Tri-specific binding molecules directed against tumor-associated antigens and uses thereof

Abstract

The present disclosure provides trispecific binding molecules that specifically bind to CD2, CD3, and tumor associated antigens, conjugates comprising the trispecific binding molecules, and pharmaceutical compositions comprising the trispecific binding molecules and the conjugates. The disclosure further provides methods of using the trispecific binding molecules to treat cancers expressing the tumor-associated antigens. The disclosure yet further provides a recombinant host cell engineered to express the trispecific binding molecule, and a method of producing the trispecific binding molecule by culturing the host cell under conditions that express the trispecific binding molecule.

Description

[0001] This application is a divisional application of Chinese application 201880086747.X, filed on November 20, 2018, entitled "Trispecific binding molecule against tumor-associated antigens and its use therein".

[0002] 1. Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 589,331, filed November 21, 2017, the contents of which are incorporated herein by reference in their entirety.

[0004] 2. Sequence List

[0005] This application contains a sequence list that has been electronically filed in ASCII format, and the entire sequence list is hereby incorporated by reference. The ASCII copy was created on November 19, 2018, and is named NOV-001WO_SL.txt, with a size of 592,187 bytes. Background Technology

[0006] Redirected-targeted T-cell lysis (RTCC) is an exciting mechanism for first-line and refractory treatment. Antibodies and antibody fragments with excellent selectivity have been successfully engineered in various forms to achieve the dual specificity required to crosslink T cells to a single receptor on target cells.

[0007] First-line treatment for certain indications or the indications themselves may promote an immunosuppressive environment that fosters T-cell unresponsiveness, thereby reducing the efficacy of existing RTCC therapies. For example, acute myeloid lymphoma has been shown to be a disease particularly difficult to evade when immune surveillance is avoided (Teague and Klein, 2013, Journal for ImmunoTherapy of Cancer 1: 13). The solid tumor microenvironment can also provide escape mechanisms and promote tumor cell survival through multiple pathways (Vaney et al., 2015, Seminars in Cancer Biology 35: S151-S184).

[0008] Therefore, an improved RTCC method is needed. Summary of the Invention

[0009] This disclosure extends the RTCC principle by providing a multispecific binding molecule that binds to tumor-associated antigens (“TAA”) and CD2, in addition to CD3 or other components of the TCR complex on T cells.

[0010] This invention is based, at least in part, on the discovery that, in addition to components of the TCR complex, conjugation of CD2 improves clinical outcomes of RTCC therapy by activating a subset of T cells that are refractory to stimulation using bispecific conjugates targeting only the TAA and TCR complex. As illustrated in the examples, treatment of tumors with a trispecific conjugate molecule that conjugates TAA, CD2, and CD3 results in enhanced antitumor activity, particularly in the presence of unresponsive T cells, compared to conjugation of only TAA and CD3. Not bound by theory, the inventors believe that conjugation of the CD2-and-TCR complex in a single multispecific molecule can stimulate both the primary signaling pathway promoting T cell-mediated tumor cell lysis (e.g., by TCR aggregation) and the secondary co-stimulatory pathway to induce T cell proliferation and potentially overcome unresponsiveness.

[0011] Therefore, this disclosure provides a trispecific binding molecule (“TBM”) that binds to (1) a tumor-associated antigen (“TAA”), (2) CD2, and (3) CD3 or other components of the TCR complex. The TBM comprises at least three antigen-binding modules (“ABMs”) capable of binding to components of the TAA, CD2, and TCR complex. In some embodiments, each antigen-binding module is capable of binding to its respective target while simultaneously binding to each of the other antigen-binding modules with its respective target. Each ABM may be immunoglobulin-based or non-immunoglobulin-based, and thus the TBM of this disclosure may include immunoglobulin-based ABMs, non-immunoglobulin-based ABMs, or combinations thereof. Immunoglobulin-based ABMs that may be used in the TBM of this disclosure are described in Section 6.2.1 below and in specific examples 31 to 406, 575 to 583, 591 to 660, 662 to 667, 671, 673 to 762, and 830 to 898. Non-immunoglobulin-based ABMs that can be used in this disclosure of TBMs are described in Section 6.2.2 below and in specific examples 2 to 30, 403 to 406, and 584 to 589. Further features of exemplary ABMs that bind to components of the TCR complex are described in Section 6.5 below and in specific examples 38 to 106, 590 to 667, and 1045. Further features of exemplary ABMs that bind to CD2 are described in Section 6.6 below and in specific examples 2 to 37, 403 to 406, 575 to 589, and 1044. Further features of exemplary ABMs that bind to TAAs are described in Section 6.7 below and in specific examples 107 to 276, 346 to 406, 668 to 762, and 1046.

[0012] The ABMs (or portions thereof) of the TBM disclosed herein can be interconnected, for example, through short peptide linkers or through Fc domains. Methods and components for linking ABMs to form the TBM are described below in Section 6.3 and in specific examples 401 to 453, 763 to 829 and 899 to 957.

[0013] The TBM disclosed herein has at least three ABMs (i.e., the TBM is at least trivalent), but may also have more than three ABMs. For example, a TBM may have four ABMs (i.e., tetravalent), five ABMs (i.e., pentavalent), or six ABMs (i.e., hexavalent), provided that the TBM has at least one ABM capable of binding TAA, at least one ABM capable of binding CD2, and at least one ABM capable of binding a component of the TCR complex. Exemplary trivalent, tetravalent, pentavalent, and hexavalent TBM configurations are shown in Figure 1 The details are described in Section 6.4 and in Specific Examples 958 to 1042 below.

[0014] This disclosure further provides nucleic acids (in the form of a single nucleic acid or multiple nucleic acids) encoding the TBM of this disclosure, and recombinant host cells and cell lines engineered to express the nucleic acids of this disclosure and the TBM. Exemplary nucleic acids, host cells, and cell lines are described in Section 6.8 below and in specific examples 570 to 573 and 1169 to 1176.

[0015] This disclosure further provides pharmaceutical conjugates incorporating the TBMs disclosed herein. For convenience, such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”, although some or all of the ABMs may be non-immunoglobulin domains. Examples of ADCs are described below in Section 6.9 and in specific examples 469 to 507 and 1054 to 1093.

[0016] This disclosure further provides formulations comprising the TBM or conjugates disclosed herein. Examples of formulations are described below in Section 6.10 and in specific examples 508 to 565 and 1094 to 1151.

[0017] Pharmaceutical compositions comprising the TBM and ADC disclosed herein are also provided. Examples of pharmaceutical compositions are described below in Section 6.11 and in specific examples 566 and 1152.

[0018] This document further provides methods for using the TBM, ADC, and pharmaceutical compositions disclosed herein, for example, for treating proliferative conditions (e.g., cancer) that express TAA. Exemplary methods are described in Section 6.12 below and in specific examples 567 to 568 and 1153 to 1155.

[0019] This disclosure further provides methods for using the TBMs, ADCs, and pharmaceutical compositions of this disclosure in combination with other agents and therapies. Exemplary agents, therapies, and methods of combination therapy are described in Section 6.13 and Specific Examples 569 and 1156 to 1168 below. Attached Figure Description

[0020] Figure 1 A-1Z: Exemplary TBM configuration. Figure 1 A shows Figure 1 The components of the exemplary TBM configuration shown in B-1Z are not shown. The entirety of the different structural domains connecting each chain is not shown (e.g., the joints connecting the VH and VL structural domains of scFv, the hinges connecting the CH2 and CH3 structural domains of Fc, etc. are omitted). Figure 1 B-1O and 1V-Z show trivalent TBMs; Figure 1 P-1R shows a tetravalent TBM; Figure 1 S shows a pentavalent TBM, and Figure 1 T-1U shows a hexavalent TBM.

[0021] Figure 2 Schematic diagram of the bispecific and trispecific constructs in Example 1.

[0022] Figure 3 A-3D: Results of retargeted T cell cytotoxicity assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 3 A-3B: Within 24 hours ( Figure 3 A) and 48 hours ( Figure 3 B) Results obtained using pan-T effector cells from the first donor after incubation. Figure 3 C-3D: Within 24 hours ( Figure 3 C) and 48 hours ( Figure 3 D) Results obtained using pan-T effector cells from a second donor after incubation.

[0023] Figure 4 A-4B: Results of retargeted T cell cytotoxicity assays using Daudi target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 4 A: Results obtained using pan-T effector cells from the first donor after 48 hours of incubation. Figure 4B: Results obtained using pan-T effector cells from a second donor after 48 hours of incubation.

[0024] Figure 5 Results of T-cell retargeting assays using negative control human K562 target cells and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19.

[0025] Figure 6 A-6E: Results of retargeted T cell cytotoxicity assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 6 A-6E shows the results obtained using pan-T effector cells from five different donors.

[0026] Figure 7 Results of T-cell retargeting assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 7 The results obtained using human cryopreserved PBMCs from patients diagnosed with acute myeloid leukemia are shown.

[0027] Figure 8 A-8D: Results of retargeted T cell cytotoxicity assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 8 A-8D shows the results obtained from retesting target cells at 48 hours, 72 hours, 96 hours, and 120 hours, respectively.

[0028] Figure 9 A-9B: Cell proliferation assay results. CD4+ sorted cells are shown in... Figure 9 In A, CD8+ sorted cells are shown in Figure 9 B in.

[0029] Figure 10 A-10F: Results of cytokine release assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19. Figure 10 A: IFNγ; Figure 10 B: TNFα; Figure 10 C: IL2; Figure 10 D: IL10; Figure 10 E: IL6; Figure 10 F: Caption explanation.

[0030] Figure 11 Results of granzyme B ELISpot assay using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19.

[0031] Figure 12 AC: Schematic diagram of the three-specific construct of Example 2. Figure 12 A: AB2-1; Figure 12 B: AB2-2; Figure 12 C: AB2-3.

[0032] Figure 13 Results of a retargeted T-cell cytotoxicity assay using Nalm6 target cells expressing human CD-19 and TBM targeting human CD3, human CD2, and human CD19 (Example 2).

[0033] Figure 14 Results of a retargeting T-cell cytotoxicity assay using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and human growth hormone, bispecific constructs targeting human CD3 and human CD19, and TBM targeting human CD3, human CD2, and human CD19 (Example 2). Figure 14 The results obtained using pan-T effector cells from cynomolgus monkeys are shown.

[0034] Figure 15 Results of NFAT activation assays using TBMs targeting human CD3, human CD2, and human CD19 (Example 2).

[0035] Figure 16 AE: Schematic diagram of the three-specific construct of Example 3. Figure 16 A: AB3-1; Figure 16 B: AB3-2; Figure 16 C: AB3-3; Figure 16 D: AB3-4; Figure 16 E: AB3-5.

[0036] Figure 17Results of a retargeted T-cell cytotoxicity assay using Nalm6 target cells expressing human CD-19 and TBM targeting human CD3, human CD2, and human CD19 (Example 3).

[0037] Figure 18 Results of NFAT activation assays using TBMs targeting human CD3, human CD2, and human CD19 (Example 3).

[0038] Figure 19 AC: Schematic diagram of the three-specific construct of Example 4. Figure 19 A: AB4-1; Figure 19 B: AB4-2; Figure 19 C: AB4-3.

[0039] Figure 20 Results of a retargeted T-cell cytotoxicity assay using Nalm6 target cells expressing human CD-19 and TBM targeting human CD3, human CD2, and human CD19 (Example 4).

[0040] Figure 21 Results of NFAT activation assays using TBMs targeting human CD3, human CD2, and human CD19 (Example 4).

[0041] Figure 22 A-22D: Results of cytokine release assays using TBM targeting human CD3, human CD2, and human CD19 (Example 4). Figure 22 A: IL-2; Figure 22 B: TNFα; Figure 22 C: IFNγ; Figure 22 D: Caption

[0042] Figure 23 A-23B: Schematic diagram of the bispecific and trispecific constructs of Example 5. Figure 23 A: Bispecific construct; Figure 23 B: Three-specific constructs.

[0043] Figure 24 Results of T-cell cytotoxicity assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and CD19 or TBMs targeting human CD3, human CD2 and human CD19 (Example 5).

[0044] Figure 25 A-25B: Results of cytokine release assays using Nalm6 target cells expressing human CD-19 and bispecific constructs targeting human CD3 and CD19, as well as TBMs targeting human CD3, human CD2, and human CD19 (Example 5). Figure 25A shows the result of a construct with a CD3 binding arm corresponding to BMA031, and Figure 25 B shows the results for constructs with CD3 binding arms corresponding to OKT3. Results for trispecific constructs are shown in black; results for bispecific constructs are shown in gray.

[0045] Figure 26 A-26B: Schematic diagram of the bispecific and trispecific constructs of Example 6. Figure 26 A: Bispecific construct; Figure 26 B: Three-specific constructs.

[0046] Figure 27 Results of T-cell cytotoxicity assays using HCC1954 target cells expressing human Her2 and bispecific constructs targeting human CD3 and human Her2 or TBMs targeting human CD3, human CD2 and human Her2 (Example 6).

[0047] Figure 28 Results of cytokine release assays using HCC1954 target cells expressing human Her2 and bispecific constructs targeting human CD3 and human Her2, as well as TBMs targeting human CD3, human CD2, and human Her2 or GH (Example 6).

[0048] Figure 29 AB: Schematic diagram of the bispecific and trispecific constructs of Example 7. Figure 29 A: Bispecific construct; Figure 29 B: Three-specific constructs.

[0049] Figure 30 Results of T-cell cytotoxicity assays using OVCAR8 target cells expressing human cortisol and bispecific constructs targeting human CD3 and human cortisol or TBM targeting human CD3, human CD2 and human cortisol (Example 7).

[0050] Figure 31 Results of cytokine release assays using Ovcar8 target cells expressing human cortisol and bispecific constructs targeting human CD3 and human cortisol (MSLN), as well as TBMs targeting human CD3, human CD2, and human cortisol or GH (Example 6).

[0051] Figure 32 : Schematic diagram of the three-specific construct of Example 8. Detailed Implementation

[0052] 6.1. Definition

[0053] As used herein, the following terms are intended to have the following meanings:

[0054] Antigen binding module As used herein, the term "antigen-binding module" or "ABM" refers to the portion of the TBM disclosed herein that has the ability to bind non-covalently, reversibly, and specifically to an antigen. An ABM may be immunoglobulin-based or non-immunoglobulin-based. As used herein, the terms "ABM1" and "CD2 ABM" (etc.) refer to an ABM that specifically binds to CD2, the terms "ABM2" and "TCR ABM" (etc.) refer to an ABM that specifically binds to a component of the TCR complex, and the terms "ABM3" and "TAA ABM" (etc.) refer to an ABM that specifically binds to a tumor-associated antigen. The terms ABM1, ABM2, and ABM3 are used merely for convenience and are not intended to convey any particular conformation of the TBM. In some embodiments, the TCR ABM binds to CD3 (referred to herein as "CD3 ABM," etc.). Therefore, disclosures relating to ABM2 and TCR ABM also apply to CD3 ABM.

[0055] AntibodyAs used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) belonging to the immunoglobulin family that binds to an antigen non-covalently, reversibly, and specifically. For example, a naturally occurring IgG-type "antibody" is a tetramer comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists 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 the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (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 (including, for example, anti-Id antibodies against the antibodies disclosed herein). These antibodies can belong to any isotype / type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0056] Both the light and heavy chains are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, 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, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0057] antibody fragmentsAs used herein, the term "antibody fragment" refers to one or more portions of an antibody. In some embodiments, these portions are part of one or more contact domains of the antibody. In some other embodiments, these portions are antigen-binding fragments (which retain the ability to bind antigens non-covalently, reversibly, and specifically), sometimes referred to herein as "antigen-binding fragments," "their antigen-binding segments," "antigen-binding moieties," etc. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of VH domains (Ward et al., 1989, Nature [Nature] 341: 544-546); and separated complementarity-determining regions (CDRs). Therefore, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv).

[0058] Antibody fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, intracellular antibodies, bisomal antibodies, tripoisome antibodies, tetrasomal antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005 Nature Biotechnology 23: 1126-1136). Antibody fragments can be grafted into peptide-based scaffolds such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin peptide monomers).

[0059] Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1) to form a pair of antigen-binding regions together with complementary light chain peptides (e.g., VL-VC-VL-VC) (Zapata et al., 1995, Protein Eng. 8: 1057-1062; and U.S. Patent No. 5,641,870).

[0060] Antigen-binding domainThe term "antigen-binding domain" refers to a portion of a molecule that has the ability to bind non-covalently, reversibly, and specifically to an antigen. Exemplary antigen-binding domains include antigen-binding fragments and portions of immunoglobulin-based and non-immunoglobulin-based scaffolds that retain the ability to bind non-covalently, reversibly, and specifically to antigens. As used herein, the term "antigen-binding domain" encompasses antibody fragments that retain the ability to bind non-covalently, reversibly, and specifically to antigens.

[0061] Haptop The term "half-antibody" refers to a molecule containing at least one ABM or an ABM chain and capable of associating with another molecule containing ABM or an ABM chain via, for example, disulfide bridging or molecular interactions (e.g., the club-and-mortar interaction between Fc heterodimers). A half-antibody can consist of one polypeptide chain or more than one polypeptide chain (e.g., ...). ， It consists of two polypeptide chains of Fab. In a preferred embodiment, the hapten contains an Fc region.

[0062] Examples of haptens are molecules comprising a heavy chain and a light chain of an antibody (e.g., an IgG antibody). Another example of a hapten is a molecule comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a VL domain and a CL domain, and the 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 ABM. Yet another example of a hapten is a polypeptide comprising an scFv domain, a CH2 domain, and a CH3 domain.

[0063] A half antibody may include more than one ABM, such as a half antibody containing (in order from N-terminus to C-terminus) an scFv domain, a CH2 domain, a CH3 domain, and another scFv domain.

[0064] The hapten may also include an ABM chain, which, when associated with another ABM chain in another hapten, forms a complete ABM.

[0065] Therefore, a TBM can contain one, more typically two, or even more than two halves, and a halves can contain one or more ABMs or one or more ABM chains.

[0066] In some TBMs, the first hapten will associate with the second hapten, for example, through heterodimerization. In other TBMs, the first hapten will be covalently linked to the second hapten, for example, through disulfide bridges or chemical cross-linking. In still other TBMs, the first hapten will associate with the second hapten through covalent attachment and non-covalent interactions, such as disulfide bridges and mortise-and-tenon interactions.

[0067] The term "half-antibody" is intended for descriptive purposes only and does not indicate a specific configuration or method of manufacture. The descriptions of half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., are merely for convenience and descriptive purposes.

[0068] Complementarity Determinant Region As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, three CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3) exist in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, and CDR-L3) exist in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described in the literature by Kabat et al. ,1991, “Sequences of Proteins of Immunological Interest”, 5th edition, National Institutes of Health, Department of Public Health, Bethesda, MD (“Carbart” numbering scheme); Al-Lazikani et al., 1997, JMB 273: 927-948 (“Josiah” numbering scheme) and ImMunoGenTics (IMGT) numbers (Lefranc, 1999, The Immunologist 7: 132-136 (1999); Lefranc et al., 2003, Dev. Comp. Immunol. 27: 55-77 (“IMGT” numbering scheme). For example, for the classical form, according to 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). According to Chothia, the CDR amino acids in VH 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). Combining the CDR definitions of Kabat and Josiah, the CDR consists 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. According to 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 region of the antibody can be determined using the IMGT / DomainGap Align procedure.

[0069] Single-chain Fv or scFvAs used herein, the term "single-chain Fv" or "scFv" refers to an antibody fragment containing the VH and VL domains of an antibody, wherein these domains are contained within 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 the desired structure for antigen binding. For a review of scFv, see Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, (1994) Springer-Verlag, New York, pp. 269–315.

[0070] bisomal antibodies As used herein, the term "bimeric antibody" refers to a small antibody fragment having two antigen-binding sites, typically formed by the pairing of scFv chains. Each scFv contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL, where VH is located at the N-terminus or C-terminus of VL). Unlike typical scFvs (where VH and VL are separated by a linker that allows VH and VL on the same polypeptide chain to pair and form an antigen-binding domain), bimeric antibodies typically contain a linker, but this linker is too short to allow the VH and VL domains on the same chain to pair, thus forcing the VH and VL domains to pair with a complementary domain on another chain, resulting in two antigen-binding sites. More comprehensive descriptions of bisomatic antibodies can be found in the following literature: e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90: 6444-6448.

[0071] FvThe term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin and contains a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) consisting of a heavy chain and a light chain variable domain tightly, non-covalently associated. In this configuration, 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 target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only the three CDRs specific to the target) may have the ability to recognize and bind to the target. The reference to the VH-VL dimer herein is not intended to convey any particular configuration. For example, but not limited to, the VH and VL can combine in any configuration described herein to form a hapten, or they can exist separately on individual haptens and combine together when the individual haptens associate to form an antigen-binding domain, such as the TBM disclosed herein. When present on a single polypeptide chain (e.g., scFv), the VH is the N-terminus or C-terminus of the VL.

[0072] Multispecific binding molecules The term "multispecific binding molecule" refers to a molecule that specifically binds to at least two antigens and contains two or more antigen-binding domains. These antigen-binding domains can each be independently an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived conjugate (e.g., fibronectin, Fynomer, DARPin).

[0073] Trispecific binding molecules The term "triple-specific binding molecule" or "TBM" refers to a molecule that specifically binds to three antigens and comprises three or more antigen-binding domains. The TBMs disclosed herein comprise at least one antigen-binding domain specific to a component of the TCR complex, at least one antigen-binding domain specific to CD2, and at least one antigen-binding domain specific to TAA. These antigen-binding domains may each be independently an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived conjugate (e.g., fibronectin, Fynomer, DARPin). Representative TBMs are shown in... Figure 1 In the middle. TBM can contain one, two, three, four, or even more polypeptide chains. For example, Figure 1 The TBM shown in M ​​contains a single polypeptide chain, which includes three scFvs and a single polypeptide chain linked by an ABM linker. Figure 1 The TBM shown in K contains two polypeptide chains, each containing three scFvs connected by Fc domains, etc. Figure 1The TBM shown in J contains three polypeptide chains forming scFv, ligands, and Fab connected by Fc domains, etc. Figure 1 The TBM shown in C contains four polypeptide chains, forming three Fabs connected by Fc domains and other structures. Figure 1 The TBM shown in T contains 6 polypeptide chains, forming four Fabs and two scFvs connected by Fc domains, etc.

[0074] VH The term "VH" refers to the variable region of the immunoglobulin heavy chain of the antibody (including the heavy chains of Fv, scFv, dsFv, or Fab).

[0075] VL The term "VL" refers to the variable region of an immunoglobulin light chain (including light chains of Fv, scFv, dsFv, or Fab).

[0076] Operable connection 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 linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of TBM disclosed herein, a single ABM (or a chain of ABM) can be linked by a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as a polypeptide chain of TBM disclosed herein, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame. In the context of transcriptional regulation, the term refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, the promoter or enhancer sequence is operably linked to the coding sequence.

[0077] Association The term "association" in the context of TBM 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, for example, through non-covalent association via molecular interactions or through covalent association via one or more disulfide bridges or chemical crosslinks, thereby creating a functional TBM where ABM1, ABM2, and ABM3 can bind to their respective targets. Examples of association that may be present in the TBMs disclosed herein include (but are not limited to) association between Fc regions in the Fc domain (homodimers, or more preferably, heterodimers as described in Section 6.3.1.5), association between VH and VL regions in Fab or Fv, and association between CH1 and CL in Fab.

[0078] ABM ChainA single ABM can exist as a single polypeptide chain (e.g., in the case of scFv) or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab). As used herein, the term "ABM chain" refers to all or part of the ABM present on a single polypeptide chain. The use of the term "ABM chain" is for convenience and descriptive purposes only and does not imply a particular configuration or method of production.

[0079] Host cells or recombinant host cells The term "host cell" or "recombinant host cell" refers, for example, a genetically engineered cell through the introduction of heterologous nucleic acids. It should be understood that this term refers not only to a specific subject cell but also to its offspring. Because certain modifications can occur in offspring due to mutations or environmental influences, such offspring may differ in fact from the parent cells but are still included within the scope of the term "host cell" as used herein. Host cells can transiently carry heterologous nucleic acids, for example, on an extrachromosomal heterologous expression vector, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. For the purposes of this disclosure of TBM, the host cell is preferably a mammalian-derived cell line or a cell line with mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, juvenile rat kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Martha's bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants. Engineered variants include, for example, glycan profile modified and / or site-specific integration site derivatives.

[0080] Sequence identityIn the context of two or more nucleic acid or polypeptide sequences, the term "identity" percentage refers to two or more identical sequences. When compared and aligned on a comparison window (or using one of the following sequence comparison algorithms or a specified region measured by manual calibration and visual inspection) to obtain maximum correspondence, two sequences are "substantially identical" if they have the same specified percentage of amino acid residues or nucleotides (e.g., 60% identity in a specified region, or, when not specified, throughout the entire sequence, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity). Optionally, identity exists in regions of at least about 50 nucleotides (or at least about 10 amino acids in the case of peptides or polypeptides), or more preferably, identity exists in regions of 100 to 500 or 1,000 or more nucleotides (or 20, 50, 200 or more amino acids).

[0081] For sequence comparisons, typically one sequence serves as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. Sequence alignment methods used for comparison are well known in the art. The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1970, Adv. Appl. Math. [Advances in Applied Mathematics] 2: 482c; by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443; by the similarity search method of Pearson and Lipman, 1988, Proc. Nat'l. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85: 2444; and by the computerized implementation of these algorithms (Wisconsin Genetics Software at the GeneticsComputer Group, 575 Science Dr., Madison, WI). GAP, BESTFIT, FASTA, and TFASTA in the Package); or by manual comparison and visual inspection (see, for example, Brent et al. 2003, Current Protocols in Molecular Biology).

[0082] Two examples of algorithms suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST2.0 algorithms, described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402; and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information.

[0083] Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm in Meyers and Miller, 1988, Comput. Appl. Biosci. [Computer Applications in Biological Sciences] 4: 11-17, which is incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm in Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 444-453, which is incorporated into the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0084] Conservative sequence modification The term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the TBM or its components (e.g., ABM or Fc region). Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the TBM disclosed herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having the following attributes: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the TBM disclosed herein can be substituted with other amino acid residues from the same side chain family, and the altered TBM's, for example, binding to target molecules and / or efficient heterodimerization and / or effector function can be tested.

[0085] Mutation or modification As used herein, in the context of polypeptides, the terms “mutation” and “modification” can include the substitution, addition, or deletion of one or more amino acids.

[0086] Antibody numbering systemIn this specification, unless otherwise stated, references to the numbered amino acid residues in the antibody domains are based on the EU numbering system (e.g., in Tables 7B and 7C). This system was originally designed by Edelman et al., 1969, Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 63: 78-85 and described in detail by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0087] dsFv The term "dsFv" refers to a disulfide-stabilized Fv fragment. In a dsFv, VH and VL are linked by interdomain disulfide bonds. To generate such molecules, one amino acid in each of the framework regions of VH and VL is mutated to a cysteine ​​residue, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine ​​residues. See Brinkmann, 2010, Antibody Engineering 181-189, DOI: 10.1007 / 978-3-642-01147-4_14. The term dsFv encompasses both dsFv (molecules in which VH and VL are linked by interchain disulfide bonds rather than linker peptides) and scdsFv (molecules in which VH and VL are linked by both linker and interchain disulfide bonds) known in the art.

