQ ID NO: 205);

[0508] (b)GGGGSGGGGSGGGGSVPLSLYSGGGGSGGSGGSGS (SEQ ID NO: 206);

[0509] (c) ISSGLLSGRSDNH (SEQ ID NO: 230); and

[0510] (d) A variant of any of the foregoing having 1-5 amino acid substitutions.

[0511] 53. The MBM proprotein of any one of embodiments 1 to 52, wherein each PCL comprises an amino acid sequence selected from any one of the PCL sequences of the substrate sequences listed in Table D.

[0512] 54. The MBM proprotein of any one of embodiments 1 to 53, wherein all linkers in the molecule except the PCL are non-cleavable linkers ("NCLs").

[0513] 55. The MBM proprotein of embodiment 54, wherein the NCL is selected from Table E.

[0514] 56. The MBM preprotein of any one of embodiments 1 to 55, wherein the TAA ABS is capable of binding to:

[0515] (a) any TAA identified in Section 4.5; or

[0516] (b) AFP, ALK, BAGE proteins, 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 ​​proteins (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MA GE-A3, hTERT, LMP2, MAGE proteins (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, P AX3, 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 urothelial-specific membrane protein 3.

[0517] 57. The MBM preprotein of any one of embodiments 1 to 56, wherein the TAA ABS (a) comprises (i) CDRs or (ii) VH and VL sequences of an antibody listed in Table F or (b) competes with an antibody listed in Table F for binding to the TAA.

[0518] 58. The MBM pre-protein of any one of embodiments 1 to 57, wherein the TAA is CEACAM5.

[0519] 59. The MBM preprotein of any one of embodiments 1 to 57, wherein the TAA is EpCAM.

[0520] 60. The MBM preprotein of any one of embodiments 1 to 57, wherein the TAA is HER2.

[0521] 61. The MBM preprotein of any one of embodiments 1 to 5571, wherein the TAA is PMSA.

[0522] 62. The MBM preprotein of any one of embodiments 1 to 57, wherein the TAA is STEAP2.

[0523] 63. The MBM preprotein of any one of embodiments 1 to 57, wherein the TAA is EGFR.

[0524] 64. The MBM preprotein of any one of embodiments 1 to 57, wherein the TCE ABS is capable of binding to a component of the T cell receptor (TCR) complex.

[0525] 65. The MBM preprotein of embodiment 64, wherein the component of the TCR complex is CD3.

[0526] 66. The MBM preprotein of embodiment 64, wherein the component of the TCR complex is TCRαβ.

[0527] 67. The MBM preprotein of embodiment 64, wherein the component of the TCR complex is TCRγδ.

[0528] 68. The MBM proprotein of any one of embodiments 1 to 67, wherein the TCE ABS (a) comprises (i) CDRs or (ii) VH and VL sequences of an antibody listed in Table G or (b) competes with an antibody listed in Table G for binding to its target.

[0529] 69. The MBM proprotein of any one of embodiments 1 to 68, which upon protease cleavage of PCL generates a bispecific binding molecule ("BBM").

[0530] 70. The MBM preprotein of embodiment 69, wherein the BBM is bivalent.

[0531] 71. The MBM proprotein of embodiment 69, wherein the BBM is trivalent.

[0532] 72. The MBM preprotein of any one of embodiments 1 to 71, wherein the Fc region is heterodimeric.

[0533] 73. The MBM preprotein of embodiment 72, wherein the Fc heterodimer comprises a knob-to-hole mutation.

[0534] 74. The MBM proprotein of any one of embodiments 1 to 72, wherein at least one Fc domain comprises a star mutation compared to a wild-type Fc domain.

[0535] 75. The MBM proprotein of any one of embodiments 1 to 74, wherein the TCE ABS comprises a VL having one or more amino acid deletions at the N-terminus.

[0536] 76. The MBM preprotein of any one of embodiments 1 to 74, wherein the TCE ABS comprises a VH having one or more amino acid deletions at the N-terminus.