[0088] cascading of VH domains As used herein, the term "tandem of VH domains (or VHs)" refers to a string of VH domains consisting of many identical VH domains of an antibody. The C-terminus of each of the VH domains (except the last one at the end of the tandem) is linked (with or without a adapter) to the N-terminus of another VH domain. The tandem has at least two VH domains, and in specific embodiments of the TBM disclosed herein, it has 3, 4, 5, 6, 7, 8, 9, or 10 VH domains. Tandems of VHs can be generated by using a recombination method (with or without an adapter) to link the coding nucleic acids of each VH domain in a desired order (this ensures that the nucleic acids are prepared as a single polypeptide chain) (e.g., as described in Section 6.3.3). The N-terminus of the first VH domain in the tandem is defined as the N-terminus of the tandem, and the C-terminus of the last VH domain in the tandem is defined as the C-terminus of the tandem.

[0089] Serialization of VL domains As used herein, the term "tandem of VL domains (or VLs)" refers to a string of VL domains consisting of many identical VL domains of an antibody. The C-terminus of each VL domain (except the last one at the end of the tandem) is connected (with or without a adapter) to the N-terminus of another VL. The tandem has at least two VL domains, and in specific embodiments of the TBM disclosed herein, it has 3, 4, 5, 6, 7, 8, 9, or 10 VL domains. Tandems of VLs can be generated by using a recombination method (with or without an adapter) to link the coding nucleic acids of each VL domain in a desired order (this ensures that the nucleic acids are prepared as a single polypeptide chain) (e.g., as described in Section 6.3.3). The N-terminus of the first VL domain in the tandem is defined as the N-terminus of the tandem, and the C-terminus of the last VL domain in the tandem is defined as the C-terminus of the tandem.

[0090] unit price In the context of antigen-binding molecules, the term "monovalent" as used herein refers to an antigen-binding molecule having a single antigen-binding domain.

[0091] Bivalent In the context of antigen-binding molecules, the term "bivalent" as used herein refers to an antigen-binding molecule having two antigen-binding domains. These domains may be the same or different. Therefore, a bivalent antigen-binding molecule can be monospecific or bispecific.

[0092] Trivalent In the context of antigen-binding molecules (e.g., TBMs), the term "trivalent" as used herein refers to an antigen-binding molecule having three antigen-binding domains. The TBMs disclosed herein are trispecific and specifically bind to CD2, components of the TCR complex, and TAA. Therefore, the trivalent TBMs disclosed herein have at least three antigen-binding domains, each binding to a different antigen. Examples of trivalent TBMs disclosed herein are schematically illustrated in… Figure 1 B-1U.

[0093] Quadrivalent In the context of antigen-binding molecules (e.g., TBMs), the term "tetravalent" as used herein refers to an antigen-binding molecule having four antigen-binding domains. The TBMs disclosed herein are trispecific and specifically bind to CD2, components of the TCR complex, and TAA. Therefore, the tetravalent TBMs disclosed herein typically have two antigen-binding domains binding to the same antigen (preferably, TAA) and two antigen-binding domains each binding to a separate antigen (preferably, components of the CD2 and TCR complexes). Examples of tetravalent TBMs disclosed herein are schematically illustrated in… Figure 1 In P-1R.

[0094] Pentavalent In the context of antigen-binding molecules (e.g., TBMs), the term "pentavalent" as used herein refers to an antigen-binding molecule having five antigen-binding domains. The TBMs disclosed herein are trispecific and specifically bind to components of the CD2, TCR complex, and TAA. Therefore, the pentavalent TBMs disclosed herein typically have (a) two pairs of antigen-binding domains, each binding to the same antigen, and a single antigen-binding domain binding to a third antigen, or (b) three antigen-binding domains binding to the same antigen, and two antigen-binding domains each binding to a separate antigen. Examples of pentavalent TBMs disclosed herein are schematically illustrated in… Figure 1 S in.

[0095] hexavalent In the context of antigen-binding molecules (e.g., TBMs), the term "hexavalent" as used herein refers to an antigen-binding molecule having six antigen-binding domains. The TBMs disclosed herein are trispecific and specifically bind to CD2, a component of the TCR complex, and a TAA. Although different configurations (e.g., three antigen-binding domains bound to a TAA, two antigen-binding domains bound to a component of the TCR complex, and one antigen-binding domain bound to CD2, or three antigen-binding domains bound to a TAA, two antigen-binding domains bound to CD2, and one antigen-binding domain bound to a component of the TCR complex) are within the scope of this disclosure, the hexavalent TBMs of this disclosure generally have three pairs of antigen-binding domains, each binding to the same antigen. Examples of hexavalent TBMs of this disclosure are schematically illustrated in… Figure 1 T-1U.

[0096] 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 biological products. This binding reaction may, but does not need to, be mediated by an antibody or antibody fragment, but may also be mediated by any type of ABM (such as ligands, DARPin, etc.) as described in Section 6.2. The ABMs disclosed herein typically also have a size of less than 5 x 10⁻⁶. -2 M, less than 10 -2 M, less than 5 x 10 -3 M, less than 10 -3 M, less than 5 x 10 -4 M, less than 10 -4 M, less than 5 x 10 -5 M, less than 10 -5 M, less than 5 x 10 -6 M, less than 10 -6 M, less than 5 x 10 -7 M, less than 10 -7 M, less than 5 x 10 -8 M, less than 10 -8 M, less than 5 x 10 -9 M, or less than 10 -9 M has a dissociation rate constant (KD) (koff / kon) and binds to the target antigen with an affinity at least twice that of binding to a nonspecific antigen (e.g., HSA). The term "specific binding" does not exclude cross-species reactivity. For example, an antigen-binding module (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species can also "specifically bind" to said antigens in one or more other species. Therefore, such cross-species reactivity does not itself change the classification of the antigen-binding module as a "specific" binder. In some embodiments, antigen-binding modules of this disclosure that specifically bind to human antigens (e.g., ABM1, ABM2, and / or ABM3) bind to one or more non-human mammal species, such as primate species (including but not limited to cynomolgus monkeys (M)). acaca fascicularis ), common macaque ( Macaca mulatta ), and pig-tailed monkeys ( Macaca nemestrina One or more of the following) or rodent species (e.g., mice) Mus musculus The antigen-binding modules disclosed herein (e.g., ABM1, ABM2, and / or ABM3) are not cross-species reactive.

[0097] Monoclonal antibodies As used herein, the term "monoclonal antibody" refers to polypeptides derived from the same genetic source, including antibodies, antibody fragments, molecules (including TBM), etc.

[0098] HumanizationThe term "humanized" form of nonhuman (e.g., mouse) antibody refers to a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from a hypervariable region (donor antibody) of a nonhuman species (such as a mouse, rat, rabbit, or nonhuman primate) with the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all of the following: at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of nonhuman immunoglobulins, and all or substantially all FRs are those of the human immunoglobulin lo sequence. Optionally, a humanized antibody also contains an immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596. See also the following commentaries and their cited references: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. 1: 105-115; Harris, 1995, Biochem. Soc. Transactions 23: 1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. 5: 428-433.

[0099] Human antibodiesAs used herein, the term "human antibody" includes antibodies having variable regions, wherein both the frame region and the CDR region are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from such human sequences, such as germline human sequences or mutant forms of germline human sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, as described, for example, in the following reference: Knappik et al., 2000, J Mol Biol [Journal of Molecular Biology] 296, 57-86. The structure and location of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes (e.g., Kabat numbering scheme, Josiah numbering scheme, or a combination of Kabat and Josiah numbering schemes) (see, for example, Lazikani et al., 1997, J. Mol. Bio. [Journal of Molecular Biology] 273: 927 948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia et al., 1987, J. Mol. Biol. [Journal of Molecular Biology] 196: 901-917; Chothia et al., 1989, Nature [Nature] 342: 877-883).

[0100] Human antibodies may include amino acid residues not encoded by human sequences (e.g., by introducing mutations to promote stability or production through random or site-specific mutagenesis in vitro, or through somatic mutations or conserved substitutions in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., a mouse) lineage has been transplanted into a human frame sequence.

[0101] chimeric antibodiesThe term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or a portion thereof is altered, replaced, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced, or replaced with a variable region having a different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen-recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0102] Effector Function The term "effective function" refers to the activity of an antibody molecule mediated by binding through the antibody's domain rather than the antigen-binding domain, typically mediated by the binding of effector molecules. Effector functions include complement-mediated effector functions, mediated by, for example, the binding of the C1 component of the complement to the antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and can participate in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the antibody's constant domain to the Fc receptor (FcR). The binding of antibodies to Fc receptors on cell surfaces 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 cytotoxic cells (referred to as 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 altered, for example, by increasing or decreasing the antibody's affinity for effector molecules such as Fc receptors or complement components. Binding affinity is typically altered by modifying the binding site of the effector molecule, and in this case, it is appropriate to target the site of interest and modify at least a portion of the site in a suitable manner. It is also envisioned that altering the binding site on an antibody against an effector molecule does not need to significantly change the overall binding affinity, but can alter the geometry of the interaction, resulting in the disabling of the effector mechanism, as in nonproductive binding. Further envisioning is that effector function can also be altered by modifying sites that do not directly participate in effector molecule binding but otherwise participate in the performance of effector function.

[0103] Identification As used herein, the term “identification” refers to the discovery of and interaction (e.g., binding) with an epitope of an ABM.

[0104] Epitope An epitope, or antigenic determinant, is a portion of an antigen that can be recognized by an antibody or other antigen-binding motif as described herein. Epitopes can be linear or conformational.

[0105] Nucleic acid The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0106] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al., (1985) J. Biol. Chem. 260: 2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98).

[0107] carrierThe term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, this disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have the same function.

[0108] Combining sequences Referring to Tables 7, 8, 9, 11, 12 or 13 (including their subsections), the term "combination sequence" means an ABM having a complete set of CDR, VH-VL pairs or scFv listed in the table.

[0109] VH-VL or VH-VL to When referring to VH-VL pairs, whether located on the same or different polypeptide chains, the terms "VH-VL" and "VH-VL pair" are used for convenience and are not intended to convey any particular orientation unless otherwise specified in the context. Therefore, an scFv containing "VH-VL" or "VH-VL pair" may have VH and VL domains in any orientation, for example, VH at the N-terminus of VL or VL at the N-terminus of VH.

[0110] polypeptides and proteins The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The phrases also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, and to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular peptide sequence also implicitly encompasses its conserved variants.

[0111] SubjectsThe term "subject" as used herein includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0112] cancer The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but 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, bile duct cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., such as any TAA-positive cancer of any of the aforementioned types.

[0113] tumor The term "tumor" may be used interchangeably with the term "cancer" as used herein, for example, both terms cover solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include both pre-existing and malignant cancers and tumors.

[0114] Tumor-associated antigens The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed fully or as a fragment (e.g., MHC / peptide) on the surface of cancer cells, and it can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, a TAA is a cell surface molecule overexpressed in cancer cells compared to normal cells, for example, 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, a TAA is a cell surface molecule inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a TAA will be expressed fully or as a fragment (e.g., MHC / peptide) only on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses antigens specific to cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSA").

[0115] Treatment (Treat, Treatment and Treating) As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder, or an improvement in one or more symptoms (preferably one or more identifiable symptoms) of the proliferative disorder resulting from the application of one or more of the TBMs disclosed herein. In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to suppressing the progression of the proliferative disorder physically, for example, by stabilizing identifiable symptoms, or physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to reducing or stabilizing tumor size or cancer cell count.

[0116] 6.2. Antigen binding module

[0117] Typically, one or more ABMs of the TBM disclosed herein contain sequences of immunoglobulin-based antigen-binding domains, such as antibody fragments or derivatives. These antibody fragments and derivatives typically include the CDR of an antibody and may include larger fragments and their derivatives, such as Fab, scFab, Fv, and scFv.

[0118] 6.2.1. Immunoglobulin-based modules

[0119] 6.2.1.1.Fab

[0120] In some respects, the ABM disclosed herein is a Fab domain. The Fab domain can be generated by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain, or through recombinant expression. The Fab domain typically includes a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain.

[0121] In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. The disulfide bond between the two constant domains further stabilizes the Fab domain.

[0122] For the TBM disclosed herein, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same ABM and minimize anomalous pairings of Fab domains belonging to different ABMs. For example, the Fab heterodimerization strategies shown in Table 1 below can be used:

[0123]

[0124]

[0125] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0126] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain and into the CL domain of the Fab, and / or into the VH domain and into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interactions, causing Fab components to preferentially pair with each other rather than with components of other Fabs.

[0127] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues with variable (VH, VL) and constant (CH1, CL) domains, 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, Josiah, and IMGT numbering schemes.

[0128] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. The complementarity of the heavy-chain and light-chain interfaces can be achieved based on spatial and hydrophobic contacts, electrostatic / charge interactions, or a combination of these interactions. Complementarity between protein surfaces is extensively described in the literature using terms such as lock-and-key fit, pestle-and-mortar structure, protrusions and cavities, donor and recipient, all of which imply the nature of the structural and chemical matching between two contacting surfaces.

[0129] In one embodiment, the one or more introduced modifications introduce novel hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more introduced modifications introduce novel salt bridges across the interface of the Fab component. Exemplary alternatives are provided in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are hereby incorporated by reference.

[0130] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, wherein a salt bridge is introduced between the CH1 and CL domains (see Golay et al., 2016, JImmunol 196: 3199-211).

[0131] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see Golay et al., 2016, J Immunol 196: 3199-211).

[0132] In some embodiments, the Fab domains may contain modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces, which facilitate proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0133] The Fab domain can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the pairing efficiency of Fab components. For example, engineered disulfide bonds can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see Mazor et al., 2015, MAbs 7: 377-89).

[0134] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7: 364-76, described replacing the CH1 domain with the constant domain of the α T cell receptor and the CL domain with the β domain of the T cell receptor, and further replacing these domains with additional charge-charge interactions between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0135] The ABM disclosed herein may comprise a single-chain Fab fragment, which is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker. In some embodiments, the antibody domains and linker have one of the following sequences in the direction from the N-terminus to the C-terminus: 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 may be a polypeptide having at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab domain is stabilized by a native disulfide bond between the CL domain and the CH1 domain.

[0136] In the embodiments, the antibody domain and adapter in the single-chain Fab fragment have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CH1-adaptor-VL-CL, or b) VL-CL-adaptor-VH-CH1, more preferably VL-CL-adaptor-VH-CH1.

[0137] In another embodiment, the antibody domain and adapter in the single-chain Fab fragment have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-CL-adaptor-VL-CH1 or b) VL-CH1-adaptor-VH-CL.

[0138] Optionally, in the single-chain Fab fragment, in addition to the native disulfide bond between the CL and CH1 domains, the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) are also stabilized by disulfide bonds introduced between: i) heavy chain variable domain position 44 and light chain variable domain position 100, ii) heavy chain variable domain position 105 and light chain variable domain position 43, or iii) heavy chain variable domain position 101 and light chain variable domain position 100 (according to the Cabart EU index numbering).

[0139] Such further disulfide bond stabilization of single-chain Fab fragments is achieved by introducing disulfide bonds between the variable domains VH and VL of the single-chain Fab fragment. Techniques for introducing non-natural disulfide bridges to stabilize single-chain Fv are described in, for example, WO 94 / 029350, Rajagopal et al., 1997, Prot. Engin. [Protein Engineering] 10: 1453-59; Kobayashi et al., 1998, Nuclear Medicine & Biology, 25: 387-393; and Schmidt et al., 1999, Oncogene [Oncogenes] 18: 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the single-chain Fab fragment is located between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, optional disulfide bonds between the variable domains of a single-chain Fab fragment are located between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (according to the Cabart EU index number).

[0140] 6.2.1.2.scFv

[0141] A single-chain Fv or “scFv” antibody fragment contains the VH and VL domains of an antibody within a single polypeptide chain, is expressible as a single-chain polypeptide, and retains the specificity of the complete antibody from which it is derived. Typically, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of scFV are the ABM linkers identified in Section 6.3.3, such as any linker designated L1 through L54.

[0142] Unless otherwise stated, as used herein, the scFv may have VL and VH variable regions in any order relative to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-connector-VH or may contain VH-connector-VL.

[0143] To generate scFv-encoded nucleic acids, the VH and VL-encoded DNA fragments are operatively ligated to another fragment encoding a linker, such as any ABM linker described in Section 6.3.3 (e.g., an amino acid sequence (Gly Ser)3 (SEQ ID NO:1) enables the VH and VL sequences to be expressed as a continuous single-chain protein (with its VL and VH regions connected by a flexible linker) (see, for example, Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 1990, Nature 348: 552-554).

[0144] 6.2.1.3. Other Immunoglobulin-Based Modules

[0145] The TBM disclosed herein may also include ABM in the form of an immunoglobulin, such as Fv, dsFv, (Fab')2, single-domain antibody (SDAB), VH or VL domain, or camel VHH domain (also referred to as nanobody) in addition to Fab or scFv.

[0146] ABMs can be single-domain antibodies consisting of a single VH or VL domain, which exhibits sufficient affinity for the target. In a specific embodiment, the single-domain antibody is a camel VHH domain (see, for example, Riechmann, 1999, Journal of Immunological Methods 231: 25-38; WO 94 / 04678).

[0147] 6.2.2. Non-immunoglobulin-based modules

[0148] In some embodiments, one or more of the ABMs disclosed herein are derived from non-antibody scaffold proteins (including, but not limited to, designed ankyrin repeat protein (DARPin), Avimer (abbreviation for affinity multimer), anticarrier protein / lipocarrier protein, Centyrin, Kunitz domain, Adnexin, Affilin, Affitin (also known as Nonfitin), Knottin, Pronectin, Versabody, Duocalin, and Fynomer), ligands, receptors, cytokines, or chemokines).

[0149] Non-immunoglobulin scaffolds that can be used for TBM disclosed in this paper include those listed below: Mintz and Crea, 2013, Bioprocess International, 11(2): 40-48 in Tables 3 and 4; Vazquez-Lombardi et al., 2015, Drug Discovery Today, 20(10): 1271-83. Figure 1 Table 1 and Figure 1; Skrlec et al., 2015, Trends in Biotechnology, 33(7): 408-18, Table 1 and Column 2. Mintz and Crea, 2013, Bioprocess International, 11(2): 40-48, Tables 3 and 4. Vazquez-Lombardi et al., 2015, Drug DiscoveryToday, 20(10): 1271-83. Figure 1Table 1 and Figure 1; the contents of Table 1 and column 2 of Skrlec et al., 2015, Trendsin Biotechnology [Biotechnology Trends] 33(7): 408-18 (collectively, the “Scaffold Disclosures”) are incorporated herein by reference. In specific embodiments, the disclosures pertain to scaffold disclosures of Adnexin are incorporated by reference. In another embodiment, the disclosures pertain to scaffold disclosures of Avimer are incorporated by reference. In another embodiment, the disclosures pertain to scaffold disclosures of Affibody are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of anticarrier proteins are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of DARPin are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of Kunitz domains are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of Knottin are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of Pronectin are incorporated by reference. In yet another embodiment, the disclosures pertain to scaffold disclosures of Nanofitin are incorporated by reference. In yet another embodiment, disclosures relating to the Affilin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Adnectin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the ABD scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Adhiron scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Affimer scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Alphabody scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Armadillo Repeat Protein scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Atrimer / Tetranectin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Obody / OB-fold scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Centyrin scaffold are incorporated by reference. In yet another embodiment, disclosures relating to the Repebody scaffold are incorporated by reference. In yet another embodiment, the disclosure relating to an anticarrier protein scaffold is incorporated by reference. In yet another embodiment, the disclosure relating to an Atrimer scaffold is incorporated by reference. In yet another embodiment, the disclosure relating to a bicyclic peptide scaffold is incorporated by reference.In yet another embodiment, disclosures relating to cys-knot stents are incorporated by reference. In yet another embodiment, disclosures relating to Fn3 stents (including adenonectin, Centryrin, Pronectin, and Tn3) are incorporated by reference.

[0150] In embodiments, the ABM disclosed herein may be a designed anchor protein repeat sequence protein (“DARPin”). DARPin is an antibody mimic protein that typically exhibits high specificity and high affinity for target protein binding. They are generally genetically engineered and derived from natural anchor proteins and consist of at least three, typically four, or five repeat motifs from these proteins. For tetra- or penta-repeating DARPin, their molecular weights are approximately 14 or 18 kDa (kilodaltons), respectively. Examples of DARPin can be found, for example, in U.S. Patent No. 7,417,130. Multispecific binding molecules comprising a DARPin binding module and an immunoglobulin-based binding module are disclosed, for example, in U.S. Publication No. 2015 / 0030596 A1.

[0151] In another embodiment, the ABM disclosed herein may be an Affibody. An Affibody is well known in the art and refers to an affinity protein based on a 58-amino acid residue protein domain derived from an IgG binding domain of staphylococcal protein A.

[0152] In another embodiment, the ABM disclosed herein may be an anticarrier protein. Anticarrier proteins are well known in the art and refer to another antibody mimicry technique, wherein the binding specificity is derived from a lipid carrier protein. Anticarrier proteins may also be formatted as dual-targeting proteins, referred to as Duocalin.

[0153] In another embodiment, the ABM disclosed herein may be a Versabody. Versabody is well known in the art and refers to another antibody mimicry technology. They are small proteins of 3-5 kDa with >15% cysteine, forming a high disulfide bond density scaffold that replaces the hydrophobic core typically found in proteins.

[0154] Other non-immunoglobulin ABMs include “A” domain oligomers (also known as Avimers) (see, for example, U.S. Patent Application Publications 2005 / 0164301, 2005 / 0048512, and 2004 / 017576), Fn3-based protein scaffolds (see, for example, U.S. Patent Application Publication 2003 / 0170753), VASP peptides, avian pancreatic polypeptides (aPP), tetraconnectins (CTLD3-based), affililin (γB-based crystal protein / ubiquitin), knotting proteins, SH3 domains, PDZ domains, tendamistat, neocarzinostatin, protein A domains, lipid transport proteins, transferrin, and Kunitz domains. In one aspect, the ABMs used to construct the TBMs disclosed herein comprise fibronectin-based scaffolds as shown in WO 2011 / 130324.

[0155] Furthermore, in some respects, ABMs contain a ligand-binding domain of the receptor or a receptor-binding domain of the ligand. For example, if TAA is an EGF receptor, then ABM3 may contain a portion of EGF that binds EGFR, and if TAA is a PDGF receptor, then ABM3 may contain a portion of PDGF that binds PDGF, and so on. In a particular embodiment, ABM1 is a CD2 ligand, particularly the CD58 portion as described in Section 6.6.2. The respective binding domains of many ligand / receptor pairs are well known in the art and can therefore be readily selected and applied to the TBMs disclosed herein.

[0156] 6.3. Connector

[0157] It is anticipated that the TBM disclosed herein may, in some cases, comprise directly interconnected pairs of ABMs or ABM chains (e.g., VH-CH1 or VL-CL components of Fab), for example, as a fusion protein without a linker. More preferably, the TBM disclosed herein comprises a linker portion connecting individual ABMs or ABM chains. The use of a linker portion can improve target binding, for example, by increasing the flexibility of the ABMs in the TBM and thus reducing steric hindrance. ABMs can be interconnected via, for example, Fc domains (each Fc domain representing a pair of associated Fc regions) and / or ABM linkers. The use of Fc domains will typically require the use of hinge regions as linkers of ABMs or ABM chains for optimal antigen binding. Therefore, the term "linker" encompasses, but is not limited to, Fc regions, Fc domains, hinge regions, and

[0158] Examples of Fc structural domains (formed by pairing two Fc regions), hinge regions, and ABM joints are described in Sections 6.3.1, 6.3.2, and 6.3.3, respectively.

[0159] 6.3.1. Fc structural domain

[0160] The TBM disclosed herein may include an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0161] The Fc domain can be derived from any suitable type of antibody, 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.

[0162] The Fc domain comprises two polypeptide chains, each referred to as a heavy chain Fc region. These two heavy chain Fc regions dimerize to generate the Fc domain. These two Fc regions within the Fc domain may be identical or different from each other. In natural antibodies, the Fc regions are typically identical, but for the purpose of generating multispecific binding molecules, such as TBM disclosed herein, the Fc regions may advantageously be different to allow heterodimerization, as described in Section 6.3.1.5 below.

[0163] Typically, each heavy chain Fc region contains or consists of two or three heavy chain constant structural domains.

[0164] In natural antibodies, the heavy chain Fc region of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the Fc region of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These antibodies dimerize to produce the Fc domain.

[0165] In this disclosure, the heavy chain Fc region may contain heavy chain constant domains from one or more different types of antibodies (e.g., one, two, or three different types).

[0166] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG1.

[0167] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG2.

[0168] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG3.

[0169] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG4.

[0170] In one embodiment, the heavy chain Fc region contains a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0171] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0172] It will be appreciated that the heavy chain constant domain used to generate the heavy chain Fc region of the TBM of this disclosure may include variants of the naturally occurring constant domain as described above. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the heavy chain Fc region of this disclosure contains at least one constant domain that differs sequentially from the wild-type constant domain. It will be appreciated 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 instance, the constant domain is at least 70% identical or similar. In another instance, the constant domain is at least 80% identical or similar. In another instance, the constant domain is at least 90% identical or similar. In yet another instance, the constant domain is at least 95% identical or similar. Exemplary Fc variants are described in sections 6.3.1.1 through 6.3.1.5 below.

[0173] IgM and IgA are naturally occurring covalent polymers of common H2L2 antibody units in humans. IgM exists as a pentamer when a J-chain is incorporated, or as a hexamer when a J-chain is absent. IgA exists as both a monomer and a dimer. The heavy chains of IgM and IgA have an 18-amino acid extension to a constant C-terminal domain, referred to as a tailpiece. The tailpiece includes cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and is believed to play an important role in polymerization. The tailpiece also contains glycosylation sites. In some embodiments, the TBM disclosed herein does not contain a tailpiece.

[0174] The Fc domain incorporated into the TBM disclosed herein may contain one or more modifications that alter the functional properties of the protein (e.g., binding to Fc receptors, such as FcRn or leukocyte receptors (e.g., as described in Section 6.3.1.1), binding to complement (e.g., as described in Section 6.3.1.2), modified disulfide bond structures (e.g., as described in Section 6.3.1.3), or altered glycosylation patterns (e.g., as described in Section 6.3.1.4)). The Fc domain may also be modified to include modifications that improve the manufacturability of asymmetric TBMs, for example by allowing heterodimerization (where heterodimerization is the preferential pairing of different Fc regions relative to the same Fc region). Heterodimerization allows for the generation of TBMs in which different ABMs are interconnected through Fc domains containing Fc regions with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.3.1.5 (and its subsections).

[0175] It will be appreciated that any of the modifications described in Sections 6.3.1.1 to 6.3.1.5 may be combined in any suitable manner to achieve the desired functional characteristics and / or any of the modifications described in Sections 6.3.1.1 to 6.3.1.5 may be combined with other modifications to change the characteristics of the TBM.

[0176] 6.3.1.1. Fc domains with modified FcR binding

[0177] Compared to the corresponding innate immunoglobulin, the Fc domain of the disclosed TBM can exhibit altered binding to one or more Fc receptors (FcRs). Binding to any specific Fc receptor can be increased or decreased. In one embodiment, the Fc domain contains one or more modifications that alter its Fc-receptor binding profile.

[0178] Human cells can express a wide range of membrane-bound FcRs, selected from FcαR, FcεR, FcγR, FcRn, and glycan receptors. Some cells are also capable of expressing soluble (extracellular domain) FcRs (Fridman et al., 1993, J Leukocyte Biology 54: 504-512 review). FcγRs can be further categorized by IgG binding affinity (high / low) and biological function (activation / inhibition). Human FcγRI is widely considered the only “high affinity” receptor, while all others are considered intermediate to low affinity. FcγRIIb is the only receptor with “inhibitory” function due to its intracellular ITIM motif, while all others are considered “activating” due to the ITIM motif or pairing with the common FcγR-γ chain. FcγRIIIb is also unique in that, although activated, it associates with the cell via a GPI anchor. In summary, humans express six "standard" FcγRs: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In addition to these sequences, numerous other sequences or allotypes are scattered throughout these families. Some of these sequences have been found to have important functional consequences and are therefore sometimes considered their own receptor subtypes. Examples include FcγRIIa. H134R 、FcγRIIb I190T 、FcγRIIIa F158V and FcγRIIIb NA1 、FcγRIIIb NA2 FcγRIII SH Each receptor sequence has been shown to have different affinities for the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 (Bruhns, 1993, Blood 113: 3716-3725). Other species have slightly different numbers and functions of FcγRs, among which the mouse system is currently the most well-studied and consists of 4 FcγRs, FcγRI, FcγRIIb, FcγRIII, and FcγRIV (Bruhns, 2012, Blood 119: 5640-5649). Due to the high affinity of human FcγRIs on cells for IgG1 / IgG3 / IgG4 (approximately 10...), the affinity of human FcγRIs for IgG1 / IgG3 / IgG4 is significantly higher. -8 Given the concentrations of these IgGs in serum (approximately 10 mg / ml), human FcγRIs on cells are generally considered to be "occupied" by monomeric IgGs under normal serum conditions. Therefore, cells carrying FcγRIs on their surfaces are thought to be able to alternatively "screen" or "sample" their antigenic environment by binding multispecific IgGs. Other receptors with lower affinity for IgG subclasses (at approximately 10 mg / ml)-5 -10 -7 Within the range of M), it is generally considered "unoccupied". Low-affinity receptors are therefore intrinsically sensitive to the detection of and activation by antibody-mediated immune complexes. The increased Fc density in antibody-mediated immune complexes results in increased functional affinity for binding affinity with low-affinity FcγR. This has been demonstrated in vitro using numerous methods (Shields et al., 2001, J Biol Chem 276(9): 6591-6604; Lux et al., 2013, J Immunol 190: 4315-4323). This is also considered one of the main mechanisms of action for the treatment of human ITP with anti-RhD (Crow, 2008, Transfusion Medicine Reviews 22:103-116).

[0179] Many cell types express multiple types of FcγR, and therefore, depending on the biological context, the binding of IgG or antibody immune complexes to FcγR-carrying cells can have a variety of complex outcomes. At its simplest, cells can receive activating, inhibitory, or mixed signals. This can lead to events such as phagocytosis (e.g., macrophages and neutrophils), antigen processing (e.g., dendritic cells), reduced IgG production (e.g., B cells), or degranulation (e.g., neutrophils, mast cells). Data support the conclusion that inhibitory signals from FcγRIIb can dominate activating signals (Proulx, 2010, Clinical Immunology 135: 422-429).

[0180] FcRn plays a crucial role in maintaining the long half-life of IgG in the serum of adults and children. The receptor binds to IgG in acidified vesicles (pH < 6.5), protecting IgG molecules from degradation, and then releases them into the bloodstream at higher pH levels (7.4).

[0181] FcRn differs from the leukocyte Fc receptor and, conversely, exhibits structural similarity to MHC class I molecules. A heterodimer composed of β2-microglobulin chains is non-covalently attached to a membrane-bound chain comprising three extracellular domains. One of these domains (including the carbohydrate chain) interacts with the site between the CH2 and CH3 domains of Fc, along with β2-microglobulin. This interaction involves a salt bridge constructed targeting histidine residues on IgG, which are positively charged at pH < 6.5. At higher pH, these histidine residues lose their positive charge, the FcRn-IgG interaction is weakened, and IgG dissociates.

[0182] In one embodiment, the TBM disclosed herein includes an Fc domain that is incorporated into a human FcRn.

[0183] In one embodiment, the Fc domain has (e.g., one or two) Fc regions containing a histidine residue at position 310, and preferably also containing a histidine residue at position 435. These histidine residues are important for human FcRn binding. In one embodiment, the histidine residues at positions 310 and 435 are native residues, i.e., positions 310 and 435 are unmodified. Alternatively, one or both of these histidine residues may be present due to modification.

[0184] The TBM disclosed herein may include one or more Fc regions that alter the binding of Fc to FcRn. The altered binding may be an enhanced or a degraded binding.

[0185] In one embodiment, the TBM includes an Fc domain, wherein at least one (and optionally both) Fc region contains one or more modifications such that it binds to FcRn with higher affinity and affinity compared to the corresponding natural immunoglobulin.

[0186] In one embodiment, the Fc region is modified by replacing the threonine residue at position 250 with a glutamine residue (T250Q).

[0187] In one embodiment, the Fc region is modified by replacing the methionine residue at position 252 with a tyrosine residue (M252Y).

[0188] In one embodiment, the Fc region is modified by replacing the serine residue at position 254 with a threonine residue (S254T).

[0189] In one embodiment, the Fc region is modified by replacing the threonine residue at position 256 with a glutamic acid residue (T256E).

[0190] In one embodiment, the Fc region is modified by replacing the threonine residue at position 307 (T307A) with an alanine residue.

[0191] In one embodiment, the Fc region is modified by replacing the threonine residue at position 307 with a proline residue (T307P).

[0192] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a cysteine ​​residue (V308C).

[0193] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a phenylalanine residue (V308F).

[0194] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a proline residue (V308P).

[0195] In one embodiment, the Fc region is modified by replacing the glutamine residue at position 311 with an alanine residue (Q311A).

[0196] In one embodiment, the Fc region is modified by replacing the glutamine residue at position 311 with an arginine residue (Q311R).

[0197] In one embodiment, the Fc region is modified by replacing the methionine residue at position 428 (M428L) with a leucine residue.

[0198] In one embodiment, the Fc region is modified by replacing the histidine residue at position 433 (H433K) with a lysine residue.

[0199] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 434 (N434F) with a phenylalanine residue.

[0200] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 434 (N434Y) with a tyrosine residue.

[0201] In one embodiment, the Fc region is modified by replacing the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, and the threonine residue at position 256 with a glutamic acid residue (M252Y / S254T / T256E).

[0202] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a proline residue and the asparagine residue at position 434 with a tyrosine residue (V308P / N434Y).

[0203] In one embodiment, the Fc region is modified by replacing the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, the threonine residue at position 256 with a glutamic acid residue, the histidine residue at position 433 with a lysine residue, and the asparagine residue at position 434 with a phenylalanine residue (M252Y / S254T / T256E / H433K / N434F).

[0204] It will be recognized that any of the modifications listed above can be combined to alter the binding of FcRn.

[0205] In one embodiment, the TBM includes an Fc domain, one or both of which contain one or more modifications such that the Fc domain binds to the FcRn with lower affinity and cohesion than the corresponding natural immunoglobulin.

[0206] In one embodiment, the Fc region contains any amino acid residues except for histidine at position 310 and / or position 435.

[0207] The TBM disclosed herein may contain an Fc domain, one or both of which contain one or more modifications that enhance their binding to FcγRIIb. FcγRIIb is the only inhibitory receptor in humans and the only Fc receptor found on B cells.

[0208] In one embodiment, the Fc region is modified by replacing the proline residue at position 238 with an aspartic acid residue (P238D).

[0209] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 258 (E258A) with an alanine residue.

[0210] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 (S267A) with an alanine residue.

[0211] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 with a glutamic acid residue (S267E).

[0212] In one embodiment, the Fc region is modified by replacing the leucine residue at position 328 with a phenylalanine residue (L328F).

[0213] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 258 with an alanine residue and the serine residue at position 267 with an alanine residue (E258A / S267A).

[0214] In one embodiment, the Fc region is modified by replacing the serine residue at position 267 with a glutamic acid residue and the leucine residue at position 328 with a phenylalanine residue (S267E / L328F).

[0215] It will be recognized that any of the modifications listed above can be combined to improve FcγRIIb binding.

[0216] In one embodiment of this disclosure, a TBM comprising an Fc domain is provided, the Fc domain exhibiting a binding with FcγR reduction.

[0217] In one embodiment, the TBM includes Fc domains, one or both of which contain one or more modifications that reduce Fc binding with FcγR.

[0218] The Fc domain can be derived from IgG1.

[0219] In one embodiment, the Fc region is modified by replacing the leucine residue (L234A) at position 234 with an alanine residue.

[0220] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 (L235A) with an alanine residue.

[0221] In one embodiment, the Fc region is modified by replacing the glycine residue at position 236 with an arginine residue (G236R).

[0222] In one embodiment, the Fc region is modified by replacing the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q).

[0223] In one embodiment, the Fc region is modified by replacing the serine residue at position 298 with an alanine residue (S298A).

[0224] In one embodiment, the Fc region is modified by replacing the leucine residue at position 328 with an arginine residue (L328R).

[0225] In one embodiment, the Fc region is modified by replacing the leucine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (L234A / L235A).

[0226] In one embodiment, the Fc region is modified by replacing the phenylalanine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (F234A / L235A).

[0227] In one embodiment, the Fc region is modified by replacing the glycine residue at position 236 with an arginine residue and the leucine residue at position 328 with an arginine residue (G236R / L328R).

[0228] It will be recognized that any of the modifications listed above can be combined to reduce FcγR binding.

[0229] In one embodiment, the TBM disclosed herein includes an Fc domain, wherein one or both Fc regions contain one or more modifications that reduce Fc binding to FcγRIIIa without affecting Fc binding to FcγRII.

[0230] In one embodiment, the Fc region is modified by replacing the serine residue at position 239 (S239A) with an alanine residue.

[0231] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 269 (E269A) with an alanine residue.

[0232] In one embodiment, the Fc region is modified by replacing the glutamic acid residue at position 293 (E293A) with an alanine residue.

[0233] In one embodiment, the Fc region is modified by replacing the tyrosine residue at position 296 with a phenylalanine residue (Y296F).

[0234] In one embodiment, the Fc region is modified by replacing the valine residue (V303A) at position 303 with an alanine residue.

[0235] In one embodiment, the Fc region is modified by replacing the alanine residue at position 327 (A327G) with a glycine residue.

[0236] In one embodiment, the Fc region is modified by replacing the lysine residue at position 338 (K338A) with an alanine residue.

[0237] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 376 (D376A) with an alanine residue.

[0238] It will be recognized that any of the modifications listed above can be combined to reduce FcγRIIIa binding.

[0239] 6.3.1.2. Fc domains with altered complement binding

[0240] The TBM disclosed herein may contain Fc domains, one or both of which contain one or more modifications that alter the binding of Fc to complement. The altered complement binding may be either enhanced or degraded.

[0241] In one embodiment, the Fc region contains one or more modifications that reduce its binding to C1q. The classical complement pathway is initiated by binding the hexameric C1q protein to the CH2 domain of antigen-binding IgG and IgM.

[0242] In one embodiment, the TBM disclosed herein includes an Fc domain, wherein one or both Fc regions contain one or more modifications that reduce the binding of Fc to C1q.

[0243] In one embodiment, the Fc region is modified by replacing the leucine residue (L234A) at position 234 with an alanine residue.

[0244] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 (L235A) with an alanine residue.

[0245] In one embodiment, the Fc region is modified by replacing the leucine residue at position 235 with a glutamic acid residue (L235E).

[0246] In one embodiment, the Fc region is modified by replacing the glycine residue at position 237 (G237A) with an alanine residue.

[0247] In one embodiment, the Fc region is modified by replacing the lysine residue at position 322 (K322A) with an alanine residue.

[0248] In one embodiment, the Fc region is modified by replacing the proline residue at position 331 (P331A) with an alanine residue.

[0249] In one embodiment, the Fc region is modified by replacing the proline residue at position 331 with a serine residue (P331S).

[0250] In one embodiment, the TBM disclosed herein comprises an Fc domain derived from IgG4. IgG4 has a naturally lower complement activation profile than IgG1 and also exhibits weaker binding to FcγR. Therefore, in one embodiment, the TBM comprises an IgG4 Fc domain and further comprises one or more modifications to enhance FcγR binding.

[0251] It will be recognized that any of the modifications listed above can be combined to reduce C1q binding.

[0252] 6.3.1.3. Fc domain with modified disulfide bond structure

[0253] The TBM disclosed herein may include an Fc domain comprising one or more modifications to generate and / or remove cysteine ​​residues. Cysteine ​​residues play an important role in the simultaneous assembly of Fc-based multispecific binding molecules by forming disulfide bridges between single pairs of peptide monomers. Therefore, it is possible to modify the TBM structure to generate proteins with improved therapeutic properties by changing the number and / or position of cysteine ​​residues.

[0254] The TBM disclosed herein may include an Fc domain, wherein one or both Fc regions (preferably both Fc regions) contain a cysteine ​​residue at position 309. In one embodiment, the cysteine ​​residue at position 309 is generated by modification, for example, for an Fc domain derived from IgG1, a cysteine ​​residue (L309C) is used to replace the leucine residue at position 309, and for an Fc domain derived from IgG2, a cysteine ​​residue (V309C) is used to replace the valine residue at position 309.

[0255] In one embodiment, the Fc region is modified by replacing the valine residue at position 308 with a cysteine ​​residue (V308C).

[0256] In one embodiment, two disulfide bonds in the hinge region are removed by mutating the core hinge sequence CPCC (SEQ ID NO: 2) to SPPS (SEQ ID NO: 3).

[0257] 6.3.1.4. Fc domain with modified glycosylation

[0258] In some respects, TBMs with improved manufacturability are provided, which contain fewer glycosylation sites than the corresponding immunoglobulins. These proteins have less complex post-translational glycosylation patterns and are therefore simpler and less expensive for manufacturers.

[0259] In one embodiment, the glycosylation site in the CH2 domain is removed by replacing the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q). In addition to improved manufacturability, these glycosylation mutants also reduce FcγR binding as described herein.

[0260] 6.3.1.5. Fc heterodimerization

[0261] Many multispecific molecular forms require dimerization between two Fc regions, unlike native immunoglobulins, where the two Fc regions are operatively linked to a non-identical antigen-binding domain (or a portion thereof, e.g., VH or VH-CH1 of Fab). Insufficient heterodimerization of the two Fc regions forming the Fc domain has been a barrier to improving the production of the desired multispecific molecule and represents a purification challenge. Various methods available in the art can be used to enhance the dimerization of Fc regions that may be present in the TBM disclosed herein, such as those disclosed in: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO2009 / 089004A1.

[0262] This disclosure provides TBMs comprising Fc heterodimers, i.e., Fc domains containing heterologous, non-identical Fc regions. Heterodimerization strategies are used to enhance dimerization of Fc regions operatively linked to different ABMs (or portions thereof, e.g., VH or VH-CH1 of Fab) and reduce dimerization of Fc regions operatively linked to the same ABM or portions thereof. Typically, each Fc region in an Fc heterodimer contains a CH3 domain of an antibody. This CH3 domain is derived from a constant region of any isotype, type, or subclass, and preferably IgG (IgG1, IgG2, IgG3, and IgG4) type antibody, as described in the preceding sections.

[0263] Typically, in addition to the CH3 domain, the TBM also contains other antibody fragments, such as the CH1 domain, CH2 domain, hinge domain, one or more VH domains, one or more VL domains, one or more CDRs, and / or antigen-binding fragments described herein. In some embodiments, the two heteropeptides are two heavy chains forming a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain produces the desired antibody or antibody-like molecule, while homodimerization of the same heavy chain reduces the production of the desired antibody or molecule. In an exemplary embodiment, the two or more heteropeptide chains comprise two chains that contain the CH3 domain and form a molecule in any of the multispecific molecular forms described above in this disclosure. In embodiments, the two heteropeptide chains containing the CH3 domain contain modifications (relative to the unmodified chain) that facilitate heterodimer association of the peptide. Various examples of modification strategies are provided in Table 2 and Sections 6.3.1.5.1 to 6.3.1.5.3 below.

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289] 6.3.1.5.1. Mortar and pestle structure (KIH)

[0290] The TBM disclosed herein may contain one or more, for example, multiple, modifications to one or more constant domains of the Fc domain, such as modifications to the CH3 domain. In one example, the TBM disclosed herein comprises two peptides, each peptide containing a heavy chain constant domain of an antibody, such as a CH2 or CH3 domain. In one example, the two heavy chain constant domains, such as the CH2 or CH3 domain of the TBM, contain one or more modifications that allow heterodimeric association between the two chains. In one aspect, the one or more modifications are disposed on the CH2 domains of two heavy chains. In one aspect, the one or more modifications are disposed on the CH3 domains of at least two peptides of the TBM. In one aspect, one or more modifications to a first polypeptide containing a heavy chain constant domain of TBM can produce a "pestle" and one or more modifications to a second polypeptide of TBM can produce a "mortar," such that heterodimerization of the polypeptide containing the heavy chain constant domain of TBM produces a "pestle" to engage with a "mortar" interface (e.g., an interaction, such as the CH2 domain of the first polypeptide interacting with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacting with the CH3 domain of the second polypeptide). As used herein, a "pestle" refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide containing the heavy chain constant domain of TBM and is therefore localized in a complementary "mortar" at the interface with the second polypeptide containing the heavy chain constant domain of TBM to stabilize the heteropolymer and thus facilitate heteropolymer formation (e.g., relative to homopolymer). The pestle may be present in the initial interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). The preferred input residues for forming the protrusion are typically naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In a preferred embodiment, the initial residues for forming the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0291] A "pot" refers to at least one amino acid side chain that is recessed into the interface of a second polypeptide containing a heavy chain constant domain (TBM) and thus accommodates a corresponding pot on the adjacent interface surface of a first polypeptide containing a TBM. The pot may be present in the initial interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Preferred input residues for forming the pot are typically naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Serine, alanine, or threonine are most preferred. In a preferred embodiment, the initial residue for forming the pot has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0292] In a preferred embodiment, the first CH3 domain is modified at residues 366, 405, or 407 to produce a "mortar" or "pestle" (as described above), and the second CH3 domain, heterodimerized with the first CH3 domain, is modified at residues 407 (if residue 366 in the first CH3 domain is modified), 394 (if residue 405 in the first CH3 domain is modified), or 366 (if residue 407 in the first CH3 domain is modified).

[0293] In another preferred embodiment, a first CH3 domain is modified at residue 366, and a second CH3 domain heterodimerized with the first CH3 domain is modified at residues 366, 368, and / or 407 to produce a "mortar" or "stick" complementary to the "mortar" or "stick" of the first CH3 domain. In one embodiment, the modification of the first CH3 domain introduces a tyrosine (Y) residue at position 366. In this embodiment, the modification of the first CH3 is T366Y. In one embodiment, the modification of the first CH3 domain introduces a tryptophan (W) residue at position 366. In this embodiment, the modification of the first CH3 is T366W. In some embodiments, the modification of the second CH3 domain, which is heterodimerized with the first CH3 domain (modified at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., containing the modification T366Y or T366W)), includes a modification at position 366, a modification at position 368, and a modification at position 407. In some embodiments, the modification at position 366 introduces a serine (S) residue, the modification at position 368 introduces alanine (A), and the modification at position 407 introduces valine (V). In some embodiments, the modification includes T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule contains the modification T366Y, and the second CH3 domain heterodimerized with the first CH3 domain contains the modifications T366S, L368A, and Y407V, and vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises a modification of T366W, and the second CH3 domain heterodimerized with the first CH3 domain comprises a modification of T366S, L368A, and Y407V, and vice versa.

[0294] Additional spatial or "offset" modifications (e.g., pestle and mortar structure) are described in PCT Publication No. WO 2014 / 145806 (e.g., WO 2014 / 145806's...). Figure 3 , Figure 4 and Figure 12 The contents of these publications are found in PCT Publications WO 2014 / 110601, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751 (the contents of which are incorporated herein by reference in their entirety). Examples of KIH variants include a first constant chain containing modifications of L368D and K370S, paired with a second constant chain containing modifications of S364K and E357Q.

[0295] Further mortar-and-pot structure modifications applicable to any multispecific molecules of the present invention are further described, for example, in WO 1996 / 027011 and Merchant et al., 1998, Nat. Biotechnol. [Nature Biotechnology], 16: 677-681 (the contents of which are hereby incorporated herein by reference in their entirety).

[0296] In a further embodiment, the CH3 domain may be additionally modified to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that introducing a pair of cysteine ​​residues capable of forming disulfide bonds provides stability to the heterodimerized TBM containing the paired CH3 domain. In some embodiments, the first CH3 domain contains a cysteine ​​residue at position 354, and the second CH3 domain, heterodimerized with the first CH3 domain, contains a cysteine ​​residue at position 349. In some embodiments, the first CH3 domain comprises cysteine ​​at position 354 (e.g., comprising modification S354C) and tyrosine (Y) at position 366 (e.g., comprising modification T366Y), and the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349 (e.g., comprising modification Y349C), serine at position 366 (e.g., comprising modification T366S), alanine at position 368 (e.g., comprising modification L368A), and valine at position 407 (e.g., comprising modification Y407V). In some embodiments, the first CH3 domain comprises cysteine ​​at position 354 (e.g., comprising modification S354C) and tryptophan (W) at position 366 (e.g., comprising modification T366W), and the second CH3 domain heterodimerized with the first CH3 domain comprises cysteine ​​at position 349 (e.g., comprising modification Y349C), serine at position 366 (e.g., comprising modification T366S), alanine at position 368 (e.g., comprising modification L368A), and valine at position 407 (e.g., comprising modification Y407V).

[0297] 6.3.1.5.2. Alternative Knob and Hole: IgG Heterodimerization

[0298] Heterodimerization of polypeptide chains containing paired CH3 domains of TBM can be improved by introducing one or more modifications into the CH3 domains derived from IgG1 antibody types. In an example, the modification comprises a K409R modification of one CH3 domain, which is paired with an F405L modification in a second CH3 domain. Additional modifications may also be, or alternatively, at positions 366, 368, 370, 399, 405, 407, and 409. Preferably, heterodimerization of polypeptides containing such modifications is achieved under reducing conditions, for example, at 25°C–37°C, for example, at 25°C or 37°C, for 1–10 hours, for example, 1.5–5 hours, for example, 5 hours.

[0299] The amino acid substitutions described herein can be introduced into the CH3 domain using techniques well known in the art (see, for example, McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2: 183).

[0300] The IgG heterodimerization strategy is further described in, for example, WO 2008 / 119353, WO 2011 / 131746 and WO2013 / 060867, the contents of which are hereby incorporated in their entirety by reference.

[0301] In any of the embodiments described in this section, the CH3 domain may be additionally modified to introduce a pair of cysteine ​​residues, as described in section 6.3.1.5.1.

[0302] 6.3.1.5.3. Polarity Bridge

[0303] Heterodimerization of TBM-containing polypeptide chains containing the Fc domain can be enhanced by introducing modifications based on the fundamental principle of "polar bridging." This principle involves creating residues at the binding interface of the two polypeptide chains to interact with residues in the heterodimeric configuration that have similar (or complementary) physical properties, while simultaneously interacting with residues in the homodimeric configuration that have different physical properties. Specifically, these modifications are designed so that, in heterodimer formation, polar residues interact with polar residues, and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, modified residues are used so that polar residues interact with hydrophobic residues. The combined effect of favorable interactions in the heterodimeric configuration and unfavorable interactions in the homodimeric configuration makes heterodimer formation in the Fc region more likely than homodimer formation.

[0304] In an exemplary embodiment, the above modifications are made at one or more of residues 364, 368, 399, 405, 409, and 411 of the CH3 domain.

[0305] In some embodiments, one or more modifications selected from S364L, T366V, L368Q, N399K, F405S, K409F, and R411K are introduced into one of the two CH3 domains. One or more modifications selected from Y407F, K409Q, and T411N may be introduced into the second CH3 domain.

[0306] In another embodiment, one or more modifications selected from the group consisting of S364L, T366V, L368Q, D399K, F405S, K409F and T411K are introduced into a CH3 domain, while one or more modifications selected from Y407F, K409Q and T411D are introduced into a second CH3 domain.

[0307] In one exemplary embodiment, the initial residue of threonine at position 366 of one CH3 domain is replaced by valine, while the initial residue of tyrosine at position 407 of another CH3 domain is replaced by phenylalanine.

[0308] In another exemplary embodiment, the initial residue of serine at position 364 of a CH3 domain is replaced by leucine, while the initial residue of leucine at position 368 of the same CH3 domain is replaced by glutamine.

[0309] In yet another exemplary embodiment, the initial residue of phenylalanine at position 405 of one CH3 domain is replaced by serine and the initial residue of lysine at position 409 of the same CH3 domain is replaced by phenylalanine, while the initial residue of lysine at position 409 of another CH3 domain is replaced by glutamine.

[0310] In yet another exemplary embodiment, the initial residue of aspartic acid at position 399 of one CH3 domain is replaced by lysine, and the initial residue of threonine at position 411 of the same CH3 domain is replaced by lysine, while the initial residue of threonine at position 411 of another CH3 domain is replaced by aspartic acid.

[0311] The amino acid substitutions described herein can be introduced into the CH3 domain using techniques well known in the art (see, for example, McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2: 183). Polar bridging strategies are described, for example, in WO 2006 / 106905, WO 2009 / 089004, and K. Gunasekaran et al., (2010) The Journal of Biological Chemistry, 285: 19637-19646, the contents of which are hereby incorporated herein by reference in their entirety.

[0312] Other polarity bridge modifications are described, for example, in PCT Publication No. WO 2014 / 145806 (e.g., WO 2014 / 145806). Figure 6 (see PCT Publications WO 2014 / 110601, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196 and WO 2016 / 182751, the contents of which are incorporated herein by reference in their entirety.) Examples of polar bridge variants include constant chains modified with N208D, Q295E, N384D, Q418E and N421D.

[0313] In any of the embodiments described herein, the CH3 domain may be additionally modified to introduce a pair of cysteine ​​residues, as described in Section 6.3.1.5.1.

[0314] Other strategies for enhancing heterodimerization are described, for example, in WO 2016 / 105450, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, WO 2016 / 141378, and WO 2014 / 145806 and WO 2014 / 110601 (the entire contents of which are hereby incorporated herein by reference). Any of these strategies may be used in the TBM described herein.

[0315] 6.3.2. Hinge Area

[0316] The TBM disclosed herein may also include a hinge region, such as a hinge region connecting the antigen-binding module to the Fc region. The hinge region may be natural or modified. Hinge regions are typically found at the N-terminus of the Fc region.

[0317] A natural hinge region is a hinge region typically found between the Fab and Fc domains in naturally occurring antibodies. A modified hinge region is any hinge that differs from a natural hinge region in length and / or composition. Such hinges can include hinge regions derived from other species, such as those of humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, alpacas, or goats. Other modified hinge regions may comprise complete hinge regions derived from antibodies of a different type or subclass than the heavy chain Fc region. Alternatively, the modified hinge region may comprise portions of a natural hinge or repeating unit, wherein each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. In this manner, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions can be entirely synthetic and can be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0318] Many modified hinge areas have been described in, for example, the following documents: U.S. Patent Nos. 5,677,425, WO9915549, WO 2005003170, WO 2005003169, WO 2005003170, WO 9825971 and WO 2005003171, which are incorporated herein by reference.

[0319] Examples of suitable hinge sequences are shown in Table 3.