[0537] 77. The MBM proprotein of any one of embodiments 1 to 76, wherein the first Fc domain comprises a deletion of one or more amino acids at the C-terminus relative to a wild-type Fc domain.

[0538] 78. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 1 amino acid at the C-terminus relative to a wild-type Fc domain.

[0539] 79. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 2 amino acids at the C-terminus relative to a wild-type Fc domain.

[0540] 80. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 3 amino acids at the C-terminus relative to a wild-type Fc domain.

[0541] 81. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 4 amino acids at the C-terminus relative to a wild-type Fc domain.

[0542] 82. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 5 amino acids at the C-terminus relative to a wild-type Fc domain.

[0543] 83. The MBM proprotein of embodiment 77, wherein the first Fc domain comprises a deletion of 6 amino acids at the C-terminus relative to a wild-type Fc domain.

[0544] 84. The MBM proprotein of any one of embodiments 1 to 83, wherein the second Fc domain comprises a deletion of one or more amino acids at the C-terminus relative to a wild-type Fc domain.

[0545] 85. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 1 amino acid at the C-terminus relative to the wild-type Fc domain.

[0546] 86. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 2 amino acids at the C-terminus relative to the wild-type Fc domain.

[0547] 87. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 3 amino acids at the C-terminus relative to the wild-type Fc domain.

[0548] 88. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 4 amino acids at the C-terminus relative to the wild-type Fc domain.

[0549] 89. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 5 amino acids at the C-terminus relative to the wild-type Fc domain.

[0550] 90. The MBM proprotein of embodiment 84, wherein the second Fc domain comprises a deletion of 6 amino acids at the C-terminus relative to the wild-type Fc domain.

[0551] 91. A tandem Fab MBM comprising (1) a first polypeptide chain, oriented from N-terminus to C-terminus, 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 a 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.

[0552] 92. A tandem Fab MBM comprising (1) a first polypeptide chain comprising, from N-terminus to C-terminus, (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.

[0553] 93. A tandem Fab MBM comprising a tandem Fab MBM, comprising (1) a first polypeptide chain, oriented from N-terminus to C-terminus, 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 a 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.

[0554] 94. A tandem Fab MBM comprising (1) a first polypeptide chain comprising, from N-terminus to C-terminus, (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.

[0555] 95. The tandem Fab MBM according to any one of embodiments 91 to 94, wherein the CH1 of the TCE ABS and the CH1 of the TAA ABS are identical.

[0556] 96. The tandem Fab MBM according to any one of embodiments 91 to 95, wherein the CL of the TCE ABS and the CL of the TAA ABS are the same.

[0557] 97. The tandem Fab MBM according to any one of embodiments 91 to 9674, wherein the VL of the TAA ABS and the TCE ABS are identical and are common light chain VL.

[0558] 98. The tandem Fab MBM according to any one of embodiments 91 to 97, wherein the TAA ABS is as defined in any one of embodiments 50 to 57.

[0559] 99. The tandem Fab MBM according to any one of embodiments 91 to 98, wherein the TCE ABS is as defined in any one of embodiments 64 to 68.

[0560] 100. A pharmaceutical composition comprising the MBM proprotein 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.

[0561] 101. A method of treating cancer, comprising administering an effective amount of the MBM preprotein 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 to a subject suffering from cancer.

[0562] 102. The method of embodiment 101, wherein the cancer is associated with expression of an epitope bound by a TAA ABS, e.g., as shown in Table K.

[0563] 103. A nucleic acid or nucleic acids encoding the MBM proprotein of any one of embodiments 1 to 90 or the tandem Fab MBM of any one of embodiments 91 to 99.

[0564] 104. A cell engineered to express the MBM proprotein of any one of embodiments 1 to 90 or the tandem Fab MBM of any one of embodiments 91 to 99.

[0565] 105. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the MBM preprotein 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.