[0320]

[0321] In one embodiment, the heavy chain Fc region has a complete hinge region at its N-end.

[0322] In one embodiment, the heavy chain Fc region and hinge region are derived from IgG4, and the hinge region contains the modified sequence CPPC (SEQ ID NO: 2). Compared to IgG1 containing the sequence CPPC (SEQ ID NO: 2), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 12). Serine residues present in the IgG4 sequence result in increased flexibility in this region, and thus a portion of the molecule forms disulfide bonds (intra-chain disulfide bonds) within the same protein chain rather than bridging to other heavy chains in the IgG molecule to form inter-chain disulfide bonds. (Angel et al., 1993, Mol lmmunol [Molecular Immunology] 30(1): 105-108). Changing the serine residue to proline to give the same core sequence as IgG1 allows for complete formation of inter-chain disulfide bonds in the IgG4 hinge region, thus reducing heterogeneity in the purified product. This modified isotype is named IgG4P.

[0323] 6.3.3. ABM connector

[0324] In some respects, this disclosure provides a TBM comprising at least three AMBs, wherein two or more components of an ABM (e.g., VH and VL of scFv), two or more ABMs, or ABM and non-ABM domains (e.g., dimerization domains, such as the Fc region) are interconnected via peptide linkers. Such linkers are referred to herein as “ABM linkers”, and are distinct from ADC linkers used for attaching drugs to TBMs as described, for example, in Section 6.9.2.

[0325] The range of peptide linkers can be from 2 amino acids to 60 or more amino acids, and in some respects, the range of peptide linkers is from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids. In specific embodiments, the length of the peptide linker is 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, or 50 amino acids.

[0326] Charged and / or flexible joints are particularly preferred.

[0327] Examples of flexible ABM connectors that can be used in the TBM disclosed herein include those disclosed in: Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. A particularly useful flexible connector is (GGGGS)n (SEQ ID NO: 25) (also known as (G4S)n (SEQ ID NO: 25)). In some embodiments, n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range with any two of the above numbers as endpoints, such as 1 to 5, 2 to 5, 3 to 6, 2 to 4, 1 to 4, etc.

[0328] Other examples of suitable TBM connectors for the TBM disclosed herein are shown in Table 4 below:

[0329]

[0330]

[0331] In various respects, this disclosure provides a TBM comprising one or more ABM linkers. Each of the ABM linkers may be of a length ranging from 2 to 60 amino acids, preferably 4 to 30, 5 to 25, 10 to 25, or 12 to 20 amino acids, optionally selected from Table 4 above. In specific embodiments, the TBM comprises two, three, four, five, or six ABM linkers. The ABM linkers may be located on one, two, three, four, or even more polypeptide chains of the TBM.

[0332] 6.4. Exemplary Trispecific Binding Molecules

[0333] An exemplary TBM configuration is shown in Figure 1 middle. Figure 1 A shows Figure 1 The components of the TBM conformation shown in B-1Z. scFv, Fab, non-immunoglobulin-based ABM, and Fc may each have the features described for these components in Sections 6.2 and 6.3. Figure 1 The components of the TBM configuration shown can associate with each other by any of the methods described in Sections 6.2 and 6.3 (e.g., via direct bonds, ABM joints, disulfide bonds, Fc domains modified with mortar-and-mortar structure interactions, etc.). Figure 1 The orientations and associations of the various components shown are merely exemplary; as those skilled in the art will understand, other orientations and associations may be suitable (e.g., as described in Sections 6.2 and 6.3).

[0334] The TBM disclosed herein is not limited to Figure 1 The configuration shown is shown. Other configurations that may be used are known to those skilled in the art. See, for example, WO 2014 / 145806; WO 2017 / 124002; Liu et al., 2017, Front Immunol. [Immunology Frontiers] 8: 38; Brinkmann & Kontermann, 2017, mAbs 9: 2, 182-212; US 2016 / 0355600; Klein et al., 2016, MAbs 8(6): 1010-20; and US 2017 / 0145116.

[0335] 6.4.1. Exemplary Trivalent TBM

[0336] The TBMs disclosed herein may be trivalent, meaning they have three antigen-binding domains, each of which binds one of the components of the CD2, TCR complex, and TAA.

[0337] An exemplary trivalent TBM configuration is shown in Figure 1 B to 10 and Figure 1 V-1Z.

[0338] like Figure 1 As depicted in B-1K, 1O, and 1V-1Z, TBM can contain two halves, one containing two ABMs and the other containing one ABM, the two halves being paired via an Fc domain.

[0339] exist Figure 1 In embodiment B, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises a Fab, an scFv, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0340] exist Figure 1 In embodiment C, the first (or left) hemibody comprises two Fab and Fc regions, and the second (or right) hemibody comprises Fab and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0341] exist Figure 1 In embodiment D, the first (or left) hemibody comprises a Fab, scFv, and Fc region, and the second (or right) hemibody comprises a Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0342] exist Figure 1 In embodiment E, the first (or left) hemibody comprises scFv and Fc regions, and the second (or right) hemibody comprises two Fab and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0343] exist Figure 1 In embodiment F, the first (or left) hemibody comprises scFv, an Fc region, and Fab, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0344] exist Figure 1 In embodiments of G, the first (or left) hemibody comprises scFv and an Fc region, and the second (or right) hemibody comprises Fab, an Fc region, and scFV. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0345] exist Figure 1In embodiment H, the first (or left) hemibody comprises two Fab and Fc regions, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and Fc region. The first and second hemibody associate by forming an Fc region with an Fc domain.

[0346] exist Figure 1 In embodiment I, the first (or left) hemibody comprises a Fab, scFv, and Fc region, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0347] exist Figure 1 In the embodiment of J, the first (or left) hemibody comprises a Fab and an Fc region, and the second (or right) hemibody comprises scFv, a non-immunoglobulin-based ABM, and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0348] exist Figure 1 In the embodiment of K, the first (or left) hemibody comprises an scFv and an Fc region, and the second (or right) hemibody comprises an scFv, an Fc region, and a second scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0349] exist Figure 1 In the embodiment of N, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0350] exist Figure 1 In the embodiment of O, the first (or left) hemibody comprises Fab, an Fc region, and scFab, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0351] exist Figure 1 In embodiment V, the first (or left) hemibody comprises ABM, Fab, and Fc regions based on non-immunoglobulins, and the second (or right) hemibody comprises Fab and Fc regions. The first and second hemibody associate by forming Fc regions of Fc domains.

[0352] exist Figure 1 In embodiments of W, the first (or left) hemibody comprises a non-immunoglobulin-based ABM, scFv, and Fc region, and the second (or right) hemibody comprises a Fab and Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0353] exist Figure 1 In embodiment X, the first (or left) hemibody comprises scFv, an Fc region, and a non-immunoglobulin-based ABM at the C-terminus of the Fc region, and the second (or right) hemibody comprises Fab and an Fc region. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0354] exist Figure 1 In embodiment Y, the first (or left) hemibody comprises an scFv, an Fc region, and an scFv at the C-terminus of the Fc region, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0355] exist Figure 1 In embodiment Z, the first (or left) hemibody comprises scFv, an Fc region, and a Fab at the C-terminus of the Fc region, and the second (or right) hemibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0356] Alternatively, such as Figure 1 As described in L, a trivalent TBM can contain two half-antibodies, each containing a complete ABM and a portion of another ABM (one being VH and the other VL). The two half-antibodies pair via an Fc domain, thus VH and VL associate to form a complete antigen-binding Fv domain.

[0357] The TBM can be a single chain, such as Figure 1 As shown in M. Figure 1 M's TBM contains three scFv structural domains connected by a connector.

[0358] exist Figure 1 In each configuration shown in B-1O and 1V-Z, each domain designated as X, Y, and Z represents a TCR ABM, a CD2 ABM, or a TAA ABM, although not necessarily in that order. In other words, X can be a TCR ABM, a CD2 ABM, or a TAA ABM, Y can be a TCR ABM, a CD2 ABM, or a TAA ABM, and Z can be a CD2 ABM, a TCR ABM, or a TAAA ABM, provided that the TBM contains at least one TCR ABM, at least one CD2 ABM, and at least one TAA ABM.

[0359] Therefore, this disclosure provides, as follows Figure 1Any of the three trivalent TBMs shown in B to 1O and 1V to Z, where X is a CD2 ABM, Y is a TCR ABM and Z is a TAA ABM (for convenience, this configuration of ABM is designated as "T1").

[0360] This disclosure also provides, as well as Figure 1 The trivalent TBM shown in any of B to 1O and 1V to Z, where X is a CD2 ABM, Y is a TAA ABM, and Z is a TCR ABM (for convenience, this configuration of ABM is designated as "T2").

[0361] This disclosure further provides, as follows Figure 1 The trivalent TBM shown in any of B to 1O and 1V to Z, where X is TCRABM, Y is CD2 ABM, and Z is TAA ABM (for convenience, this configuration of ABM is designated as "T3").

[0362] This disclosure further provides information such as Figure 1 The trivalent TBM shown in any one of B to 1O and 1V to Z, where X is TCRABM, Y is TAA ABM, and Z is CD2 ABM (for convenience, this configuration of ABM is designated as "T4").

[0363] This disclosure further provides information such as Figure 1 The trivalent TBM shown in any one of B to 1O and 1V to Z, where X is TAAABM, Y is CD2 ABM, and Z is TCR ABM (for convenience, this configuration of ABM is designated as "T5").

[0364] This disclosure further provides information such as Figure 1 The trivalent TBM shown in any of B to 1O and 1V to Z, where X is TAAABM, Y is TCR ABM, and Z is CD2 ABM (for convenience, this configuration of ABM is designated as "T6").

[0365] 6.4.2. Exemplary tetravalent TBM

[0366] The TBMs disclosed herein may be tetravalent, that is, they have four antigen-binding domains, one or two of which bind CD2, one or two of which bind components of the TCR complex, and one or two of which bind TAA.

[0367] An exemplary tetravalent TBM configuration is shown in Figure 1 In P-1R.

[0368] like Figure 1The tetravalent TBM described by P-1R can contain two halves, each containing two complete ABMs, which are paired via an Fc domain.

[0369] exist Figure 1 In the embodiment of P, the first (or left) hemibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) hemibody comprises a Fab, an Fc region, and a second Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0370] exist Figure 1 In the embodiment of Q, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab, an Fc region, and scFv. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0371] exist Figure 1 In the embodiment of R, the first (or left) hemibody comprises Fab, an Fc region, and scFv, and the second (or right) hemibody comprises scFv, an Fc region, and Fab. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0372] exist Figure 1 In the configuration shown in P-1R, each of X, Y, Z, and A represents a TCR ABM, a CD2 ABM, or a TAAA ABM (although not necessarily in such an order), provided that the TBM contains at least one TCR ABM, one CD2 ABM, and one TAA ABM. Therefore, the tetravalent ABM disclosed herein will comprise two ABMs targeting the TCR complex, one of CD2, and one of TAA. Preferably, the tetravalent TBM has two TAA ABMs.

[0373] Therefore, this disclosure provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X is a CD2 ABM, Y is a TCR ABM, and both Z and A are TAA ABMs (for convenience, this configuration of ABM is designated as "Tv 1").

[0374] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X is a CD2 ABM, Y and A are both TAA ABMs, and Z is a TCR ABM (for convenience, this configuration of ABM is designated as "Tv 2").

[0375] This disclosure further provides information such as Figure 1Any of the tetravalent TBMs shown in P-1R, where X is a TCR ABM, Y is a CD2 ABM, and both Z and A are TAA ABMs (for convenience, this configuration of ABM is designated as "Tv 3").

[0376] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X is a TCR ABM, Y and A are both TAA ABMs, and Z is a CD2 ABM (for convenience, this configuration of ABM is designated as "Tv 4").

[0377] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and A are both TAAABMs, Y is a CD2 ABM, and Z is a TCR ABM (for convenience, this configuration of ABM is designated as "Tv 5").

[0378] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and A are both TAAABMs, Y is a TCR ABM, and Z is a CD2 ABM (for convenience, this configuration of ABM is designated as "Tv 6").

[0379] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X is a CD2 ABM, Y and Z are both CD2 ABMs, and Z is a TCR ABM (for convenience, this configuration of ABM is designated as "Tv 7").

[0380] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and Z are both TAAABMs, Y is a CD2 ABM, and A is a TCR ABM (for convenience, this configuration of ABM is designated as "Tv 8").

[0381] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X is a TCR ABM, Y and A are both TAA ABMs, and Z is a CD2 ABM (for convenience, this configuration of ABM is designated as "Tv 9").

[0382] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and Y are both TAAABMs, Z is a CD2 ABM, and A is a TCR ABM (for convenience, this configuration of ABM is designated as "Tv 10").

[0383] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and Z are both TAAABMs, Y is a TCR ABM, and A is a CD2 ABM (for convenience, this configuration of ABM is designated as "Tv 11").

[0384] This disclosure further provides, as follows Figure 1 Any of the tetravalent TBMs shown in P-1R, where X and Y are both TAAABMs, Z is a TCR ABM, and A is a CD2 ABM (for convenience, this configuration of ABM is designated as "Tv 12").

[0385] 6.4.3. Exemplary pentavalent TBM

[0386] The TBMs disclosed herein may be pentavalent, that is, they have five antigen-binding domains, one, two, or three of which bind CD2, one, two, or three of which bind components of the TCR complex, and one, two, or three of which bind TAA.

[0387] An exemplary pentavalent TBM configuration is shown in Figure 1 S in.

[0388] like Figure 1 As depicted in S, a pentavalent TBM may contain two halves, one containing two complete ABMs and the other containing one complete ABM, the two halves being paired via an Fc domain.

[0389] exist Figure 1 In the embodiment of S, the first (or left) hemibody comprises Fab, scFv, and an Fc region, and the second (or right) hemibody comprises Fab, an Fc region, and scFv. The first and second hemibody associate by forming an Fc region of an Fc domain.

[0390] exist Figure 1 In the configuration shown by S, each of X, Y, Z, A, and B represents a TCR ABM, a CD2 ABM, or a TAA ABM (although not necessarily in such an order), provided that the TBM contains at least one TCR ABM, one CD2 ABM, and one TAA ABM. Therefore, the pentavalent TBM disclosed herein can comprise two ABMs for a component of the TCR complex, two of CD2 and TAA, or three ABMs for a component of the TCR complex, one of CD2 and TAA. Preferably, the pentavalent TBM has two or three TAA ABMs. In a specific embodiment, the pentavalent TBM has three TAA ABMs.

[0391] Therefore, this disclosure provides as follows Figure 1 S represents the pentavalent TBM, where X, Y, Z, A, and B are components of the CD2, TCR complex, and ABMs of TAA, as shown in Table 5.

[0392]

[0393]

[0394]

[0395] 6.4.4. Exemplary Hexavalent TBM

[0396] The TBMs disclosed herein may be hexavalent, that is, they have six antigen-binding domains, one, two, three, or four of which bind CD2, one, two, three, or four of which bind components of the TCR complex, and one, two, three, or four of which bind TAA.

[0397] An exemplary hexavalent TBM configuration is shown in Figure 1 T-1U.

[0398] like Figure 1 As depicted in T-1U, a pentavalent TBM may contain two halves, one containing two complete ABMs and the other containing one complete ABM, the two halves being paired via an Fc domain.

[0399] exist Figure 1 In embodiments of T, the first (or left) hemibody comprises Fab, a second Fab, an Fc region, and scFv, and the second (or right) hemibody comprises Fab, a second Fab, an Fc region, and scFv. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0400] exist Figure 1 In the embodiment of U, the first (or left) hemibody comprises a first Fv, a second Fv, a third Fv, and an Fc region, and the second (or right) hemibody comprises a first Fv, a second Fv, a third Fv, and an Fc region. The first and second hemibody associate by forming the Fc region of the Fc domain.

[0401] exist Figure 1In the configuration shown in T-1U, each of X, Y, Z, A, B, and C represents a TCR ABM, a CD2 ABM, or a TAA ABM (although not necessarily in such an order), provided that the TBM contains at least one TCR ABM, one CD2 ABM, and one TAA ABM. Therefore, the hexavalent TBM disclosed herein may include (i) two ABMs for each of the components of the TCR complex, CD2, and TAA; (ii) three ABMs for one of the components of the TCR complex, CD2, and TAA; or (iii) four ABMs for one of the components of the TCR complex, CD2, and TAA. For example, a hexavalent ABM may include three ABMs for TAA, two ABMs for CD2, and one ABM for the components of the TCR complex. As another example, a hexavalent ABM may include three ABMs for TAA, two ABMs for the components of the TCR complex, and one ABM for CD2. Preferably, the hexavalent TBM has two, three, or four TAA ABMs. In a specific embodiment, the hexavalent TBM has three TAA ABMs. In other embodiments, the hexavalent TBM has four TAA ABMs.

[0402] Therefore, this disclosure provides, as follows Figure 1 Any of the hexavalent TBMs shown in T-1U, where X, Y, Z, A, B, and C are components of the CD2, TCR complex, and ABMs of TAA, as shown in Table 6.

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413] 6.5.TCR ABM

[0414] The TBM disclosed herein contains an ABM containing a component that specifically binds to the TCR complex. The TCR is a disulfide-linked membrane-anchored heterodimer protein, typically composed of highly variable α and β chains expressed as a portion of a complex with an invariant CD3 chain molecule. T cells expressing this receptor are called α:β (or αβ) T cells, although a minority of T cells (called γδ T cells) express alternative receptors (formed by variable γ and δ chains).

[0415] In a preferred embodiment, the TBM disclosed herein contains ABM that specifically binds to CD3.

[0416] 6.5.1.CD3 ABM

[0417] The TBM disclosed herein may contain ABM that specifically binds to CD3. The term "CD3" refers to the cluster 3 co-receptor of the T cell receptor (or a co-receptor complex, or a polypeptide chain of the co-receptor complex). The amino acid sequence of the human CD3 polypeptide chain is provided in NCBI accessions P04234, P07766, and P09693. CD3 proteins may also include variants. CD3 proteins may also include fragments. CD3 proteins may also include post-translational modifications to the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation.

[0418] In some embodiments, the TBM disclosed herein may comprise an ABM, which is an anti-CD3 antibody (e.g., as described in US2016 / 0355600, WO 2014 / 110601, and WO 2014 / 145806, the contents of which are hereby incorporated by reference) or its antigen-binding domain. Exemplary anti-CD3 VH, VL, and scFV sequences that may be used in the TBM disclosed herein are provided in Table 7A.

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] The CDR sequences of many CD3 binding compounds defined by the Kabat numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, Department of Public Health, National Institutes of Health, Bethesda, MD), the Josiah numbering scheme (Al-Lazikani et al., 1997, J. Mol. Biol, 273: 927-948), and combinations of Kabat and Josiah numbering are provided in Tables 7B-7D.

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] In some embodiments, the TBM disclosed herein may include a CD3 ABM containing a CDR such as any one of CD3-1 to CD3-128 as defined by Kabat numbering (e.g., as listed in Table 7B). In other embodiments, the TBM disclosed herein may include a CD3 ABM containing a CDR such as any one of CD3-1 to CD3-128 as defined by Josiah numbering (e.g., as listed in Table 7C). In still other embodiments, the TBM disclosed herein may include a CD3ABM containing a CDR such as any one of CD3-1 to CD3-128 as defined by a combination of Kabat and Josiah numbering (e.g., as listed in Table 7D).

[0449] In some embodiments, a CD3 ABM contains a CDR sequence of CD3-1. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-2. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-3. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-4. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-5. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-6. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-7. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-8. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-9. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-10. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-11. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-12. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-13. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-14. In some embodiments, a CD3 ABM contains a CDR sequence of CD3-15. In some embodiments, a CD3 ABM contains CDR sequences of CD3-16. In some embodiments, a CD3 ABM contains CDR sequences of CD3-17. In some embodiments, a CD3 ABM contains CDR sequences of CD3-18. In some embodiments, a CD3 ABM contains CDR sequences of CD3-19. In some embodiments, a CD3 ABM contains CDR sequences of CD3-20. In some embodiments, a CD3 ABM contains CDR sequences of CD3-21. In some embodiments, a CD3 ABM contains CDR sequences of CD3-22. In some embodiments, a CD3 ABM contains CDR sequences of CD3-23. In some embodiments, a CD3 ABM contains CDR sequences of CD3-24. In some embodiments, a CD3 ABM contains CDR sequences of CD3-25. In some embodiments, a CD3 ABM contains CDR sequences of CD3-26. In some embodiments, a CD3 ABM contains CDR sequences of CD3-27. In some embodiments, a CD3 ABM contains CDR sequences of CD3-28. In some embodiments, a CD3 ABM contains CDR sequences of CD3-29. In some embodiments, a CD3 ABM contains CDR sequences of CD3-30. In some embodiments, a CD3 ABM contains CDR sequences of CD3-31. In some embodiments, a CD3 ABM contains CDR sequences of CD3-32. In some embodiments, a CD3 ABM contains CDR sequences of CD3-33.In some embodiments, a CD3 ABM contains CDR sequences of CD3-34. In some embodiments, a CD3 ABM contains CDR sequences of CD3-35. In some embodiments, a CD3 ABM contains CDR sequences of CD3-36. In some embodiments, a CD3 ABM contains CDR sequences of CD3-37. In some embodiments, a CD3 ABM contains CDR sequences of CD3-38. In some embodiments, a CD3 ABM contains CDR sequences of CD3-39. In some embodiments, a CD3 ABM contains CDR sequences of CD3-40. In some embodiments, a CD3 ABM contains CDR sequences of CD3-41. In some embodiments, a CD3 ABM contains CDR sequences of CD3-42. In some embodiments, a CD3 ABM contains CDR sequences of CD3-43. In some embodiments, a CD3 ABM contains CDR sequences of CD3-44. In some embodiments, a CD3 ABM contains CDR sequences of CD3-45. In some embodiments, a CD3 ABM contains CDR sequences of CD3-46. In some embodiments, a CD3 ABM contains CDR sequences of CD3-47. In some embodiments, a CD3 ABM contains CDR sequences of CD3-48. In some embodiments, a CD3 ABM contains CDR sequences of CD3-49. In some embodiments, a CD3 ABM contains CDR sequences of CD3-50. In some embodiments, a CD3 ABM contains CDR sequences of CD3-51. In some embodiments, a CD3 ABM contains CDR sequences of CD3-52. In some embodiments, a CD3 ABM contains CDR sequences of CD3-53. In some embodiments, a CD3 ABM contains CDR sequences of CD3-54. In some embodiments, a CD3 ABM contains CDR sequences of CD3-55. In some embodiments, a CD3 ABM contains CDR sequences of CD3-56. In some embodiments, a CD3 ABM contains CDR sequences of CD3-57. In some embodiments, a CD3 ABM contains CDR sequences of CD3-58. In some embodiments, a CD3 ABM contains CDR sequences of CD3-59. In some embodiments, a CD3 ABM contains CDR sequences of CD3-60. In some embodiments, a CD3 ABM contains CDR sequences of CD3-61. In some embodiments, a CD3 ABM contains CDR sequences of CD3-62. In some embodiments, a CD3 ABM contains CDR sequences of CD3-63. In some embodiments, a CD3 ABM contains CDR sequences of CD3-64. In some embodiments, a CD3 ABM contains CDR sequences of CD3-65. In some embodiments, a CD3 ABM contains CDR sequences of CD3-66.In some embodiments, a CD3 ABM contains CDR sequences of CD3-67. In some embodiments, a CD3 ABM contains CDR sequences of CD3-68. In some embodiments, a CD3 ABM contains CDR sequences of CD3-69. In some embodiments, a CD3 ABM contains CDR sequences of CD3-70. In some embodiments, a CD3 ABM contains CDR sequences of CD3-71. In some embodiments, a CD3 ABM contains CDR sequences of CD3-72. In some embodiments, a CD3 ABM contains CDR sequences of CD3-73. In some embodiments, a CD3 ABM contains CDR sequences of CD3-74. In some embodiments, a CD3 ABM contains CDR sequences of CD3-75. In some embodiments, a CD3 ABM contains CDR sequences of CD3-76. In some embodiments, a CD3 ABM contains CDR sequences of CD3-77. In some embodiments, a CD3 ABM contains CDR sequences of CD3-78. In some embodiments, a CD3 ABM contains CDR sequences of CD3-79. In some embodiments, a CD3 ABM contains CDR sequences of CD3-80. In some embodiments, a CD3 ABM contains CDR sequences of CD3-81. In some embodiments, a CD3 ABM contains CDR sequences of CD3-82. In some embodiments, a CD3 ABM contains CDR sequences of CD3-83. In some embodiments, a CD3 ABM contains CDR sequences of CD3-84. In some embodiments, a CD3 ABM contains CDR sequences of CD3-85. In some embodiments, a CD3 ABM contains CDR sequences of CD3-86. In some embodiments, a CD3 ABM contains CDR sequences of CD3-87. In some embodiments, a CD3 ABM contains CDR sequences of CD3-88. In some embodiments, a CD3 ABM contains CDR sequences of CD3-89. In some embodiments, a CD3 ABM contains CDR sequences of CD3-90. In some embodiments, a CD3 ABM contains CDR sequences of CD3-91. In some embodiments, a CD3 ABM contains CDR sequences of CD3-92. In some embodiments, a CD3 ABM contains CDR sequences of CD3-93. In some embodiments, a CD3 ABM contains CDR sequences of CD3-94. In some embodiments, CD3 ABM contains CDR sequences of CD3-95. In some embodiments, CD3 ABM contains CDR sequences of CD3-96. In some embodiments, CD3 ABM contains CDR sequences of CD3-97. In some embodiments, CD3 ABM contains CDR sequences of CD3-98. In some embodiments, CD3 ABM contains CDR sequences of CD3-99.In some embodiments, a CD3 ABM contains CDR sequences of CD3-100. In some embodiments, a CD3 ABM contains CDR sequences of CD3-101. In some embodiments, a CD3 ABM contains CDR sequences of CD3-102. In some embodiments, a CD3 ABM contains CDR sequences of CD3-103. In some embodiments, a CD3 ABM contains CDR sequences of CD3-104. In some embodiments, a CD3 ABM contains CDR sequences of CD3-105. In some embodiments, a CD3 ABM contains CDR sequences of CD3-106. In some embodiments, a CD3 ABM contains CDR sequences of CD3-107. In some embodiments, a CD3 ABM contains CDR sequences of CD3-108. In some embodiments, a CD3 ABM contains CDR sequences of CD3-109. In some embodiments, a CD3 ABM contains CDR sequences of CD3-110. In some embodiments, a CD3 ABM contains CDR sequences of CD3-111. In some embodiments, a CD3 ABM contains CDR sequences of CD3-112. In some embodiments, a CD3 ABM contains CDR sequences of CD3-113. In some embodiments, a CD3 ABM contains CDR sequences of CD3-114. In some embodiments, a CD3 ABM contains CDR sequences of CD3-115. In some embodiments, a CD3 ABM contains CDR sequences of CD3-116. In some embodiments, a CD3 ABM contains CDR sequences of CD3-117. In some embodiments, a CD3 ABM contains CDR sequences of CD3-118. In some embodiments, a CD3 ABM contains CDR sequences of CD3-119. In some embodiments, a CD3 ABM contains CDR sequences of CD3-120. In some embodiments, a CD3 ABM contains CDR sequences of CD3-121. In some embodiments, a CD3 ABM contains CDR sequences of CD3-122. In some embodiments, a CD3 ABM contains CDR sequences of CD3-123. In some embodiments, a CD3 ABM contains CDR sequences of CD3-124. In some embodiments, a CD3 ABM contains CDR sequences of CD3-125. In some embodiments, a CD3 ABM contains CDR sequences of CD3-126. In some embodiments, a CD3 ABM contains CDR sequences of CD3-127. In some embodiments, a CD3 ABM contains CDR sequences of CD3-128.

[0450] The TBM disclosed herein may contain complete heavy and light variable sequences of any one of CD3-1 to CD3-128. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-1. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-1. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-2. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-3. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-4. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-5. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-6. In some embodiments, the TBM disclosed herein contains a CD3 ABM containing VH and VL sequences of CD3-7. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-8. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-9. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-10. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-11. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-12. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-13. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-14. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-15. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-16. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-17. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-18. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-19. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-20. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-21.In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-22. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-23. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-24. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-25. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-26. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-27. In some embodiments, the TBM disclosed herein comprises a CD3 ABM containing VH and VL sequences of CD3-28.

[0451] 6.5.2. TCR-α / β ABM

[0452] The TBM disclosed herein may contain an ABM that specifically binds to the TCR-α chain, TCR-β chain, or TCR-αβ dimer. Exemplary anti-TCR-α / β antibodies are known in the art (see, for example, US 2012 / 0034221; Borst et al., 1990, HumImmunol. [Human Immunology] 29(3): 175-88 (describing antibody BMA031), the contents of which are incorporated herein by reference). The VH, VL, and cabat CDR sequences of antibody BMA031 as described in US 2012 / 0034221 are provided in Table 8.

[0453]

[0454] In one embodiment, the TCR ABM may contain the CDR sequence of antibody BMA031. In other embodiments, the TCR ABM may contain the VH and VL sequences of antibody BMA031.

[0455] 6.5.3.TCR-γ / δ ABM

[0456] The TBM disclosed herein may contain an ABM that specifically binds to the TCR-γ chain, TCR-δ chain, or TCR-γδ dimer. Exemplary anti-TCR-γ / δ antibodies are known in the art (see, for example, U.S. Patent No. 5,980,892 (which describes δTCS1, produced by a hybridoma deposited at ATCC with accession number HB 9578), the contents of which are incorporated herein by reference).

[0457] 6.6.CD2 ABM

[0458] 6.6.1. Immunoglobulin-based CD2 ABM

[0459] In some embodiments, the TBM disclosed herein may comprise an ABM of an anti-CD2 antibody or its antigen-binding domain. Exemplary anti-CD2 antibodies are known in the art (see, for example, US 6,849,258, CN 102827281A, US 2003 / 0139579 A1, and US 5,795,572). Table 9 provides exemplary CDR, VH, and VL sequences that may be included in an anti-CD2 antibody or its antigen-binding fragment for use in the TBM disclosed herein.

[0460]

[0461] In some embodiments, the CD2 ABM comprises the CDR sequence of CD2-1 (SEQ ID NO: 247-252). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of CD2-1 (SEQ ID NO: 253-254). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of hu1CD2-1 (SEQ ID NO: 255-256). In some embodiments, the CD2 ABM comprises variable sequences of the heavy and light chains of hu2CD2-1 (SEQ ID NO: 257 and 256, respectively).

[0462] In other embodiments, the CD2 ABM may comprise the CDR sequence of antibody 9D1 generated by a hybridoma deposited on May 16, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC6132, and described in CN 102827281A. In other embodiments, the CD2 ABM may comprise the CDR sequence of antibody LO-CD2b generated by a hybridoma deposited on June 22, 1999, at the United States Type Culture Collection (PTA-802), and described in US 2003 / 0139579 A1. In yet another embodiment, the CD2 ABM may comprise the CDR sequence of CD2 SFv-Ig produced by a construct expressing a clone in recombinant Escherichia coli, deposited on April 9, 1993 at ATCC with accession number 69277, and described in US 5,795,572.

[0463] In other embodiments, the CD2 ABM may comprise the VH and VL sequences of antibody 9D1. In other embodiments, the CD2 ABM may comprise the VH and VL sequences of antibody LO-CD2b. In yet another embodiment, the CD2 ABM may comprise the VH and VL sequences of CD2 SFv-Ig generated from a construct of a clone expressed in recombinant E. coli having ATCC accession number 69277.

[0464] 6.6.2. CD2 ABM based on CD58

[0465] In some respects, this disclosure provides a TBM comprising a CD2 ABM as a ligand. The CD2 ABM specifically binds to human CD2, whose natural ligand is CD58, also known as LFA-3. The CD58 / LFA-3 protein is a glycoprotein expressed on the surface of multiple cell types (Dustin et al., 1991, Annu. Rev. Immunol. [Annual Review of Immunology] 9: 27) and plays a role in mediating T cell-APC interactions in both antigen-dependent and antigen-independent modes (Wallner et al., 1987, J. Exp. Med. [Journal of Experimental Medicine] 166: 923). Therefore, in some respects, the CD2 ABM is a CD58 moiety. As used herein, the CD58 portion comprises an amino acid sequence having at least 70% sequence identity with the CD2-binding portion of CD58 (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the CD2-binding portion of CD58). The sequence of human CD58 has the Uniprot identifier P19256 (www.uniprot.org / uniprot / P19256). It has been determined that a CD58 fragment containing amino acid residues 30-123 of the full-length CD58 (i.e., the sequence designated as CD58-4 in Table 10 below) is sufficient to bind to CD2. Wan et al., 1999, Cell [Cell] 97: 791-803. Therefore, in some respects, the CD58 portion contains an amino acid sequence that has at least 70% sequence identity with amino acids 30-123 of CD58 (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence designated as CD58-4).

[0466] The interaction between CD58 and CD2 has been mapped using X-ray crystallography and molecular modeling. Substitution of residues E25, K29, K30, K32, D33, K34, E37, D84, and K87 (where the numbers refer to those in the mature polypeptide) reduces binding to CD2. Ikemizu et al., 1999, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 96:4289-94. Therefore, in preferred embodiments, the CD58 portion disclosed herein retains wild-type residues at E25, K29, K30, K32, D33, K34, E37, D84, and K87.

[0467] In contrast, the following substitutions (where the numbers refer to the full-length polypeptide) do not affect binding to CD2: F29S; V37K; V49Q; V86K; T113S; and L121G. Therefore, the CD58 portion of this disclosure may include one, two, three, four, five, or all six of the aforementioned substitutions.

[0468] An exemplary CD58 portion is provided in the following Table 10:

[0469]

[0470] 6.6.3. CD2 ABM based on CD48

[0471] In some respects, this disclosure provides a TBM comprising a CD2 ABM as a CD48 portion. As used herein, the CD48 portion comprises an amino acid sequence having at least 70% sequence identity with the CD2-binding portion of CD48 (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the CD2-binding portion of CD48). The sequence of human CD48 has the Uniprot identifier P09326 (www.uniprot.org / uniprot / P09326), which includes a signal peptide (amino acids 1-26) and a GPI anchor (amino acids 221-243). In some respects, the CD48 moiety contains an amino acid sequence that has at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the amino acid sequence comprising amino acids 27-220 of the Uniprot identifier P09326. Human CD48 has an Ig-like C2I type domain (amino acids 29-127 of the Uniprot identifier P09326) and an Ig-like C22 type domain (amino acids 132-212 of the Uniprot identifier P09326). Therefore, in some embodiments, the CD48 portion comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the Ig-like C2 type 2 domain (amino acids 132-212 with Uniprotector identifier P09326). In some embodiments, the CD48 portion may comprise one or more natural variants relative to the sequence having Uniprotector identifier P09326. For example, the CD48 portion may include an E102Q replacement.For example, the CD48 portion may contain an amino acid sequence corresponding to the CD-48 isotype or its CD2 binding portion (e.g., the isotype with Uniprot identifier P09326-2 or its CD2 binding portion).

[0472] 6.7. Tumor-associated antigen ABM

[0473] The TBM disclosed herein comprises at least one ABM that specifically binds to a tumor-associated antigen (TAA). Preferably, the TAA is a human TAA. The antigen may or may not be present on normal cells. In some embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0474] It is anticipated that any type of tumor and any type of TAA can be targeted by the TBM disclosed herein. Exemplary types of cancer that can be targeted include acute lymphoblastic 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 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 carcinoma, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, pulmonary tract cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma, and other bladder cancers. However, those skilled in the art will recognize that TAAs for virtually any type of cancer are known.

[0475] Exemplary TAAs that can generate the TBM disclosed herein include ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; ADRB3; Aggregate proteoglycan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; ALK; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-α-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MD R15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (reticulin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; cadherin 17; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TAR) C); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 / eosinophil activation chemokine-2); CCL25 (TECK); CCL26 (eosinophil activation chemokine-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3) ; CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA); CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C;CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD32b; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8 ; CD80; CD81; CD83; CD86; CD97; CD179a; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; 6; CDK7; CDK9; CDKN1A (p21Wap1 / Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN6; CLDN7 (sealing protein-7); CLN3; CLU (cluster protein); CMKLR1; CMKOR1 (RDC1); CNR1; CO L18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (β-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (1-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 / LIX) ); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR / STRL33 / Bonzo); CYB5; CYC1; CYSLTR1; CGRP; C1q; C1r; C1; C4a; C4b; C2a; C 2b;C3a;C3b;DAB2IP;DES;DKFZp451J0118;DNCL1;DPP4;E-selectin;E2F1;ECGF1;EDG1;EFNA1;EFNA3;EFNB2;EGF;EGFR;EGFRvIII;ELAC2;ENG;ENO1;ENO2;ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); Factor VII; Factor IX; Factor V; Factor VIIa; Factor X; Factor XII; Factor XIII; FADD; FasL; FASN; FCE R1A; FCE R2; Fc γ receptor; FCGR3A; FCRL5; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20 ;FGF21;FGF22;FGF23;FGF3(int-2);FGF4(HST);FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR3;FIGF(VEGFD);FIL1(EPSILON) FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; folate receptor α; folate receptor β; FOS; FOSL1 (FRA-1); fucose GM1; FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GloboH; GNAS1; GNRH1; GPNMB; GPR2 (CCR10); GPR20; GPR31; GPR44; GPR 64; GPR81 (FKSG80); GPRC5D; GRCC10 (C10); GRP; GSN (gelatin); GSTP1; Glycoprotein (gP) IIb / IIIa; HAVCR1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; Her2; HER3; HGF; HIF1A; HIP1; Histamine and histamine receptor; HLA-A; HLA-DRA; HM74; HMGB1; HMOX1; HMWMAA; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFN A1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFN-γ;IFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-1;IL-α;IL-1- β; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16;IL17;IL17B;IL17C;IL17R;IL18;IL18BP;IL18R1;IL18RAP;IL19;IL1A;IL1B;IL1F10;IL1F5;IL1F6;IL1F7;IL1F8;IL1F9;IL1HY1;IL1R 1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2; IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (α6 integrin); ITGAV; ITGB3; ITGB4 (β4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC BoxBP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); L-selectin; LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LRP6; LPS; LTA (TNF-β); LTB; LTB4R (GPR16); LTB4R2; LTBR; LY6K; LYPD8; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun ); MDK; mesothelin; MIB1; intermediate factor; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neuroprotein polysaccharide; NKG2D; NFKB1; NFKB2; NGF; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2;NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A 2; NR6A1; NRP1; NRP2; NT5E; NTN4; NY-BR-1; o-acetyl-GD2; ODZ1; OPRD1; OR51E2; P2RX7; PANX3; PAP; PART1; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1 ;PF4 (CXCL4);PGE2;PGF;PGR;phosphatase proteoglycan;PIAS2;PIK3CG;PLAC1; Plasminogen activator;PLAU (uPA);PLG;PLXDC1; Polysialic acid;PPBP (CXCL7);PPID;PR1;PRKCQ;PRKD1;PRL;PROC; Protein C;PROK2;PSAP;PSCA;PTAFR;PTEN;PTGS2 (COX-2);PTN;RAC2 (p21Rac2);RAGE;RARB;RGS1;RGS13;RGS3;RNF110 (ZNF144);ROBO2;SIO0A2;S CGB1D2 (lipophilic B); SCGB2A1 (mammary globin 2); SCGB2A2 (mammary globin 1); SCYE1 (endothelial monocyte activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINAB5 (mammary filament inhibitory protein); SERPINE1 (PAI-1); SERPINAF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC34A2; SLC39A6; SLC43A1; SLIT2; SLITRK6; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; Substance P; TACSTD2; TB4R2; TBX21; TCP10; TDGF1; TEK; TEM1 / CD248; TEM7R; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (platelet-reactive protein-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-α; TNFAIP2 (B94); TNFAIP3;TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE ); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TN FSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase ha); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSHR; TSLP; TWEAK; thrombomodulin; thrombin; UPK2; VEGF; VEGFB; VEGFC; multifunctional proteoglycan; VHL C5; VLA-4; XCL1 (lymphocyte chemokine); XCL2 (SCM-1b); XCR1 (GPRS / CCXCR1); YY1; and ZFPM2.

[0476] In some embodiments, the TAA is ADRB3. In some embodiments, the TAA is AKAP-4. In some embodiments, the TAA is ALK. In some embodiments, the TAA is an androgen receptor. In some embodiments, the TAA is B7H3. In some embodiments, the TAA is BCMA. In some embodiments, the TAA is BORIS. In some embodiments, the TAA is BST2. In some embodiments, the TAA is Cadherin17. In some embodiments, the TAA is CAIX. In some embodiments, the TAA is CD171. In some embodiments, the TAA is CD179a. In some embodiments, the TAA is CD19. In some embodiments, the TAA is CD20. In some embodiments, the TAA is CD22. In some embodiments, the TAA is CD24. In some embodiments, the TAA is CD30. In some embodiments, the TAA is CD300LF. In some embodiments, the TAA is CD32b. In some embodiments, the TAA is CD33. In some embodiments, the TAA is CD38. In some embodiments, the TAA is CD44v6. In some embodiments, the TAA is CD72. In some embodiments, the TAA is CD79a. In some embodiments, the TAA is CD79b. In some embodiments, the TAA is CD97. In some embodiments, the TAA is CEA. In some embodiments, the TAA is CLDN6. In some embodiments, the TAA is CLEC12A. In some embodiments, the TAA is CLL-1. In some embodiments, the TAA is CS-1. In some embodiments, the TAA is CXORF61. In some embodiments, the TAA is cyclin B1. In some embodiments, the TAA is CYP1B1. In some embodiments, the TAA is EGFR. In some embodiments, the TAA is EGFRvIII. In some embodiments, the TAA is EMR2. In some embodiments, the TAA is EPCAM. In some embodiments, the TAA is EphA2. In some embodiments, the TAA is EphB2. In some embodiments, the TAA is ERBB2. In some embodiments, the TAA is ERG (TMPRSS2 ETS fusion gene). In some embodiments, the TAA is ETV6-AML. In some embodiments, the TAA is FAP. In some embodiments, the TAA is FCAR. In some embodiments, the TAA is FCRL5. In some embodiments, the TAA is FLT3. In some embodiments, the TAA is FLT3. In some embodiments, the TAA is folate receptor α.In some embodiments, the TAA is folate receptor β. In some embodiments, the TAA is Fos-associated antigen 1. In some embodiments, the TAA is fucosyl GM1. In some embodiments, the TAA is GD2. In some embodiments, the TAA is GD2. In some embodiments, the TAA is GD3. In some embodiments, the TAA is GloboH. In some embodiments, the TAA is GM3. In some embodiments, the TAA is gp100Tn. In some embodiments, the TAA is GPC3. In some embodiments, the TAA is GPNMB. In some embodiments, the TAA is GPR20. In some embodiments, the TAA is GPRC5D. In some embodiments, the TAA is GPR64. In some embodiments, the TAA is HAVCR1. In some embodiments, the TAA is HER3. In some embodiments, the TAA is HMWMAA. In some embodiments, the TAA is hTERT. In some embodiments, the TAA is IgF-I receptor. In some embodiments, the TAA is IGLL1. In some embodiments, the TAA is IL-11Ra. In some embodiments, the TAA is IL-13Ra2. In some embodiments, the TAA is KIT. In some embodiments, the TAA is LAIR1. In some embodiments, the TAA is LCK. In some embodiments, the TAA is LewisY. In some embodiments, the TAA is LILRA2. In some embodiments, the TAA is LMP2. In some embodiments, the TAA is LRP6. In some embodiments, the TAA is LY6K. In some embodiments, the TAA is LY75. In some embodiments, the TAA is LYPD8. In some embodiments, the TAA is MAD-CT-1. In some embodiments, the TAA is MAD-CT-2. In some embodiments, the TAA is mesothelin. In some embodiments, the TAA is ML-IAP. In some embodiments, the TAA is MUC1. In some embodiments, the TAA is MYCN. In some embodiments, the TAA is NA17. In some embodiments, the TAA is NCAM. In some embodiments, the TAA is NKG2D. In some embodiments, the TAA is NY-BR-1. In some embodiments, the TAA is o-acetyl-GD2. In some embodiments, the TAA is OR51E2. In some embodiments, the TAA is OY-TES1. In some embodiments, the TAA is a p53 mutant. In some embodiments, the TAA is PANX3. In some embodiments, the TAA is PAX3. In some embodiments, the TAA is PAX5.In some embodiments, the TAA is PDGFR-β. In some embodiments, the TAA is PLAC1. In some embodiments, the TAA is polysialic acid. In some embodiments, the TAA is PRSS21. In some embodiments, the TAA is PSCA. In some embodiments, the TAA is RhoC. In some embodiments, the TAA is ROR1. In some embodiments, the TAA is sarcoma translocation inflection point protein. In some embodiments, the TAA is SART3. In some embodiments, the TAA is SLC34A2. In some embodiments, the TAA is SLC39A6. In some embodiments, the TAA is sLe. In some embodiments, the TAA is SLITRK6. In some embodiments, the TAA is spermin 17. In some embodiments, the TAA is SSEA-4. In some embodiments, the TAA is SSX2. In some embodiments, the TAA is TAAG72. In some embodiments, the TAA is TAARP. In some embodiments, the TAA is TACSTD2. In some embodiments, the TAA is TEM1 / CD248. In some embodiments, the TAA is TEM7R. In some embodiments, the TAA is TGS5. In some embodiments, the TAA is Tie 2. In some embodiments, the TAA is Tn Ag. In some embodiments, the TAA is TSHR. In some embodiments, the TAA is a tyrosinase. In some embodiments, the TAA is UPK2. In some embodiments, the TAA is VEGFR2. In some embodiments, the TAA is WT1. In some embodiments, the TAA is XAGE1.

[0477] TAA ABMs can contain, for example, ligand- or antibody-based portions. For instance, in the case of BCMA as the TAA, the ABM can be APRIL, a BCMA ligand, or a portion thereof that binds to BCMA, or an anti-BCMA antibody or its antigen-binding fragment. Ligands and antibodies binding to TAAs are well known in the art. In the case of antibody-based portions, the anti-TAA antibody or antigen-binding fragment can contain, for example, the CDR sequence of the antibodies listed in Table 11. In some embodiments, the anti-TAA antibody or its antigen-binding domain has the heavy chain and light chain variable region sequences of the antibodies listed in Table 11.

[0478]

[0479]

[0480]

[0481]

[0482]

[0483] 6.7.1. BCMA

[0484] In some respects, this disclosure provides TBM, where ABM3 BCMA is a member of the tumor necrosis family receptor (TNFR) expressed on cells of the B cell lineage. BCMA expression is highest on terminally differentiated B cells with long-lived plasma cell fates, including plasma cells, plasmablasts, and subsets of activated and memory B cells. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has recently been associated with many cancers, autoimmune disorders, and infectious diseases. Cancers with increased BCMA expression include several hematologic malignancies such as multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, various leukemias, and glioblastoma.

[0485] The TBM containing an ABM that binds to BCMA may include, for example, an anti-BCMA antibody or its antigen-binding domain. The anti-BCMA antibody or its antigen-binding domain may contain, for example, CDR, VH, VL, or scFV sequences listed in Tables 12A-12G.

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511] In some embodiments, the ABM contains a CDR sequence of BCMA-1. In some embodiments, the ABM contains a CDR sequence of BCMA-2. In some embodiments, the ABM contains a CDR sequence of BCMA-3. In some embodiments, the ABM contains a CDR sequence of BCMA-4. In some embodiments, the ABM contains a CDR sequence of BCMA-5. In some embodiments, the ABM contains a CDR sequence of BCMA-6. In some embodiments, the ABM contains a CDR sequence of BCMA-7. In some embodiments, the ABM contains a CDR sequence of BCMA-8. In some embodiments, the ABM contains a CDR sequence of BCMA-9. In some embodiments, the ABM contains a CDR sequence of BCMA-10. In some embodiments, the ABM contains a CDR sequence of BCMA-11. In some embodiments, the ABM contains a CDR sequence of BCMA-12. In some embodiments, the ABM contains a CDR sequence of BCMA-13. In some embodiments, the ABM contains a CDR sequence of BCMA-14. In some embodiments, the ABM contains a CDR sequence of BCMA-15. In some embodiments, the ABM contains a CDR sequence of BCMA-16. In some embodiments, the ABM contains a CDR sequence of BCMA-17. In some embodiments, the ABM contains a CDR sequence of BCMA-18. In some embodiments, the ABM contains a CDR sequence of BCMA-19. In some embodiments, the ABM contains a CDR sequence of BCMA-20. In some embodiments, the ABM contains a CDR sequence of BCMA-21. In some embodiments, the ABM contains a CDR sequence of BCMA-22. In some embodiments, the ABM contains a CDR sequence of BCMA-23. In some embodiments, the ABM contains a CDR sequence of BCMA-24. In some embodiments, the ABM contains a CDR sequence of BCMA-25. In some embodiments, the ABM contains a CDR sequence of BCMA-26. In some embodiments, the ABM contains a CDR sequence of BCMA-27. In some embodiments, the ABM contains a CDR sequence of BCMA-28. In some embodiments, the ABM contains a CDR sequence of BCMA-29. In some embodiments, the ABM contains a CDR sequence of BCMA-30. In some embodiments, the ABM contains a CDR sequence of BCMA-31. In some embodiments, the ABM contains a CDR sequence of BCMA-32. In some embodiments, the ABM contains a CDR sequence of BCMA-33. In some embodiments, the ABM contains a CDR sequence of BCMA-34.In some embodiments, the ABM contains a CDR sequence of BCMA-35. In some embodiments, the ABM contains a CDR sequence of BCMA-36. In some embodiments, the ABM contains a CDR sequence of BCMA-37. In some embodiments, the ABM contains a CDR sequence of BCMA-38. In some embodiments, the ABM contains a CDR sequence of BCMA-39. In some embodiments, the ABM contains a CDR sequence of BCMA-40.

[0512] In some embodiments, the CDR is defined by a Kabat number, as listed in Tables 12B and 10E. In other embodiments, the CDR is defined by a Josiah number, as listed in Tables 12C and 10F. In still other embodiments, the CDR is defined by a combination of Kabat and Josiah numbers, as listed in Tables 12D and 10G.

[0513] In some embodiments, a TBM comprising an ABM bound to a BCMA may comprise a variable sequence of heavy and light chains of any one of BCMA-1 to BCMA-40.

[0514] In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-1, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-2, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-3, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-4, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-5, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-6, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-7, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-8, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-9, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-10, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-11, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-12, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-13, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-14, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-15, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-16, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-17, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-18, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-19, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-20, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-21, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-22, as listed in Table 12A.

[0515] In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-23, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-24, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-25, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-26, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-27, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-28, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-29, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-30, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-31, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-32, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-33, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-34, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-35, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-36, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-37, as listed in Table 12A. In some embodiments, the ABM comprises variable sequences of heavy and light chains of BCMA-38, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-39, as listed in Table 12A. In some embodiments, the ABM comprises a variable sequence of heavy and light chains of BCMA-40, as listed in Table 12A.

[0516] 6.7.2.CD19

[0517] B cell expression of cell surface proteins that can be used as markers for differentiation and identification. One such human B cell marker is the CD19 antigen, which is found on mature B cells but not on plasma cells. CD19 is expressed during early pre-B cell development and persists until plasma cell differentiation. CD19 is expressed on both normal B cells and malignant B cells whose abnormal growth can lead to B-cell lymphoma. For example, CD19 is expressed in B-cell lineage malignancies, including but not limited to non-Hodgkin's lymphoma (B-NHL), chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

[0518] In some respects, the TBM disclosed herein comprises ABM3 that specifically binds to CD19. Table 13 below lists exemplary CDR and variable domain sequences that can be incorporated into ABM3 that specifically binds to CD19.

[0519]

[0520]

[0521]

[0522] In some aspects, the ABM3 comprises a heavy chain CDR having the amino acid sequences of CD19-H1, CD19-H2A, and CD19-H3 as listed in Table 13; and a light chain CDR having the amino acid sequences of CD19-L1, CD19-L2, and CD19-L3 as listed in Table 13. In a specific embodiment, the ABM3 comprises a heavy chain variable region having the amino acid sequence of VHA as listed in Table 13; and a light chain variable region having the amino acid sequence of VLA as listed in Table 13.

[0523] In other respects, the ABM3 comprises a heavy chain CDR having the amino acid sequences of CD19-H1, CD19-H2B, and CD19-H3 as listed in Table 13; and a light chain CDR having the amino acid sequences of CD19-L1, CD19-L2, and CD19-L3 as listed in Table 13. In a specific embodiment, the ABM3 comprises a heavy chain variable region having the amino acid sequence of VHB as listed in Table 13; and a light chain variable region having the amino acid sequence of VLB as listed in Table 13.

[0524] In other respects, the ABM3 comprises a heavy chain CDR having the amino acid sequences of CD19-H1, CD19-H2C, and CD19-H3 as listed in Table 13; and a light chain CDR having the amino acid sequences of CD19-L1, CD19-L2, and CD19-L3 as listed in Table 13. In a specific embodiment, the ABM3 comprises a heavy chain variable region having the amino acid sequence of VHC as listed in Table 13; and a light chain variable region having the amino acid sequence of VLB as listed in Table 13.

[0525] In other respects, the ABM3 comprises a heavy chain CDR having the amino acid sequences of CD19-H1, CD19-H2D, and CD19-H3 as listed in Table 13; and a light chain CDR having the amino acid sequences of CD19-L1, CD19-L2, and CD19-L3 as listed in Table 13. In a specific embodiment, the ABM3 comprises a heavy chain variable region having the amino acid sequence of VHD as listed in Table 13; and a light chain variable region having the amino acid sequence of VLB as listed in Table 13.

[0526] In other respects, the ABM3 is in the form of scFV. An exemplary anti-CD19 scFv comprises an amino acid sequence of any one of CD19-scFv1 to CD19-scFv12 as listed in Table 13.

[0527] 6.8. Nucleic Acids and Host Cells

[0528] On the other hand, this disclosure provides nucleic acids encoding the TBM of this disclosure. In some embodiments, the TBM is encoded by a single nucleic acid. In other embodiments, the TBM is encoded by multiple nucleic acids (e.g., two, three, four or more).

[0529] A single nucleic acid can encode a TBM containing a single polypeptide chain, a TBM containing two or more polypeptide chains, or a portion of a TBM containing more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a TBM containing three, four, or more polypeptide chains, or three polypeptide chains of a TBM containing four or more polypeptide chains). For individual control of expression, read frames encoding two or more polypeptide chains can be under the control of individual transcriptional regulatory elements (e.g., promoters and / or enhancers). Read frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by internal ribosome entry site (IRES) sequences to allow translation into different polypeptides.