[0566] 106. A method for producing a MBM proprotein or a tandem Fab MBM, the method comprising:

[0567] (a) culturing the cell according to Example 104 or Example 105 under conditions where the MBM is expressed; and

[0568] (b) recovering the MBM proprotein or tandem Fab MBM, if applicable, from the cell culture.

[0569] 107. The method according to embodiment 106, further comprising enriching the MBM preprotein or tandem FabMBM, as applicable.

[0570] 108. The method of embodiment 106 or embodiment 107, further comprising purifying the MBM proprotein or tandem Fab MBM, as applicable.

[0571] 8. Examples

[0572] 8.1. Example 1: Effect of linker length on Fc masking of the C-terminal antigen binding site

[0573] Produce Figure 3BThe anti-TAAxCD3 bispecific antibodies of the configuration shown in FIG. 5 , wherein the C-terminus of the Fc domain has different linker lengths (5, 15 and 25 amino acids), the difference being (ref. Figure 3B ) Linker B is a non-cleavable linker. The purpose of this study was to determine the effect of linkers that separate the Fc domain from the C-terminal Abs or Abs chains on the ability of the Abs to bind to a target (in this case CD3) on JURKAT cells. A conventional bispecific anti-TAAxCD3 bispecific antibody was used as a control for CD3 binding.

[0574] Binding of the TAAxCD3 bispecific antibody to JURKAT cells was determined by flow cytometry analysis followed by detection with an anti-human IgG antibody labeled with allophycocyanin (APC). 5 Cells / well were incubated with serially diluted anti-TAAxCD3 bispecific antibodies at 4°C for 30 minutes. After incubation, cells were washed twice with cold PBS containing 1% filtered FBS, and APC-labeled anti-human secondary antibodies were added to the cells and incubated for another 30 minutes. Wells containing only secondary antibodies were used as controls.

[0575] After incubation, cells were washed, resuspended in 200 μL cold PBS containing 1% filtered FBS, and analyzed by flow cytometry on a BD FACSCanto II.

[0576] Results are shown in Fig. 9 In. Fig. 9 As shown in , the ability of CD3 Abs to bind their targets is correlated with linker length, where shorter linker lengths maximize steric hindrance of the Fc and result in maximal inhibition of CD3 binding, while longer linker lengths are more permissive for CD3 binding (but still reduced compared to traditional bispecific antibodies).

[0577] 8.2. Example 2: Fc steric hindrance reduces target binding of anti-TAAxCD3 bispecific antibodies

[0578] Produce Figure 7 Anti-TAAxCD3 bispecific antibodies of the configuration depicted in , wherein linkers A and B are cleavable linkers as shown or replaced by non-cleavable G4S linkers (SEQ ID NO: 1) by transient transfection in Expi293 cells. Fig.10 Shown are the expressions of anti-TAAxCD3 bispecific antibodies with cleavable linkers A and B (Fc-PCL-CD3-TAA) and anti-TAAxCD3 bispecific antibodies without cleavable linkers A and B (Fc-G4S-CD3-TAA) after single-step Pro A purification.

[0579] Binding of TAAxCD3 bispecific antibodies to JURKAT cells was assessed using flow cytometric analysis as described in Section 8.1.

[0580] All anti-TAAxCD3 bispecific antibodies with Fc steric hindrance showed inhibition of CD3 binding on JURKAT cells relative to anti-TAAxCD3 bispecific antibodies in IgG configuration ( Fig.11 A) Similar constructs with 4 or 6 amino acid truncations at the C-terminus of Fc and / or 1 or 2 amino acid truncations at the N-terminus of VH / VL similarly inhibited CD3 binding to JURKAT cells (data not shown).

[0581] 9. References

[0582] 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 was individually indicated to be incorporated by reference for all purposes. If there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended to control.

Claims

1. A binding molecule comprising: (a) the first Fc domain; (b) a second Fc domain; and (c) a first component linked to the first antigen binding site (ABS) of said first Fc domain via a protease cleavable linker (PCL), wherein (i) the PCL is 25 amino acids or less in length, and / or (ii) the first ABS is sterically hindered from binding its target.