[0530] In some embodiments, a TBM comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the TBM may be equal to or less than the number of polypeptide chains in the TBM (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0531] The nucleic acid disclosed herein may be DNA or RNA (e.g., mRNA).

[0532] On the other hand, this disclosure provides a host cell and a vector containing the nucleic acid disclosed herein. The nucleic acid may be present in a single vector or in different vectors, and the vectors may be present in the same host cell or different host cells, as described in more detail below.

[0533] 6.8.1. Carrier

[0534] This disclosure provides vectors comprising nucleotide sequences encoding the TBM or TBM components described herein. In one embodiment, the vector comprises nucleotides encoding the immunoglobulin-based ABM described herein. In one embodiment, the vector comprises nucleotides encoding the Fc domain described herein. In one embodiment, the vector comprises nucleotides encoding the recombinant non-immunoglobulin-based ABM described herein. The vectors of this disclosure may encode one or more ABMs, one or more Fc domains, one or more non-immunoglobulin-based ABMs, or combinations thereof (e.g., when multiple components or sub-components are encoded as a single polypeptide chain). In one embodiment, the vector comprises the nucleotide sequences described herein. The vectors include, but are not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs).

[0535] A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Laurel's sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern Equine Encephalitis virus, and flaviviruses.

[0536] Furthermore, cells that stably integrate DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. These markers can provide proton shifts to, for example, auxotrophic hosts, provide resistance to antimicrobial agents (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Optimal mRNA synthesis may also require additional elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0537] Once an expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into suitable host cells. This can be achieved using various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used for culturing the resulting transfected cells and recovering the expressed peptide are known to those skilled in the art and can be modified or optimized based on the specific expression vector and mammalian host cells used in this specification.

[0538] 6.8.2. Cells

[0539] This disclosure also provides host cells containing the nucleic acids disclosed herein.

[0540] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0541] In one embodiment, host cell genetic engineering is achieved by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of influencing gene expression in a host compatible with such a sequence. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Other factors necessary or helpful in achieving expression, such as inducible promoters, may also be used.

[0542] This disclosure also provides host cells containing the vectors described herein.

[0543] The cells may 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.

[0544] 6.9. Antibody-drug conjugates

[0545] The TBMs disclosed herein can be conjugated to a drug component, for example, via a linker. For convenience, such conjugates are referred to herein as antibody-drug conjugates (or “ADCs”), although it is true that one or more (or all) of the said ABMs may be based on a non-immunoglobulin scaffold.

[0546] In some respects, the drug portion exerts cytotoxic or cell-inhibiting activity. In one embodiment, the drug portion is selected from maytansine alkaloids, kinin-like protein KIF11 inhibitors, and V-ATPase (vacuole H+). -ATPase inhibitors, apoptosis-promoting agents, Bcl2 (B-cell lymphoma 2) inhibitors, MCL1 (myeloid leukemia 1) inhibitors, HSP90 (heat shock protein 90) inhibitors, IAP (inhibitor of apoptosis) inhibitors, mTOR (mechanistic target of rapamycin) inhibitors, microtubule stabilizers, microtubule destabilizers, orestatine, dolalastatin, MetAP (methionine aminopeptidase), CRM1 (chromosome maintenance 1) inhibitors, DPPIV (dipeptidyl peptidase IV) inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 (cyclin-dependent kinase 2) inhibitors, CDK9 (cyclin-dependent kinase 9) inhibitors, kinin inhibitors, HDAC (histone deacetylase) inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, RNA polymerase inhibitors, topoisomerase inhibitors, or DHFR (dihydrofolate reductase) inhibitors.

[0547] In one embodiment, the connector is selected from cuttable connectors, non-cuttable connectors, hydrophilic connectors, procharged connectors, or dicarboxylic acid-based connectors.

[0548] In a specific embodiment, the ADC is a compound according to structural formula (I):

[0549] [DL-XY] n -Ab

[0550] Or its salts, wherein each “D” independently represents a cytotoxic agent and / or a cell inhibitor (“drug”); each “L” independently represents a linker; “Ab” represents the TBM described herein; each “XY” represents the functional group R on the linker. x And the complementary functional group R on the antibody y The connection formed between them, and n represents the number of drugs connected to the ADC, or the drug-to-antibody ratio (DAR) of the ADC.

[0551] Specific embodiments of various antibodies (Abs) that may contain ADCs include the various embodiments of TBM described above.

[0552] In some specific embodiments of the ADC and / or salt of structural formula (I), each D is the same and / or each L is the same.

[0553] Specific embodiments of the cytotoxic agent and / or cell inhibitor (D) and adapter (L) of the ADC disclosed herein, as well as the number of cytotoxic agents and / or cell inhibitors linked to the ADC, are described in more detail below.

[0554] 6.9.1. Cytotoxic agents and / or cell inhibitors

[0555] The cytotoxic agents and / or cell inhibitors may be any agents known to inhibit cell growth and / or cell replication and / or kill cells, particularly cancer and / or tumor cells. Many agents with cytotoxic and / or cell-inhibiting properties are known in the literature. Non-limiting examples of various types of cytotoxic agents and / or cell inhibitors include, for example, but not limited to, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalating agents (e.g., groove-binding agents, such as minor groove binders), RNA / DNA antimetabolites, cell cycle regulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents.

[0556] The following are specific, non-limiting examples of some of these various types of pharmaceuticals.

[0557] Alkylating agents: asaley ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC 167780; CAS Registry No. 3577897)); AZQ ((1,4-cyclohexadiene-1,4-diaminocarboxylic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CAS Registry No. 57998682)); BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC 409962; CAS Registry No. 154938)); busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS Registry No. 55981); (carboxyphthalic acid)platinum (NSC 27164; CAS Registry No. 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxyl)diammineplatin(II)); NSC 241240; CAS Registry No. 41575944)); CCNU ((N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS Registry No. 13010474)); CHIP (isopropylplatinum; NSC 256927); chlorambucil (NSC 3088; CAS Registry No. 305033); chloramphenicol ((2-[[[(2-chloroethyl)nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC 178248; CAS Registry No. 54749905)); cisplatin (cisplatin; NSC 119875; CAS registration number 15663271); Clomesone (NSC 338947; CAS registration number 88343720); Cyanomorpholine doxorubicin (NCS 357704; CAS registration number 88254073); Cyclodisone (NSC 348948; CAS registration number 99591738); Dihydrogalactitol (5,6-diepoxygalactitol; NSC 132313; CAS registration number 23261203); Fluorodopa ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil; NSC 73754; CAS registration number 834913); Heptanylsulfamethoxazole (NSC 329680; CAS registration number 96892578); Heinzone (NSC 329680; CAS registration number 96892578); 142982; CAS registration number 23255938); melphalan (NSC 8806; CAS registration number 3223072); methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC 95441; 13909096); mitomycin C (NSC 26980; CAS registration number 50077);Mitozolamide (NSC353451; CAS Registry No. 85622953); Nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride; NSC 762; CAS Registry No. 55867); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidinyl)-1-nitrosourea; NSC 95466; CAS Registry No. 13909029)); Piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC344007)); Piperazine dione (NSC 135758; CAS Registry No. 41109802); Pipobroman ((N,N-bis(3-bromopropionyl)piperazine; NSC 135758; CAS Registry No. 41109802); Pipobroman ((N,N-bis(3-bromopropionyl)piperazine; NSC 135758; CAS Registry No. 41109802); 25154; CAS registration number 54911); N-methylmitomycin C (NSC56410; CAS registration number 801525); Spiral hydantoin mustard (NSC 172112; CAS registration number: 56605164); Tiroxitron (triglycidyl isocyanurate; NSC 296934; CAS registration number 2451629); Tetrachlorocyclohexylplatin (NSC 363812; CAS registration number 62816982); Thiotepa (N,N',N''-tris-1,2-ethylenedimethylthiophosphoramide; NSC6396; CAS registration number 52244); Triethylene melamine (NSC 9706; CAS registration number 51183); Uracil nitrogen mustard (desmethyldopan; NSC 34462; CAS Registry No. 66751); Yoshi-864 ((bis(3-methoxymethoxypropyl)amine hydrochloride; NSC 102627; CAS Registry No. 3458228).

[0558] Topoisomerase I inhibitorsCamptothecin (NSC 94600; CAS Registry No. 7689-03-4); various camptothecin derivatives and analogues (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC295500, NSC 249910, NSC 606985, NSC 74028, NSC 176323, NSC 295501, NSC 606172, NSC606173, NSC 610458, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC364830, and NSC...). 606497); morpholinisoxorubicin (NSC 354646; CAS registration number 89196043); SN-38 (NSC 673596; CAS registration number 86639-52-3).

[0559] Topoisomerase II inhibitorsDoxorubicin (NSC 123127; CAS Registry No. 25316409); Benzisoquinolinedione (NSC 308847; CAS Registry No. 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxymethanesulfonylaniline; NSC 249992; CAS Registry No. 51264143)); Anthraquinone derivatives ((NSC 355644); Etoposide (VP-16; NSC 141540; CAS Registry No. 33419420); Pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propylamine, 9-methoxy-N,N-dimethoxy-5-nitro-,monomethylsulfonate; NSC 366140; CAS Registry No. 99009219); Bisantrene hydrochloride (NSC 337766; CAS Registry No. 71439684); Daunorubicin (NSC 821151; CAS Registry No. 23541506); Doxorubicin (NSC 267469; CAS Registry No. 63950061); Mitoxantrone (NSC 301739; CAS Registry No. 70476823); Minoril (NSC 269148; CAS Registry No. 71628961); N,N-Dibenzyl benzoylmycin (NSC 268242; CAS Registry No. 70878512); Oxanthrazole (NSC 366140; CAS Registry No. 99009219); Bisantrene hydrochloride (NSC 337766; CAS Registry No. 71439684); Daunorubicin (NSC 821151; CAS Registry No. 23541506); Deoxydoxime (NSC 267469; CAS Registry No. 63950061); Mitoxantrone (NSC 301739; CAS Registry No. 70476823); Minoril (NSC 269148; CAS Registry No. 71628961); N,N-Dibenzyl benzoylmycin (NSC 268242; CAS Registry No. 70878512); Oxanthrazole (NSC 366140; CAS Reg 349174; CAS registration number 105118125); rubidazone (NSC164011; CAS registration number 36508711); teniposide (VM-26; NSC 122819; CAS registration number 29767202).

[0560] DNA intercalating agentTriamcinolone acetonide (CAS registration number 4803274); Antimycin A (CAS registration number 89675376); Tomamycin (CAS registration number 35050556); DC-81 (CAS registration number 81307246); Siberiamycin (CAS registration number 12684332); Pyrrolobenzodiazepines (CAS registration number 945490095); SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl)-- 5-Oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5(11aH)-one); SG2000 (SJG-136; (11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3--dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5(11aH)-one); NSC 694501; CAS registration number 232931576).

[0561] RNA / DNA antimetabolites: L-Aranoxin (NSC 153353; CAS Registry No. 59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672); 5-Fluorouracil (NSC 19893; CAS Registry No. 51218); Acevitin (NSC 163501; CAS Registry No. 42228922); aminopterin derivatives N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazoline)methyl]amino]benzoyl-]L-aspartic acid (NSC 132483); aminopterin derivatives N-[4-[[(2,4-diamino-5-ethyl-6-quinazoline)methyl]amino]benzoyl]L-aspartic acid (NSC 153353; CAS Registry No. 59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672); 5-Fluorouracil (NSC 19893; CAS Registry No. 51218); Acevitin (NSC 163501; CAS Registry No. 42228922); aminopterin derivatives N-[2-chloro-5-[[(2,4-diamino-5-ethyl-6-quinazoline)methyl]amino]benzoyl]L-aspartic acid (NSC 153353; CAS Registry No. 59163416); 5-azacytidine (NSC 102816; CAS Registry No. 320672); 5-fluorouracil (NSC 19893; CAS Registry No. 51 184692); aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC 134033); folic acid antagonist (antifo) ((N α -(4-amino-4-deoxypteroyl)-N 7-Hymenphthaloyl-L-ornithine; NSC 623017); Baker's soluble antifol (NSC 139105; CAS registration number 41191042); Dichloroallyl quinone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC126771; CAS registration number 36417160); brequinar (NSC 368390; CAS registration number 96201886); ftorafur ((prodrug; 5-fluoro-1-(tetrahydro-2-furanyl)-uracil; NSC 148958; CAS registration number 37076689); 5,6-dihydro-5-azacytidine (NSC 264880; CAS registration number 62402317); Methotrexate (NSC740; CAS registration number 59052); Methotrexate derivatives (N-[[4-[[(2,4-diamino-6-pteridyl)methyl]methylamino]-1-naphthyl]carbonyl]L-glutamic acid; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartic acid ester; NSC224131; CAS registration number 603425565); Pyrazofurin (NSC 143095; CAS registration number 30868305); Trimetrexate (NSC 352122; CAS registration number 82952645).

[0562] DNA antimetabolites: 3-HP (NSC 95678; CAS Registry No. 3814797); 2'-deoxy-5-fluorouracil (NSC27640; CAS Registry No. 50919); 5-HP (NSC 107392; CAS Registry No. 19494894); α-TGDR (α-2'-deoxy-6-thioguanine; NSC 71851 CAS Registry No. 2133815); aphidicolin glycinate (NSC 303812; CAS Registry No. 92802822); ara C (cytarabine; NSC 63878; CAS Registry No. 69749); 5-aza-2'-deoxycytidine (NSC 127716; CAS registration number 2353335); β-TGDR (β-2'-deoxy-6-thioguanine; NSC71261; CAS registration number 789617); Cyclocytosine (NSC 145668; CAS registration number 10212256); Guanidine (NSC 1895; CAS registration number 1455772); Hydroxyurea (NSC 32065; CAS registration number 127071); Inosine glycodialdehyde (NSC 118994; CAS registration number 23590990); Macbecin II (NSC330500; CAS registration number 73341738); Pyrazoloimidazole (NSC 51143; CAS registration number 6714290); Thioguanine (NSC 127716; CAS registration number 2353335); β-TGDR (β-2'-deoxy-6-thioguanine; NSC71261; CAS registration number 789617); Cyclocytosine (NSC 145668; CAS registration number 10212256); Guanidine (NSC 1895; CAS registration number 1455772); Inosine urea (NSC 32065; CAS registration number 127071); Inosine glycodialdehyde (NSC 118994; CAS registration number 23590990); Macbecin II (NSC 330500; CAS registration number 73341738); Pyrazoloimidazole (NSC 51143; CAS registration number 6714290); Thioguanine (NSC 127716; CAS registration number 789617); Cyclocytosine ( 752; CAS Registry No. 154427); Thiopurine (NSC 755; CAS Registry No. 50442).

[0563] Cell cycle regulatorsSilymarin (CAS registration number 22888-70-6); Epigallocatechin gallate (EGCG; CAS registration number 989515); Proanthocyanidin derivatives (e.g., proanthocyanidin A1 [CAS registration number 103883030], proanthocyanidin B1 [CAS registration number 20315257], proanthocyanidin B4 [CAS registration number 29106512], arecotannin B1 [CAS registration number 79763283]); Isoflavones (e.g., genistein [4',5,7-trihydroxyisoflavone; CAS registration number 446720], daidzein [4',7-dihydroxyisoflavone, CAS registration number 486668]); Indole-3-carbinol (CAS registration number 700061); Quercetin (NSC) 9219; CAS registration number 117395); estradiol (NSC 89201; CAS registration number 2998574); nocodazole (CAS registration number 31430189); podophyllotoxin (CAS registration number 518285); vinorelbine tartrate (NSC 608210; CAS registration number 125317397); candidacin (NSC 667642; CAS registration number 124689652).

[0564] Kinase inhibitors:Afatinib (CAS registration number 850140726); Axitinib (CAS registration number 319460850); ARRY-438162 (binimetinib) (CAS registration number 606143899); Bosutinib (CAS registration number 380843754); Cabozantinib (CAS registration number 1140909483); Ceritinib (CAS registration number 1032900256); Crizotinib (CAS registration number 877399525); Dabrafenib (CAS registration number 1195765457); Dasatinib (NSC 732517; CAS registration number 302962498); Erlotinib (NSC 718781; CAS registration number 183319699); Everolimus (NSC 733504; CAS Registry No. 159351696); Fortenatinib (NSC 745942; CAS Registry No. 901119355); Gefitinib (NSC 715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC 716051; CAS Registry No. 220127571); Lapatinib (CAS Registry No. 388082788); Lenvatinib (CAS Registry No. 857890392); Muritinib (CAS 366017096); Nilotinib (CAS Registry No. 923288953); Nintedanib (CAS Registry No. 656247175); Palbociclib (CAS Registry No. 571190302); Pazopanib (NSC 733504; CAS Registry No. 159351696); Fortenatinib (NSC 745942; CAS Registry No. 901119355); Gefitinib (NSC 715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC 716051; CAS Registry No. 220127571); Lapatinib (CAS Registry No. 388082788); Lenvatinib (CAS Registry No. 857890392); Muritinib (CAS 366017096); Ni 737754; CAS Registration No. 635702646); Pigastanib (CAS Registration No. 222716861); Panatinib (CAS Registration No. 1114544318); Rapamycin (NSC 226080; CAS Registration No. 53123889); Regorafenib (CAS Registration No. 755037037); AP 23573 (Desfomolix) (CAS Registration No. 572924540); INCB018424 (Ruxolitinib) (CAS Registration No. 1092939177); ARRY-142886 (Selmetinib) (NSC 741078; CAS Registration No. 606143-52-6); Sirolitinib (NSC 226080; CAS Registration No. 53123889); Sorafenib (NSC 226080; CAS Registration No. 53123889); 724772; CAS Registration No. 475207591); Sunitinib (NSC 736511; CAS Registration No. 341031547); Tofacitinib (CAS Registration No. 477600752); Tessirolimus (NSC 683864; CAS Registration No. 163635043); Trametinib (CAS Registration No. 871700173); Vandetanib (CAS Registration No. 443913733);Vemurafenib (CAS Registry No. 918504651); SU6656 (CAS Registry No. 330161870); CEP-701 (lesaurtinib) (CAS Registry No. 111358884); XL019 (CAS Registry No. 945755566); PD-325901 (CAS Registry No. 391210109); PD-98059 (CAS Registry No. 167869218); ATP-competitive TORC1 / TORC2 inhibitors, including PI-103 (CAS Registry No. 371935749), PP242 (CAS Registry No. 1092351671), PP30 (CAS Registry No. 1092788094), and Torin. 1 (CAS Registration No. 1222998368), LY294002 (CAS Registration No. 154447366), XL-147 (CAS Registration No. 934526893), CAL-120 (CAS Registration No. 870281348), ETP-45658 (CAS Registration No. 1198357797), PX 866 (CAS Registration No. 502632668), GDC-0941 (CAS Registration No. 957054307), BGT226 (CAS Registration No. 1245537681), BEZ235 (CAS Registration No. 915019657), XL-765 (CAS Registration No. 934493762).

[0565] Protein synthesis inhibitorsAcriflavine (CAS registration number 65589700); Amikacin (NSC 177001; CAS registration number 39831555); Abakacin (CAS registration number 51025855); Asmicin (CAS registration number 55779061); Azithromycin (NSC 643732; CAS registration number 83905015); Kanamycin (CAS registration number 4696768); Chlortetracycline (NSC 13252; CAS registration number 64722); Clarithromycin (NSC 643733; CAS registration number 81103119); Clindamycin (CAS registration number 18323449); Clomocycline (CAS registration number 1181540); Cycloheximide (CAS registration number 66819); Dactinomycin (NSC 177001; CAS registration number 39831555); 3053; CAS registration number 50760); dalfopridin (CAS registration number 112362502); demecycline (CAS registration number 127333); dibekacin (CAS registration number 34493986); dihydrostreptomycin (CAS registration number 128461); erythromycin (CAS registration number 62013041); doxycycline (CAS registration number 17086281); emetine (NSC 33669; CAS registration number 483181); erythromycin (NSC 55929; CAS registration number 114078); fluerythromycin (CAS registration number 83664208); framycetin (neomycin B; CAS registration number 119040); gentamicin (NSC 3053; CAS registration number 50760); neomycin (CAS registration number 119040); gentamicin (NSC 3053; CAS registration number 50760); dalfopridin (CAS registration number 112362502); demecycline (CAS registration number 127333); dibekacin (CAS registration number 34493986); dihydrostreptomycin (CAS registration number 128461); erythromycin (NSC 3053; CAS registration number 407661); erythromycin (NSC 3053; CAS registration number 50760); dalfopridin (CAS registration number 112362502); dibekacin (CAS registration number 1273 ... 82261; CAS registration number 1403663); glycine derivatives, such as tigecycline (CAS registration number 220620097); hygromycin B (CAS registration number 31282049); isapamicin (CAS registration number 67814760); josamycin (NSC 122223; CAS registration number 16846245); kanamycin (CAS registration number 8063078); ketone lactones, such as telithromycin (CAS registration number 191114484), quinephedrine (CAS registration number 205110481), and sophormycin (CAS registration number 760981837); gentamicin (CAS registration number 154212); lysine (CAS registration number 992212); methylcycline ...992212); methylcycline (NSC 122223; CAS registration number 992212); methylcycline (NSC 122223; CAS registration number 992212); methylcycline (NSC 122223; CAS registration number 992212); methylcycline (NSC 122223; CAS registration 78502; CAS Registration No. 2013583); Metacycline (metacycline hydrochloride (rondomycin); NSC 356463; CAS Registration No. 914001); Midecamycin (CAS Registration No. 35457808); Minocycline (NSC 141993;CAS Registration No. 10118908; Mevocalycin (CAS Registration No. 55881077); Neomycin (CAS Registration No. 119040); Netilmicin (CAS Registration No. 56391561); Prunobycin (CAS Registration No. 3922905); Oxazolidinones, such as Ipizodil (CAS Registration No. 165800044), Linezolid (CAS Registration No. 165800033), Preszolid (CAS Registration No. 252260029), Radezolid (CAS Registration No. 869884786), Ranbezolid (CAS Registration No. 392659380), Sutezolid (CAS Registration No. 168828588), Terizolamide (CAS Registration No. 856867555); Oxytetracycline (NSC) 9169; CAS Registry No. 2058460); Paromomycin (CAS Registry No. 7542372); Piperazine (CAS Registry No. 4599604); Peptidyl transferase inhibitors, such as chloramphenicol (NSC 3069; CAS Registry No. 56757) and derivatives, such as chloramphenicol azidochloramphenicol (CAS Registry No. 13838089), florfenicol (CAS Registry No. 73231342), and thiamphenicol (CAS Registry No. 15318453), and pleuromutilins such as retaparin (CAS Registry No. 224452668), tiamulin (CAS Registry No. 55297955), and voremurin (CAS Registry No. 101312929); Pirimibromycin (CAS Registry No. 79548735); Puromycin (NSC 3069; CAS Registry No. 79548735); 3055; CAS registration number 53792); Quinupordine (CAS registration number 120138503); Ribomycin (CAS registration number 53797356); Rotamycin (CAS registration number 74014510); Rolimycin (CAS registration number 751973); Roxithromycin (CAS registration number 80214831); Sisomicin (CAS registration number 32385118); Spectinomycin (CAS registration number 1695778); Spiramycin (CAS registration number 8025818); Streptomycin, such as Punamycin (CAS registration number 270076603), Quinupordine / Dalfopristin (CAS registration number 126602899), and Vitamex (CAS registration number 11006761); Streptomycin (CAS registration number 57921); Tetracycline (NSC 108579; CAS Registry No. 60548); Tobramycin (CAS Registry No. 32986564); Acetylosin (CAS Registry No. 2751099); Tylosin (CAS Registry No. 1401690); Vimalamicin (CAS Registry No. 49863481).

[0566] Histone deacetylase inhibitors abexilinostat (CAS registration number 783355602); belistat (NSC 726630; CAS registration number 414864009); chidamide (CAS registration number 743420022); entenoxetine (CAS registration number 209783802); givinostat (CAS registration number 732302997); mocetinostat (CAS registration number 726169739); pabistat (CAS registration number 404950807); quisic Quisinostat (CAS registration number 875320299); Resminostat (CAS registration number 864814880); Romidesin (CAS registration number 128517077); Sulforaphane (CAS registration number 4478937); Thioureidobutyronitrile (Kevetrin™; CAS registration number 6659890); Valproic acid (NSC93819; CAS registration number 99661); Vorinostat (NSC93819; CAS registration number 99661); 701852 (CAS Registration No. 149647789); ACY-1215 (rocilinostat; CAS Registration No. 1316214524); CUDC-101 (CAS Registration No. 1012054599); CHR-2845 (tefinostat; CAS Registration No. 914382608); CHR-3996 (CAS Registration No. 1235859138); 4SC-202 (CAS Registration No. 910462430); CG200745 (CAS Registration No. 936221339); SB939 (pracinostat; CAS Registration No. 929016966).

[0567] Mitochondrial inhibitorsPancratistatin (NSC 349156; CAS Registry No. 96281311); Rhodamine-123 (CAS Registry No. 63669709); Edifosine (NSC 324368; CAS Registry No. 70641519); d-α-tocopherol succinate (NSC 173849; CAS Registry No. 4345033); Compound 11β (CAS Registry No. 865070377); Aspirin (NSC 123) 406186; CAS registration number 50782); Rosacetic acid (CAS registration number 519233); Berberine (CAS registration number 633658); Phytostyramine (CAS registration number 17397896); GX015-070 (Obatoclax®; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrolo-2-yl)methylene)-3-methoxy-2H-pyrrolo-5-yl)-; NSC 729280; CAS registration number 803712676); Celastrol (Tripterygium wilfordii; CAS registration number 34157830); Metformin (NSC 91485; CAS registration number 1115704); Brilliant Green (NSC 5011; CAS registration number 633034); ME-344 (CAS registration number 1374524556).

[0568] Antimitotic agents:Colchicine (NSC 406042); orlistatine, such as MMAE (monomethyl orlistatine E; CAS registration number 474645-27-7) and MMAF (monomethyl orlistatine F; CAS registration number 745017-94-1); Soft sponge B (NSC 609395); Colchicine (NSC 757; CAS registration number 64868); Colchicine derivatives (N-benzoyl-deacetylated benzamide; NSC 33410; CAS registration number 63989753); Tail slug 10 (NSC 376128; CAS registration number 110417-88-4); Maytansine (NSC 609395); 153858; CAS Registration No. 35846-53-8); rhozoxin (NSC332598; CAS Registration No. 90996546); paclitaxel (NSC 125973; CAS Registration No. 33069624); paclitaxel derivatives ((2'-N-[3-(dimethylamino)propyl]glutaric acid paclitaxel; NSC 608832); colchicine (3-demethyl colchicine; NSC 361792); triphenylmethylcysteine ​​(NSC 49842; CAS Registration No. 2799077); vincristine sulfate (NSC 49842; CAS Registration No. 143679); vincristine sulfate (NSC 67574; CAS Registration No. 2068782).

[0569] Any of these agents, including or potentially modified to include attachment sites with TBM, may be included in the ADCs disclosed herein.

[0570] In a specific embodiment, the cytotoxic agent and / or cell inhibitor is an antimitotic agent.

[0571] In another specific embodiment, the cytotoxic agent and / or cell inhibitor is orlistatine, for example, monomethylorlistatine E (“MMAE:”) or monomethylorlistatine F (“MMAF”).

[0572] 6.9.2. ADC Connector

[0573] In the ADC disclosed herein, the cytotoxic agent and / or cell inhibitor are linked to the TBM via ADC adapters. The ADC adapters linking the cytotoxic agent and / or cell inhibitor to the ADC's TBM can be short, long, hydrophobic, hydrophilic, flexible, or rigid, or can be composed of fragments each independently possessing one or more of the aforementioned properties, such that the adapters can include fragments with different properties. The adapters can be multivalent, such that they covalently link more than one agent to a single site on the TBM, or the adapters can be monovalent, such that they covalently link a single agent to a single site on the TBM.

[0574] As those skilled in the art will understand, the ADC adapter connects the cytotoxic agent and / or cell inhibitor to the TBM by forming a covalent link at one location with the cytotoxic agent and / or cell inhibitor and a covalent link at another location with the TBM. The covalent link is formed through a reaction between functional groups on the ADC adapter and functional groups on the agent and the TBM. As used herein, the expression “ADC adapter” is intended to include (i) an unconjugated form of the ADC adapter comprising functional groups capable of covalently linking the ADC adapter to the cytotoxic agent and / or cell inhibitor and functional groups capable of covalently linking the ADC adapter to the TBM; (ii) a partially conjugated form of the ADC adapter comprising functional groups capable of covalently linking the ADC adapter to the TBM and covalently linking the cytotoxic agent and / or cell inhibitor, and vice versa; and (iii) a fully conjugated form of the ADC adapter covalently linked to both the cytotoxic agent and / or cell inhibitor and the TBM. In some specific embodiments of the ADC connector and ADC disclosed herein, and the synthon for conjugating the connector-drug agent to the TBM, the portion comprising the functional group on the ADC connector and the covalent connection formed between the ADC connector and the TBM are specifically represented as R, respectively. x And XY.

[0575] The ADC adapter preferably (but not necessarily) is chemically stable to extracellular conditions and can be designed to specifically cleave, die, and / or otherwise degrade within cells. Alternatively, ADC adapters not designed to specifically cleave or degrade within cells can be used. The choice of a stable or unstable ADC adapter may depend on the toxicity of the cytotoxic agent and / or cell inhibitor. For agents toxic to normal cells, a stable adapter is preferred. For selective or targeted agents with low toxicity to normal cells, the chemical stability of the ADC adapter to the extracellular environment is less important. A variety of ADC adapters that can be used to link drugs to TBMs in the context of an ADC are known in the art. Any of these and other ADC adapters can be used to link cytotoxic agents and / or cell inhibitors to the TBM of the ADC disclosed herein.

[0576] Exemplary multivalent ADC connectors that can be used to link numerous cytotoxic agents and / or cell inhibitors to a single TBM molecule are described, for example, in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; WO2014 / 093640, the contents of which are incorporated herein by reference in their entirety. For example, the Fleximer connector technology developed by Mersana et al. has the potential to enable high-DAR ADCs with favorable physicochemical properties. As shown below, the Mersana technology is based on incorporating drug molecules into a solubilized polyacetal backbone via a series of ester bonds. The method can provide a high-load ADC (DAR up to 20) while maintaining good physicochemical properties.

[0577] Further examples of dendritic junctions can be found in the following literature: US 2006 / 116422; US 2005 / 271615; de Groot et al., 2003, Angew. Chem. Int. Ed. [Applied Chemistry - International Edition] 42: 4490-4494; Amir et al., 2003, Angew. Chem. Int. Ed. [Applied Chemistry - International Edition] 42: 4494-4499; Shamis et al., 2004, J. Am. Chem. Soc. [Journal of the American Chemical Society] 126: 1726-1731; Sun et al., 2002, Bioorganic & Medicinal Chemistry Letters [Bioorganic & Medicinal Chemistry Letters] 12: 2213-2215; Sun et al., 2003, Bioorganic & Medicinal Chemistry [Bioorganic & Medicinal Chemistry Letters] 11: 1761-1768; King et al., 2002, Tetrahedron Letters 43: 1987-1990, each incorporated herein by reference.

[0578] Exemplary monovalent ADC connectors that can be used are described in, for example, Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045: 71-100; Kitson et al., 2013, CROs-MOs--Chemica-ggi--Chemistry Today 31(4): 30-38; Ducry et al., 2010, BioconjugateChem. 21: 5-13; Zhao et al., 2011, J. Med. Chem. 54: 3606-3623; US Patent Nos. 7,223,837, 8,568,728, and 8,535,678; and WO In 2004010957, each of them is incorporated into this article through citation.

[0579] For example, but not limited to, some cuttable and non-cuttable ADC connectors described in this disclosure may be included as follows.

[0580] 6.9.2.1. Cuttable ADC connector

[0581] In some embodiments, the selected ADC adapter is cleavable in vivo. A cleavable ADC adapter may include chemically or enzymatically unstable or degradable links. Cleavable ADC adapters typically rely on intracellular processes to release drugs, such as reduction of cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. Cleavable ADC adapters typically incorporate one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the ADC adapter is incleavable. In some embodiments, the ADC adapter contains chemically unstable groups, such as hydrazones and / or disulfide groups. Adapters containing chemically unstable groups utilize the differential properties between plasma and certain cytoplasmic compartments. Intracellular conditions that promote drug release from hydrazone-containing ADC adapters are the acidic environment of endosomes and lysosomes, while disulfide-containing ADC adapters are reduced in cytosols containing high concentrations of thiols, such as glutathione. In some embodiments, the plasma stability of ADC adapters containing chemically unstable groups can be improved by introducing steric hindrance using substituents near the chemically unstable groups.

[0582] Acid-labile groups, such as hydrazones, remain intact during systemic circulation in a neutral blood pH environment (pH 7.3–7.5) and are hydrolyzed to release the drug once the ADC is internalized into the compartments of the cell's mildly acidic endosomes (pH 5.0–6.5) and the lysosomes (pH 4.5–5.0). This pH-dependent release mechanism is associated with non-specific drug release. To improve the stability of the hydrazone groups at the ADC linker, the ADC linker can be modified chemically (e.g., by substitution), allowing for more efficient release in lysosomes while minimizing cyclic losses.

[0583] The hydrazone-containing ADC adapter may contain additional cleavage sites, such as additional acid-labile and / or enzyme-labile cleavage sites. An ADC including an exemplary hydrazone-containing ADC adapter comprises the following structure:

[0584]

[0585] Where D and Ab represent cytotoxic agents and / or cell inhibitors (drugs) and Ab, respectively, and n represents the number of drug-ADC connectors linked to TBM. In some ADC connectors, such as connector (Ig), the ADC connector comprises two cleavable groups—a disulfide and an hydrazone moiety. For such ADC connectors, efficient release of the unmodified free drug requires an acidic pH or a disulfide reduction and acidic pH. Connectors such as (Ih) and (Ii) have been shown to be effective at a single hydrazone cleavage site.

[0586] When ADCs are internalized into acidic cellular compartments, additional ADC linkers that remain intact during systemic circulation and undergo hydrolysis to release the drug include carbonates. Such ADC linkers can be used in situations where cytotoxic agents and / or cell inhibitors can be covalently attached to oxygen.

[0587] Other acid-destabilizing groups that may be included in the ADC connector include ADC connectors containing a cis-aconitol group. Cis-aconitol chemicals use a carboxylic acid juxtaposed with the amide bond to accelerate the hydrolysis of the amide under acidic conditions.

[0588] Cleavable ADC connectors can also include disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs upon internalization within cells, where the cytoplasm provides a significantly more reducing environment compared to the extracellular environment. Cleavage of disulfide bonds typically requires the presence of cytoplasmic thiol cofactors, such as (reduced) glutathione (GSH), making disulfide-containing ADC connectors fairly stable in circulation and selectively releasing drugs from the cytosol. Intracellular enzymatic proteins disulfide isomerases or similar enzymes capable of cleaving disulfide bonds can also promote preferential cleavage of intracellular disulfide bonds. GSH has been reported to exist in cells at concentrations ranging from 0.5–10 mM, compared to significantly lower concentrations of circulating GSH or cysteine ​​(the most abundant low-molecular-weight thiol) in approximately five types of tumor cells, where irregular blood flow leads to hypoxia, resulting in enhanced reductase activity and thus even higher glutathione concentrations. In some embodiments, the in vivo stability of the ADC connector containing disulfide can be enhanced by chemical modification of the ADC connector, for example, by using steric hindrance adjacent to the disulfide bond.

[0589] An ADC including an exemplary ADC connector containing disulfide has the following structure:

[0590]

[0591] Where D and Ab represent the drug and TBM, respectively, n represents the number of drug-ADC connectors linked to the TBM, and R is independently selected, for example, from hydrogen or alkyl groups each time it appears. In some embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the ADC connector. When one or more R groups are selected from lower alkyl groups such as methyl, structures such as (Ij) and (Il) exhibit improved in vivo stability.

[0592] Another type of cleavable ADC adapter that can be used is the ADC adapter, which is specifically cleaved by an enzyme. Such ADC adapters are typically peptide-based or include peptide regions that act as substrates for the enzyme. Peptide-based ADC adapters tend to be more stable in plasma and extracellular environments compared to chemically unstable ADC adapters. Peptide bonds generally exhibit good serum stability because lysosomal proteases have very low activity in the blood due to endogenous inhibitors and have a higher blood pH, which is unfavorable compared to lysosomes. Drug release from TBM occurs particularly due to the action of lysosomal proteases, such as cathepsins and plasmin. These proteases can be present at elevated levels in some tumor cells.

[0593] In an exemplary embodiment, the cleavable peptide is selected from tetrapeptides, such as Gly-Phe-Leu-Gly (SEQ ID NO: 716) and Ala-Leu-Ala-Leu (SEQ ID NO: 717); or dipeptides, such as Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, phenyl Gly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Met-(D)Lys, and Asn-(D)Lys. In some embodiments, polypeptides with longer dipeptides are preferred due to the hydrophobicity of longer peptides.

[0594] Several dipeptide-based cleavable ADC linkers have been described for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepine, tamethasone, and orlistatine / orlistatine family members to TBM (see Dubowchik et al., 1998, J. Org. Chem [Organic Chemistry Journal] 67: 1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. [Bioorganic Chemistry and Medicinal Chemistry Communications] 8(21): 3341-3346; Walker et al., 2002, Bioorg. Med. Chem. Lett. [Bioorganic Chemistry and Medicinal Chemistry Communications] 12: 217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett. [Bioorganic Chemistry and Medicinal Chemistry Communications] 14: 4323-4327; Sutherland et al., 2013, Blood 122: 1455-1463; and Francisco et al., 2003, Blood 102: 1458-1465, each incorporated herein by reference. All of these dipeptide ADC adapters or modified forms of these dipeptide ADC adapters may be used in the ADCs disclosed herein. Other dipeptide ADC adapters that can be used include those found in ADCs, such as Seattle Genetics' SGN-35 (Adcetris™), Seattle Genetics' SGN-75 (anti-CD-70, Val-Cit-monomethylorestatin F (MMAF)), Seattle Genetics' SGN-CD33A (anti-CD-33, Val-Ala-(SGD-1882)), Celldex Therapeutics' glenoidumab (CDX-011) (anti-NMB, Val-Cit-monomethylorestatin E (MMAE)), and cytokinin PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[0595] Enzyme-cleavable ADC linkers can include suicide spacers to spatially separate the drug from the cleavage site. Direct attachment of the drug to the peptide ADC linker can lead to the proteolytic release of the drug's amino acid adducts, thereby impairing its activity. Using suicide spacers allows for the elimination of fully active, unmodified drugs during amide bond hydrolysis.

[0596] One type of suicide spacer is a bifunctional p-aminobenzyl alcohol group that links to a peptide via an amino group, forming an amide bond. The amine-containing drug can then attach to the benzyl hydroxyl group (PABC) of the ADC linker via a carbamate functional group. The resulting prodrug is activated upon protease-mediated cleavage, leading to a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and residues of the ADC linker group. The following protocol describes the fragmentation of p-aminobenzyl ether and the release of the drug:

[0597]

[0598] XD represents the unmodified drug.

[0599] Heterocyclic variants of this suicide group are also described. See, for example, U.S. Patent No. 7,989,434, which is incorporated herein by reference.

[0600] In some embodiments, the enzymatically cleavable ADC linker is a β-glucuronide-based ADC linker. Drug release can be achieved by cleaving the β-glucuronide glycosidic bond with the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and overexpressed in some tumor types, while its extracellular enzyme activity is low. β-glucuronide-based ADC linkers can be used to avoid the tendency of ADCs to aggregate due to the hydrophilicity of β-glucuronide. In some embodiments, β-glucuronide-based ADC linkers are preferably used as ADC linkers for ADCs linked to hydrophobic drugs. The following scheme describes drug release from an ADC containing a β-glucuronide-based ADC linker:

[0601]

[0602] Several β-glucuronic acid-based cleavable ADC linkers have been described for linking drugs such as orlistatine, camptothecin and doxorubicin analogs, CBI minor groove conjugates, and psymberin to the TBM (see, Nolting, Chapter 5, “Linker Technology in Antibody-Drug Conjugates” in: Antibody-Drug Conjugates: Methods in Molecular Biology, Vol. 1045, pp. 71-100, Laurent Ducry (ed.), Springer Science & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug. Chem. 17: 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett.). [Bioorganic Chemistry and Pharmaceutical Chemistry Communications] 17: 2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. [Journal of the American Chemical Society] 127: 11254-11255, each incorporated herein by reference. All of these β-glucuronic acid-based ADC adapters can be used in the ADCs disclosed herein.

[0603] Additionally, cytotoxic agents and / or cell inhibitors containing phenolic groups can be covalently bonded to the ADC linker via phenol oxygen. One such ADC linker described in WO 2007 / 089149 relies on a method in which a diamino-ethane “SpaceLink” is used in conjunction with a conventional “PABO”-based suicide group to deliver phenol. The cleavage of the ADC linker is illustrated below, where D represents a cytotoxic agent and / or cell inhibitor having a phenolic hydroxyl group.

[0604]

[0605] A cleavable ADC connector may include uncleavable portions or segments, and / or cleavable segments or portions may be included in another uncleavable ADC connector to make it cleavable. For example only, polyethylene glycol (PEG) and related polymers may include cleavable groups in the polymer backbone. For instance, a polyethylene glycol or polymer ADC connector may include one or more cleavable groups, such as disulfides, hydrazones, or dipeptides.

[0606] Other degradable linkages that may be included in the ADC linker include ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Hydrolyzable linkages include, but are not limited to, carbonate linkages; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by the reaction of alcohols with phosphate groups; acetal bonds as products of the reaction of aldehydes and alcohols; orthoester bonds as products of the reaction of formate esters and alcohols; and oligonucleotide bonds formed by phosphoramidite groups, including but not limited to 5' hydroxyl groups located at the polymer ends and on the oligonucleotides.

[0607] In some embodiments, the ADC adapter includes an enzymatically cleavable peptide moiety, for example, an ADC adapter comprising structure (IVa) or (IVb):

[0608]

[0609] Or its salts, wherein: peptide represents a peptide that can be cleaved by lysosomal enzymes (illustrated as C→N with the carboxyl and amino "termini" not shown); T represents a polymer containing one or more ethylene glycol units or alkylene chains or combinations thereof; R a Selected from hydrogen, alkyl, sulfonates, and methanesulfonates; P is an integer from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; Represents the attachment point of the ADC adapter to cytotoxic agents and / or cell inhibitors; and This represents the attachment point to the rest of the ADC connector.

[0610] In some embodiments, the peptide is selected from tripeptides or dipeptides. In specific embodiments, the dipeptide is selected from: Val-Cit, Cit-ValAla-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Ala-Val, Val-Ala, Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, and Trp-Cit. In some embodiments, the dipeptide is selected from: Cit-Val and Ala-Val.

[0611] Specific exemplary embodiments of the ADC connector according to structural formula (IVa) that may be included in the ADC disclosed herein include the ADC connector shown below (as shown, the ADC connector includes groups suitable for covalently linking the ADC connector to TBM):

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619] Specific exemplary embodiments of the ADC connector according to structural formula (IVb) that may be included in the ADC disclosed herein include the ADC connector shown below (as shown, the ADC connector includes groups suitable for covalently linking the ADC connector to TBM):

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639] In some embodiments, the ADC adapter includes an enzymatically cleavable peptide moiety, for example, an ADC adapter comprising the structure (IVc) or (IVd):

[0640]

[0641] Or its salts, wherein: peptide represents a peptide that can be cleaved by lysosomal enzymes (illustrated as C→N with the carboxyl and amino "termini" not shown); T represents a polymer containing one or more ethylene glycol units or alkylene chains or combinations thereof; R a Selected from hydrogen, alkyl, sulfonates, and methanesulfonates; P is an integer from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; x Represents the attachment point of the ADC adapter to cytotoxic agents and / or cell inhibitors; and This represents the attachment point to the rest of the ADC connector.

[0642] Specific exemplary embodiments of the ADC connector according to structural formula (IVc) that may be included in the ADC disclosed herein include the ADC connector shown below (as shown, the ADC connector includes groups suitable for covalently linking the ADC connector to TBM):

[0643]

[0644]

[0645]

[0646]

[0647]

[0648] Specific exemplary embodiments of the ADC connector according to structural formula (IVd) that may be included in the ADC disclosed herein include the ADC connector shown below (as shown, the ADC connector includes groups suitable for covalently linking the ADC connector to TBM):

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658] In some embodiments, the ADC connector comprising structures (IVa), (IVb), (IVc), or (IVd) further comprises a carbonate portion that is cleavable by exposure to an acidic medium. In specific embodiments, the ADC connector is attached to a cytotoxic agent and / or a cell inhibitor via oxygen attachment.

[0659] 6.9.2.2. Non-cuttable joints

[0660] While cleavable ADC linkers can offer certain advantages, the ADC linker comprising the ADC disclosed herein need not be cleavable. With incleavable ADC linkers, drug release is independent of differential properties between plasma and certain cytoplasmic compartments. It is assumed that drug release occurs after the ADC is internalized via antigen-mediated endocytosis and delivered to a lysosomal compartment, where the TBM is degraded to the amino acid level via intracellular proteolytic degradation. This process releases a drug derivative consisting of the drug, the ADC linker, and amino acid residues covalently attached to the ADC linker. Amino acid drug metabolites from conjugates with incleavable ADC linkers are more hydrophilic and generally have lower membrane permeability, resulting in fewer bystander effects and less nonspecific toxicity (compared to conjugates with cleavable ADC linkers). Generally, ADCs with incleavable ADC linkers exhibit greater stability in circulation than ADCs with cleavable ADC linkers. The indestructible ADC connector can be an alkylene chain, or it can be polymeric in nature, such as based on polyalkylene glycol polymers, amide polymers, or may include segments of alkylene chains, polyalkylene glycols and / or amide polymers.

[0661] Various non-cleavable ADC connectors for linking drugs to TBMs have been described. See Jeffrey et al., 2006, Bioconjug. Chem. 17: 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17: 2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127: 11254-11255, each incorporated herein by reference. All of these ADC connectors may be included in the ADCs disclosed herein.

[0662] In some embodiments, the ADC connector is inoperable in vivo, for example, an ADC connector according to structural formulas (VIa), (VIb), (VIc), or (VId) (as shown in the figure, the ADC connector includes groups suitable for covalently linking the ADC connector to the TBM:

[0663]

[0664]

[0665]

[0666] or its salt, wherein: R a Selected from hydrogen, alkyl, sulfonates and methanesulfonates; R x It is a portion containing functional groups capable of covalently connecting the ADC connector to the TBM; and (Represents the attachment point of the ADC connector to cytotoxic agents and / or cell inhibitors).

[0667] Specific exemplary embodiments of the ADC connector according to structural formulas (VIa)-(VId) that may be included in the ADC disclosed herein include the ADC connector shown below (as shown, the ADC connector includes groups suitable for covalently linking the ADC connector to the TBM, and...). (Representing the attachment point with cytotoxic agents and / or cell inhibitors).

[0668]

[0669]

[0670]

[0671]

[0672]

[0673] 6.9.2.3. Groups used to attach the connector to the TBM

[0674] A variety of groups can be used to attach ADC linkers-drug synthonants to TBMs to generate ADCs. Attachment groups can be inherently electrophilic and include: maleimide groups, activated disulfides, active esters (e.g., NHS esters and HOBt esters), haloformates, acyl halides, alkyl halides, and benzyl halides (e.g., haloacetamides). Emerging techniques related to “self-stabilizing” maleimides and “bridging disulfides” also exist, as discussed below, and can be used according to this disclosure. The specific groups used will depend in part on the attachment site with the TBM.

[0675] The schematic diagram below illustrates an example of spontaneous hydrolysis under TBM conjugation conditions to yield a “self-stabilizing” maleimide group for an ADC substance with improved stability. See US20130309256 A1; also see Lyon et al., Nature Biotech published online, doi: 10.1038 / nbt.2968.

[0676]

[0677]

[0678]

[0679] Polytherics has disclosed a method for bridging a pair of thiol groups derived from the reduction of a natural hinge disulfide bond. See Badescu et al., 2014, Bioconjugate Chem. [Bioconjugate Chemistry] 25:1124-1136. The reaction is illustrated in the schematic diagram below. One advantage of this method is the ability to synthesize enriched DAR4 ADCs by completely reducing IgG (to give 4 pairs of thiol groups) and then reacting it with 4 equivalents of an alkylating agent. ADCs containing “bridging disulfides” are also said to have improved stability.

[0680]

[0681] Similarly, as described below, maleimide derivatives capable of bridging a pair of thiol groups have been developed (see 1 below). See WO 2013 / 085925.

[0682]

[0683] 6.9.2.4. Considerations for ADC connector selection

[0684] As those skilled in the art will know, the ADC linker selected for a particular ADC can be influenced by a variety of factors, including but not limited to the attachment site to the TBM (e.g., lys, cys, or other amino acid residues), the structural constraints of the drug pharmacophore, and the lipophilicity of the drug. The specific ADC linker selected for an ADC should seek to balance these different factors for a particular TBM / drug combination. For a review of the factors influencing the selection of ADC linkers in an ADC, see Nolting, Chapter 5, “Linker Technology in Antibody-Drug Conjugates,” in: Antibody-Drug Conjugates: Methods in Molecular Biology, Vol. 1045, pp. 71–100, Laurent Ducry (ed.), Springer Science & Business Medica, LLC, 2013.

[0685] For example, ADCs have been observed to affect the killing of neighboring antigen-negative cells present near antigen-positive tumor cells. The mechanism of ADC-mediated killing of neighboring cells suggests that metabolites formed during the intracellular processing of ADCs can play a role. Neutral cytotoxic metabolites produced by ADC metabolism in antigen-positive cells appear to play a role in neighboring cell killing while preventing charged metabolites from diffusing across the membrane into the mediator, thus not affecting neighboring cell killing. In some embodiments, ADC adapters are selected to attenuate the neighboring cell killing effect caused by cellular metabolites of ADCs. In some embodiments, ADC adapters are selected to increase the neighboring cell killing effect.

[0686] The properties of the ADC linker can also affect ADC aggregation under usage and / or storage conditions. Typically, ADCs reported in the literature contain no more than 3–4 drug / antibody molecules (see, for example, Chari, 2008, Acc Chem Res [Chemical Research Reports] 41: 98–107). Attempts to obtain a higher drug-to-antibody ratio (“DAR”) often fail due to ADC aggregation, especially if both the drug and the ADC linker are hydrophobic (King et al., 2002, J Med Chem [Journal of Medicinal Chemistry] 45: 4336–4343; Hollander et al., 2008, Bioconjugate Chem [Bioconjugate Chemistry] 19: 358–361; Burke et al., 2009 Bioconjugate Chem [Bioconjugate Chemistry] 20: 1242–1250). In many cases, a DAR higher than 3–4 can be beneficial as a means of increasing potency. In cases where cytotoxic agents and / or cell inhibitors are inherently hydrophobic, it may be desirable to select relatively hydrophilic ADC connectors as a means of reducing ADC aggregation, especially where a DARS greater than 3-4 is desired. Therefore, in some embodiments, the ADC connector is incorporated with a chemical moiety that reduces ADC aggregation during storage and / or use. The ADC connector may be incorporated with polar or hydrophilic groups, such as charged groups, or groups that become charged at physiological pH, to reduce ADC aggregation. For example, the ADC connector may be incorporated with charged groups, such as salts or groups that deprotonate, for example, carboxylates or protonated compounds (e.g., amines) at physiological pH.

[0687] Exemplary multivalent ADC adapters that can generate up to 20 DARs and can be used to link various cytotoxic agents and / or cell inhibitors to TBMs are described in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO2014 / 093394; WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety.

[0688] In specific embodiments, such as as determined by size exclusion chromatography (SEC), the aggregation of the ADC during storage or use is less than about 10%. In specific embodiments, such as as determined by size exclusion chromatography (SEC), the aggregation of the ADC during storage or use is less than 10%, for example less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, or even lower.

[0689] 6.9.3. Methods for manufacturing ADCs

[0690] The disclosed ADC can be synthesized using well-known chemical substances. The selected chemical substances will depend in particular on the properties of one or more cytotoxic agents and / or cell inhibitors, the ADC adapter, and the groups used to attach the ADC adapter to the TBM. Typically, the ADC according to formula (I) can be prepared according to the following scheme:

[0691] DLR x +Ab-R y →[DL-XY] n -Ab (I)

[0692] Where D, L, Ab, XY, and n are as defined above, and R x and R y These represent complementary groups that can form covalent bonds with each other, as discussed above.

[0693] Group R x and R y The properties will depend on the synthetic DL-R used. xChemical substances conjugated to TBM. Generally, the chemicals used should not alter the integrity of the TBM, such as its ability to bind to its target. Preferably, the binding properties of the conjugated antibody will be very similar to those of the unconjugated TBM. A variety of chemicals and techniques for conjugating molecules to biomolecules, particularly immunoglobulins whose components are typically structural units of the TBM disclosed herein, are well known.See, for example, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy” in Monoclonal Antibodies And Cancer Therapy, edited by Reisfeld et al., Alan R. Liss, Inc., 1985; Hellstrom et al., “Antibodies For Drug Delivery” in Controlled Drug Delivery, edited by Robinson et al., Marcel Dekker, Inc., 2nd ed., 1987; Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies '84: Biological And Clinical Applications. Pinchera et al. (eds.), 1985; “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62: 119-58; PCT Publication WO 89 / 12624. Any of these chemicals can be used to link the synthon to TBM.

[0694] Many functional groups R used to attach synthons to accessible lysine residues xThe chemicals are known, and include, for example, but not limited to, NHS-esters and isothiocyanates.

[0695] Many functional groups R used to attach synthons to accessible free thiol groups of cysteine ​​residues x The chemicals are known and include, for example, but not limited to, haloacetyl and maleimide.

[0696] However, conjugated chemicals are not limited to available side-chain groups. By linking a suitable small molecule to an amine, the side chain of an amine can be converted into other useful groups, such as a hydroxyl group. This strategy can be used to increase the number of available linking sites on an antibody by conjugating a multifunctional small molecule to the side chain of an accessible amino acid residue of TBM. Then, a functional group R suitable for covalently linking the synthon to these "converted" functional groups is selected. x Included in the synthon.

[0697] TBMs can also be engineered to include amino acid residues for conjugation. Methods for engineering TBMs to include non-genetically encoded amino acid residues that can be used to conjugate drugs in the context of ADCs are described in Axup et al., 2012, Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences] 109(40): 16101-16106, as are the chemicals and functional groups used to link synthons to non-coding amino acids.

[0698] Typically, the synthon is attached to the side chain of an amino acid residue of TBM, including, for example, a primary amino group of an accessible lysine residue or a thiol group of an accessible cysteine ​​residue. The free thiol group can be obtained by reducing the interchain disulfide bond.