2. The binding molecule of claim 1, wherein the binding of the first ABS to its target is enhanced upon protease cleavage of the PCL.

3. The binding molecule of claim 1, wherein the first ABS is sterically hindered from binding its target prior to protease cleavage of the PCL, and the steric hindrance is released after protease cleavage of the PCL.

4. The binding molecule according to any one of claims 1 to 3, wherein the PCL is 20 amino acids or less in length.

5. The binding molecule according to any one of claims 1 to 3, wherein the PCL is 15 amino acids or less in length.

6. The binding molecule according to any one of claims 1 to 3, wherein the PCL is 10 amino acids or less in length.

7. The binding molecule of any one of claims 1 to 6, wherein the binding of the first ABS to its target is enhanced at least 10-fold following protease cleavage of the PCL.

8. The binding molecule of any one of claims 1 to 6, wherein the binding of the first ABS to its target is enhanced at least 100-fold following protease cleavage of the PCL.

9. The binding molecule according to any one of claims 1 to 8, wherein the first ABS is a Fab.

10. The binding molecule according to any one of claims 1 to 8, wherein the first ABS is Fv.

11. The binding molecule according to any one of claims 1 to 10, wherein the first component is VH.

12. The binding molecule according to any one of claims 1 to 10, wherein the first component is VL.

13. The binding molecule according to any one of claims 1 to 12, wherein the binding molecule comprises at least two polypeptide chains, wherein a first polypeptide chain comprises the first Fc domain and the first component of the first ABS, and a second polypeptide chain comprises the second Fc domain.

14. The binding molecule of claim 13, wherein the second polypeptide chain comprises the second component of the first ABS.

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

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

17. The binding molecule of claim 16, wherein (1) the first polypeptide chain further comprises the CH1 C-terminus of the VH, and (2) the second polypeptide chain further comprises the CL C-terminus of the VL.

18. The binding molecule according to any one of claims 13 to 15, further comprising a third polypeptide chain comprising the 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 of claim 19, wherein (a) the first ABS is TCE ABS and the second ABS is TAA ABS or (b) the first ABS is TAA ABS and the second ABS is TCE ABS.

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

22. The binding molecule of claim 21, wherein the TCE ABS is capable of binding to CD3, TCRαβ or TCRγδ.

23. The binding molecule according to any one of claims 20 to 22, wherein the TAA ABS is capable of binding to: (a) any TAA identified in Section 6.6; or (b) AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-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, EGF RvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (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 proteins (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 proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, or urothelial-specific membrane protein 3.

24. The binding molecule of 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 of claim 24, wherein the Fc heterodimer comprises knob-hole mutations, for example, wherein (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 of 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 Fabs are generated following protease cleavage of said PCL.

28. The binding molecule of any one of claims 1 to 26, wherein a multispecific binding molecule comprising the first Fc domain and the second Fc domain is generated following 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 listed 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 listed in Table B.

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

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

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

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

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

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

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

38. A binding molecule according to any one of claims 1 to 31, wherein the binding molecule has the configuration depicted in Figure 7.

39. The binding molecule of 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 with any one of SEQ ID NOs: 335, 336, 337 and 338.

40. The binding molecule of 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 of 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 of 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 (eg, as described in Section 6.10.1).

43. The binding molecule of 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 (eg, as described in Section 6.10.2).

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

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

46. ​​The method of claim 45, wherein the cancer is associated with expression of an epitope bound by a TAA ABS, e.g., as shown in Table K.

47. A nucleic acid or multiple nucleic acids encoding a 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 a binding molecule according to any one of claims 1 to 43 under the control of one or more promoters.

49. A method for producing a binding molecule, the method comprising: (a) culturing the cell according to claim 48 under conditions where the binding molecule is expressed; as well as (b) recovering the binding molecule from the cell culture.

50. The method of claim 49, further comprising enriching the binding molecule and / or purifying the binding molecule.

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