[0699] For R y It is the linkage of thiol groups (e.g., when R...) x When it is maleimide, the TBM is usually first completely or partially reduced to destroy the interchain disulfide bridges between cysteine ​​residues.

[0700] Cysteine ​​residues that do not participate in disulfide bridging can be engineered into TBMs by modifying one or more codons. Reduction of these unpaired cysteine ​​residues produces thiol groups suitable for conjugation. Preferred positions for incorporating engineered cysteine ​​residues include, for example, but not limited to: positions S112C, S113C, A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbers) on the human IgG1 heavy chain and positions V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbers) on the human Igκ light chain (see, for example, U.S. Patent Nos. 7,521,541, 7,855,275, and 8,455,622).

[0701] As those skilled in the art will understand, the number of cytotoxic agents and / or cell inhibitors linked to TBM molecules can vary, making the collection of ADCs inherently heterogeneous, with some TBMs containing one linked agent, some containing two linked agents, some containing three linked agents, etc. (and some containing no linked agents). The degree of heterogeneity will depend in particular on the chemicals used to link the cytotoxic agents and / or cell inhibitors. For example, when the TBM is reduced to produce a thiol group for attachment, a heterogeneous mixture of TBMs with 0, 2, 4, 6, or 8 linked agents per molecule is typically produced. Furthermore, by limiting the molar ratio of the attachment compounds, TBMs with 0, 1, 2, 3, 4, 5, 6, 7, or 8 linked agents per molecule are typically produced. Therefore, it should be understood that, depending on the context, the drug-to-TBM ratio (DTR) can be the average of the collection of TBMs. For example, "DTR4" can refer to an ADC formulation that has not undergone purification to separate a specific DTR peak and can contain a heterogeneous mixture of ADC molecules with varying amounts of attached cell inhibitors and / or cytotoxic agents / TBMs (e.g., 0, 2, 4, 6, 8 reagents / TBMs), but with an average drug-to-TBM ratio of 4. Similarly, in some embodiments, "DTR2" refers to a heterogeneous ADC formulation in which the average drug-to-TBM ratio is 2.

[0702] When it is desired to enrich a formulation, a TBM with a defined number of linked cytotoxic agents and / or cell inhibitors can be obtained by purifying a heterogeneous mixture, for example by column chromatography, such as hydrophobic interaction chromatography.

[0703] Purity can be assessed using a variety of methods known in the art. As a specific example, ADC formulations can be analyzed by HPLC or other chromatographic methods, and purity can be assessed by analyzing the area under the curve of the resulting peak.

[0704] 6.10. Formulation

[0705] This disclosure provides formulations comprising multiple TBMs and / or multiple TBM conjugates (e.g., at least 100, at least 1,000, at least 10,000, or at least 100,000 TBMs and / or TBM conjugates). Formulations include, for example, compositions comprising, a cell culture supernatant containing TBM molecules and enriched or purified TBM molecules (e.g., TBM fractionated or purified from the cell culture supernatant).

[0706] The formulation may include, for example, multiple TBMs or conjugates, wherein at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) of the trispecific molecules in the formulation have the same primary amino acid sequence. In various embodiments, 50% to 99% of the trispecific molecules in the formulation have the same primary amino acid sequence (e.g., 50% to 95%, 50% to 80%, 50% to 70%, 60% to 95%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 80%, 80% to 95%, 95% to 99%, or any range with any two of the above values ​​as endpoints).

[0707] In some embodiments, most of the trispecific molecules in the formulation have the same interchain crosslinks (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%). As used herein, "interchain crosslinks" refers to crosslinks between two linear polypeptide chains, such as crosslinks formed by disulfide bridges. In various embodiments, 50% to 99% of the trispecific molecules in the formulation have the same interchain crosslinks (e.g., 50% to 95%, 50% to 80%, 50% to 70%, 60% to 95%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 80%, 80% to 95%, 95% to 99%, or any range with any two of the above values ​​as endpoints).

[0708] In some embodiments, most of the trispecific molecules in the formulation have the same ABM1:ABM2:ABM3 ratio (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%). In various embodiments, 50% to 99% of the trispecific molecules in the formulation have the same ABM1:ABM2:ABM3 ratio (e.g., 50% to 95%, 50% to 80%, 50% to 70%, 60% to 95%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 80%, 80% to 95%, 95% to 99%, or any range with any two of the above values ​​as endpoints).

[0709] 6.11. Pharmaceutical Compositions

[0710] The TBMs disclosed herein (and their conjugates; unless the context otherwise requires, references to TBMs herein also refer to conjugates containing TBMs, such as ADCs) can be formulated into pharmaceutical compositions containing said TBMs, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition containing the TBMs disclosed herein, the TBM formulation may be combined with one or more pharmaceutically acceptable excipients or carriers.

[0711] For example, formulations of TBM can be prepared by mixing TBM with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, 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 Wilkins, New York, NY; Avis et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Decker, NY; Lieberman et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Decker, NY; Lieberman et al. (eds.), 1990; Pharmaceutical Dosage Forms: Disperse Systems, Marcel Decker, NY; Weiner and Kotkoskie, 2000; Excipient Toxicity and Safety, Marcel Decker, NY.

[0712] The selection of an administration regimen for TBM depends on several factors, including the serum or tissue turnover rate of TBM, symptom level, immunogenicity of TBM, and accessibility of target cells. In some embodiments, the administration regimen maximizes the amount of TBM delivered to the subject, consistent with acceptable levels of side effects. Therefore, the amount of TBM delivered depends in part on the specific TBM and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies and small molecules are available (see, for example, Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Decker, New York, NY; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Decker, NY; Baert et al., 2003, New England Journal of Medicine 348: 601-608; Milgrom et al., 1999, New England Journal of Medicine 341: 1966–1973; Slamon et al., 2001, New Engl. J. Med. 344:783–792; Beniaminovitz et al., 2000, New Engl. J. Med. 342:613–619; Ghosh et al., 2003, New Engl. J. Med. 348:24–32; Lipsky et al., 2000, New Engl. J. Med. 343:1594–1602).

[0713] The appropriate dosage is determined by a clinician, for example, using parameters or factors known or suspected to affect or expected to affect treatment. Typically, the dosage begins at an amount slightly less than the optimal dose and is then increased in small increments until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic values ​​include those for symptoms (e.g., inflammation) or the levels of inflammatory cytokines produced.

[0714] The actual dose level of TBM in the pharmaceutical compositions disclosed herein can be varied to obtain a specific amount of TBM that effectively achieves a desired therapeutic response for a particular subject, composition, and route of administration without toxicity to said subject. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific TBM, route of administration, time of administration, excretion rate of the specific TBM used, duration of treatment, other agents combined with the specific TBM used (e.g., active agents, such as therapeutic drugs or compounds and / or inert materials used as carriers), the age, sex, weight, condition, general health status, and prior medical history of the treated subject, and similar factors known in the medical field.

[0715] Compositions containing the TBM disclosed herein may be administered by continuous infusion or by dose at intervals such as daily, weekly, or 1-7 times per week. Dosage may be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dosing regimens are those involving the maximum dose or frequency of administration to avoid significant undesirable side effects.

[0716] The effective dose for a particular subject may vary depending on 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, for example, Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Florida; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0717] The route of administration can be, for example, local or dermal application, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional injection or infusion, or via sustained-release systems or implants (see, for example, Sidman et al., 1983, Biopolymers 22: 547-556; Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12: 98-105; Epstein et al., 1985 Proc. Natl. Acad. Sci. USA 82: 3688-3692; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA 77: 4030-4034; U.S. Patent Nos. 6,350,466 and 6,316,024. Where necessary, the composition may also contain a solubilizer and a local anesthetic (such as lidocaine) to reduce pain at the injection site. Alternatively, pulmonary administration may be used, for example, by using an inhaler or nebulizer and a formulation containing a nebulizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0718] The compositions disclosed herein can also be administered via one or more of the various methods known in the art through one or more routes of administration. As those skilled in the art will understand, the route and / or mode of administration will vary depending on the desired outcome. Selected routes of administration for TBM include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other general routes of administration, such as by injection or infusion. General administration can represent administration methods other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions disclosed herein can be administered via non-general routes, such as local, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration. In one embodiment, the TBM is administered by infusion. In another embodiment, the multispecific epitope-binding protein of the present disclosure is administered subcutaneously.

[0719] If TBM is administered using a controlled-release or sustained-release system, a pump can be used to achieve controlled-release or sustained-release (see Langer, ibid.; Sefton, 1987, CRC Crit. Ref Biomed. Eng. [CRC Reference Review in Biomedical Engineering] 14: 20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. [New England Journal of Medicine] 321: 574). Polymer materials can be used to achieve controlled or sustained release of the therapeutic agents disclosed herein (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. [Journal of Neurosurgery] 71: 105); US Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl acrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolic acid) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, stable during storage, sterile, and biodegradable. Controlled-release or sustained-release systems can be placed near a preventative or therapeutic target, thus requiring only a portion of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115–138 (1984)).

[0720] Controlled-release systems are discussed in Langer's review (1990, Science 249: 1527-1533). Sustained-release formulations comprising one or more TBMs disclosed herein can be produced using any technique known to those skilled in the art. See, for example, U.S. Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al., 1996, Radiotherapy & Oncology 39: 179-189; Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50: 372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety.

[0721] If TBMs are applied topically, they can be formulated as ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical DosageForms, 19th edition, Mack Pub. Co., Easton, Pennsylvania (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms are typically used, containing a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some cases greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, etc., and may be sterile or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to influence various properties, such as osmotic pressure. Other suitable topical dosage forms include aerosol formulations, in which, in some cases, the active ingredient is packaged in combination with a solid or liquid inert carrier in a mixture or squeeze bottle containing a pressurized volatile substance (e.g., a gaseous propellant such as Freon). Humectants or wetting agents may also be added to the pharmaceutical composition and dosage form if desired. Examples of such other ingredients are well known in the art.

[0722] If a composition containing TBM is administered intranasally, the TBM can be formulated as an aerosol, spray, nebulizer, or drops. In particular, preventative and therapeutic agents used according to this disclosure can be conveniently delivered using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas) in the form of an aerosol spray from a pressurized package or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a measured amount. Capsules and cartridges (composed of, for example, gelatin) used in inhalers or blowpipes can be formulated as a powder mixture containing the compound and a suitable powder base (such as lactose or starch).

[0723] The TBM disclosed herein can be administered in combination therapy regimens, as described in Section 6.13 below.

[0724] In some embodiments, TBMs can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds disclosed herein cross the BBB (if desired), they can be formulated, for example, as liposomes. For methods of preparing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may include one or more portions that are selectively transported to specific cells or organs, thereby enhancing the delivery of targeted drugs (see, for example, Ranade, 1989, J. Clin. Pharmacol. [Journal of Clinical Pharmacology] 29:685). Exemplary targeting components include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988, Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 153: 1038); antibodies (Bloeman et al., 1995, FEBS Lett. [Federation of European Biochemical Societies Letters] 357: 140; Owais et al., 1995, Antimicrob. Agents Chemother. [Antimicrobial Agents Chemotherapy] 39: 180); surfactant protein A receptor (Briscoe et al., 1995, Am. J. Physiol. [American Journal of Physiology] 1233: 134); p120 (Schreier et al., 1994, J. Biol. Chem. [Journal of Biochemistry] 269: 9090); see also Keinanen and Laukkanen, 1994, FEBS Lett. [Federation of European Biochemical Societies Letters] 346: 123; Killion and Fidler, 1994, Immunomethods [Immunomethods] 4: 273.

[0725] When used in combination therapy, for example, as described in Section 6.13 below, the TBM disclosed herein and one or more other agents may be administered to a subject in the same pharmaceutical composition. Alternatively, the TBM and one or more other agents of combination therapy may be administered to a subject in parallel as separate pharmaceutical compositions.

[0726] The treatment methods described herein may further include performing a “companion diagnostic” test, thereby testing a sample from a subject who is a candidate for the TBM therapy disclosed herein for expression of a TAA targeted by ABM3. The companion diagnostic test may be performed before the initiation of the TBM therapy disclosed herein and / or during the TBM treatment regimen disclosed herein to monitor the subject’s continued suitability for the TBM therapy. The agent used in the companion diagnostic may be TBM itself or another diagnostic agent, such as a monospecific antibody labeled against a TAA recognized by ABM3 or a nucleic acid probe for detecting TAA RNA. Samples that can be tested in the companion diagnostic assay may be any sample in which cells targeted by TBM may be present, such as tumor (e.g., solid tumor) biopsies, lymph, feces, urine, blood, or any other bodily fluid that may contain circulating tumor cells.

[0727] 6.12. Therapeutic Indications

[0728] The TBM disclosed herein can be used to treat any proliferative disorder expressing TAA (e.g., cancer). In specific embodiments, the cancer is HER2+ carcinoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloproliferative neoplasm (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, and gestational trophoblastic disease. Glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal 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, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin in the neck. Squamous neck cancer with occult primary, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome. (syndrome), skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or nephroblastoma.

[0729] Table 14 below shows exemplary indications for which TBM targeting a specific TAA can be used.

[0730]

[0731]

[0732]

[0733]

[0734] Therefore, this disclosure provides a method of treating cancer, comprising administering a TBM to a subject with cancer, wherein ABM3 (i.e., TAA ABM) binds to a TAA expressed on that type of cancer. In some embodiments, a TBM targeting a TAA identified in Table 14 may be administered to a subject with cancer expressing a TAA as shown in Table 14. For example, but not limited to, a TBM targeting EPCAM or folate receptor α may be administered to a subject with colorectal cancer; a TBM targeting BCMA or CD19 may be administered to a subject with hematologic malignancies such as multiple myeloma; a TBM targeting PSCA or PCMA may be administered to a subject with prostate cancer; a TBM targeting tyrosinase or GP3 may be administered to a subject with melanoma; and a TBM targeting CD33, CLL-1, or FLT3 may be administered to a subject with hematologic malignancies such as acute myeloid leukemia.

[0735] 6.13. Combination Therapy

[0736] The TBM disclosed herein can be used in combination with other known drugs and therapies. For example, the TBM disclosed herein can be used in treatment regimens in combination with surgery, chemotherapy, antibodies, radiation, peptide vaccines, steroids, cytotoxins, or combinations thereof.

[0737] For convenience, agents used in combination with the TBM disclosed herein are referred to herein as “other” agents.

[0738] As used herein, “combined” administration means the delivery of two (or more) different treatments to a subject during the course of illness, such as after the subject has been diagnosed with a disorder and before the disorder is cured or cleared, or before treatment is terminated for other reasons. In some embodiments, the delivery of the first treatment is still in progress when the delivery of the second treatment begins, so there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous delivery” or “parallel delivery.” The term “parallel” is not limited to administering therapies (e.g., TBM and other agents) at exactly the same time, but rather means administering a pharmaceutical composition containing the TBM of this disclosure to a subject in a sequence and at time intervals such that the TBM of this disclosure can work in conjunction with one or more other therapies to provide an increased benefit (compared to if they were administered in other ways). For example, each therapy may be administered to the subject at the same time or in any order at different time points; however, if not administered at the same time, they should be administered sufficiently close in time to provide the desired therapeutic effect.

[0739] The TBM disclosed herein and one or more other agents may be administered simultaneously or sequentially in the same or separate compositions. For sequential administration, the TBM may be administered first, followed by another agent, or the order of administration may be reversed.

[0740] The TBM and one or more other agents may be administered to the subject in any suitable form and via any suitable route. In some embodiments, the route of administration is the same. In other embodiments, the route of administration is different.

[0741] In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins.

[0742] In some embodiments of each scenario, the treatment is more effective due to combined administration. For example, the second treatment may be more effective than observed when the first treatment is administered in its absence, such as by observing an equivalent effect with less second treatment, or by reducing symptoms to a greater extent, or by observing a similar effect to the first treatment. In some embodiments, delivery results in a greater reduction in symptoms or other parameters associated with the disorder compared to the results observed when one treatment is not administered. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effect of the first treatment remains detectable when the second treatment is delivered.

[0743] The TBM and / or other agents disclosed herein may be administered during periods of impairment or during remission or low activity of the disease. The TBM may be administered prior to treatment with one or more other agents, concurrently with treatment with one or more other agents, after treatment with one or more other agents, or during periods of impairment remission.

[0744] When used in combination, TBM and / or one or more additional agents may be administered at a higher, lower, or same amount or dose than each agent used alone (e.g., as a monotherapy).

[0745] One or more additional agents of the combination therapy disclosed herein may be administered to a subject in parallel. The term "parallel" is not limited to administering the therapy (e.g., a prophylactic or therapeutic agent) at exactly the same time, but rather means administering the pharmaceutical composition comprising the TBM of this disclosure to a subject in a sequence and at time intervals, such that the molecules of this disclosure can work together with one or more additional therapies to provide an increased benefit (compared to if they were administered in other ways). For example, each therapy may be administered to a subject at the same time or in any order at different time points; however, if not administered at the same time, the therapies should be administered sufficiently close in time to provide the desired therapeutic or preventative effect. Each therapy may be administered to a subject separately in any suitable form and via any suitable route.

[0746] The TBM and one or more other agents may be administered to the subject via the same or different routes of administration.

[0747] The TBM and one or more other agents can be cyclically administered. Cyclic therapy involves administering a first therapy (e.g., a first preventative or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second preventative or therapeutic agent) for a period of time, optionally followed by administering a third therapy (e.g., a preventative or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., cyclic, to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and / or to improve the efficacy of the therapies.

[0748] In some cases, the one or more additional agents mentioned are other anticancer agents, antiallergic agents, antinausea agents (or antiemetics), analgesics, cell protectants, and combinations thereof.

[0749] In one embodiment, the TBM disclosed herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinblastine alkaloids (e.g., vincristine, vinorelbine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alenzusmab, gemtuzumab, rituximab, tosimomab, obbituzumab, famumab, daratumumab, ileotuzumab), antimetabolites (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclarubicin A, mycotoxin, or bortezomib), and immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0750] Common chemotherapy agents considered for combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), and cisplatin (Platinol®). ), Leustatin®, Cyclophosphamide (Cytoxan® or Neosar®), Cytarabine, Cytosar-U®, Cytarabine Liposome Injection (DepoCyt®), Dacarbazine (DTIC-Dome®), Cosmegan (actinomycin D), Cerubidine®, DaunoXome®, Dexamethasone, Taxotere® Doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), and ifosfamide (IFEX®). Irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), gemtuzumab (mylotarg), paclitaxel (Taxol®), phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 cocarmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0751] Of particular interest are anticancer agents used in combination with the TBM disclosed herein, including: anthracycline; alkylating agents; antimetabolites; inhibitors of calcium-dependent phosphatase (calcineurin or p70S6 kinase FK506) or drugs that inhibit p70S6 kinase; mTOR inhibitors; immunomodulators; anthracycline; vinca alkaloids; proteasome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; CDK4 kinase inhibitors; BTK inhibitors; MKN kinase inhibitors; DGK kinase inhibitors; or oncolytic viruses.

[0752] Exemplary alkylating agents include, but are not limited to, nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazines: uramustine (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen). Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®, nitrogen mustard (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), piperobromidine (Amedel®, Vercyte®), triethylene melamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphamide, temozolomide (Temodar®), thioplex®, busilvex®, myleran®, carmustine (BiCNU®), lomustine (CeeNU®), streptozotocin (Zanosar®), and dacarbazine (DTIC-Dome®).Other exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); actinomycin D (also known as actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcomain, and phenylalanine mustard, Alkeran®); hexalen® (also known as hexamethylmelamine (HMM)); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); and lomustine (also known as CCN). U, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Leukeran® chlorambucil; Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazocarbamide, DTIC-Dome®); Hexalen® hexamethylmelamine (also known as hexamethylmelamine (HMM)); Ifex®; Prednumustine; Matulane®; Mechlorethamine (also known as nitrogen mustard) Mustard, nitrogen mustard and nitrogen mustard hydrochloride (Mustargen®); streptozotocin (Zanosar®); thiophosphoamide (also known as thiophosphoamide, TESPA and TSPA, Thioplasty®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine hydrochloride (Treanda®).

[0753] Exemplary mTOR inhibitors include, for example, tamsulosin; defotiolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-Hexamethyl-2,3,10,14,20-pentaoxa-11,36-diaza-4-azatricyclo[30.3.1.04,9]hexadecane-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinic acid ester, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS) 164301-51-3); emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H...

Claims

1. A trispecific binding molecule (TBM) comprising: (a) Antigen-binding module 1 (ABM1) specifically binds to human CD2. (b) Antigen-binding module 2 (ABM2) that specifically binds to components of the human T-cell receptor (TCR) complex; and (c) Antigen binding module 3 (ABM3) that specifically binds to human tumor-associated antigens (TAAs).

2. The TBM of claim 1, wherein each antigen-binding module is capable of binding its respective target while binding to each of the other antigen-binding modules with its respective target.

3. The TBM as described in claim 1, wherein ABM1 is: (a) An immunoglobulin scaffold-based ABM, optionally comprising an anti-CD2 antibody, an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain; or (b) ABM based on a non-immunoglobulin scaffold, optionally being a Kunitz domain, Adnexin, affinity, DARPin, Avimer, Anticalin, lipid transporter, Centyrin, Versabody, Knottin, Adnectin, Pronectin, Affitin / Nanofitin, Affilin, Atrimer / tetraconnector, bicyclic peptide, cys-knot, Fn3 scaffold, Obody, Tn3, Aan Affimer, BD, Adhiron, Duocalin, Alphabody, Armadillo repeat protein, Repebody, or Fynomer.

4. The TBM as claimed in claim 3, wherein ABM1 is scFv or Fab.

5. The TBM of claim 3, wherein ABM1 comprises any binding sequence listed in Table 9.

6. The TBM of claim 1, wherein ABM1 comprises a receptor-binding domain of a CD2 ligand.

7. The TBM as claimed in claim 1, wherein ABM1 is a CD58 portion.

8. The TBM of claim 7, wherein ABM1 comprises amino acids 1-94 of CD58-2.

9. The TBM as claimed in claim 1, wherein ABM1 is a CD48 portion.

10. The TBM of claim 1, wherein the component of the human TCR complex is CD3.

11. The TBM of claim 10, wherein ABM2 is: (a) An immunoglobulin scaffold-based ABM, optionally comprising an anti-CD3 antibody, an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain; or (b) ABM based on a non-immunoglobulin scaffold, optionally being a Kunitz domain, Adnexin, affinity, DARPin, Avimer, Anticalin, lipid transporter, Centyrin, Versabody, Knottin, Adnectin, Pronectin, Affitin / Nanofitin, Affilin, Atrimer / tetraconnector, bicyclic peptide, cys-knot, Fn3 scaffold, Obody, Tn3, Aan Affimer, BD, Adhiron, Duocalin, Alphabody, Armadillo repeat protein, Repebody, or Fynomer.

12. The TBM of claim 11, wherein ABM2 is scFv or Fab.

13. The TBM of claim 11, wherein ABM2 comprises any binding sequence listed in any one of Tables 7A to 7D.

14. The TBM of claim 13, wherein ABM2 comprises VH and VL sequences of CD3-21 as listed in Table 7A.

15. The TBM of claim 1, wherein the component of the human TCR complex is the α subunit of TCR.

16. The TBM of claim 15, wherein ABM2 is an anti-CD3 antibody, an antibody fragment, scFv, Fv, dsFv, Fab, scFab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, a camel VHH domain, DARPin, Avimer, Anticalin / lipotransporter, Centyrin, Versabody, Duocalin, or Fynomer.

17. The TBM of claim 1, wherein if TAA is a receptor, then ABM3 comprises a receptor-binding domain of a ligand of the receptor, and if TAA is a ligand, then ABM3 comprises a ligand-binding domain of a receptor of the ligand.

18. The TBM of claim 1, wherein ABM3 is: (a) An immunoglobulin scaffold-based ABM, optionally comprising an anti-TAA antibody, an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, or a camel VHH domain; or (b) ABM based on a non-immunoglobulin scaffold, optionally being a Kunitz domain, Adnexin, affinity, DARPin, Avimer, Anticalin, lipid transporter, Centyrin, Versabody, Knottin, Adnectin, Pronectin, Affitin / Nanofitin, Affilin, Atrimer / tetraconnector, bicyclic peptide, cys-knot, Fn3 scaffold, Obody, Tn3, Aan Affimer, BD, Adhiron, Duocalin, Alphabody, Armadillo repeat protein, Repebody, or Fynomer.

19. The TBM of claim 18, wherein the TAA is TSHR, CD171, CS-1, CLL-1, GD3, TnAg, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, EGFRvIII, NCAM, CAIX, LMP2, EphA2, fucose GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, G D2, folate receptor α, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, spermin 17, XAGE1, Tie 2. MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD19, CD20, CD30, ERBB2, ROR1, FLT3, TAAG72, CD22, CD33, GD2, BCMA, gp100Tn, FAP, tyrosinase, EPCAM, CEA, IgF-I receptor, cadherin 17, CD32b, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, TACSTD2, or EphB2.

20. The TBM of claim 18, wherein the TAA is a BCMA.

21. The TBM of claim 20, wherein ABM3 comprises any binding sequence listed in any one of Tables 12A, 12B, 12C, 12D, 12E, or 12F.

22. The TBM of claim 18, wherein the TAA is CD19.

23. The TBM of claim 22, wherein ABM3 comprises any binding sequence listed in Table 13.

24. The TBM of claim 18, wherein the TAA is Her2.

25. The TBM of claim 18, wherein the TAA is mesothelin.

26. The TBM of claim 18, wherein ABM3 is scFv or Fab.

27. The TBM as described in claim 1, wherein it is a trivalent TBM.

28. The TBM of claim 27, wherein the trivalent TBM has any of the configurations depicted in Figures 1B-1U and 1V-1Z.

29. The TBM of claim 28, having the configuration described in FIG1I.

30. The TBM of claim 29, wherein the ABM has a configuration designated as T6.

31. The TBM as claimed in claim 1, wherein it is a tetravalent TBM.

32. The TBM as described in claim 1, wherein it is a pentavalent TBM.

33. The TBM as described in claim 1, wherein it is a hexavalent TBM.

34. A conjugate comprising TBM as claimed in any one of claims 1 to 33, and a cytotoxic or cytotoxic inhibitor.

35. A pharmaceutical composition comprising TBM as claimed in any one of claims 1 to 33, and an excipient.

36. A method of treating a subject with cancer, comprising administering to the subject with cancer an effective amount of TBM as described in any one of claims 1 to 33.

37. The method of claim 36, wherein the cancer is selected from HER2+ carcinoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, primary unknown cancer, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colon Rectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip cancer And oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, primary metastatic squamous cell carcinoma of the neck, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, breast cancer Head tumors, paragangliomas, parathyroid carcinomas, penile cancer, laryngeal cancer, pheochromocytoma, pituitary tumors, pleural pulmonary blastomas, primary central nervous system lymphomas, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumors, salivary gland cancer, Cezari syndrome, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, spinal cord tumors, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and nephroblastoma.

38. One or more nucleic acids, said one or more nucleic acids encoding TBM as claimed in any one of claims 1 to 33.

39. A cell engineered to express TBM as claimed in any one of claims 1 to 33.

40. A cell transfected with one or more expression vectors under the control of one or more promoters, the expression vectors comprising one or more nucleic acid sequences encoding TBM as described in any one of claims 1 to 33.

41. A method for generating TBM, comprising: (a) The cells as described in claim 40 are cultured under the conditions of TBM expression; as well as (b) The TBM was recovered from the cell culture.

Images